kidney-replenishing herb induces socs-3 expression via erk...

Research ArticleKidney-Replenishing Herb Induces SOCS-3Expression via ERKMAPK Pathway and Improves Growth ofthe First-Trimester Human Trophoblast Cells

Chang-ying Xing1 De-xiang Zhang2 Sui-qi Gui3 andMin-fang Tao1

1Shanghai Jiao Tong University Affiliated Sixth Peoplersquos Hospital Shanghai 200233 China2Affiliated Hospital of Nantong University Nantong 226001 China3The Hospital of Obstetrics amp Gynecology Shanghai 200011 China

Correspondence should be addressed to Chang-ying Xing xingchangying8163com

Received 9 February 2017 Revised 18 July 2017 Accepted 2 August 2017 Published 28 September 2017

Academic Editor Kuang C Lai

Copyright copy 2017 Chang-ying Xing et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Kidney-replenishing herb is a traditional medicine formula in China which has been widely used for clinical treatment of recurrentmiscarriage Our previous study showed that Kidney-replenishing herb could promote proliferation and inhibit apoptosis of thehuman first-trimester trophoblasts In the present study we further explored the potential mechanism and signal pathway ofKidney-replenishing herb on human trophoblast cells Our research showed that Kidney-replenishing herb stimulated proliferationand reduced apoptosis of human trophoblast cells in vitro and this appeared to be positive correlation with SOCS-3 transcriptionsuggesting that Kidney-replenishing herb regulated biological functions of human trophoblast cells by inducing SCOS-3 expressionFurthermore the Kidney-replenishing herb treatment stimulated the phosphorylation of ERK12 and blocking the signalingpathway by mitogen-activated protein MAPK (MEK) inhibitor U0126 inhibited Kidney-replenishing herb-induced SOCS-3transcription depressed proliferation and promoted apoptosis of human trophoblasts Kidney-replenishing herbs still inducedERK12 phosphorylation after SOCS-3 siRNA silence Overexpression of SOCS-3 stimulated the proliferation of trophoblastThesefindings suggest that SOCS-3 expression is induced by Kidney-replenishing herbs via activation of MAPK pathways and this maypossibly be involved in promoting human trophoblast cells growth which is contributed to embryo development

1 Introduction

A successful pregnancy requires perfect placenta conditionsThe early placental development is defined by an intri-cate balance between cellular proliferation and apoptosis oftrophoblast cells [1 2] Proliferation markers are stronglyexpressed in cytotrophoblast in early stages of gestation [3]At the same time apoptosis is present in trophoblast cellsthroughout gestation and is believed to be physiologicallyimportant for normal placental development and fetal growth[4] It is well accepted that the insufficient trophoblast growthor excessive apoptosis occurs in the placenta of humanfirst-trimester spontaneous pregnancy loss and fetal growthrestriction Our previous studies demonstrated the positiveinfluence of Kidney-replenishing herb on the proliferation

and inhibition of IFN-120574 induced apoptosis of human first-trimester trophoblasts [5] However the roles of Kidney-replenishing herb in regulation of trophoblast cells functionand relative molecular mechanisms remain unclear

The extracellular signal-regulated kinase 12 (ERK12)mitogen-activated protein kinase (MAPK) pathway is one ofthe important signaling cascades which is involved in the cellproliferation and embryo development furthermore ERK12MAPK signal transduction also provides protection againstapoptosis in several cell types when they are challenged bycellular stresses or chemotherapeutic drugs [6] ERK12 iswidely expressed and markedly activated in the villous cytot-rophoblasts throughout early embryonic development [7]Mice embryos which lacked ERK2 died early during embryo-genesis because of the defection in trophoblast development

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 2473431 12 pageshttpsdoiorg10115520172473431

2 Evidence-Based Complementary and Alternative Medicine

Mutant embryos fail to form the ectoplacental cone andextraembryonic ectoderm which derives from the polar tro-phectoderm [8] These indicate that ERK2 is required for theproliferation of trophoblast stem cells in the polar tro-phectoderm and ERK12MAPK pathway has an importantfunction in the regulation of trophoblasts growth

Suppressors of cytokine signaling (SOCS) protein are afamily of intracellular proteins that control cytokine signaling[9] The SOCS family consists of at least eight membersincluding cytokine induced SH2 protein (CIS) and SOCS-1ndash7 SOCS proteins contain a central Src homology 2 domainprotein (SH2) domain a conserved C-terminus referred toas the SOCS box and unique N-terminal region [10] SOCSfamily proteins can be induced by cytokines growth factorsand several immunomodulators Roberts and colleaguesshowed that mice with a deletion of SOCS-3 gene died atmidgestation because of the placental defects They observedthat SOCS-3(minusminus) embryos were slightly smaller than thewild type but appeared otherwise normal However theplacental spongiotrophoblast layer was significantly reducedand accompanied by increased numbers of giant trophoblastcells The network of embryonic vessels and maternal sinuseswas inadequately developed and yolk sac erythropoiesiswas normal They concluded that the embryonic lethalityis not caused by anatomical defects of the embryo butrather poor placental development that results in the embryodevelopmental arrest and death [11] Therefore it is believedthat SOCS-3 is critical for a successful pregnancy outcome byregulating trophoblast function during the placental develop-ment An interesting study reported by Isobe and colleaguesshowed that MEK antagonist could inhibit SOCS-3 expres-sion of the undifferentiated rat trophoblast-like cell line [12]Another study has demonstrated that SOCS-3 binds and inac-tivates RasGAP a negative regulator of Ras signaling leadingto increased RasMAPK pathway activity in human JEG-3trophoblastic cells [13] These data suggest that the activationof ERK12MAPK pathway signal pathway may be relatedto SOCS-3 expression However the roles of SOCS-3 in theinduction of proliferation processes and relative molecularmechanisms in primary first-trimester human trophoblastsare still unclear

In this study we observed the effects of Kidney-replenish-ing herbs on the proliferation and apoptosis of human first-trimester trophoblasts we therefore investigated the effects ofthe herbs on the expression of SOCS-3 in human trophoblastsby reverse transcription-polymerase chain reaction (RT-PCR) and Western blot In order to elucidate the intracel-lular signal pathway mediating the upregulation in SOCS-3expression by Kidney-replenishing herb we investigated thephosphorylation of ERK12 signal pathway involved in theproliferative and apoptosis of human trophoblasts by Kidney-replenishing herb

2 Materials and Methods

21 Isolation and Cultivation of Human First-TrimesterCytotrophoblast First-trimester human villous tissues (5ndash10weeks of gestation) were collected from clinically normalpregnancies which were terminated for nonmedical reasons

at the Hospital of Obstetrics and Gynecology Fudan Univer-sity Shanghai Medical College The study was approved bythe Fudan University Ethical Review Board and each patientsigned the consent form Villous tissues were immediatelysuspended in ice-cold DMEM and transmitted to the labora-tory where they were washed 2-3 times in sterile phosphate-buffered saline (PBS) to remove the excess blood Primarytrophoblast cells were isolated by trypsin-DNase type I diges-tion and layered over a discontinuous Percoll gradient asdescribed by our previous study [14] This assay suppliesa 95 purity of trophoblast cells These isolated humantrophoblast cells and the choriocarcinoma JAR cell line werecultured in DMEM supplemented with 2mMglutamine 10FCS at 37∘C in 5 CO

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22 Preparation of Serum Kidney-replenishing herbs aretraditional medicines which have satisfactory effects onthreatenedmiscarriages includingDangshen 12 g Tusizi 15 gBaishu 6 g Baishao 9 g Duzhong 12 g Sangjisheng 12 gSugeng 6 g and Tiaohuangqin 15 g Herb serum was madeaccording to our previous study [14] All of the herbs werecollected and made by the scholars of Shanghai GraduateSchool of Medication (Chinese Academy of Science)

23 Materials Histochemical ABC kit was purchased fromSino-American Bio-Technology (Luoyang China) Rever-tAid First Strand cDNA Synthesis Kit and Taq DNApolymerase were purchased from Fermentas (Thermo FisherScientificMAUSA) BCA-100 ProteinQuantitative AnalysisKit was purchased from Sangon Biotechnology (ShanghaiChina) Mouse anti-GAPDH monoclonal antibodies andplasmid extraction kit were purchased from KangchengBiotechnology (Shanghai China) Rabbit anti-human SOCS-3 antibodies mouse anti-phosphoERK monoclonal antibod-ies rabbit anti-ERKmonoclonal antibodies and mouse anti-GAPDH were purchased from Santa Cruz Biotechnology(Santa Cruz CA USA) Annexin V-FITC apoptosis kitPRK5 plasmid and pRNAT-U61 plasmid were purchasedfrom RampD Systems (Minneapolis MN USA) HRP-goatanti-rabbit IgG was purchased from Dingguo Biotechnol-ogy (Shanghai China) TRIzol reagent BLOCK-iT oligo[13750062] and Lipofectamine 2000 Kit were purchasedfrom Invitrogen (Carlsbad CA USA) SOCS-3 forwardprimer (51015840-CGCCTCAAGACCTTCAGCTC-31015840) and reverseprimer (51015840-ATCCAGGAACTCCCGAA-31015840) product 650 bpGAPDH forward primer (51015840-GAAGGTGAAGGTCGGAGT-C-31015840) and reverse primer (51015840-GATGGTGATGGGATTTC-31015840) and product 220 bp were designed and integrated byKangcheng Biotechnology (Shanghai China)

