adenosine attenuates lps-induced cardiac dysfunction by...

Research ArticleAdenosine Attenuates LPS-Induced Cardiac Dysfunction byInhibition of Mitochondrial Function via the ER Pathway

Mengnan Zeng12 Beibei Zhang12 Benke Li12 Yuxuan Kan12 ShengchaoWang12

Weisheng Feng 12 and Xiaoke Zheng 12

1Henan University of Chinese Medicine Zhengzhou 450046 China2Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment amp Chinese Medicine Development ofHenan Province Zhengzhou 450046 China

Correspondence should be addressed to Weisheng Feng fwshhactcmeducn and Xiaoke Zheng zhengxk2006163com

Received 27 September 2018 Revised 14 November 2018 Accepted 28 November 2018 Published 10 January 2019

Academic Editor Lucindo Quintans-Junior

Copyright copy 2019 Mengnan Zeng et alThis 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

Sepsis is a life-threatening organ dysfunction syndrome with a high rate of mortality It is caused by an abnormal immune responseto infection and the occurrence of sepsis-induced cardiomyopathy is the primary cause of death The present study was designedto examine the effects of adenosine on lipopolysaccharide- (LPS-) induced cardiac anomalies and the underlying mechanismsinvolved Adenosine (25 50 and 100 mgkg ig 2 timesday) was administered for three days followed by the induction of sepsisby intraperitoneal injection of LPS (10 mgkg2h) The effects of adenosine on inflammatory factors LVEF LVFS and MAPK inseptic rats (half male and half female) were observed Subsequently the effect of adenosine (10 120583M) on the mitochondrial functionof H9c2 cells stimulated with LPS (20 120583gmL 24 h) was observed in the presence and absence of the estrogen receptor-specificantagonist ICI182780 The results show that medium to high doses of adenosine can significantly promote cardiac function (LVEFand LVFS) and reduce the levels of inflammatory factors (TNF-120572 IL-6 PCT and cTnI) and p-JNK in septic rats with a significantdifference seen between male and female rats The results of flow cytometry show that adenosine significantly inhibited increasesin ROS levels mitochondrial membrane potential and the swelling degree of mitochondria in H9c2 cells stimulated with LPSbut this effect could be blocked by ICI182780 indicating that adenosine attenuated LPS-induced cardiac dysfunction by inhibitingmitochondrial function via the ER pathway

1 Introduction

Sepsis is a life-threatening organ dysfunction syndromecaused by an abnormal immune response to infection [1]and is one of the major causes of death in critically illpatients According to the latest statistical data [2] themortality rate of patients with sepsis is as high as 243and the occurrence of sepsis-induced cardiomyopathy is theprimary cause of death In a study by Pulido et al [3] therecorded incidence of sepsis-induced cardiomyopathy wasreported to be as high as 64 In recent years with thedevelopment of technology monitoring cardiac function agreater understanding of sepsis-induced cardiomyopathy hasbeen reachedTheheart is an organ rich inmitochondria andthemitochondrial dysfunction in sepsis is therefore receivingincreasing attention [4] Studies have shown that many

factors are associated with the development of myocardialdamage in sepsis such as excessive production of ROS anddestruction of mitochondrial membrane potential [5 6]however the specific underlying mechanism of myocardialdamage in patients with sepsis remains unclear Gram-negative bacteria which could secrete lipopolysaccharide arethe main pathogens that induce sepsis In this study LPS wasused to induce sepsis

Adenosine a compound produced by linking the N-9of adenine to the C-1 of D-ribose with a 120573-glycosidic bondis an endogenous nucleoside distributed throughout humancells and can be directly phosphorylated in the myocardiumforming adenylate [7] Adenosine has a physiological effectin many systems (eg cardiovascular system) and tissuesFor instance adenosine slows atrioventricular node conduc-tion blocks atrioventricular node reentry and restores the

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 1832025 10 pageshttpsdoiorg10115520191832025

