kangquan recipe regulates the expression of bambi protein...

Research ArticleKangquan Recipe Regulates the Expression ofBAMBI Protein via the TGF-120573Smad Signaling Pathway toInhibit Benign Prostatic Hyperplasia in Rats

Wenfan Chen12 Xiaoqing Huang12 Axiang Peng1 Tingting Chen2 Renzhi Yang12

Yuanpeng Huang 1 Zongbao Yang2 and Shengyan Xi 2

1Department of Traditional Chinese Medicine Zhongshan Hospital Xiamen University Xiamen 361004 Fujian Province China2Department of Traditional Chinese Medicine School of Medicine Xiamen University Xiamen 361102 Fujian Province China

Correspondence should be addressed to Yuanpeng Huang huangyp998xmueducn and Shengyan Xi xishengyanxmueducn

Received 23 January 2019 Revised 13 March 2019 Accepted 25 March 2019 Published 2 May 2019

Academic Editor Cheorl-Ho Kim

Copyright copy 2019 Wenfan Chen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Kangquan Recipe (KQR) is a traditional Chinese medicine compound made by our research group for the treatmentof benign prostatic hyperplasia (BPH) Whether KQR can treat BPH as a single drug or play a role in the treatment of BPH incombination therapy needs further study Aim of the Study To investigate the effect of KQR on the expression of TGF-120573Smadsignaling pathway-related factors in ratswith BPH In-depth analysis revealed the relevant signal transductionmechanismbywhichKQR acts to treat BPHMaterials and Methods Forty-eight male Sprague-Dawley rats were randomly divided into six groups of 8rats each In addition to the control group 40 rats were castrated and then injected with testosterone propionate to form a prostatichyperplasia model After 30 days three groups received different concentrations of KQR (14 gkg 7 gkg and 35 gkg) and thefinasteride group received 05mgkg finasteride The BPH group and the control group received the same volume of saline Allgroups were treated for a total of 30 days Rat body weight prostate volume wet weight index histology and the mRNA andprotein levels of TGF-120573 TGF-120573R1 TGF-120573R2 p-Smad2 p-Smad3 BAMBI E-cadherin and N-cadherin in the prostate tissue weremeasured after the end of treatment Results Compared with the control group the BPH group had increased prostate wet weightvolume and index and the histology showed significant BPH Compared with the BPH group the three KQR groups and thefinasteride group all had varying levels of reduction in the prostate wet weight volume and index of the prostate and varying degreesof improvement in the histological manifestations of BPH KQR downregulates the mRNA andor protein expression of TGF-120573TGF-120573R1 TGF-120573R2 p-Smad2 p-Smad3 andN-cadherin protein in prostate tissue and increases themRNA andprotein expressionof BAMBI and E-cadherin proteinConclusions In the model of BPH induced by testosterone propionate after castration KQR caninhibit the conduction of the TGF-120573Smad signaling pathway by upregulating the expression of BAMBI protein and reversing EMTin rat prostate tissue

1 Introduction

Benign prostatic hyperplasia (BPH) is a common diseasein elderly men with lower urinary tract symptoms (LUTS)caused by hyperplasia of the prostatic epithelium and stromalcells [1] At present the main therapeutic drugs for BPHare 5120572-reductase inhibitors and 120572-blockers [2 3] such asfinasteride and terazosin However they can cause adversereactions such as fatigue hypotension ejaculation disorderssexual dysfunction and an increased risk of prostate fibrosis

[3 4] Surgical treatment of BPH requires strict surgical indi-cations and carries with it the inevitable complications andrisks of surgery and the possibility of recurrence In fact thenumber of patients undergoing surgery for BPH is graduallydecreasing [5] Therefore it is particularly important to finda therapeutic drug that can effectively treat BPH with fewadverse reactions

Traditional Chinese medicine (TCM) treatment of dis-eases including BPH has been reported [6 7] In this studyKangquan Recipe (KQR) has a unique effect in treating

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 6281819 12 pageshttpsdoiorg10115520196281819

2 Evidence-Based Complementary and Alternative Medicine

BPH with mild effects and small side effects Clinicalstudies have shown that [8] KQR can effectively reduceInternational Prostate Symptom Score (I-PSS) scores resid-ual urine volume (RUV) and total prostatic volume(TPV)and improve the maximum urinary flow rate (Qmax) andaverage urine flow rate (Qave) levels in patients with BPHPrevious experimental studies found that [9] the mecha-nism of KQR treatment of BPH may be to regulate thebalance of plasma testosterone and estradiol and reducethe expression of proliferating cell nuclear antigen (PCNA)mRNA to inhibit prostate cell proliferation However thepotential mechanism by which KQR acts in the treatment ofBPH remains to be further studied Recently the influenceof the transforming growth factor-120573 (TGF-120573)suppressor ofmothers against decapentaplegic (Smad) signaling pathwayon BPH has attracted the attention of many scholars

In BPH activation of the TGF-120573Smad signaling pathwayoccurswhenTGF-1205731 bindswith the receptors on the envelopeand then combines with Smad to form a complex [10]The Smad complex enters the nucleus and binds to theSmad binding element in the promoter region of epithelial-mesenchymal transition (EMT)-related genes regulatingthe expression of 120572-smooth muscle actin connective tissuegrowth factor and other EMT-related target genes therebycausing EMT in prostate tissue [11] Therefore the TGF-120573Smad signaling pathway is considered one of the keypathways leading to EMT in prostate epithelial cells

In the process of the TGF-120573 family members binding tothe receptors there is a pseudoreceptor namely bone mor-phogenetic protein and activin membrane-bound inhibitor(BAMBI) It does not possess kinase activity due to the lack ofa serinethreonine kinase domain in the intracellular region[12] However it has a similar structure to the extracellulardomain of TGF-120573 type I receptor meaning that it cancompete with type I receptors and bind to type II receptorsto form the ligand-receptor complex thereby blocking thetransduction of the signal along the TGF-120573Smad signalingpathway [13 14]

Therefore this study aimed to observe the regulation byKQR of the expression of TGF-120573Smad signaling pathway-related factors in prostate tissue and to further explore themechanism of action of KQR which is helpful in elucidatingthe related signal transduction mechanism of BPH to providea basis for clinical treatment At the same time we searchedthe existing literature and found that few studies haveinvestigated the effect of BPH on the TGF-120573Smad signalingpathway This study is a prospective study of traditionalChinese medicine The aims of this study were to exploremultitarget and multichannel regulation by a traditionalChinese medicine to promote the in-depth application oftraditional Chinese medicine to treat BPH and to form aneffective systematic and standardized treatment plan

2 Materials and Methods

21 Experimental Animals In total 48 male Sprague-Dawley(SD) rats (200 plusmn 20 g) were purchased from Shanghai SlackLaboratory Animals Co Ltd These animals were placed

in cages in the animal room of Xiamen University MedicalCollege [License No SYXK (Min) 2013-0006]The rats werekept under the following conditions room temperature 20-22∘C relative humidity 65-70 12 h natural light dark cycleand ad libitum food [Beijing Huakang Biotechnology CoLtd Beijing Feed Certificate (2014) 06057] and water Thestudy was reviewed and approved by the Animal EthicsCommittee of Xiamen University and conducted accordingto the guidelines for the care and use of laboratory animals

22 Experimental Drug Preparation Techniques The medic-inal components of KQR [Morinda officinalis (vouchernumber 170713) Pheretima aspergillum (voucher number170814) Eupolyphaga sinensis Walker (voucher number1703310162) Rheum palmatum (voucher number 170526)Trachelospermum jasminoides (voucher number 170601)and Cinnamomum cassia Presl (voucher number 171117)]were purchased from Yanlaifu Co Ltd (production licensenumber Fujian 20112046) and approved by ProfessorYingkun Qiu (Xiamen University China) All the herbals arestored in the Experimental Center of Traditional ChineseMedicine of Medical College of Xiamen University Then15 g of Morinda officinalis 15 g of Pheretima aspergillum 10 gof Eupolyphaga sinensis Walker 5 g of Rheum palmatum20 g of Trachelospermum jasminoides 5 g of Cinnamomumcassia Presl and 700mL of distilled water were directlymixedThemixturewas left to soak in the decocting containerfor 30 minutes and then boiled with high heat simmeredfor 30 minutes and filtered through 8 layers of filter gauzeNext 450mL of distilled water was added to the decoctingcontainer and cooked for another 30minutes at low heat Thetwo combined filtrates were concentrated to 50mL at 58∘Cin a rotary evaporator (RE-52AA Shanghai Yarong) whichcorresponds to a high dose of 14 gmL of the crude drugA middle dose of 07 gmL and a low dose of 035 gml wereprepared by dilution with distilled water