24 RT-PCR Analysis of SOCS-3 Expression in Human First-Trimester Trophoblast Total cellular RNA ofhuman first-trimester trophoblast was extracted using TRIzol reagentand cDNA Synthesis Kit according to the manufacturerrsquosprotocol The total RNA of 3 120583g was denatured and RT wasperformed for 1 h at 42∘Cwith 05mgOligo dT 05 120583g 10mMdNTP 2 120583l RNase inhibitor 1120583l 200UMoloney virus-reversetranscriptase and 10 reaction buffer in a total volume of20ml After 5min precycle at 95∘C 30 cycles of denaturation

Evidence-Based Complementary and Alternative Medicine 3

at 94∘C (50 s) the reaction was followed by 30 cycles of 50 s at55∘C and 50 s at 72∘CWhen the final cycle was over sampleswere kept at 72∘C for 15min for the further extension ThePCR products were separated using a 2 agarose gel andethidium bromide-stained bands were photographed Therelative intensity of SOCS-3 is the ratio of the absorbancevalue of SOCS-3 to that of GAPDH

25 Western Blot Cells were harvested and lysed for 30minin 1ml of RIPA buffer (50mM TrisndashHCl pH 74 150mMNaCl 1 NP-40 10mM NaF 025 sodium deoxycholate1mM EDTA 1mM PMSF and phosphatase inhibitors) and10 120583l cocktail the extracts were incubated for 20min on iceand cleared by centrifugation Samples were incubated inSDS-PAGE (SDS-polyacrylamide gel electrophoresis) samplebuffer at 95∘C for 10minThe proteins were separated in a gelcontaining 10 acrylamide After that the separated proteinswere transferred onto PVDF membrane The membraneswere incubated with 5 nonfat milk powder in Tris-bufferedsaline (05M Tris 15M NaCl) supplemented with 01Tween (TBST) for 1 h Incubation overnight at 4∘C with theprimary antibody SOCS-3 diluted 1 500 for detection ofSOCS-3 anti-GAPDH diluted 1 5000 for detection ofGAPDH anti-ERK12 diluted 1 1500 for detection of ERK12and antiphosphorylated ERK12 diluted 1 1000 for detectionof phosphorylated ERK12 After washing with TBST threetimes the membrane was incubated with horseradish perox-idase secondary antibodies at a dilution of 1 2000 in 1 BSA-TBST at 37∘C for 1 h The proteins were detected using ECLWestern blotting detection reagents

26 Immunohistochemistry for SOCS-3 in Villous TissueParaffin-embedded sections of human first-trimester placen-tal bed samples were deparaffinized rehydrated and antigenretrieved Endogenous peroxides activity was quenched with3 H2O2 Samples were covered with normal blocker serum

and incubated with rabbit anti-human SOCS-3 antibodyat 1 100 dilution The sections were then treated withappropriate avidin-biotin histostain kit according to themanufacturerrsquos instructions Slides were stained with DABand counterstained with haematoxylinThe isotype-matchedcontrol antibodies were used to exclude nonspecific staining

27 Flow Cytometry (FCM) FCM was applied to measuredcells proliferation Human trophoblasts were harvested andfixed 30min with 70 ethanol at 4∘C and then resuspendedin 05ml of PBS containing PE-PCNA mouse anti-humanantibody (20120583l106 cells) incubated at 37∘C for 30min A totalof 20000 cells were routinely acquired in a FACScan flowcytometer (BD Biosciences CA USA)

In order to measure cells apoptosis the trophoblast cellswere treated by Kidney-replenishing herb serum and controlserum and then washed with PBS in binding buffer at a den-sity of 4 times 105 cells (10MmHepes pH 74 140mMNaCl and25mM CaCl2) Fluorescein isothiocyanate (FITC)-AnnexinV and PI were added to a final concentration of 1 120583gml Themixture was incubated for 10min and then analyzed by FCMas described above

28 Detection of Cell Viability The trophoblast cells wereplated in a 96-well plates at a density of 2 times 104 cells perwell After cells were treated with a series of concentrationsof Kidney-replenishing herb for 48 h and incubated at 37∘CMTT (5mgml) 20120583l was added to the cells After 4 hincubation the absorbance was measured with a wavelengthof 450 nm

29 SOCS-3 Plasmid Transfection The SOCS-3-PRK5 plas-mid constructwas provided byDrMiuraOsamu fromTokyoMedical andDental University JAR choriocarcinoma cell linewas recognized a model to study placental functions JARcells were seeded in a six-well culture plate of 2 times 104wellWhen cells reached 80confluent theywere transfectedwithSOCS-3 expression plasmids using Lipofectamine 2000 plusreagent according to the manufacturerrsquos instructions PRK5were used as a control Successful transfection of SOCS-3genes into trophoblasts was confirmed by positive green fluo-rescence reviewed under fluorescent microscope After 6 h ofincubation cells were incubated with DMEM containing 10FBS for 48 h their mRNA expression detected by RT-PCRand protein expression by SDS-PAGE

210 SOCS-3 Stealth siRNA Transfection SOCS-3 StealthsiRNA was procured from Invitrogen The siRNA sequencetargeting SOCS-3 is 51015840-AGUAGAUGUAAUAGGCUC-UUCUGGG-31015840 A random siRNA (51015840-CCCAGAAGAGCC-UAU-31015840) which does not have any target region in humangenes served as negative control JAR cells were seeded in asix-well plate of 2 times 104well When cells reached 80 conflu-entmediumwas changed toOPTIMEMThe siRNAoligonu-cleotides targeting NME1 (Invitrogen Carlsbad CA) andLipofectamine 2000 were mixed in OPTIMEM and thenadded to the cells at room temperature Efficiency of silencingof the target gene was assessed byWestern blot after transfec-tion 72 h

211 Data Analysis Data were expressed as the mean plusmn SDData were analyzed with application of the two-way ANOVAusing the SPSS 130 software package differences were con-sidered as statistically significant at 119901 lt 005

3 Results

31 Kidney-Replenishing Herb Enhanced Proliferation ofHuman First-Trimester Trophoblasts and JAR Cell Lines Totestify the effects of Kidney-replenishing herb on the prolifer-ation of human trophoblast we first explored its effect on cellviability Primary human trophoblasts and JARwere culturedin DMEM with 1 FBS for 12 h and then cultured in DMEMwith 10 and 20ofKidney-replenishing herb for 48 hwhilecells were simultaneously treated with rat serum (10 20)for a duration of 48 h as a positive control Analysis of thedemographic data revealed that control serum enhanced cellviability compared with vehicle control group (119901 lt 001)and suggested that serum alone stimulated cell viability butthere is no significant difference in different concentrationof control serum As shown in Figure 1 the proliferation ofcells was significantly higher in Kidney-replenishing herb


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Figure 1 Kidney-replenishing herb promoted human first-trimester trophoblast proliferation activity We first evaluated themodulation of Kidney-replenishing herb on proliferation activity ofhuman trophoblast by MTT as shown in this figure within 10ndash20ranges of concentrations Kidney-replenishing herb stimulated thetrophoblast proliferation in a dose-dependent manner comparedcontrol Likewise Kidney-replenishing herb affected JAR cellproliferation in a similar manner lowastlowast119901 lt 001 versus the vehiclecontrol

compared with the same concentration control serum (119901 lt001) 20ofKidney-replenishing herb activated cell viabilityhigher than that of 10 Kidney-replenishing herb exposure(119901 lt 005) Kidney-replenishing herb promoted the tropho-blast proliferation in a dose-dependent manner LikewiseKidney-replenishing herb stimulated JAR cell proliferation ina similar manner

32 Kidney-Replenishing Herb Promoted Growth of HumanFirst-Trimester Trophoblast Cells through MAPKERK12Signaling Pathway MAPKERK12 signaling pathway isinvolved in regulation of proliferation and apoptosis ofhuman trophoblasts we wondered whether Kidney-replen-ishing herb improved the growth of human first-trimestertrophoblasts via MAPKERK12 signaling pathway or not

After being incubated in DMEM with 1 FBS for 12 hcells were treated with 10 or 20 of kidney-replenishingherb for 05 1 2 and 4 h to analyze the phosphorylation ofERK12 by Western blot analysis The results in Figures 2(a)and 2(b) showed that Kidney-replenishing herb stimulationevoked dose-dependent ERK12 phosphorylation and theactivation persisted for at least 4 hours the maximal effectwas achieved at 2 h Otherwise the slight effect was alsoobserved at 20 of control serum as shown in Figure 2(c) itsuggested that serum could also induce trophoblasts ERK12phosphorylationU0126 a specific inhibitor of ERKupstreamkinase MEK12 almost significantly inhibited activation ofERK induced by Kidney-replenishing herb It could be con-cluded that Kidney-replenishing herb activated the ERK12MAPK signaling pathway in human trophoblast cells