2 Evidence-Based Complementary and Alternative Medicine

normal sinus rhythm of patients with paroxysmal supraven-tricular tachycardia (PSVT) (with or without preexcitationsyndrome) [8 9] Whether adenosine can contribute toprotection of cardiomyopathy induced by LPS has not beenreported In our previous studies it has been reported thatadenosine has estrogen-like effects mediated by the estrogenreceptor (ER) [10] Other studies have shown that drugswith estrogen-like activity can protect the immune system ofmice with traumatic blood loss and reduce the occurrence ofimmunosuppression [11 12] Furthermore estradiol (E2) hasbeen demonstrated to modulate the function of LPS-treatedmacrophages and H9c2 cells [13 14] suggesting a possiblelink between estrogen-like activity and sepsis Neverthelessit remains to be clarified whether the estrogen-like effects ofadenosine can contribute to protection of cardiac dysfunctionassociated with LPS-induced sepsis This study was designedto examine the effects of adenosine on LPS-induced cardiacanomalies and the underlying mechanisms involved

Astragali Radix (named Huang Qi in China called HQ inthis study) is a traditional Chinesemedicine that has the effectof supplementing qi mainly containing astragalus saponinssucrose astragalus polysaccharides amino acids seleniumzinc copper and so on [15] Researches have reported thatHQ have many kinds of pharmacological activities such asstrong heart diuresis and antithrombotic fight free radicaldamage protect myocardium and enhance the immunefunction [16] HQ is used for treatment of myocardial injurysuch as sepsis diabetes and hypertension [17] In additionHQ has estrogen-like activity In our study HQ is a positivecontrol drug

2 Materials and Methods

21 Plant Material and Reagents Adenosine and LPS werepurchased from Sigma (St Louis MO USA) HuangqiZhusheye (referred to HQ positive control group in thisstudy) was purchased from Zbd Pharmaceutical Co Ltd(HeiLongJiang China) Dulbeccorsquos modified Eagle medium(DMEM Gibco Pittsburgh PA USA) heat-inactivatedfetal calf serum (HyClone Logan UT USA) methyl thi-azolyl tetrazolium (MTT) and dimethyl sulfoxide (DMSO)(Amresco Seattle WA USA) ICI182780 (Tocris BristolUK) and Salirasib (FST APExBIO Amazon USA) Amicroplate reader was also used (Bio-Rad Hercules CAUSA) were used

22 Cell Culture and Treatment H9c2 cells for in vitro exper-iments were purchased from ATCC (Rockville MD USA)and cultured in DMEM supplemented with 50 unitsmLpenicillin 50 120583gmL streptomycin and 10 heat-inactivatedfetal calf serum at 37∘C in a humidified incubator with 5CO2(Thermo Scientific Waltham MA USA) There were

four experimental groups in the present study control model(LPS 20 120583gmL) HQ (01 mgmL LPS 20 120583gmL positivecontrol) and adenosine (10120583M LPS 20 120583gmL) groups H9c2cells were seeded on a 100 mm times 20 mm cell culture flaskat a density of 2times105 cellsmL Following treatment withHQ and adenosine for 24 h the cells were collected For

antagonism the estrogen antagonist Faslodex (ICI182780 1120583M ER nonspecific) was added 30 min prior to treatmentwith HQ and adenosine to evaluate whether the observedeffects were mediated by the ER Cells from each groupwere used for mitochondrial swelling membrane potential(ΔΨm) and ROS detection assays

23 Evaluation of the Degree of Mitochondrial Swelling [18]To determine the large amplitude swelling of H9c2 cells theisolation of the mitochondria and the cytosol was performedusing a Cell Mitochondria Isolation kit (SM0020 BeijingSolarbio Science amp Technology Co Ltd China) BrieflyH9c2 cells in each group were incubated in ice-cold mito-chondrial lysis buffer for 20 min The cell suspension wastransferred to a glass homogenizer and homogenized for 20strokes The homogenate was subjected to centrifugation at800 x g for 15 min at 4∘C to remove the nuclei and unbrokencells The supernatant was collected and centrifuged againat 12000 x g for 15 min at 4∘C to obtain the mitochondrialfraction Samples of mitochondria were dissolved in bufferand subjected to flow cytometry (FCM BD FACS Aria IIIBD Biosciences USA)