23 HPLC Chemical analysis of KQR was performed usinga liquid chromatograph model Shimadzu LC-20A (ShimadzuCorporation Japan) The separation column model is YMC-Pack ODS column (YMC Japan) and the mobile phaseis 01 phosphoric acid aqueous solution (A)-acetonitrile(B) 5-45 acetonitrile gradient system elution time 0-60minutes and 100 B 60-70 minutes The flow rate was1mLmin and the test solution injection volume was 20 120583L

24 Main Reagents The reagents used were as followsFinasteride tablets (product code NO22063) was supplied byMerck SharpampDohmeLimitedUK Testosterone propionateinjection (batch number 170512) was provided by ChinaNingbo Second Hormone Factory TRIZOL reagent (3070-250) was provided by Beijing Tian Enze Gene TechnologyCo Ltd China ReverTra Ace qPCR RT Master Mix (FSQ-201) was supplied by the TOYOBO Corporation of Japan FSUniversal SYBR Green Master (4913914001) was supplied byROCHE SwitzerlandAnti-TGF120573R1 antibody (product codeab31013) anti-TGF-120573R2 antibody (product code ab186838)

Evidence-Based Complementary and Alternative Medicine 3

anti-BAMBI antibody (product code ab203070) anti-E-cadherin antibody (product code ab133597) and N-cadherin(product code ab18203) were provided by Abcam UK Anti-TGF 1205731 (product code ARG55096) p-Smad2 (product codeARG51796) and p-Smad3 (product code ARG51797) werepurchased from Arigo Company China

25 Instruments The instruments used are listed belowThe precision balance (PL601-L) was produced by MET-TLER TOLEDO Switzerland The Intelligent Biomicroscope(BX53) was produced by the Olympus Corporation ofJapan The paraffin slicer (RM2235) embedding machine(EG1150H) and fully automatic tissue dewatering machine(ASP200S) were produced by Leica Germany The gelelectrophoresis apparatus (PowerPac Basic) and the elec-trophoresis tank (Mini-PROTEAN) were manufactured bythe Bio-Rad Corporation in the United States The fullyautomatic chemiluminescence image analysis system (5200Stype) was produced by Tanon Company China The PCRinstrument (Model ABI7500) was produced by AppliedBiosystems USA The PRO Multi-Function Label (M200)was produced by Tecan Switzerland

26 Establishment of Benign Prostatic Hyperplasia AnimalModel The rat model of BPH was established according tothe protocol used in previous experiments [9] After oneweekof adaptation to the environment rats in all groups exceptthe control group were anesthetized via abdominal injectionof chloral hydrate (3mLkg) and the scrotums of the ratswere cut layer by layer under sterile conditions The testisand epididymis were exfoliated to avoid the effects of residualtestosterone in the body The stump was ligated and thensutured layer by layer The cages were kept clean and dry toprevent infection of the incision After a week of recoverythe animals were injected subcutaneously with testosteronepropionate at a dose of 35mgkg once daily for 30 days Thecontrol groupwas injected subcutaneously with physiologicalsaline

27 Experimental Design A total of forty-eight SD male ratswere randomly divided into 6 groups by random numbertable method 8 rats were as the control group and theother 40 prostate hyperplasia rat models were randomlydivided into the following groups with 8 rats in each groupKQR high dose group KQR middle dose group KQR lowdose group finasteride group (positive control) and BPHgroup These groups of rats were injected subcutaneouslywith 35mgkg testosterone propionate daily and the controlgroup was injected with the same amount of normal salinefor 30 consecutive days According to the equivalent doseconversion formula for humans and rats the KQR highdose group received 14 gkg (corresponding to 12 times theclinical dose) the KQR middle dose group received 7 gkg(equivalent to 6 times the clinical dose) the KQR low dosegroup received 35 gkg (equivalent to 3 times the clinicaldose) and the finasteride control group received 05mgkg(equivalent to 6 times the clinical dose) the medicine wasdissolved in 10mLkg of normal saline immediately before

use In the control group and theBPHgroup only 10 mLkg ofnormal saline was administered The administration volumewas 1mL100 g and it was administered intragastrically oncea day for 30 consecutive days (during the administrationperiod the body weight was measured once a week and theamount was adjusted accordingly)

28 Measurement of Rat Body Weight Prostate Volume WetWeight and Index After 4 weeks of treatment the bodyweight of the rats was measured with a balance Afterthe rats were euthanized the wet weights of the bilateralventral lobes of the prostate tissue were measured and theprostate volume was measured by the volumetric methodThe prostate index (PI = prostate wet weightbody weight times100) was calculated

29 Prostate Tissue Pathological Changes A portion of ratprostate tissue was immersed in 10 formalin for fixationdehydrated and embedded in paraffin Hematoxylin andeosin (HampE) staining was performed after the tissue was cutinto 4-micron-thick slices and pathological changes in the ratprostate tissue were observed under a microscope

210 Western Blotting Detected the Protein Expression Levelsof TGF-120573 TGF-120573R1 TGF-120573R2 pSmad2 p-Smad3 BAMBIE-Cadherin and N-Cadherin in Rat Prostate Tissue Westernblotting was performed with reference to the method inXi et al [15] in which the following antibodies were usedrabbit anti-anti-TGF 1205731 antibody (11000 dilution) rabbitanti-anti-TGF-120573 receptor 1 antibody (11000 dilution) rabbitanti-anti-TGF-120573 receptor 2 antibody (11000 dilution) rabbitanti-anti-Smad 2 antibody (11000 dilution) rabbit anti-anti-Smad 3 antibody (11000 dilution) rabbit anti-anti-BAMBIantibody (11000 dilution) rabbit anti-E-cadherin antibody(11000 dilution) rabbit anti-N-cadherin antibody (11000dilution) and murine anti-GAPDH monoclonal antibody(15000 dilution)

211 qRT-PCR Was Used to Detect the mRNA Expressionof TGF-120573 TGF-120573R1 BAMBI Smad2 E-Cadherin and N-Cadherin in Rat Prostate Tissue The qRT-PCR was per-formed with reference to the protocol in Xi et al andprevious experiments [9 15] The primers were synthesizedby Shanghai Shenggong Co Ltd and the primer sequenceswere as follows GAPDH F-ACCA CAGT CCAT GCCATCAC R-TCCA CCAC CCTG TTGC TG 168 bp TGF-120573 F-CATT GCTG TCCC GTGC AGA R-AGGT AACGCCAG GAAT TGTT GCTA 103 bp TGF120573R1 F-CACACAGT CAGT GCGG TGAG AG R-AGAG GCCC ACAAGAGT TTCA ACA 88 bp BAMBI F-GCAA TTAC CGAGGACT GCAT GA R-TCCT GCAC CTTA GTGA TGAGGTTT C 112 bp Smad2 F-TTAC AGAT CCAT CGAACTCG GAGA R-CACT TAGG CACT CGGC AAAC AC150 bp E-cadherin F-CACA CTGA TGGT GAGG GTACAAGG R-GGGC TTCA GGAA CACA TACA TGG 123 bpN-cadherin F-GACT GCAC CGAC GTAG ACAG GA R-ATCC ATAC CACG AACA TGAG GACA 116 bp Eachexperiment was repeated three times


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Figure 1 Chemical structure of KQR active ingredients identified by HPLC Nortrachelogenin-81015840-O-120573-glucoside (a) Nortrachelogenin-51015840-C-120573-glucoside (b) Luteoloside (c) Apigenin-71015840-glucoside (d) Tracheloside (e) Nortrachelogenin (f) Isoquercitrin (g) and Arctigenin (h)