To test the effects of Kidney-replenishing herb on humantrophoblast cell proliferation the flow cytometry was carriedout to measure PCNA We cultured cells in the serum ofconcentrations of Kidney-replenishing herb (0 10 and 20)for 48 h the same concentration of control serum (10 20)as a control At the same time we treated cells with absence or

presence of 20 herb in the presence of 30 120583molL U0126 Asshown in Figure 2(f) Kidney-replenishing herb substantiallyincreased proliferation of human first-trimester trophoblastcells in a dose-responsive manner the proliferation of humanfirst-trimester trophoblast cells was significantly higher withKidney-replenishing herb concentrations of 10 and 20(119901 lt 001) compared with either concentration control serumand this proliferation reached a peak in the 20 of Kidney-replenishing herb Combined with the results of the aboveMTT and our other study [8] it suggested that Kidney-replenishing herb can stimulate proliferation of the first-trimester trophoblast cells in an appropriate concentrationrange by inducing alterations in both cell viability andmorphology After treatment with U0126 cell proliferationwas inhibited almost 80 and U0126 repressed the herbeffect on the proliferation relative to that of 20 of herb(119901 lt 001) Our results suggested that ERK12 pathway wasinvolved in Kidney-replenishing herb regulation of tropho-blast proliferation

In addition we measured the early apoptotic events bysimultaneous detection ofAnnexin-V-FITCPI staining usingflowcytometryAs shown in Figure 2(g) control serumgroupinhibited apoptoticcell to a certain degree compared withvehicle control (119901 lt 005) but there was no significant differ-ence between different concentration of control serumKidney-replenishing herb repressed the apoptotic effect onthe U0126 relative to that vehicle control (119901 lt 001) Further-more Kidney-replenishing herb prevented apoptosis of tro-phoblastic cells compared with the same concentration con-trol serum (119901 lt 005) It suggested that Kidney-replenishingherb prevents early apoptotic events in trophoblastic cells byactivating ERK12 pathway

33 Human First-Trimester Trophoblasts Expressed SOCS-3In Vivo We detected SOCS-3 protein expression in humanfirst-trimester villus by immunohistochemistry SOCS-3 wasexpressed in the nucleus and cytoplasmof human trophoblastcells as shown in Figure 3 Immunohistochemical proce-dures were performed two times and similar results wereobtained

To study the influence of Kidney-replenishing herb on theexpression of SOCS-3 in human trophoblasts the cells werecultured in DMEM with 1 FBS for 12 h and then treatedwith 0 10 and 20 Kidney-replenishing herb and 10 and20control serumWedetected dimSOCS-3 transcription invehicle control group (Figure 4(a)) which suggested littleendogenous SOCS-3 expression in primary cultured humantrophoblasts 10 or 20 serum induced SOCS-3 expressionat 2 hoursrsquo stimulation (Figures 4(d) and 4(e)) Howeverthere is no increased expression of SOCS-3 between treat-ment by 10 and 20 group of control serum (119901 gt 005)suggesting that SOCS-3 may be transient reduced by serumMeantime we found that SOCS-3 was upregulated at signif-icant levels only 1 h after Kidney-replenishing herb adminis-tration (Figures 4(b) and 4(c)) then was enhanced rapidlyreached its peak at 2 h and was slowly declined but after 4 hof treatment it was still above the basal level As shown inFigure 4(f) treatmentwith various concentrations ofKidney-replenishing herb increased the transcription of SOCS-3 in


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Figure 2 Modulation of Kidney-replenishing herb on growth of the primary cultured human first-trimester trophoblast cells To verify rapidactivation of ERK12 cells were starved for 12 h subjected to 10 or 20 of Kidney-replenishing herb treatments for 4 hThe phosphorylationof ERK12 was observed in response to Kidney-replenishing herb treatment ((a) (b)) The phosphorylation of ERK12 induced by 20 ofcontrol serum was also detected (c) The samples were controlled by immunoblot against ERK (d) and GAPDH (e) The primary culturedtrophoblasts were starved with 1 FBS for 12 h and then treated with vehicle 10 Kidney-replenishing herb 20 Kidney-replenishingherb 10 control serum 20 control serum and Kidney-replenishing herb combined with U0126 (30mmoll) or U0126 (30mmoll) alonerespectively for 48 h The percent of PCNA expression (f) or Annexin-V-FITCPI staining (g) was observed by flow cytometry lowast119901 lt 005lowastlowast119901 lt 001 when compared with 20 control serum

trophoblast cells in a dose-dependent manner There is astatistically significant SOCS-3 transcriptionwhich increasedin various concentrations of Kidney-replenishing herb groupcompared with the either control serum group or vehiclegroup (119901 lt 001)

34 Kidney-Replenishing Herb Enhanced SOCS-3 ProteinExpression In Vitro in Human First-Trimester Trophoblasts

After observing increased SOCS-3 transcription in the first-trimester human trophoblast cells treated with Kidney-replenishing herb we analyzed SOCS-3 protein expression introphoblast cells cultured for 4 h byWestern blot As shown inFigure 5(c) there appeared SCOS3 band in the serum treatedprimary human trophoblasts and the protein levels of SOCS-3 was significantly increased after treatment with Kidney-replenishing herb (Figures 5(a) and 5(b)) suggesting that


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Figure 3 SOCS-3 expression in vivo in human first-trimester placental bed tissue Specific brown-coloured staining for SOCS-3 was detectedin the cytoplasm and nucleus of villous trophoblasts (a) No background staining was observed in the isotype control experiments (b)Magnification times200

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Figure 4 SOCS-3 transcription in the Kidney-replenishing herb-treated human first-trimester trophoblast As shown in (a) there wereweak SOCS-3 transcription in group of vehicle control and no increased expression of SOCS-3 within 4 h Then we further observed thetranscription of SOCS-3 in the primary cultured first-trimester trophoblast cells after treatment with 10 (b) or 20 (c) Kidney-replenishingherb for different times The slight SOCS-3 mRNA amount was determined in human trophoblasts response to treatment by 10 and 20group of serum control at 2 h treatment ((d) (e)) We founded that SOCS-3 transcription increased slightly after Kidney-replenishing herbtreatment for 1 h and reached its peak at 2 h and Kidney-replenishing herb increased the transcription of SOCS-3 in trophoblast cells in adose-dependent manner (f) lowastlowast119901 lt 001 lowast119901 lt 005 versus 20 control serum


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Figure 5 Kidney-replenishing herb promoted SOCS-3 proteinexpression in human first-trimester trophoblasts Primary humantrophoblasts were incubated for 4 h with 10 to 20 Kidney-replenishing herb As shown in (a) (b) there is also an increase ofSOCS-3 protein expression (05 1 2 and 4 h) Control cells weretreated with serum in the same way without herb and detected atrace SOCS-3 expression at 2 h (c)The levels of GAPDHwere exam-ined as an internal control (d) The experiments were performed atleast three times and representative result was presented

SOCS-3 was successfully induced by Kidney-replenishingherb in human trophoblast cells

35 U0126 Influence on SOCS-3 Expression in theKidney-Replenishing Herb-Treated Human First-TrimesterTrophoblast After starved with 1 FBS for 12 h the primarycultured trophoblasts were treated with 20 Kidney-replenishing herb 20 Kidney-replenishing herb or 20control serum combined with U0126 (30120583molL) and U0126(30 120583molL) alone for 8 h respectively As shown in Figure 6U0126 remarkably inhibited the SOCS-3 transcriptioninduced byKidney-replenishing herb in trophoblast cells (1 h4 h and 8 h) except U0126 effect on the SOCS-3 expressionto that of 20 Kidney-replenishing herb at 2 h (119901 lt 005)shown in Figure 6(c) The results indicated that Kidney-replenishing herb has the potential to stimulate theERK12MAPK signaling to induce the transcription ofSOCS-3 in human trophoblast cells (Figure 6(e)) but thereis one else signaling to induce SOCS-3 transcription Basedon this finding we next explored the effects of U0126 onthe level of SOCS-3 protein expression in trophoblast cellsAfter treatment of trophoblast with U0126 for 20minwe evaluated the expression of SOCS-3 in the presence ofKidney-replenishing herb (1 2 4 and 8 h) butwe did not findthe SOCS-3 protein byWestern blot analysisThe experiment

was performed in triplicate with essentially similar resultsThese results indicate that Kidney-replenishing herb canupregulate SOCS-3 expression via ERK12 pathway

36 Kidney-Replenishing Herb Induced ERK12 Phosphoryla-tion and SOCS-3 Protein Expression In Vitro in JAR CellsAfter observing increased ERK12 phosphorylation andSOCS-3 expression in the first-trimester human trophoblastcells treated with Kidney-replenishing herb we detected thepERK12 and SOCS-3 protein in the human placenta chori-ocarcinoma JAR cells by Western blot After serum beingdeprived in DMEM for 24 h JAR cells were cultured andthen treated with 10 Kidney-replenishing herb As shownin Figure 7 The phosphorylation of ERK12 was observedin response to herb treatment and the maximal effect wasachieved at 2 h and then slowly declined until 8 h herbstimulation Expression of SOCS-3 was detected at 10minand peaked at two hours in response to Kidney-replenishingherb treatments the significant SOCS-3 protein amount wasdetermined after 8 h herb treatment The results suggestedthat Kidney-replenishing herb could also induce ERK12phosphorylation and SOCS-3 protein expression in JAR cells