24 Evaluation of Mitochondrial Membrane Potential(ΔΨm) [18] ΔΨm was assessed using FCM with 551015840661015840-tetrachloro-111015840331015840-tetraethylbenzimidazole-carbocyanideiodine staining (JC-1 GK3610 Genview Beijing China)H9c2 cells in each group were stained with JC-1 for 20min at 37∘C and subjected to FCM JC-1 (red fluorescence)represented normal cells and monomeric JC-1 (greenfluorescence) represented cells in which ΔΨmwas increased

25 Determination of Intracellular ROS Levels The produc-tion of ROS in H9C2 cells was fluorometrically monitoredusing the nonfluorescent probe 2101584071015840-dichlorofluoresceindiacetate (DCFH-DA) (CA1410 Beijing Solarbio Science ampTechnology Co Ltd China) DCFH-DA passively diffusesinto cells and is deacetylated becoming a fluorescent com-pound 2101584071015840-dichlorofluorescein (DCFH) DCFH reacts withROS to form the fluorescent product DCF which is trappedinside the cells Cells in each group cultured on 6-well disheswere trypsinized and collected by centrifugation DCFH-DA diluted in DMEM to a final concentration of 1 120583Mwas added to the H9c2 cells and incubated at 37∘C for20 min Subsequently H9c2 cells were washed three timeswith PBS and DCF fluorescence was measured by FCMMean fluorescence intensity values represented the levels ofintracellular ROS

26 Animals Thepresent studywas conducted in accordancewith theRegulations of Experimental AnimalAdministrationissued by the State Committee of Science and Technologyof the Peoplersquos Republic of China Male (6 weeks old 200plusmn 20 g) and female (6 weeks old 180 plusmn 20 g) Wistar ratswere obtained from Beijing Vital River Laboratory AnimalTechnology Co Ltd (Ethical approval reference numberSCXK2016-0011) All rats were housed in cages on a 12 h12h light-dark schedule at a controlled temperature (22∘C)

Evidence-Based Complementary and Alternative Medicine 3

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Figure 1 Effects of adenosine on heart function in LPS-induced septic rats as assessed by ultrasound Female and male rats received LPS (10mgkg ip 2 h) prior to gavage with different doses of adenosine (a) Effects of different doses of adenosine on LVEF (b) Effects of differentdoses of adenosine on LVFS X plusmn SD n = 8 rats per group lowastP lt 005 lowastlowastP lt 001 versus female model group P lt 001 P lt 001 versus malemodel group ampP lt 005 versus male rats in each group

with free access to food and water at the Laboratory AnimalResearch Center of Henan University of Chinese MedicineAll the procedures for the care of the rats were in accordancewith the institutional guidelines for animal use in researchFollowing adaptive feeding for one week 75 male and 75female rats received LPS (10 mgkg ip) induced the sepsisdisease and were divided into five groups with 15 femaleand 15 male rats in each model (LPS 10 mgkg ip) HQ(2000 mgkg + LPS 10 mgkg ip) low-dose adenosine (Ad25 mgkg + LPS 10 mgkg ip) mid-dose adenosine (Ad 50mgkg+ LPS 10 mgkg ip) and high-dose adenosine (Ad100 mgkg+ LPS 10 mgkg ip) groups In addition 10 femaleand 10 male rats received the same volume of saline (ip) asa control group Two hours later treatment rats were gavagedwith different doses of adenosine while control rats weregavagedwithwaterThedoseswere administered as describedabove twice a day for 3 consecutive days Eighteen hoursafter the final administration the animals were anesthetizedwith ketamine hydrochloride to measure heart function andblood was subsequently collected using the abdominal aorticmethod The carefully dissected hearts were weighed on asensitive torsion balance and immediately frozen in liquidnitrogen at -80∘C