212 Statistical Analysis The parameter data were expressedas means plusmn SD (119909 plusmn s) Experimental data were analyzedby using GraphPad Prism 7 software (GraphPad SoftwareInc La Jolla United States) Statistical significance wasdetermined by using one-way analysis of variance [ANOVA]Differences of Plt005 were considered significant

3 Results

31 Analysis of KQR As shown in Figure 1 KQR wasseparated with High Performance Liquid Chromatogra-phy (HPLC) system and its chromatographic fingerprintingestablished Comparing the retention time and UV spec-tra with reference samples the following 8 major chem-ical compositions were identified Nortrachelogenin-81015840-O-120573-glucoside (peak 1 Rt = 1501min) Nortrachelogenin-51015840-C-120573-glucoside (peak 2 Rt = 2578 min) Luteoloside (peak3 Rt = 2869min) Apigenin-71015840-glucoside (peak 4 Rt =3623min) Tracheloside (peak 5 Rt = 4081min) Nortrach-elogenin (peak 6 Rt = 4627min) Isoquercitrin (peak 7 Rt =4905min) and Arctigenin (peak 8 Rt = 5491min)

32 Effect of KQR on Wet Weight Prostate Volume andPI in Rats As shown in Figures 2(a)ndash2(d) compared withthe control group the weight prostate wet weight prostatevolume and PI were significantly different in the BPHgroup (Plt001) Compared with the BPH group (Figures2(b)ndash2(d)) the three KQR group and finasteride (control)group had significantly lower prostate wet weights prostate

volumes and PI values (Plt005 or Plt001) Compared withthe finasteride group the KQR high and middle dose groupshad no significant differences in prostate wet weight prostatevolume and PI values (Pgt005) KQR could significantlyreduce the prostate wet weight prostate volume and PI of ratswith BPH its effect had a positive relationship with the drugconcentration

33 Effect of KQR on the Pathological Morphology of ProstateTissue in Rats with BPH Pathological observation was con-ducted on prostate tissue in each group The prostate glandof the control group (Figure 3(a)) showed no hyperplasiaand the glandular epithelial cells were mostly cubic andarranged in a single layer And interstitial and fibrous tissuecontent did not increaseThe prostate gland of the BPHgroup(Figure 3(b)) had proliferation with an increased numberof glandular epithelial cells and an increased volume withhigh columnarity The cells were arranged in a single layeror multiple layers and the glandular epithelial cell layerformed folds into the cavity showing papillary processesinterstitial fibrous tissue and smooth muscle tissue increaseIn the finasteride group (Figure 3(c)) the prostate glandwas dilated some epithelial cells still proliferated but thepapillary processes were significantly reduced and inter-stitial fibrous tissue was not obvious Prostate hyperplasiawas significantly alleviated in the KQR high dose group(Figure 3(d)) and epithelial cells were mostly in a single layerwith few papillary processes And interstitial fibrous tissueand smooth muscle tissue were significantly reduced In the


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Figure 3 Effects of KQR on pathology of prostate tissue in rats Sections were stained with hematoxylin and eosin (HampE) and viewed ata magnification of times200 (a) Control group (b) BPH group (c) finasteride group (05mgkg) (d) KQR high dose group (14 gkg) (f) KQRmiddle dose group (7 gkg) (f) KQR low dose group (35 gkg)


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Figure 4 Effects of KQR on the protein expression of TGF-120573 TGF-120573R1 TGF-120573R2 p-Smad2 and p-Smad3 in the prostate tissue Data wererepresented asmean plusmn SD (n = 6mice per group) Statistical analysis lowastPlt005 lowastlowastPlt001 compared with the control group Plt005 Plt001compared with the BPH group

KQR middle dose group (Figure 3(e)) the prostate glandwas shrunken the glandular epithelial cells were slightlyproliferated the wrinkles were reduced and only a smallpart showed papillary processes a small amount of interstitialtissue and vascular fibrous tissue The prostate glands of theKQR low dose group (Figure 3(f)) were slightly dilated andthe folds of the glandular epithelial cells into the cavity wereslightly reduced the epithelial cells were enlarged and closelyarranged And partial interstitial fibrous tissue and smoothmuscle tissue were showed

34 Effects of KQR on the Protein Expression of TGF-120573 TGF-120573R1 TGF-120573R2 p-Smad2 p-Smad3 BAMBI E-Cadherin andN-Cadherin in the Prostate Tissue of Rats with BPH As

shown in Figure 4 the expression levels of TGF-120573 TGF-120573R1 TGF-120573R2 p-Smad2 and p-Smad3 which were relatedto the TGF-120573Smad signaling pathway were upregulated inthe BPH group compared with the control group (Plt005or Plt001) The expression levels of TGF-120573 TGF-120573R1 TGF-120573R2 p-Smad2 and p-Smad3 were significantly decreasedafter finasteride treatment (Plt005 or Plt001) Among theKQR groups the high dose group also downregulated theexpression levels of TGF-120573 TGF-120573R1 TGF-120573R2 p-Smad2and p-Smad3 relative to the model group (Plt005 or Plt001)(see the Supplementary Figure 1) and therewas no significantdifference in protein levels compared with the finasteridegroups (Pgt005) However as the concentration of KQRdecreased its downregulation effects were weakened



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Figure 5 Effects of KQR on the protein expression of BAMBI E-cadherin and N-cadherin in the prostate tissue Data were represented asmean plusmn SD (n = 6 mice per group) Statistical analysis lowastPlt005 lowastlowastPlt001 compared with the control group Plt005 Plt001 comparedwith the BPH group

BAMBI is a pseudoreceptor in the TGF-120573 signalingpathway which is structurally similar to TGF-120573R1 andcompetitively binds to TGF-120573R2 thereby blocking the TGF-120573Smad signaling pathway As shown in Figure 5 comparedwith the control group the BPH group had decreasedexpression of BAMBI (Plt005) Interestingly the expressionof BAMBIwas significantly upregulated after KQRhigh dosesor finasteride treatment (Plt005) As the concentration ofKQR decreased the level of upregulation decreased (seethe Supplementary Figure 2) E-cadherin and N-cadherinare epithelial-mesenchymal transition (EMT)-related factorsCompared with the control group the BPH group hadincreased expression ofN-cadherin and decreased expressionof E-cadherin (Plt005) KQR high doses or finasteride wereable to downregulate the expression of N-cadherin andupregulate the expression of E-cadherin (Plt005) but as theconcentration of KQR increased its effect is strengthened (seethe Supplementary Figure 2)

35 Effects of KQR on the mRNA Expression of TGF-120573 TGF-120573R1 BAMBI Smad2 E-Cadherin and N-Cadherin in theProstate Tissue of Rats with BPH Figure 6 shows that com-pared with the control group the BPH group had increasedmRNAexpression levels of TGF-120573 TGF-120573R1 p-SMAD2 andN-cadherin (Figures 6(a)ndash6(c) and 6(f)) and the expressionlevels of BAMBI and a decreased mRNA expression levelof E-cadherin (Plt001) (Figures 6(d) and 6(e)) Compared

with the BPH group the KQR and finasteride groups haddecreased mRNA expression levels of TGF-120573 TGF-120573R1Smad2 and N-cadherin and increased expression mRNAexpression levels of BAMBI and E-cadherin (Plt005 orPlt001) Compared with the finasteride group the KQRmiddle and low dose groups had no significant differencesin the level of BAMBI mRNA expression and the KQRhigh and middle dose groups had no significant different inmRNA expression level of E-cadherin (Pgt005) Comparedwith the KQR high dose group the KQRmiddle and low dosegroups had significantly difference mRNA expression levelsof BAMBI Smad2 N-cadherin and E-cadherin (Plt005or Plt001) These results suggest that KQR could decreasethe mRNA expression of TGF-120573 TGF-120573R1 Smad2 and N-cadherin and increase the mRNA expression of BAMBI andE-cadherin in the prostates of rats with BPHThe relationshipwas dose-dependent

4 Discussion

BPH is a common disease in older men With the increasein the aging population the BPH has attracted increasingattention [16] There is a wealth of experience with usingtraditional Chinese medicine in the treatment of this diseaseTraditional Chinese medicine states that the pathologicalfeatures of BPH are based on kidney deficiencyThe incidenceis caused by hot and humid bets qi stagnation blood stasis