37 Kidney-Replenishing Herb Induced ERK12 Phosphoryla-tion after the SOCS-3 siRNA Interference in JAR Cells In thepresent work we aimed to study the influence of knockdownof SOCS-3 expression by RNA interference (siRNA) on theSOCS-3 expression and ERK12 phosphorylation in JAR cellsCells transfected with BLOCK-iT vector only were used asa negative control RT-PCR analysis showed that SOCS-3 mRNA in JAR cells was inhibited successfully by 70(Figure 8) and Western blot analysis showed that SOCS-3 protein expression in JAR cells was silenced We furtherinvestigated the effect of SOCS-3 silence on the ERK12phosphorylation After 48 h of transfection pERK12 proteinexpression was still induced by exposure of cells to 10Kidney-replenishing herb (1 2 4 and 8 h) pERK12 proteinlevels peaked at 4 h and declined thereafter (Figure 9(c))suggesting that ERK12 phosphorylation in trophoblastmightbe not dependent on SOCS-3 expression

38 SOCS-3 Overexpression Enhanced Proliferation of JARCells RT-PCR showed that SOCS-3 mRNA expression inJAR cells was increase after transfection SOCS-3-PRK5 plas-mid for 48 h (Figure 10(a)) We further observed the SOCS-3 transcription increased obviously after treatment with10 Kidney-replenishing herb (Figure 10(b)) Since Kidney-replenishing herb could stimulate human trophoblast prolif-eration we further determined the influence of SOCS-3 over-expression on the cell proliferation in JAR cells Treatmentof JAR cells with serum starve for 72 h and JAR cells wastransfected with PRK5 empty vector or SOCS-3 expressionplasmid the flow cytometry was carried out to measure theexpression of PCNAThe percent of empty vector-transfectedcells showed no increase in PCNA expression (Figure 10(c))whereas cells transfected with SOCS-3 plasmid were 3324 plusmn682 (119901 lt 005) suggesting that SOCS-3 overexpressionmight partially increase JAR cells proliferation Furthermoretreatment with Kidney-replenishing herb stimulated JAR


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Figure 6 Kidney-replenishing herb induced SOCS-3 transcription of the first-trimester human in the presence of U0126 for 8 h The resultssuggested that Kidney-replenishing herb could stimulate the SOCS-3 transcription of human trophoblast cells in a time-dependent mannerand the SOCS-3 induced by Kidney-replenishing herb could even be sustained at least for 4 h U0126 as a MEK inhibitor remarkablyeliminated SOCS-3 transcription induced by Kidney-replenishing herb Data shown are representative of three independent experimentslowastlowast119901 lt 001 lowast119901 lt 005 versus 20 control serum

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SOCS-3

GAPDH

Figure 7 Kidney-replenishing herb induces phosphorylation ofERK12 and increases SOCS-3 protein JAR cells were serum-starvedfor 24 h and stimulated with 10 Kidney-replenishing herb for8 h Western blot analysis was performed using antibodies againstpERK12 ERK12 SOCS-3 and GAPDH

cells proliferation compared with the same concentrationcontrol serum after SOCS-3 overexpression (119901 lt 005) Thusit is likely that SOCS-3 expression might be involved in theproliferation of human trophoblasts

M 1 2 43

250

750

GAPDH (220 bp)

SOCS-3 (650 bp)(bp)

Figure 8 RT-PCR analysis of SOCS-3 mRNA expression aftertransfected with Stealth siRNA at 48 h M marker lane 1 2 cellstransfected with Stealth siRNA lane 3 cells transfected negativecontrol and lanes 4 untransfected cells GAPDH expression wasused as an internal control Molecular weight is shown on the left

4 Discussion

It is known that the placenta plays an important role in thesuccessful pregnancy The placenta supply nutrition to thegrowing fetus of which development is vital for fetal survivaland growth [15] Placental function is essential for thedevelopment of the mammalian embryo during pregnancy[16] Maintenance placental normal function depends on theperfect balance among trophoblast layer proliferation matu-ration and apoptosis [17] There is evidence of predominant


8421

(h)

(a)

8421

(h)

(b)

8421

(h)

(c)

8421

(h)

(d)

8421

(h)

(e)

Figure 9 10Kidney-replenishing herb-induced ERK12 phospho-rylation was not affected by SOCS-3 interference in JAR cells JARcells were starved for 24 h subjected to 10 Kidney-replenishingherb treatments for 8 h The phosphorylation of ERK12 wasobserved in response to vehicle control (a) and 10 control serum(b) the protein levels of pERK12 was significantly increased aftertreatment with 10 Kidney-replenishing herb (c)The samples werecontrolled by immunoblot against ERK (d) and GAPDH (e)

proliferation in normal human first-trimester villous pla-cental trophoblast especially in cytotrophoblast Meantimeapoptosis is thought to play physiological roles in placentalgrowth [18] the rate of apoptosis is low throughout normalearly pregnancy The inadequate proliferation or excessiveapoptosis is reported in human placenta complicated witha few abnormal pregnancies such as spontaneous abortionpreeclampsia preterm delivery and intrauterine growthrestriction [19ndash21] For this reason how to enhance thebiological functions of trophoblast cells is of great interest forresearchers on reproductive medication

Kidney-replenishing herb is a traditional drug whichwidely used to treat spontaneous abortion Our previousstudies demonstrated that Kidney-replenishing herb canmodulate the balance of Th1Th2 cytokines at the mater-nofetal interface and maintains pregnancy by accelerating itsgrowth and fusion and reducing the rate of apoptosis [14]Our present study founded that exposure of serum-starved

SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(a)

SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(b)

Empt

y ve

ctor

PRK5

PRK5

-SO

CS-3

PRK5

-SO

CS-3

+ 1

0he

rb se

rum

PRK5

-SO

CS-3

+ 1

0co

ntro

l ser

um

0

10

20

30

40

50

60

Perc

enta

ge o

f PCN

A

lowast

lowast

lowast

(c)

Figure 10 Transfection of SOCS-3 gene into JAR trophoblasts RT-PCR analysis of SOCS-3 mRNA expression after treatment with10 of control serum (a) and 10 Kidney-replenishing herb (b)M marker lane 1 SOCS-3 expression which was detectable inuntransfected cells lane 2 cells transfected with liposome lanes3 cells transfected with PRK5 lanes 4 cells transfected withpRNAT-U6 and lanes 5 cells transfected with SOCS-3-PRK51at 48 h (c) JAR cells proliferation was measured by using flowcytometry trophoblast cells proliferation was increased after SOCS-3 overexpression lowast119901 lt 005 versus untransfected cells

normal human trophoblast cells to Kidney-replenishingherb for 48 h resulted in a dose-dependent promotion ofcell proliferation activity proliferating cell nuclear antigen(PCNA) expression was also induced by exposure of cellsto herb PCNA is a well-known cell-cycle marker that hasbeen proved as a useful tool for determining the proportionof proliferating cells [22] The cell cycle is divided intodistinct phases in mammalian Quiescent cells exist in theG0 phase when they enter the cell cycle at G1 cells beginDNA replication and PCNA expression increases quicklyat the same time If cells pass through the S phase PCNAreached the peak as DNA replication is completed and thencells progress through the G2 phase before entering mitosis(M-phase) meanwhile PCNA expression correspondinglydecreases


Our previous studies have showed that Kidney-replenish-ing herb reduced the percentage of normal human cytotro-phoblast cells entering sub-G1-phase and shifted cell-cyclephase from G1 phases to S phase In agreement with ourprevious finding our study found that Kidney-replenishingherb can promote PCNA expression of human first-trimestertrophoblast in a dose-dependent manner Furthermore wehave assayed the early phase of apoptosis triggered by serumdeprivation by using Annexin V-propidium iodide (PI)labeling and flow cytometric analysis Kidney-replenishingherb showed significant decrease apoptosis of primary tro-phoblasts in this study In conclusion our study observedthat administrationwithKidney-replenishing herb promotedproliferation and diminished cell apoptosis of human first-trimester trophoblasts even in the presence of U0126 How-ever less is known about how Kidney-replenishing herbcontrols trophoblast cell proliferation and apoptosis

Cytokines and growth factors regulate the growth anddifferentiation of cells by binding to cell-surface receptorsand activating intracellular signal transduction cascadesThesuppressors of cytokine signaling (SOCS) family of pro-teins (SOCS-1simSOCS-7 and CIS) regulate cytokine signalingSOCS-3 is regulated in multiple cell types and induced by IL-6 IL-11 leptin LIF and so on [23] Many reports confirmedthat SOCS-3 have profound effects on the regulation ofimmunity and inflammation by affecting the activationdevelopment and homeostatic functions of all lineagesinvolved in immune and inflammatory responses SOCS-3expression also has been detected in trophoblast cells More-over it seems that SOCS-3 may be involved in the process ofplacentation

SOCS-3 knockout gene in themouse presents several pla-cental anomalies such as poorly formed spongiotrophoblastand labyrinthine layers and is accompanied by increasednumbers of mature trophoblast giant cells These severalplacental insufficiencies ultimately result in mice which dieduring the embryonic stage of development however theseembryos can be saved by a tetraploid rescue approach [11 1224] These observations gave some useful clues for exploringthe effect of SOCS-3 on human trophoblast cells