27 Cytometric Bead Array (CBA) CBA was performedaccording to themanufacturerrsquos instructionsHeart tissuewasincubated with lysate treatment reagent (6299995 BD Bio-sciences New York USA) on ice for 15 minutes to release theintracellular phosphorylated extracellular-regulated proteinkinases 12 (p-ERK12) c-Jun N-terminal kinase (p-JNK)and p-p38 proteins into the supernatant A total of 50 120583Lheart tissue lysate from each sample was incubated with 50120583L anti-p-ERK12 (7205548 BD) anti-p-JNK (7170701 BD)or anti-p-p38 (7202572 BD) antibody conjugated to beadsand 50 120583L PE-labeled anti-p-ERK12 anti-p-JNK and anti-p-p38 in the dark at room temperature for 2 hours with

sufficient shaking Following washing with CBA wash bufferthe sampleswere resuspended in 500120583LCBAwash buffer andanalyzed immediately by FCM using the Cell Quest software(BD) The Tracking Beads system was used according to themanufacturerrsquos guidelines

28 ELISA Plasmawas collected and used to detect the levelsof interleukin 6 (IL-6 R180109-003a Neobioscience Co LtdShenzhen China) tumor necrosis factor 120572 (TNF-120572 R180109-102a Neobioscience Co Ltd Shenzhen China) procalci-tonin (PCT CSB-E13419r CUSABIO BIOTECH CO LtdWuhan China) and cardiac troponin I (cTnI 2HSPA1018Life Diagnostics lnc West Chester PA) according to therespective manufacturerrsquos instructions

29 Statistical Analysis Analyses were performed using theSPSS 200 software (IBM New York NY USA) Statisticalsignificance was assessed in comparison with the respectivecontrol for each experiment using one-way ANOVA A pvalue less than 005 was accepted as significant

3 Results

31 Effect of Adenosine on Heart Function in LPS-InducedSeptic Rats Ultrasound revealed that LPS challenge signif-icantly decreased left ventricular ejection fraction (LVEF)and left ventricular fractional shortening (LVFS) the effect ofwhich was abrogated by adenosine As a positive control HQalso increased LVEF and LVFS in LPS-induced septic ratsInterestingly the effects of HQ and adenosine were differentbetween male and female rats HQ and adenosine displayeda better effect on heart function in female rats as shown inFigure 1

32 Effects of Adenosine on Proinflammatory Cytokines inLPS-Induced Septic Rats To assess the effect of adenosine on


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Figure 2 Effects of adenosine on proinflammatory cytokine levels in LPS-induced septic rats as assessed by ELISA Female and male ratsreceived LPS (10 mgkg ip 2 h) prior to gavage with different doses of adenosine (a) Effects of different doses of adenosine on TNF-120572 (b)Effects of different doses of adenosine on IL-6 (c) Effects of different doses of adenosine on PCT (d) Effects of different doses of adenosineon cTnI X plusmn SD n = 8 rats per group lowastP lt 005 lowastlowastP lt 001 versus female model group P lt 001 P lt 001 versus male model group ampP lt005 versus male rats in each group

proinflammatory cytokine production the levels of TNF120572IL-6 PCT and cTnI were evaluated by ELISA As shown inFigure 2 LPS significantly elevated the levels of all the mea-sured proinflammatory cytokines while HQ and adenosineattenuated this increase thereby conferring cardioprotectionMoreover we also observed that the effect of HQ andadenosine showed better effects of mitigated cytokines infemale rats

33 Effects of Adenosine on the MAPK Signaling Pathwayin LPS-Induced Septic Rats To investigate whether theMAPK signaling pathway contributes to the cardioprotectionoffered by adenosine against LPS treatment the levels ofphosphorylated stress signaling molecules p38 ERK andJNK were evaluated by CBA using flow cytometry Theresults demonstrated that HQ and adenosine decreasedthe phosphorylation of JNK and p38 and increased thephosphorylation of ERK In addition the effects of HQ and

adenosine on the phosphorylation levels of all three kinaseswere different between male and female rats the latter ofwhich had a better prognosis The results are shown inFigure 3