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Figure 6 Effects of KQR on the mRNA expression of TGF-120573 TGF-120573R1 BAMBI Smad2 E-cadherin and N-cadherin in the prostate tissueData were represented as mean plusmn SD (n = 6 or 7 mice per group) Statistical analysis lowastPlt005 lowastlowastPlt001 compared with the control groupPlt005 Plt001 compared with the BPH group 998771Plt005 998771998771Plt001 compared with the KQR high dose group

and heat accumulation [17] KQR consists of morinda offic-inalis pheretima aspergillum eupolyphaga sinensis Walkerrheum palmatum trachelospermum jasminoides and cin-namomum cassia Presl

Morinda officinalis has anti-inflammatory and antioxida-tion activities [18] inhibiting the production of prostaglandinE2 and tumor necrosis factor-120572 (TNF-120572) is beneficial forthe treatment of BPH [19] Pheretima aspergillum has avariety of beneficial pharmacological activities includingdiuretic fibrinolytic anticoagulant anti-inflammatory and

antioxidation [20] Eupolyphaga sinensis Walker have anti-coagulant antithrombotic and antitumor effects [21] Rheumpalmatumhas been shown to have anti-inflammatory antiox-idation anticancer and improved renal function [22] Thepharmacological properties of the trachelospermum jas-minoides include anti-inflammatory analgesic antitumorantiviral and antibacterial activities [23] The combinationof various medicines both attacking and supplementingtonifies the kidneys and replenishing the qi promotingblood circulation and clearing and dissipating the heat The


results of this experiment show that KQR can reduce thePI value prostate volume and wet weight in rats Tissuesections also confirmed that KQR can significantly inhibitprostate gland dilation in rats This indicates that KQR hasa good therapeutic effect on rats with BPH This findingfurther proves the effectiveness of KQR in the treatment ofBPH

It is worth mentioning that eight active substances inthe KQR component that are beneficial for the treatmentof BPH have been detected Specifically NortrachelogeninNortrachelogenin-51015840-C-120573-glucoside and Nortrachelogenin-81015840-O-120573-glucoside are a group of plant-derived polyphenoliccompounds that induce cell death and inhibit cancer cell pro-liferation and reduce hormone dependenceThe risk of sexualcancer [24] Luteoloside has anti-inflammatory antioxidantand antitumor effects [25] Apigenin-71015840-glucoside has antiox-idant anti-inflammatory and antitumor activities [26] notonly has Trachelogenin liver protection but also antitumorviral activity is involved in inhibiting the proliferation oftumor cells [27] both in vivo and in vitro studies of Iso-quercitrin have been shown to treat cancer cardiovasculardisease and allergic reactions [28] Arctigenin studies haveshown strong antiprostate cancer activity [29]

BPH is a disease that most men face The incidence ofBPH in men aged 61-70 is 70 and the incidence after 80years is 90 [30] TGF-120573 plays an important role in thedevelopment of BPH Excessive expression of TGF-120573 caninduce local vascular formation and inflammatory responsesand can also increase the incidence of BPH [31 32] Studieshave shown that the expression of TGF-1205731 is elevated in BPH[33] and the high expression level of TGF-120573 is also associatedwith poor clinical outcomes [34] In prostatic hyperplasiacell lines the expression level of TGF-120573 is associated withthe migration of BPH-1 cells [35] TGF-120573 is able to exertits biological effects by binding to receptors type 1 and2 [36] TGF-120573R1 and TGF-120573R2 are important factors inTGF-120573Smad signaling [37] The TGF-120573 receptors containa serinethreonine kinase that is directly involved in TGF-120573 signaling [38] The experimental results were as expectedKQR can downregulate the expression level of TGF-1205731 andthe expression levels of TGF120573R-1 and TGF120573R-2 are alsodecreased One of the mechanisms by which KQR treats BPHmay be through the inhibition of the TGF-120573Smad signalingpathway

The main purpose of this study was to analyze the effectof KQR on TGF-120573Smad signaling in rat prostate tissue Inthe Smad family Smad2 and Smad3 are receptor-activatedSmads (R-SMADs) that can be phosphorylated and activatedby TGF-120573 binding to type 1 and type 2 receptors [39]Phosphorylated Smad is responsible for transducing TGF-120573 signaling from the cell membrane to the nucleus wherethe transcription of the target gene is either induced orinhibited thereby completing the signal transduction [40]Phosphorylation of Smad2 or Smad3 (p-Smad) representsthe activation of the TGF-120573Smad signaling pathway Theabnormal expression of the Smad protein has been confirmedto be closely related to the occurrence and development ofBPH [41ndash43] Our data showed that the levels of p-Smad2and p-Smad3 and the levels of Smad2 mRNA in the prostate

tissue of rats treated with KQR were lower than those inthe model group This may be one of the reasons for thedecrease in the activation of the TGF-120573Smad signalingpathway Furthermore KQR reduced the expression of TGF-1205731 and inhibited the phosphorylation of Smad2 and Smad3blocking the TGF-120573Smad signaling pathway and therebyinhibiting the development of BPHNext we investigated themechanism bywhichKQR blocked the activation of the TGF-120573Smad signaling pathway

The BAMBI protein is a transmembrane glycoprotein anda pseudoreceptor of the TGF-120573Smad signaling pathway [44]Studies have shown that inhibition of BAMBI expression incolorectal cancer can upregulate TGF-120573 signaling leadingto elevated levels of p-Smad2 and p-Smad3 [45] Yan et alfound that the complex formed by BAMBI protein Smad7and TGF-1205731 receptor can prevent the phosphorylation ofSmad3 and block the TGF-120573Smad signaling pathway [46] Inaddition there is a therapeutic effect on the development ofprostate cancer [47] Although the BAMBI protein has beenstudied in many diseases such as tumors and tissue fibrosis[48 49] the influence of traditional Chinese medicine onBAMBI in BPH is poorly understood In the present studywe found that BAMBI mRNA and protein were expressed inrat prostate specimens and upregulated with KQR treatmentThese data indicate that overexpression of BAMBI proteininhibits TGF-120573 signaling and prevents the activation andphosphorylation of Smad2 and Smad3

In EMT the expression levels of molecular markers onepithelial cells such as E-cadherin are decreased while theexpression levels of molecular markers in interstitial cellssuch as N-cadherin are increased [50] Alonso-Magdalena etal also found that cells proliferating in BPH tissue are derivedfrom EMT [51] In recent years studies have shown [52] thatEMT occurs in BPH cells and is associated with the TGF-120573Smad signaling pathway These studies suggest that EMTmay be involved in the development of BPH and play animportant role Our results indicate that in prostate tissueoverexpressing BAMBI protein the protein expression levelsof E-cadherin are upregulated while the protein expressionlevel of N-cadherin is downregulated This suggests thatKQR can regulate the expression of BAMBI and therebyintervention of the TGF-120573Smad signaling pathway Upreg-ulation of BAMBI expression may be the main mechanismby KQR to inhibit EMT in prostate tissue In addition theresults showed that KQR high dose can effectively inhibit thepathological changes of rat prostate tissue inhibit the expres-sion of TGF reduce the downstream signal transduction ofthe TGF-120573Smad signaling pathway and reverse EMT inBPH

Although our results suggest that KQR can be used as apotential drug for future clinical treatment of BPH due tothe complexity of traditional Chinese medicine ingredientstheremay be some shortcomings in this experiment First themechanism by which KQR increases the protein expressionof BAMBI remains unclear and further research is neededto confirm the role of KQR Second Chinese medicineformula has multipathway and multitarget effects Thereforethe TGF-120573Smad signaling pathway may not reflect the mainmechanisms involved in BPH


5 Conclusion

In summary this study demonstrates that KQR has theeffect of treating BPH in rats It can upregulate the proteinexpression of BAMBI and inhibit the expression of TGF-120573TGF-120573R1 TGF-120573R2 Smad2 p-Smad2 and p-Smad3 relatedfactors to block the transmission of TGF-120573Smad signalingpathway and reverse EMT phenomenon in rat prostate tissueFurther research into the mechanism of this formulation willhelp to demonstrate the potential value of traditional Chinesemedicine in the treatment of BPH