Lee and colleagues reported cytoplasmic and nuclearexpression of SOCS in vitro and SOCS-3 nuclear transloca-tion only when its levels increase [10] White et al demon-strated that none of SOCS-3 expression was found in thenucleus while little SOCS-3 expression was found in cyto-plasm [25] Our data demonstrated cytoplasmic and nuclearlocalization of SOCS proteins in the human placenta of thefirst trimester of pregnancy by immunohistochemical anal-yses in vivo suggesting there are sufficient expressions ofSOCS-3 in placental trophoblasts which play a crucial role inregulating placenta function Our data further demonstratedthat SOCS-3 is expressed in purified human trophoblastsand JAR trophoblast cell line by RT-PCR and Western blotAccording to Goren and colleagues SOCS-3 has a novel roleas a regulator of keratinocyte proliferation and differenti-ation in vitro and in vivo [26] We founded that Kidney-replenishing herb to human trophoblast cells showed a dose-responsive stimulation of proliferation Meanwhile treat-mentwithKidney-replenishing herb induced SOCS-3mRNA

expression in trophoblast in a dose-dependent manner Weobserved that SOCS-3 overexpression increases basal prolif-eration of JAR Thus it was reasonable to guess that SOCS-3 may be related to the trophoblastic proliferation and thedevelopment of placenta We further observed that signalingpathways induced SOCS-3 in order to find its precise physio-logical function

The mitogen-activated protein kinase (MAPK) is one ofrevolutionary signaling pathways inmammalian cells includ-ing the extracellular signal-regulated kinases (ERK12) thep38 MAP kinases (p38a p38b p38g and p38d) the c-Jun-NH2-terminal kinases (JNK1 JNK2 and JNK3) and ERK5[27] The activation of the ERK signaling pathway has beenassociated with cell proliferation differentiation and cell sur-vival and provides protection against apoptosis [8] It is wellknown that ERK12MAPK cascade is one of the major sig-naling pathways involved in placenta development ERK12is also widely expressed in villous cytotrophoblasts in thefirst-trimester gestation [28]The proliferation of trophoblastcells is dependent on activation of ERK 12 and disruptionof the ERK2 locus leads to embryonic lethality early inmouse development after implantation [8]Our study showedthat Kidney-replenishing herb could induce the activationof ERK12 in a dose-dependent manner and promote thegrowth of trophoblast cells in vitro U0126 a MEK inhibitorcould significantly inhibit this enhanced phosphorylation ofERK12 and proliferation of trophoblast cells induced byKidney-replenishing herb In addition we have found thatKidney-replenishing herb prevents the apoptotic process bymeans of the activation of ERK pathway It suggested thatKidney-replenishing herb promotes proliferation and pre-vents the early events of apoptosis via ERK pathway in humantrophoblasts On the other hand we found that Kidney-replenishing herb increased SOCS-3 expression and activatedthe ERK12 pathway U0126 could significantly abrogatethe mRNA and protein expression of SOCS-3 induced byKidney-replenishing herb in trophoblast cells It indicatedthat Kidney-replenishing herb enhanced SOCS-3 expressionthrough activation of the ERK12 pathway in trophoblastcells Several studies demonstrated that the activation ofERK12 is involved in the regulation of SOCS-3 expressionin response to cytokine [29 30] However the mechanismsgoverning the ERKMAPK-dependent regulation of SOCS-3 expression are not well understood We hypothesized thatKidney-replenishing herb may affect trophoblast prolifera-tion by regulating SOCS-3 expression To confirm the roleof SOCS-3 expression in herb-induced ERK pathway JARhuman choriocarcinoma cells are used to study cellularsignaling which has the characteristics of early placentaltrophoblast cells [31] We knocked down SOCS expression byRNA interference (RNAi) in JAR cells and reevaluated theeffect of herb on activation of ERK pathway Our datashowed that ERK phosphorylation stimulated by Kidney-replenishing herb has no change by SOCS-3 siRNA silenceWe further founded that the SOCS-3 overexpression in JARtrophoblast cells triggers the proliferation of themselvesThus it is proposed that the intracellular ERK phosphoryla-tion is an upstream signal of SOCS-3 expression andKidney-replenishing herb enhanced SOCS-3 expression through


activation of the ERK12 pathway thereby inducing tropho-blast proliferation

Collectively our findings imply that the stimulatory effectof Kidney-replenishing herb on trophoblast proliferation andSOCS-3 expression depends on the ERK12 pathway andSOCS-3 may be involved placentation and embryonic devel-opment Interestingly U0126 only partly suppressed SOCS-3 mRNA and proliferation of the trophoblasts upregulatedby Kidney-replenishing herb suggesting that besides ERKsignals there might be other signal pathways which were alsoinvolved in the Kidney-replenishing herb-induced growth ofhuman trophoblasts

SOCS-3 have been identified as important regulators ofERKMAPK pathway and STAT3 signaling in undifferenti-ated Rcho-1 cells which were derived from rat choriocarci-noma [12] Other studies showed that STAT3 signal transduc-tion pathway induces SOCS-3 in acutemyeloid leukemia cells[32] and in rat astrocytes [33] We wonder if Kidney-replen-ishing herb upregulates SOCS-3 expression through activa-tion of STAT3 pathway in human trophoblast cells BecauseKidney-replenishing herb in cytotrophoblasts could promotethe expression of SOCS-3 in the first-trimester human tro-phoblast at one-hour stimulation we detected the phospho-rylation of STAT3 in response to herb treatment in first-trimester human trophoblast cells at 10min 20min 30minand one hour respectively however we did not detect anyphosphorylation of STAT3 (data not shown) It suggests thatSTAT3 pathway is not involved in the regulation of SOCS-3expression induced by Kidney-replenishing herb Huang andcolleagues study also indicates that there was SOCS-3 expres-sion but no STAT3 activation by IFN-120574 at one hour [34]Other data show that there is an inverse correlation betweenthe expression of SOCS-3 and IL-6-induced phosphorylationof STAT3 in prostate cancer cells furthermore downregula-tion of SOCS-3 by a short interfering RNA approach resultedin inhibition of proliferation and an increased apoptoticrate [35] It is not known whether various types of STAT3expression relative to SOCS-3 might be different stimulationor different cultured cells

Manypublications suggested that STAT3 andERKMAPKpathways are involved in the regulation of cell proliferationapoptosis and angiogenesis and participants in the processesof induction progression and metastasis of carcinomaActivation of STAT3 may prevent death of tumor cells andstimulates tumor proliferation invasion and migration [35ndash37] It is possible that the activation of ERK-12 but notSTAT3 which was induced upon Kidney-replenishing herbtreatment in trophoblast is themechanism that herb regulatestrophoblast cell proliferation and avoids its overgrowth

5 Conclusions

Our study has shown that Kidney-replenishing herb mightpromoted survival and antiapoptosis via ERK12 signalingpathway and provides the first evidence for SOCS-3 inthe regulation of human first-trimester trophoblast Furtherwork on Kidney-replenishing herb will need to be donewhich could be useful in therapeutics of recurrent sponta-neous abortion and other pregnant complications It could

be inferred that elucidating the role of SOCS-3 in ERK12pathways might provide clues as to how trophoblast cellscan be controlled by Kidney-replenishing herb as well asinsight into how SOCS-3 regulates survival and growth introphoblasts

Conflicts of Interest

All authors have no conflicts of interest to disclose

Acknowledgments

The authors are grateful to Dr Miura Osamu of the TokyoMedical and Dental University for providing SOCS-3 plas-mid This study was supported by Research Program ofShanghai Municipal Commission of Health and FamilyPlanning 201440004 (to Chang-ying Xing)

References

[1] Y-Y Fu W-L Gao M Chen K X Chai Y-L Wang and L-M Chen ldquoProstasin regulates human placental trophoblast cellproliferation via the epidermal growth factor receptor signalingpathwayrdquoHumanReproduction vol 25 no 3 pp 623ndash632 2010

[2] J E Ray J Garcia A Jurisicova and I Caniggia ldquoMtdBoktakes a swing Proapoptotic MtdBok regulates trophoblastcell proliferation during human placental development and inpreeclampsiardquo Cell Death and Differentiation vol 17 no 5 pp846ndash859 2010

[3] L Danihel P Gomolcak M Korbel et al ldquoExpression ofproliferation and apoptotic markers in human placenta duringpregnancyrdquo Acta Histochemica vol 104 no 4 pp 335ndash3382002

[4] Y Huang F Dong Q Du et al ldquoSwainsonine induces apoptosisthrough mitochondrial pathway and caspase activation in goattrophoblastsrdquo International Journal of Biological Sciences vol 10no 7 pp 789ndash797 2014

[5] H-Y Wang S-Q Gui and C-J Xu ldquoEffect of bushen yiqirecipe on bioactivity behavior of human cytotrophoblast ofearly pregnancyrdquo Chinese Journal of Integrated Traditional andWestern Medicine vol 24 no 6 pp 525ndash528 2004 (Chinese)

[6] B A Ballif and J Blenis ldquoMolecular mechanisms mediatingmammalian mitogen-activated protein kinase (MAPK) kinase(MEK)-MAPK cell survival signalsrdquoCell Growth andDifferenti-ation the Molecular Biology Journal of the American Associationfor Cancer Research vol 12 no 8 pp 397ndash408 2001

[7] M-R DuW-H Zhou L Dong et al ldquoCyclosporin A promotesgrowth and invasiveness in vitro of human first-trimestertrophoblast cells via MAPK3MAPK1-mediated AP1 and Ca2+calcineurinNFAT signaling pathwaysrdquo Biology of Reproduc-tion vol 78 no 6 pp 1102ndash1110 2008