Abbreviations

KQR Kangquan RecipeBPH Benign prostatic hyperplasiaLUTS Lower urinary tract symptomsI-PSS International Prostate Symptom ScoreRUV Residual urine volumeTPV Total prostatic volumeQmax Maximum urinary flow rateQave Average urine flow ratePCNA Proliferating cell nuclear antigenTGF-120573 Transforming growth factor-120573Smad Suppressor of mothers against

decapentaplegicBAMBI Bone morphogenetic protein and activin

membrane-bound inhibitorEMT Epithelial-mesenchymal transitionSD Sprague-DawleyPI Prostate indexHE Hematoxylin and eosinTCM Traditional Chinese medicine

Data Availability

All the data are available in the manuscript

Conflicts of Interest

All the authors have stated that there are nopotential conflictsof interest

Authorsrsquo Contributions

Wenfan Chen contributed to the compilation of data andthe writing of manuscripts Yuanpeng Huang Zongbao Yangand ShengyanXi contributed to data analysis and administra-tive support Xiaoqing Huang Axiang Peng Tingting Chenand Renzhi Yang participated in the experimental processand contributed to the collection of data Wenfan Chen andXiaoqing Huang contributed equally to this work All theauthors read the paper and approved its publication

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (no 81674041) the Xiamen Science andTechnology Bureau Research Project (no 3502Z20164013)

and the Natural Science Foundation of Fujian Province (no2018J01395)

Supplementary Materials

Supplementary Figure 1 effects of different doses of KQR onprotein expression of TGF-120573 TGF-120573R1 TGF-120573R2 p-Smad2and p-Smad3 in the prostate tissue Supplementary Figure2 effects of different doses of KQR on protein expression ofBAMBI E-cadherin and N-cadherin in the prostate tissueData were represented as mean plusmnSD (n = 6 mice per group)Statistical analysis Plt005 Plt001 comparedwith theBPHgroup 998771Plt005 998771998771Plt001 compared with the KQR high dosegroup (Supplementary Materials)

References

[1] M Saito P Tsounapi R Oikawa et al ldquoProstatic ischemiainduces ventral prostatic hyperplasia in the SHR possiblemechanism of development of BPHrdquo Scientific Reports vol 4article 3822 2014

[2] C Saez A C Gonzalez-Baena M A Japon et al ldquoRegressivechanges in finasteride-treated human hyperplastic prostatescorrelate with an upregulation of TGF-beta receptor expres-sionrdquo Prostate vol 37 pp 84ndash90 1998

[3] K T FooH S S HoM Y CWong et al ldquoSingapore urologicalassociation clinical guidelines for male lower urinary tractsymptomsbenign prostatic hyperplasiardquo Singapore MedicalJournal vol 58 no 8 pp 473ndash480 2017

[4] H K Kim C Zhao B R Choi et al ldquoIs transforminggrowth factor-beta signaling activated in human hypertrophiedprostate treated by 5-alpha reductase inhibitorrdquo Disease Mark-ers vol 35 no 6 Article ID 783287 pp 679ndash685 2013

[5] L Minutoli M Rinaldi H Marini et al ldquoApoptotic pathwayslinked to endocrine system as potential therapeutic targets forbenign prostatic hyperplasiardquo International Journal ofMolecularSciences vol 17 article 1311 2016

[6] Q Bian W Wang N Wang Y Peng W Ma and R DaildquoArachidonic acid metabolomic study of BPH in rats andthe interventional effects of Zishen pill a traditional Chinesemedicinerdquo Journal of Pharmaceutical and Biomedical Analysisvol 128 pp 149ndash157 2016

[7] S K Kim H Seok H J Park et al ldquoInhibitory effect ofcurcuminon testosterone induced benign prostatic hyperplasiarat modelrdquo BMC Complementary and Alternative Medicine vol15 article 380 2015

[8] Y P Huang Y H Wen G J Wu Z F Hong S W Xu andA X Peng ldquoClinical study on Kangquan Recipe for benignprostatic hyperplasia patients a randomized controlled trialrdquoChinese Journal of Integrative Medicine vol 20 no 12 pp 949ndash954 2014

[9] Y-P Huang J Du Z-F Hong Z-Q Chen J-F Wu and J-Y Zhao ldquoEffects of Kangquan Recipe on sex steroids and cellproliferation in rats with benign prostatic hyperplasiardquo ChineseJournal of Integrative Medicine vol 15 no 4 pp 289ndash292 2009

[10] J Zhang X Zhang F Xie et al ldquoThe regulation of TGF-betaSMAD signaling by protein deubiquitinationrdquo Protein andCell vol 5 no 7 pp 503ndash517 2014

[11] J Zhang X Tian and J Xing ldquoSignal transduction pathwaysof EMT induced by TGF-beta SHH and WNT and theircrosstalksrdquo Journal of Clinical Medicine vol 5 article 41 2016


[12] J Chen J O Bush C E Ovitt Y Lan and R Jiang ldquoTheTGF-120573 pseudoreceptor gene Bambi is dispensable for mouseembryonic development and postnatal survivalrdquo Genesis vol45 no 8 pp 482ndash486 2007

[13] J Tang C C Gifford R Samarakoon and P J HigginsldquoDeregulation of negative controls on TGF-1205731 signaling intumor progressionrdquo Cancers vol 10 article 159 2018

[14] Y He Z Ou X Chen et al ldquoLPSTLR4 signaling enhancesTGF-120573 response through downregulating BAMBI during pro-static hyperplasiardquo Scientific Reports vol 6 article 27051 2016

[15] S Xi Y Peng G Y Minuk et al ldquoThe combination effects ofShen-Ling-Bai-Zhu on promoting apoptosis of transplanted H-22 hepatocellular carcinoma in mice receiving chemotherapyrdquoJournal of Ethnopharmacology vol 190 pp 1ndash12 2016

[16] S A Kaplan ldquoRe cost-effectiveness analysis of six therapies forthe treatment of lower urinary tract symptoms due to benignprostatic hyperplasiardquo e Journal of Urology vol 200 no 2pp 220-220 2018

[17] C H Ma W L Lin S L Lui et al ldquoEfficacy and safety ofChinese herbal medicine for benign prostatic hyperplasia sys-tematic review of randomized controlled trialsrdquo Asian Journalof Andrology vol 15 no 4 pp 471ndash482 2013

[18] J-H ZhangH-L Xin Y-MXu et al ldquoMorinda officinalis howndash a comprehensive review of traditional uses phytochemistryand pharmacologyrdquo Journal of Ethnopharmacology vol 213 pp230ndash255 2018

[19] I-T Kim H-J Park J-H Nam et al ldquoIn-vitro and in-vivoanti-inflammatory and antinociceptive effects of the methanolextract of the roots of Morinda officinalisrdquo Journal of Pharmacyand Pharmacology vol 57 no 5 pp 607ndash615 2005

[20] Y T Fu K Y Chen Y S Chen and C H Yao ldquoEarthworm(Pheretima aspergillum) extract stimulates osteoblast activityand inhibits osteoclast differentiationrdquo BMC Complementaryand Alternative Medicine vol 14 article 440 2014

[21] BDai J Qi R Liu andY Zhang ldquoEupolyphaga sinensisWalkerdemonstrates angiogenic activity and inhibits A549 cell growthby targeting the KDR signaling pathwayrdquo Molecular MedicineReports vol 10 no 3 pp 1590ndash1596 2014

[22] M B Rokaya Z Munzbergova B Timsina and K R BhattaraildquoRheum australe D Don a review of its botany ethnobotanyphytochemistry and pharmacologyrdquo Journal of Ethnopharma-cology vol 141 no 3 pp 761ndash774 2012

[23] Z F Zhao X R He Y H Zhao et al ldquoPhytochemistrypharmacology and traditional uses of plants from the genusTrachelospermumLrdquoMolecules vol 22 article 1406 2017

[24] E Peuhu P Paul M Remes et al ldquoThe antitumor lignanNortrachelogenin sensitizes prostate cancer cells to TRAIL-induced cell death by inhibition of the Akt pathway and growthfactor signalingrdquo Biochemical Pharmacology vol 86 no 5 pp571ndash583 2013

[25] X Wang T Yuan N Yin et al ldquoLuteoloside protects the uterusfrom Staphylococcus aureus-induced inflammation apoptosisand injuryrdquo Inflammation vol 41 no 5 pp 1702ndash1716 2018

[26] N Nasr Bouzaiene F Chaabane A Sassi L Chekir-Ghediraand K Ghedira ldquoEffect of apigenin-7-glucoside genkwaninand naringenin on tyrosinase activity and melanin synthesis inB16F10 melanoma cellsrdquo Life Sciences vol 144 pp 80ndash85 2016

[27] A F Moura K S B Lima T S Sousa et al ldquoIn vitro antitumoreffect of a lignan isolated from Combretum fruticosum trach-elogenin in HCT-116 human colon cancer cellsrdquo Toxicology inVitro vol 47 pp 129ndash136 2018

[28] K Valentova J Vrba M Bancırova J Ulrichova and V KrenldquoIsoquercitrin Pharmacology toxicology and metabolismrdquoFood and Chemical Toxicology vol 68 pp 267ndash282 2014

[29] P Wang W Solorzano T Diaz C E Magyar S M Henningand J V Vadgama ldquoArctigenin inhibits prostate tumor cellgrowth in vitro and in vivordquo Clinical Nutrition Experimentalvol 13 pp 1ndash11 2017

[30] J M Hu L M Zhang L J Zou et al ldquoRole of inflammation inbenign prostatic hyperplasia development amongHanChinesea population-based and single-institutional analysisrdquo Interna-tional Journal of Urology vol 22 no 12 pp 1138ndash1142 2015

[31] X Wang M Li M Hu P Wei and W Zhu ldquoBAMBI over-expression together with beta-sitosterol ameliorates NSCLCvia inhibiting autophagy and inactivating TGF-betaSmad23pathwayrdquo Oncology Reports vol 37 no 5 pp 3046ndash3054 2017

[32] A Starsıchova E Hruba E Slabakova et al ldquoTGF-1205731 signalingplays a dominant role in the crosstalk between TGF-1205731 and thearyl hydrocarbon receptor ligand in prostate epithelial cellsrdquoCellular Signalling vol 24 no 8 pp 1665ndash1676 2012

[33] J A Eastham L D Truong E Rogers et al ldquoTransform-ing growth-factor-beta-1 - comparative immunohistochemicallocalization in human primary and metastatic prostate-cancerrdquoLaboratory Investigation vol 73 pp 628ndash635 1995

[34] S T dos Reis J Pontes-Junior A A Antunes et al ldquoTgf-beta 1expression as a biomarker of poor prognosis in prostate cancerrdquoClinics vol 66 no 7 pp 1143ndash1147 2011

[35] E Slabakova Z Pernicova E Slavıckova A Starsıchova AKozubık and K Soucek ldquoTGF-1205731-induced EMT of non-transformed prostate hyperplasia cells is characterized by earlyinduction of SNAI2Slugrdquoe Prostate vol 71 no 12 pp 1332ndash1343 2011

[36] J F Santibanez M Quintanilla and C Bernabeu ldquoTGF-betaTGF-beta receptor system and its role in physiological andpathological conditionsrdquo Clinical Science vol 121 pp 233ndash2512011

[37] X Huang and C Lee ldquoRegulation of stromal proliferationgrowth arrest differentiation and apoptosis in benign prostatichyperplasia by TGF-120573rdquo Frontiers in Bioscience vol 8 pp s740ndashs749 2003

[38] R Derynck and X H Feng ldquoTGF-beta receptor signalingrdquoBiochimica et Biophysica Acta vol 1333 pp F105ndashF150 1997

[39] H Ji Y Li F Jiang et al ldquoInhibition of transforming growthfactor betaSMAD signal by MiR-155 is involved in arsenictrioxide-induced anti-angiogenesis in prostate cancerrdquo CancerScience vol 105 no 12 pp 1541ndash1549 2014

[40] A Alcaraz P Hammerer A Tubaro F H Schroder and R Cas-tro ldquoIs there evidence of a relationship between benign prostatichyperplasia and prostate cancer findings of a literature reviewrdquoEuropean Urology vol 55 no 4 pp 864ndash875 2009

[41] E Gold N Jetly M K OrsquoBryan et al ldquoActivin C antagonizesactivin A in vitro and overexpression leads to pathologies invivordquoeAmerican Journal of Pathology vol 174 no 1 pp 184ndash195 2009

[42] L Du Y Lei J Chen H Song and X Wu ldquoPotential ameliora-tive effects of Qing Ye Dan against cadmium induced prostaticdeficits via regulating Nrf-2HO-1 and TGF-beta1smad path-waysrdquo Cellular Physiology and Biochemistry vol 43 no 4 pp1359ndash1368 2017

[43] B Kou W Liu W Zhao et al ldquoThymoquinone inhibitsepithelial-mesenchymal transition in prostate cancer cells bynegatively regulating the TGF-120573Smad23 signaling pathwayrdquoOncology Reports vol 38 no 6 pp 3592ndash3598 2017


[44] LWang L Xie F Tintani et al ldquoAberrant transforming growthfactor-120573 activation recruits Mesenchymal stem cells duringprostatic hyperplasiardquo Stem Cells Translational Medicine vol 6no 2 pp 394ndash404 2017

[45] W Yu and H Chai ldquoInhibition of BAMBI reduces the viabilityand motility of colon cancer via activating TGF-120573Smad path-way in vitro and in vivordquo Oncology Letters vol 14 no 4 pp4793ndash4799 2017

[46] X Yan Z Lin F Chen et al ldquoHuman BAMBI cooperates withSmad7 to inhibit transforming growth factor-120573 signalingrdquo eJournal of Biological Chemistry vol 284 no 44 pp 30097ndash30104 2009

[47] E Gold and G Risbridger ldquoActivins and activin antagonistsin the prostate and prostate cancerrdquo Molecular and CellularEndocrinology vol 359 no 1-2 pp 107ndash112 2012

[48] C-L Yuan R Liang Z-H Liu et al ldquoBone morphogeneticprotein and activin membrane-bound inhibitor overexpressioninhibits gastric tumor cell invasion via the transforming growthfactor-120573epithelial-mesenchymal transition signaling pathwayrdquoExperimental anderapeutic Medicine vol 15 no 6 pp 5422ndash5430 2018

[49] M Katoh ldquoMultilayered prevention and treatment of chronicinflammation organ fibrosis and cancer associatedwith canon-ical WNTbetacatenin signaling activation (Review)rdquo Interna-tional Journal of Molecular Medicine vol 42 pp 713ndash725 2018

[50] S A Broster and N Kyprianou ldquoEpithelial-mesenchymal tran-sition in prostatic diseaserdquo Future Oncology vol 11 no 23 pp3197ndash3206 2015

[51] P Alonso-Magdalena C Brossner A Reiner et al ldquoA rolefor epithelial-mesenchymal transition in the etiology of benignprostatic hyperplasiardquo Proceedings of the National Acadamy ofSciences of the United States of America vol 106 no 8 pp 2859ndash2863 2009

[52] S HuW Yu T-J Lv C-S Chang X Li and J Jin ldquoEvidence ofTGF-1205731mediated epithelial-mesenchymal transition in immor-talized benign prostatic hyperplasia cellsrdquoMolecularMembraneBiology vol 31 no 2-3 pp 103ndash110 2014

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


BPH with mild effects and small side effects Clinicalstudies have shown that [8] KQR can effectively reduceInternational Prostate Symptom Score (I-PSS) scores resid-ual urine volume (RUV) and total prostatic volume(TPV)and improve the maximum urinary flow rate (Qmax) andaverage urine flow rate (Qave) levels in patients with BPHPrevious experimental studies found that [9] the mecha-nism of KQR treatment of BPH may be to regulate thebalance of plasma testosterone and estradiol and reducethe expression of proliferating cell nuclear antigen (PCNA)mRNA to inhibit prostate cell proliferation However thepotential mechanism by which KQR acts in the treatment ofBPH remains to be further studied Recently the influenceof the transforming growth factor-120573 (TGF-120573)suppressor ofmothers against decapentaplegic (Smad) signaling pathwayon BPH has attracted the attention of many scholars