[8] M K Saba-El-Leil F D J Vella B Vernay et al ldquoAn essentialfunction of themitogen-activated protein kinase Erk2 inmousetrophoblast developmentrdquo EMBO Reports vol 4 no 10 pp964ndash968 2003

[9] J C Tan and R Rabkin ldquoSuppressors of cytokine signaling inhealth and diseaserdquo Pediatric Nephrology vol 20 no 5 pp 567ndash575 2005

[10] K-H Lee K-J Moon H S Kim et al ldquoIncreased cytoplasmiclevels of CIS SOCS1 SOCS2 or SOCS3 are required for nuclear


translocationrdquo FEBS Letters vol 582 no 15 pp 2319ndash23242008

[11] A W Roberts L Robb S Rakar et al ldquoPlacental defects andembryonic lethality in mice lacking suppressor of cytokinesignaling 3rdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 98 no 16 pp 9324ndash9329 2001

[12] A Isobe T Takeda M Sakata et al ldquoSTAT3-mediated Con-stitutive Expression of SOCS3 in an Undifferentiated RatTrophoblast-like Cell Linerdquo Placenta vol 27 no 8 pp 912ndash9182006

[13] D M Morales-Prieto S Ospina-Prieto W Chaiwangyen etal ldquoIntranuclear crosstalk between extracellular regulatedkinase12 and signal transducer and activator of transcription3 regulates JEG-3 choriocarcinoma cell invasion and prolifera-tionrdquoThe Scientific World Journal vol 2013 Article ID 25984510 pages 2013

[14] X Li S Gui and HWang ldquoEffect of Kidney-replenishing herbon the indoleamine 23-dioxygenase of human syncytiotro-phoblasts cultured in vitro and the balance of helper T-cellcytokinesrdquoGynecological Endocrinology vol 23 no 11 pp 653ndash661 2007

[15] J Rossant and J C Cross ldquoPlacental development lessons frommouse mutantsrdquo Nature Reviews Genetics vol 2 no 7 pp 538ndash548 2001

[16] MDe Falco V Fedele L Cobellis et al ldquoImmunohistochemicaldistribution of proteins belonging to the receptor-mediatedand the mitochondrial apoptotic pathways in human placentaduring gestationrdquo Cell and Tissue Research vol 318 no 3 pp599ndash608 2004

[17] M Kar D Ghosh and J Sengupta ldquoHistochemical and mor-phological examination of proliferation and apoptosis in humanfirst trimester villous trophoblastrdquo Human Reproduction vol22 no 11 pp 2814ndash2823 2007

[18] H Yamauchi K-I Katayama M Ueno K Uetsuka HNakayama and K Doi ldquoInvolvement of p53 in 1-120573-D-arabi-nofuranosylcytosine-induced trophoblastic cell apoptosis andimpaired proliferation in rat placentardquo Biology of Reproductionvol 70 no 6 pp 1762ndash1767 2004

[19] W-H Zhou L Dong M-R Du X-Y Zhu and D-JLi ldquoCyclosporin A improves murine pregnancy outcome inabortion-prone matings Involvement of CD8086 and CD28CTLA-44rdquo Reproduction vol 135 no 3 pp 385ndash395 2008

[20] B L Sheppard and J Bonnar ldquoUteroplacental hemostasis inintrauterine fetal growth retardationrdquo Seminars in Thrombosisand Hemostasis vol 25 no 5 pp 443ndash446 1999

[21] I P Crocker S Cooper S C Ong and P N Baker ldquoDifferencesin apoptotic susceptibility of cytotrophoblasts and syncytiotro-phoblasts in normal pregnancy to those complicated withpreeclampsia and intrauterine growth restrictionrdquo AmericanJournal of Pathology vol 162 no 2 pp 637ndash643 2003

[22] M Klingler A Blaschitz C Campoy et al ldquoThe effect ofdocosahexaenoic acid and folic acid supplementation on pla-cental apoptosis and proliferationrdquo British Journal of Nutritionvol 96 no 1 pp 182ndash190 2006

[23] J J Babon and N A Nicola ldquoThe biology and mechanism ofaction of suppressor of cytokine signaling 3rdquo Growth Factorsvol 30 no 4 pp 207ndash219 2012

[24] L Larsen and C Ropke ldquoSuppressors of cytokine signallingSOCSrdquo APMIS vol 110 no 12 pp 833ndash844 2002

[25] G E White A Cotterill M R Addley E J Soilleux and DR Greaves ldquoSuppressor of cytokine signalling protein SOCS

3

expression is increased at sites of acute and chronic inflamma-tionrdquo Journal of Molecular Histology vol 42 no 2 pp 137ndash1512011

[26] I Goren A Linke E Muller J Pfeilschifter and S Frank ldquoThesuppressor of cytokine signaling-3 is upregulated in impairedskin repair Implications for keratinocyte proliferationrdquo Journalof Investigative Dermatology vol 126 no 2 pp 477ndash485 2006

[27] K C Moon J S Park E R Norwitz et al ldquoExpression of extra-cellular signal-regulated kinase12 and p38 mitogen-activatedprotein kinase in the invasive trophoblasts at the humanplacental bedrdquo Placenta vol 29 no 5 pp 391ndash395 2008

[28] J Liu B Cao Y-X Li X-Q Wu and Y-L Wang ldquoGnRHI and II up-regulate MMP-26 expression through the JNKpathway in human cytotrophoblastsrdquo Reproductive Biology andEndocrinology vol 8 article no 5 2010

[29] J G Bode S Ludwig C A Correia Freitas et al ldquoTheMKK6p38 mitogen-activated protein kinase pathway is capa-ble of inducing SOCS3 gene expression and inhibits IL-6-induced transcriptionrdquoBiological Chemistry vol 382 no 10 pp1447ndash1453 2001

[30] J G Bode A Nimmesgern J Schmitz et al ldquoLPS and TNF120572induce SOCS3 mRNA and inhibit IL-6-induced activation ofSTAT3 in macrophagesrdquo FEBS Letters vol 463 no 3 pp 365ndash370 1999

[31] A Nilkaeo and S Bhuvanath ldquoInterleukin-1 modulationof human placental trophoblast proliferationrdquo Mediators ofInflammation vol 2006 Article ID 79359 2006

[32] J-J Schuringa A T J Wierenga W Kruijer and E VellengaldquoConstitutive Stat3 Tyr705 and Ser727 phosphorylation inacute myeloid leukemia cells caused by the autocrine secretionof interleukin-6rdquo Blood vol 95 no 12 pp 3765ndash3770 2000

[33] R P Donnelly H Dickensheets and D S Finbloom ldquoTheinterleukin-10 signal transduction pathway and regulation ofgene expression in mononuclear phagocytesrdquo Journal of Inter-feron and Cytokine Research vol 19 no 6 pp 563ndash573 1999

[34] Y Huang J J Feld R K Sapp et al ldquoDefective hepatic responseto interferon and activation of suppressor of cytokine signaling3 in chronic Hepatitis Crdquo Gastroenterology vol 132 no 2 pp733ndash744 2007

[35] I Bellezza H Neuwirt C Nemes et al ldquoSuppressor of cytokinesignaling-3 antagonizes cAMP effects on proliferation andapoptosis and is expressed in human prostate cancerrdquo TheAmerican Journal of Pathology vol 169 no 6 pp 2199ndash22082006

[36] B E Barton J G Karras T F Murphy A Barton and H F-S Huang ldquoSignal transducer and activator of transcription 3(STAT3) activation in prostate cancer direct STAT3 inhibitioninduces apoptosis in prostate cancer linesrdquo Molecular CancerTherapeutics vol 3 no 1 pp 11ndash20 2004

[37] L B Mora R Buettner J Seigne et al ldquoConstitutive activationof Stat3 in human prostate tumors and cell lines direct inhi-bition of Stat3 signaling induces apoptosis of prostate cancercellsrdquo Cancer Research vol 62 no 22 pp 6659ndash6666 2002

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Mutant embryos fail to form the ectoplacental cone andextraembryonic ectoderm which derives from the polar tro-phectoderm [8] These indicate that ERK2 is required for theproliferation of trophoblast stem cells in the polar tro-phectoderm and ERK12MAPK pathway has an importantfunction in the regulation of trophoblasts growth

Suppressors of cytokine signaling (SOCS) protein are afamily of intracellular proteins that control cytokine signaling[9] The SOCS family consists of at least eight membersincluding cytokine induced SH2 protein (CIS) and SOCS-1ndash7 SOCS proteins contain a central Src homology 2 domainprotein (SH2) domain a conserved C-terminus referred toas the SOCS box and unique N-terminal region [10] SOCSfamily proteins can be induced by cytokines growth factorsand several immunomodulators Roberts and colleaguesshowed that mice with a deletion of SOCS-3 gene died atmidgestation because of the placental defects They observedthat SOCS-3(minusminus) embryos were slightly smaller than thewild type but appeared otherwise normal However theplacental spongiotrophoblast layer was significantly reducedand accompanied by increased numbers of giant trophoblastcells The network of embryonic vessels and maternal sinuseswas inadequately developed and yolk sac erythropoiesiswas normal They concluded that the embryonic lethalityis not caused by anatomical defects of the embryo butrather poor placental development that results in the embryodevelopmental arrest and death [11] Therefore it is believedthat SOCS-3 is critical for a successful pregnancy outcome byregulating trophoblast function during the placental develop-ment An interesting study reported by Isobe and colleaguesshowed that MEK antagonist could inhibit SOCS-3 expres-sion of the undifferentiated rat trophoblast-like cell line [12]Another study has demonstrated that SOCS-3 binds and inac-tivates RasGAP a negative regulator of Ras signaling leadingto increased RasMAPK pathway activity in human JEG-3trophoblastic cells [13] These data suggest that the activationof ERK12MAPK pathway signal pathway may be relatedto SOCS-3 expression However the roles of SOCS-3 in theinduction of proliferation processes and relative molecularmechanisms in primary first-trimester human trophoblastsare still unclear