In BPH activation of the TGF-120573Smad signaling pathwayoccurswhenTGF-1205731 bindswith the receptors on the envelopeand then combines with Smad to form a complex [10]The Smad complex enters the nucleus and binds to theSmad binding element in the promoter region of epithelial-mesenchymal transition (EMT)-related genes regulatingthe expression of 120572-smooth muscle actin connective tissuegrowth factor and other EMT-related target genes therebycausing EMT in prostate tissue [11] Therefore the TGF-120573Smad signaling pathway is considered one of the keypathways leading to EMT in prostate epithelial cells

In the process of the TGF-120573 family members binding tothe receptors there is a pseudoreceptor namely bone mor-phogenetic protein and activin membrane-bound inhibitor(BAMBI) It does not possess kinase activity due to the lack ofa serinethreonine kinase domain in the intracellular region[12] However it has a similar structure to the extracellulardomain of TGF-120573 type I receptor meaning that it cancompete with type I receptors and bind to type II receptorsto form the ligand-receptor complex thereby blocking thetransduction of the signal along the TGF-120573Smad signalingpathway [13 14]

Therefore this study aimed to observe the regulation byKQR of the expression of TGF-120573Smad signaling pathway-related factors in prostate tissue and to further explore themechanism of action of KQR which is helpful in elucidatingthe related signal transduction mechanism of BPH to providea basis for clinical treatment At the same time we searchedthe existing literature and found that few studies haveinvestigated the effect of BPH on the TGF-120573Smad signalingpathway This study is a prospective study of traditionalChinese medicine The aims of this study were to exploremultitarget and multichannel regulation by a traditionalChinese medicine to promote the in-depth application oftraditional Chinese medicine to treat BPH and to form aneffective systematic and standardized treatment plan

2 Materials and Methods

21 Experimental Animals In total 48 male Sprague-Dawley(SD) rats (200 plusmn 20 g) were purchased from Shanghai SlackLaboratory Animals Co Ltd These animals were placed

in cages in the animal room of Xiamen University MedicalCollege [License No SYXK (Min) 2013-0006]The rats werekept under the following conditions room temperature 20-22∘C relative humidity 65-70 12 h natural light dark cycleand ad libitum food [Beijing Huakang Biotechnology CoLtd Beijing Feed Certificate (2014) 06057] and water Thestudy was reviewed and approved by the Animal EthicsCommittee of Xiamen University and conducted accordingto the guidelines for the care and use of laboratory animals

22 Experimental Drug Preparation Techniques The medic-inal components of KQR [Morinda officinalis (vouchernumber 170713) Pheretima aspergillum (voucher number170814) Eupolyphaga sinensis Walker (voucher number1703310162) Rheum palmatum (voucher number 170526)Trachelospermum jasminoides (voucher number 170601)and Cinnamomum cassia Presl (voucher number 171117)]were purchased from Yanlaifu Co Ltd (production licensenumber Fujian 20112046) and approved by ProfessorYingkun Qiu (Xiamen University China) All the herbals arestored in the Experimental Center of Traditional ChineseMedicine of Medical College of Xiamen University Then15 g of Morinda officinalis 15 g of Pheretima aspergillum 10 gof Eupolyphaga sinensis Walker 5 g of Rheum palmatum20 g of Trachelospermum jasminoides 5 g of Cinnamomumcassia Presl and 700mL of distilled water were directlymixedThemixturewas left to soak in the decocting containerfor 30 minutes and then boiled with high heat simmeredfor 30 minutes and filtered through 8 layers of filter gauzeNext 450mL of distilled water was added to the decoctingcontainer and cooked for another 30minutes at low heat Thetwo combined filtrates were concentrated to 50mL at 58∘Cin a rotary evaporator (RE-52AA Shanghai Yarong) whichcorresponds to a high dose of 14 gmL of the crude drugA middle dose of 07 gmL and a low dose of 035 gml wereprepared by dilution with distilled water

23 HPLC Chemical analysis of KQR was performed usinga liquid chromatograph model Shimadzu LC-20A (ShimadzuCorporation Japan) The separation column model is YMC-Pack ODS column (YMC Japan) and the mobile phaseis 01 phosphoric acid aqueous solution (A)-acetonitrile(B) 5-45 acetonitrile gradient system elution time 0-60minutes and 100 B 60-70 minutes The flow rate was1mLmin and the test solution injection volume was 20 120583L

24 Main Reagents The reagents used were as followsFinasteride tablets (product code NO22063) was supplied byMerck SharpampDohmeLimitedUK Testosterone propionateinjection (batch number 170512) was provided by ChinaNingbo Second Hormone Factory TRIZOL reagent (3070-250) was provided by Beijing Tian Enze Gene TechnologyCo Ltd China ReverTra Ace qPCR RT Master Mix (FSQ-201) was supplied by the TOYOBO Corporation of Japan FSUniversal SYBR Green Master (4913914001) was supplied byROCHE SwitzerlandAnti-TGF120573R1 antibody (product codeab31013) anti-TGF-120573R2 antibody (product code ab186838)












200

250

300

35020000

40000

0

0

60000

400

800

1600

1200

Time

Wavelength

mAU

(a)

(b)

(e)

(f)

(h)

(g)

(d)

(c)

HOOH

OH

OH

OH

OH

H

OH

OHOH

OH

OHOH

OH

OH

OH

OHOH

HO

HO

HO

HOHO

HOHO

H

H

H

HH

O

OO

O

O O

O

O

O

OHOH

OHOH

OH

OH

OH

HO

HO

HO

HOHO

HO

HO

OO

O

O

O

O

O

O

O

OO

O

O

O

HO

HOHO

HO HO

O

（3

（3

（3

（3

（3

（3

（3

（3CO

（3CO

（3CO

（3CO

（3CO

Arctigenin

Nortrachelogenin

Tracheloside


Luteoloside



Isoquercitrin

O--glucoside



3 Results







100

200

300

400

500

lowastlowast

Body

wei

ght (

g)

(a)


05

10

15

20

lowastlowast

Pros

tate

wet

wei

ght (

g)

(b)


10

05

15

lowastlowast

Pros

tate

vol

ume (

cm

3 )

(c)


01

02

03

04

05 lowastlowast

Pros

tate

inde

x (

)

(d)


(a) (b) (c)

(d) (e) (f)



-44

-56

-65

-58

-65

-36

TGF- 1

TGF- R1

TGF- R2

p-Smad 2

p-Smad 3

GAPDH


(a)


02

04

06

08

10

lowast

TGF-

1

Prot

ein

Leve

l

(b)


02

04

06

08

10lowast

TGF-

R1

Pro

tein

Lev

el

(c)


05

10

15

20

lowast

TGF-

R2

Pro

tein

Lev

el

(d)


02

04

06

08

10

p-Sm

ad2

Prot

ein

Leve

l

lowastlowast

(e)


05

10

15

p-Sm

ad3

Prot

ein

Leve

l

lowastlowast

(f)







-29BAMBI

E-cadherin -97

-125

-36

N-cadherin

GAPDH

(a)


05

10

15

20

lowastlowast

BAM

BI P

rote

in L

evel

(b)


02

04

06

08

10

E-ca

dher

in P

rote

in L

evel

lowastlowast

(c)


02

04

06

08

10

N-c

adhe

rin P

rote

in L

evel

lowastlowast

(d)





4 Discussion




05

10

15

20TG

F-

1 m

RNA

Lev

el

lowastlowast

(a)


05

10

15

20

TGF

-R1

mRN

A L

evel

lowastlowast

(b)


30

15

45

60

75

Smad

2 m

RNA

Lev

el

lowastlowast

(c)


05

10

15

20

BAM

BI m

RNA

Lev

el

lowastlowast

(d)


05

10

15

E-Ca

dher

in m

RNA

Lev

el

lowastlowast

(e)


30

15

45

60

75

N-C

adhe

rin m

RNA

Lev

el lowastlowast

(f)















5 Conclusion


Abbreviations




Data Availability






Acknowledgments





References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






























200

250

300

35020000

40000

0

0

60000

400

800

1600

1200

Time

Wavelength

mAU

(a)

(b)

(e)

(f)

(h)

(g)

(d)

(c)