In this study we observed the effects of Kidney-replenish-ing herbs on the proliferation and apoptosis of human first-trimester trophoblasts we therefore investigated the effects ofthe herbs on the expression of SOCS-3 in human trophoblastsby reverse transcription-polymerase chain reaction (RT-PCR) and Western blot In order to elucidate the intracel-lular signal pathway mediating the upregulation in SOCS-3expression by Kidney-replenishing herb we investigated thephosphorylation of ERK12 signal pathway involved in theproliferative and apoptosis of human trophoblasts by Kidney-replenishing herb

2 Materials and Methods

21 Isolation and Cultivation of Human First-TrimesterCytotrophoblast First-trimester human villous tissues (5ndash10weeks of gestation) were collected from clinically normalpregnancies which were terminated for nonmedical reasons

at the Hospital of Obstetrics and Gynecology Fudan Univer-sity Shanghai Medical College The study was approved bythe Fudan University Ethical Review Board and each patientsigned the consent form Villous tissues were immediatelysuspended in ice-cold DMEM and transmitted to the labora-tory where they were washed 2-3 times in sterile phosphate-buffered saline (PBS) to remove the excess blood Primarytrophoblast cells were isolated by trypsin-DNase type I diges-tion and layered over a discontinuous Percoll gradient asdescribed by our previous study [14] This assay suppliesa 95 purity of trophoblast cells These isolated humantrophoblast cells and the choriocarcinoma JAR cell line werecultured in DMEM supplemented with 2mMglutamine 10FCS at 37∘C in 5 CO

2

22 Preparation of Serum Kidney-replenishing herbs aretraditional medicines which have satisfactory effects onthreatenedmiscarriages includingDangshen 12 g Tusizi 15 gBaishu 6 g Baishao 9 g Duzhong 12 g Sangjisheng 12 gSugeng 6 g and Tiaohuangqin 15 g Herb serum was madeaccording to our previous study [14] All of the herbs werecollected and made by the scholars of Shanghai GraduateSchool of Medication (Chinese Academy of Science)

23 Materials Histochemical ABC kit was purchased fromSino-American Bio-Technology (Luoyang China) Rever-tAid First Strand cDNA Synthesis Kit and Taq DNApolymerase were purchased from Fermentas (Thermo FisherScientificMAUSA) BCA-100 ProteinQuantitative AnalysisKit was purchased from Sangon Biotechnology (ShanghaiChina) Mouse anti-GAPDH monoclonal antibodies andplasmid extraction kit were purchased from KangchengBiotechnology (Shanghai China) Rabbit anti-human SOCS-3 antibodies mouse anti-phosphoERK monoclonal antibod-ies rabbit anti-ERKmonoclonal antibodies and mouse anti-GAPDH were purchased from Santa Cruz Biotechnology(Santa Cruz CA USA) Annexin V-FITC apoptosis kitPRK5 plasmid and pRNAT-U61 plasmid were purchasedfrom RampD Systems (Minneapolis MN USA) HRP-goatanti-rabbit IgG was purchased from Dingguo Biotechnol-ogy (Shanghai China) TRIzol reagent BLOCK-iT oligo[13750062] and Lipofectamine 2000 Kit were purchasedfrom Invitrogen (Carlsbad CA USA) SOCS-3 forwardprimer (51015840-CGCCTCAAGACCTTCAGCTC-31015840) and reverseprimer (51015840-ATCCAGGAACTCCCGAA-31015840) product 650 bpGAPDH forward primer (51015840-GAAGGTGAAGGTCGGAGT-C-31015840) and reverse primer (51015840-GATGGTGATGGGATTTC-31015840) and product 220 bp were designed and integrated byKangcheng Biotechnology (Shanghai China)

24 RT-PCR Analysis of SOCS-3 Expression in Human First-Trimester Trophoblast Total cellular RNA ofhuman first-trimester trophoblast was extracted using TRIzol reagentand cDNA Synthesis Kit according to the manufacturerrsquosprotocol The total RNA of 3 120583g was denatured and RT wasperformed for 1 h at 42∘Cwith 05mgOligo dT 05 120583g 10mMdNTP 2 120583l RNase inhibitor 1120583l 200UMoloney virus-reversetranscriptase and 10 reaction buffer in a total volume of20ml After 5min precycle at 95∘C 30 cycles of denaturation












3 Results



lowastlowast


lowastlowast



020406080

100120140160180

Prol

ifera

tion

of ce

ll


10 controlserum











4205 1(h)

(a)

4205 1(h)

(b)

4205 1(h)

(c)

4205 1(h)

(d)

4205 1(h)

(e)

Vehi

cleco

ntro

l

10

her

bse

rum

20

her

bse

rum

10

cont

rol

seru

m

20

cont

rol

seru

m

U01

26 +

20

herb

seru

m

U01

26

0102030405060

Perc

enta

ge o

f PCN

A

lowastlowast

lowastlowast

(f)

Vehi

cleco

ntro

l

10

her

bse

rum

20

her

bse

rum

10

cont

rol

seru

m

20

cont

rol

seru

m

U01

26 +

20

herb

seru

m

U01

26

lowastlowast

lowast

lowast

lowast

Perc

enta

ge o

f

0102030405060

apop

tosis

(g)






(a) (b)


Marker 4 h2 h0 1 h

GAPDH (220 bp)

SOCS-3 (650 bp)

lowastlowast


lowastlowast

lowast

0

01

02

03

04

05

06

SOCS

-3G

APD

H

4 h2 h1 h0



(a) vehicle control





(f)



4205 1

(h)

(a)

4205 1

(h)

(b)

4205 1

(h)

(c)

4205 1

(h)

(d)











20001000

250100

750500(b

p)

0

01

02

03

04

05SO

CS-3

GA

PDH

lowast

lowastlowast

lowast

lowastlowast

4 h 8 h0 1 h 2 h



(a) U0126




(e)


pERK12

10

min

20

min

30

min

1h

2h

4h

8h

RRK12

SOCS-3

GAPDH



M 1 2 43

250

750

GAPDH (220 bp)

SOCS-3 (650 bp)(bp)


4 Discussion



8421

(h)

(a)

8421

(h)

(b)

8421

(h)

(c)

8421

(h)

(d)

8421

(h)

(e)




SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(a)

SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(b)

Empt

y ve

ctor

PRK5

PRK5

-SO

CS-3

PRK5

-SO

CS-3

+ 1

0he

rb se

rum

PRK5

-SO

CS-3

+ 1

0co

ntro

l ser

um

0

10

20

30

40

50

60

Perc

enta

ge o

f PCN

A

lowast

lowast

lowast

(c)















5 Conclusions





Acknowledgments


References




























3



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results



lowastlowast


lowastlowast



020406080

100120140160180

Prol

ifera

tion

of ce

ll


10 controlserum











4205 1(h)

(a)

4205 1(h)

(b)

4205 1(h)

(c)

4205 1(h)

(d)

4205 1(h)

(e)

Vehi

cleco

ntro

l

10

her

bse

rum

20

her

bse

rum

10

cont

rol

seru

m

20

cont

rol

seru

m

U01

26 +

20

herb

seru

m

U01

26

0102030405060

Perc

enta

ge o

f PCN

A

lowastlowast

lowastlowast

(f)

Vehi

cleco

ntro

l

10

her

bse

rum

20

her

bse

rum

10

cont

rol

seru

m

20

cont

rol

seru

m

U01

26 +

20

herb

seru

m

U01

26

lowastlowast

lowast

lowast

lowast

Perc

enta

ge o

f

0102030405060

apop

tosis

(g)






(a) (b)


Marker 4 h2 h0 1 h

GAPDH (220 bp)

SOCS-3 (650 bp)

lowastlowast


lowastlowast

lowast

0

01

02

03

04

05

06

SOCS

-3G

APD

H

4 h2 h1 h0



(a) vehicle control





(f)



4205 1

(h)

(a)

4205 1

(h)

(b)

4205 1

(h)

(c)

4205 1

(h)

(d)











20001000

250100

750500(b

p)

0

01

02

03

04

05SO

CS-3

GA

PDH

lowast

lowastlowast

lowast

lowastlowast

4 h 8 h0 1 h 2 h



(a) U0126




(e)


pERK12

10

min

20

min

30

min

1h

2h

4h

8h

RRK12

SOCS-3

GAPDH



M 1 2 43

250

750

GAPDH (220 bp)

SOCS-3 (650 bp)(bp)


4 Discussion



8421

(h)

(a)

8421

(h)

(b)

8421

(h)

(c)

8421

(h)

(d)

8421

(h)

(e)




SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(a)

SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(b)