HOOH

OH

OH

OH

OH

H

OH

OHOH

OH

OHOH

OH

OH

OH

OHOH

HO

HO

HO

HOHO

HOHO

H

H

H

HH

O

OO

O

O O

O

O

O

OHOH

OHOH

OH

OH

OH

HO

HO

HO

HOHO

HO

HO

OO

O

O

O

O

O

O

O

OO

O

O

O

HO

HOHO

HO HO

O

（3

（3

（3

（3

（3

（3

（3

（3CO

（3CO

（3CO

（3CO

（3CO

Arctigenin

Nortrachelogenin

Tracheloside


Luteoloside



Isoquercitrin

O--glucoside



3 Results







100

200

300

400

500

lowastlowast

Body

wei

ght (

g)

(a)


05

10

15

20

lowastlowast

Pros

tate

wet

wei

ght (

g)

(b)


10

05

15

lowastlowast

Pros

tate

vol

ume (

cm

3 )

(c)


01

02

03

04

05 lowastlowast

Pros

tate

inde

x (

)

(d)


(a) (b) (c)

(d) (e) (f)



-44

-56

-65

-58

-65

-36

TGF- 1

TGF- R1

TGF- R2

p-Smad 2

p-Smad 3

GAPDH


(a)


02

04

06

08

10

lowast

TGF-

1

Prot

ein

Leve

l

(b)


02

04

06

08

10lowast

TGF-

R1

Pro

tein

Lev

el

(c)


05

10

15

20

lowast

TGF-

R2

Pro

tein

Lev

el

(d)


02

04

06

08

10

p-Sm

ad2

Prot

ein

Leve

l

lowastlowast

(e)


05

10

15

p-Sm

ad3

Prot

ein

Leve

l

lowastlowast

(f)







-29BAMBI

E-cadherin -97

-125

-36

N-cadherin

GAPDH

(a)


05

10

15

20

lowastlowast

BAM

BI P

rote

in L

evel

(b)


02

04

06

08

10

E-ca

dher

in P

rote

in L

evel

lowastlowast

(c)


02

04

06

08

10

N-c

adhe

rin P

rote

in L

evel

lowastlowast

(d)





4 Discussion




05

10

15

20TG

F-

1 m

RNA

Lev

el

lowastlowast

(a)


05

10

15

20

TGF

-R1

mRN

A L

evel

lowastlowast

(b)


30

15

45

60

75

Smad

2 m

RNA

Lev

el

lowastlowast

(c)


05

10

15

20

BAM

BI m

RNA

Lev

el

lowastlowast

(d)


05

10

15

E-Ca

dher

in m

RNA

Lev

el

lowastlowast

(e)


30

15

45

60

75

N-C

adhe

rin m

RNA

Lev

el lowastlowast

(f)















5 Conclusion


Abbreviations




Data Availability






Acknowledgments





References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















100

200

300

400

500

lowastlowast

Body

wei

ght (

g)

(a)


05

10

15

20

lowastlowast

Pros

tate

wet

wei

ght (

g)

(b)


10

05

15

lowastlowast

Pros

tate

vol

ume (

cm

3 )

(c)


01

02

03

04

05 lowastlowast

Pros

tate

inde

x (

)

(d)


(a) (b) (c)

(d) (e) (f)



-44

-56

-65

-58

-65

-36

TGF- 1

TGF- R1

TGF- R2

p-Smad 2

p-Smad 3

GAPDH


(a)


02

04

06

08

10

lowast

TGF-

1

Prot

ein

Leve

l

(b)


02

04

06

08

10lowast

TGF-

R1

Pro

tein

Lev

el

(c)


05

10

15

20

lowast

TGF-

R2

Pro

tein

Lev

el

(d)


02

04

06

08

10

p-Sm

ad2

Prot

ein

Leve

l

lowastlowast

(e)


05

10

15

p-Sm

ad3

Prot

ein

Leve

l

lowastlowast

(f)







-29BAMBI

E-cadherin -97

-125

-36

N-cadherin

GAPDH

(a)


05

10

15

20

lowastlowast

BAM

BI P

rote

in L

evel

(b)


02

04

06

08

10

E-ca

dher

in P

rote

in L

evel

lowastlowast

(c)


02

04

06

08

10

N-c

adhe

rin P

rote

in L

evel

lowastlowast

(d)





4 Discussion




05

10

15

20TG

F-

1 m

RNA

Lev

el

lowastlowast

(a)


05

10

15

20

TGF

-R1

mRN

A L

evel

lowastlowast

(b)


30

15

45

60

75

Smad

2 m

RNA

Lev

el

lowastlowast

(c)


05

10

15

20

BAM

BI m

RNA

Lev

el

lowastlowast

(d)


05

10

15

E-Ca

dher

in m

RNA

Lev

el

lowastlowast

(e)


30

15

45

60

75

N-C

adhe

rin m

RNA

Lev

el lowastlowast

(f)















5 Conclusion


Abbreviations




Data Availability






Acknowledgments





References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















-44

-56

-65

-58

-65

-36

TGF- 1

TGF- R1

TGF- R2

p-Smad 2

p-Smad 3

GAPDH


(a)


02

04

06

08

10

lowast

TGF-

1

Prot

ein

Leve

l

(b)


02

04

06

08

10lowast

TGF-

R1

Pro

tein

Lev

el

(c)


05

10

15

20

lowast

TGF-

R2

Pro

tein

Lev

el

(d)


02

04

06

08

10

p-Sm

ad2

Prot

ein

Leve

l

lowastlowast

(e)


05

10

15

p-Sm

ad3

Prot

ein

Leve

l

lowastlowast

(f)







-29BAMBI

E-cadherin -97

-125

-36

N-cadherin

GAPDH

(a)


05

10

15

20

lowastlowast

BAM

BI P

rote

in L

evel

(b)


02

04

06

08

10

E-ca

dher

in P

rote

in L

evel

lowastlowast

(c)


02

04

06

08

10

N-c

adhe

rin P

rote

in L

evel

lowastlowast

(d)





4 Discussion




05

10

15

20TG

F-

1 m

RNA

Lev

el

lowastlowast

(a)


05

10

15

20

TGF

-R1

mRN

A L

evel

lowastlowast

(b)


30

15

45

60

75

Smad

2 m

RNA

Lev

el

lowastlowast

(c)


05

10

15

20

BAM

BI m

RNA

Lev

el

lowastlowast

(d)


05

10

15

E-Ca

dher

in m

RNA

Lev

el

lowastlowast

(e)


30

15

45

60

75

N-C

adhe

rin m

RNA

Lev

el lowastlowast

(f)















5 Conclusion


Abbreviations




Data Availability






Acknowledgments





References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















-29BAMBI

E-cadherin -97

-125

-36

N-cadherin

GAPDH

(a)


05

10

15

20

lowastlowast

BAM

BI P

rote

in L

evel

(b)


02

04

06

08

10

E-ca

dher

in P

rote

in L

evel

lowastlowast

(c)


02

04

06

08

10

N-c

adhe

rin P

rote

in L

evel

lowastlowast

(d)





4 Discussion




05

10

15

20TG

F-

1 m

RNA

Lev

el

lowastlowast

(a)


05

10

15

20

TGF

-R1

mRN

A L

evel

lowastlowast

(b)


30

15

45

60

75

Smad

2 m

RNA

Lev

el

lowastlowast

(c)


05

10

15

20

BAM

BI m

RNA

Lev

el

lowastlowast

(d)


05

10

15

E-Ca

dher

in m

RNA

Lev

el

lowastlowast

(e)


30

15

45

60

75

N-C

adhe

rin m

RNA

Lev

el lowastlowast

(f)















5 Conclusion


Abbreviations




Data Availability






Acknowledgments





References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















05

10

15

20TG

F-

1 m

RNA

Lev

el

lowastlowast

(a)


05

10

15

20

TGF

-R1

mRN

A L

evel

lowastlowast

(b)


30

15

45

60

75

Smad

2 m

RNA

Lev

el

lowastlowast

(c)


05

10

15

20

BAM

BI m

RNA

Lev

el

lowastlowast

(d)


05

10

15

E-Ca

dher

in m

RNA

Lev

el

lowastlowast

(e)


30

15

45

60

75

N-C

adhe

rin m

RNA

Lev

el lowastlowast

(f)















5 Conclusion


Abbreviations




Data Availability






Acknowledgments





References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





























5 Conclusion


Abbreviations




Data Availability






Acknowledgments





References



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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