Empt

y ve

ctor

PRK5

PRK5

-SO

CS-3

PRK5

-SO

CS-3

+ 1

0he

rb se

rum

PRK5

-SO

CS-3

+ 1

0co

ntro

l ser

um

0

10

20

30

40

50

60

Perc

enta

ge o

f PCN

A

lowast

lowast

lowast

(c)















5 Conclusions





Acknowledgments


References




























3



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast


lowastlowast



020406080

100120140160180

Prol

ifera

tion

of ce

ll


10 controlserum











4205 1(h)

(a)

4205 1(h)

(b)

4205 1(h)

(c)

4205 1(h)

(d)

4205 1(h)

(e)

Vehi

cleco

ntro

l

10

her

bse

rum

20

her

bse

rum

10

cont

rol

seru

m

20

cont

rol

seru

m

U01

26 +

20

herb

seru

m

U01

26

0102030405060

Perc

enta

ge o

f PCN

A

lowastlowast

lowastlowast

(f)

Vehi

cleco

ntro

l

10

her

bse

rum

20

her

bse

rum

10

cont

rol

seru

m

20

cont

rol

seru

m

U01

26 +

20

herb

seru

m

U01

26

lowastlowast

lowast

lowast

lowast

Perc

enta

ge o

f

0102030405060

apop

tosis

(g)






(a) (b)


Marker 4 h2 h0 1 h

GAPDH (220 bp)

SOCS-3 (650 bp)

lowastlowast


lowastlowast

lowast

0

01

02

03

04

05

06

SOCS

-3G

APD

H

4 h2 h1 h0



(a) vehicle control





(f)



4205 1

(h)

(a)

4205 1

(h)

(b)

4205 1

(h)

(c)

4205 1

(h)

(d)











20001000

250100

750500(b

p)

0

01

02

03

04

05SO

CS-3

GA

PDH

lowast

lowastlowast

lowast

lowastlowast

4 h 8 h0 1 h 2 h



(a) U0126




(e)


pERK12

10

min

20

min

30

min

1h

2h

4h

8h

RRK12

SOCS-3

GAPDH



M 1 2 43

250

750

GAPDH (220 bp)

SOCS-3 (650 bp)(bp)


4 Discussion



8421

(h)

(a)

8421

(h)

(b)

8421

(h)

(c)

8421

(h)

(d)

8421

(h)

(e)




SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(a)

SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(b)

Empt

y ve

ctor

PRK5

PRK5

-SO

CS-3

PRK5

-SO

CS-3

+ 1

0he

rb se

rum

PRK5

-SO

CS-3

+ 1

0co

ntro

l ser

um

0

10

20

30

40

50

60

Perc

enta

ge o

f PCN

A

lowast

lowast

lowast

(c)















5 Conclusions





Acknowledgments


References




























3



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4205 1(h)

(a)

4205 1(h)

(b)

4205 1(h)

(c)

4205 1(h)

(d)

4205 1(h)

(e)

Vehi

cleco

ntro

l

10

her

bse

rum

20

her

bse

rum

10

cont

rol

seru

m

20

cont

rol

seru

m

U01

26 +

20

herb

seru

m

U01

26

0102030405060

Perc

enta

ge o

f PCN

A

lowastlowast

lowastlowast

(f)

Vehi

cleco

ntro

l

10

her

bse

rum

20

her

bse

rum

10

cont

rol

seru

m

20

cont

rol

seru

m

U01

26 +

20

herb

seru

m

U01

26

lowastlowast

lowast

lowast

lowast

Perc

enta

ge o

f

0102030405060

apop

tosis

(g)






(a) (b)


Marker 4 h2 h0 1 h

GAPDH (220 bp)

SOCS-3 (650 bp)

lowastlowast


lowastlowast

lowast

0

01

02

03

04

05

06

SOCS

-3G

APD

H

4 h2 h1 h0



(a) vehicle control





(f)



4205 1

(h)

(a)

4205 1

(h)

(b)

4205 1

(h)

(c)

4205 1

(h)

(d)











20001000

250100

750500(b

p)

0

01

02

03

04

05SO

CS-3

GA

PDH

lowast

lowastlowast

lowast

lowastlowast

4 h 8 h0 1 h 2 h



(a) U0126




(e)


pERK12

10

min

20

min

30

min

1h

2h

4h

8h

RRK12

SOCS-3

GAPDH



M 1 2 43

250

750

GAPDH (220 bp)

SOCS-3 (650 bp)(bp)


4 Discussion



8421

(h)

(a)

8421

(h)

(b)

8421

(h)

(c)

8421

(h)

(d)

8421

(h)

(e)




SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(a)

SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(b)

Empt

y ve

ctor

PRK5

PRK5

-SO

CS-3

PRK5

-SO

CS-3

+ 1

0he

rb se

rum

PRK5

-SO

CS-3

+ 1

0co

ntro

l ser

um

0

10

20

30

40

50

60

Perc

enta

ge o

f PCN

A

lowast

lowast

lowast

(c)















5 Conclusions





Acknowledgments


References




























3



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)


Marker 4 h2 h0 1 h

GAPDH (220 bp)

SOCS-3 (650 bp)

lowastlowast


lowastlowast

lowast

0

01

02

03

04

05

06

SOCS

-3G

APD

H

4 h2 h1 h0



(a) vehicle control





(f)



4205 1

(h)

(a)

4205 1

(h)

(b)

4205 1

(h)

(c)

4205 1

(h)

(d)











20001000

250100

750500(b

p)

0

01

02

03

04

05SO

CS-3

GA

PDH

lowast

lowastlowast

lowast

lowastlowast

4 h 8 h0 1 h 2 h



(a) U0126




(e)


pERK12

10

min

20

min

30

min

1h

2h

4h

8h

RRK12

SOCS-3

GAPDH



M 1 2 43

250

750

GAPDH (220 bp)

SOCS-3 (650 bp)(bp)


4 Discussion



8421

(h)

(a)

8421

(h)

(b)

8421

(h)

(c)

8421

(h)

(d)

8421

(h)

(e)




SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(a)

SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(b)

Empt

y ve

ctor

PRK5

PRK5

-SO

CS-3

PRK5

-SO

CS-3

+ 1

0he

rb se

rum

PRK5

-SO

CS-3

+ 1

0co

ntro

l ser

um

0

10

20

30

40

50

60

Perc

enta

ge o

f PCN

A

lowast

lowast

lowast

(c)















5 Conclusions





Acknowledgments


References




























3



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4205 1

(h)

(a)

4205 1

(h)

(b)

4205 1

(h)

(c)

4205 1

(h)

(d)











20001000

250100

750500(b

p)

0

01

02

03

04

05SO

CS-3

GA

PDH

lowast

lowastlowast

lowast

lowastlowast

4 h 8 h0 1 h 2 h



(a) U0126




(e)


pERK12

10

min

20

min

30

min

1h

2h

4h

8h

RRK12

SOCS-3

GAPDH



M 1 2 43

250

750

GAPDH (220 bp)

SOCS-3 (650 bp)(bp)


4 Discussion



8421

(h)

(a)

8421

(h)

(b)

8421

(h)

(c)

8421

(h)

(d)

8421

(h)

(e)




SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(a)

SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(b)

Empt

y ve

ctor

PRK5

PRK5

-SO

CS-3

PRK5

-SO

CS-3

+ 1

0he

rb se

rum

PRK5

-SO

CS-3

+ 1

0co

ntro

l ser

um

0

10

20

30

40

50

60

Perc

enta

ge o

f PCN

A

lowast

lowast

lowast

(c)















5 Conclusions





Acknowledgments


References




























3



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















20001000

250100

750500(b

p)

0

01

02

03

04

05SO

CS-3

GA

PDH

lowast

lowastlowast

lowast

lowastlowast

4 h 8 h0 1 h 2 h



(a) U0126




(e)


pERK12

10

min

20

min

30

min

1h

2h

4h

8h

RRK12

SOCS-3

GAPDH



M 1 2 43

250

750

GAPDH (220 bp)

SOCS-3 (650 bp)(bp)


4 Discussion



8421

(h)

(a)

8421

(h)

(b)

8421

(h)

(c)

8421

(h)

(d)

8421

(h)

(e)




SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(a)

SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(b)

Empt

y ve

ctor

PRK5

PRK5

-SO

CS-3

PRK5

-SO

CS-3

+ 1

0he

rb se

rum

PRK5

-SO

CS-3

+ 1

0co

ntro

l ser

um

0

10

20

30

40

50

60

Perc

enta

ge o

f PCN

A

lowast

lowast

lowast

(c)















5 Conclusions





Acknowledgments


References




























3



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















8421

(h)

(a)

8421

(h)

(b)

8421

(h)

(c)

8421

(h)

(d)

8421

(h)

(e)




SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(a)

SOCS-3

GAPDH

M 21 3 4 5

250

750

(bp)

(b)

Empt

y ve

ctor

PRK5

PRK5

-SO

CS-3

PRK5

-SO

CS-3

+ 1

0he

rb se

rum

PRK5

-SO

CS-3

+ 1

0co

ntro

l ser

um

0

10

20

30

40

50

60

Perc

enta

ge o

f PCN

A

lowast

lowast

lowast

(c)















5 Conclusions





Acknowledgments


References




























3



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























5 Conclusions





Acknowledgments


References




























3



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































3



















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















kidney-replenishing herb induces socs-3 expression via erk...

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