34 The Antagonistic Activity of ICI182780 against Adenosineon the Mitochondrial Swelling in LPS-Stimulated H9c2 CellsWith the aim of understanding mitochondrial function moreobjectively mitochondrial swelling was evaluated by FCMthe following determination of the FSC-SSC parameters FSCcorrelates highlywith cell size or volume and SSC is related togranularity and the refractive index The quantitative degreeof mitochondrial swelling was represented by the FSCSSCratio LPS significantly elevated the levels of mitochondrialswelling in H9c2 cells while HQ and adenosine attenuatedthis increase Moreover ICI182780 could block the effect ofHQ and adenosine on mitochondrial swelling but had noeffect alone The results are shown in Figure 4


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Figure 3 Effects of adenosine on the MAPK signaling pathway in LPS-induced septic rats as assessed by CBA Female and male ratsreceived LPS (10 mgkg ip 2 h) prior to gavage with different doses of adenosine (a) Effects of different doses of adenosine on thephosphorylation of ERK (b) Effects of different doses of adenosine on the phosphorylation of JNK (c) Effects of different doses of adenosineon the phosphorylation of p38 X plusmn SD n = 8 rats per group lowastP lt 005 lowastlowastP lt 001 versus female model group P lt 001 P lt 001 versusmale model group ampP lt 005 versus male rats in each group

35 The Antagonistic Activity of ICI182780 against Adenosineon the MMP (ΔΨm) in LPS-Stimulated H9c2 Cells For amore objective understanding of MMP FCM was used todetect ΔΨm JC-1 aggregates in the mitochondria and emitsred fluorescence which can be easily monitored Treatmentof H9c2 cells with LPS for 24 h resulted in the dissipationof MMP as shown by reduced JC-1 staining The red fluores-cence intensity in the HQ and adenosine groups was strongerthan that in the LPS group indicating that LPS significantlyelevated the levels of ΔΨm in H9c2 cells while HQ andadenosine attenuated this increase In addition ICI182780could block the effect of HQ and adenosine on ΔΨm but hadno effect alone The results are shown in Figure 5

36 The Antagonistic Activity of ICI182780 against Adeno-sine on ROS Production in LPS-Stimulated H9c2 Cells Toexplore the molecular mechanism of adenosine the levelsof ROS were detected by FCM As shown in Figure 6LPS significantly elevated the levels of ROS in H9c2 cellswhile HQ and adenosine attenuated this increase MoreoverICI182780 could block the effects of HQ and adenosine onROS production to differing degrees but had no effect alone

4 Discussion

Sepsis-induced myocardial dysfunction (SIMD) is an impor-tant predictive factor for the prognosis of sepsis [19] SIMDis a common complication of severe sepsis with clinicalmanifestations including myocardial damage and cardiacdysfunction The mechanism of SIMD is very complicatedexcessive release of cytokines [20] excessive activation ofcomplement [21] apoptosis of myocardial cells [22] and animbalance in energy metabolism are all considered relatedfactors [23] Through clinical research Edo Y et al observeda reduction in LVEF and left ventricular dilatation in patientsin the early stage of sepsis [24] Due to the portability and

noninvasiveness of the echocardiograph it has become themost commonly used tool for the assessment of cardiacfunction Therefore in the present study the effect of adeno-sine on cardiac function in septic rats was first assessed byechocardiography The results show that HQ (2000 mgkg)and adenosine (50 and 100 mgkg) can significantly improveLVEF and LVFS in septic rats thus promoting their cardiacfunction Interestingly the therapeutic effect of HQ andadenosine in female septic rats was significantly superior tothat in male septic rats (P lt 005)

Gram-negative bacteria [25] are the main pathogens thatinduce sepsis The lipopolysaccharides (LPS) located in thecell wall of these bacteria are lipid and polysaccharide com-plexes that can activate mononuclear cells and macrophagescausing many pathophysiological changes including therelease of a large number of proinflammatory factors suchas TNF-120572 and IL-6 Procalcitonin (PCT) is a nonhormoneglycoprotein that is released into the circulatory system ofpatients with severe systemic infections particularly thosecaused by bacteria and is used as the most accurate serologi-cal diagnostic marker for sepsis to judge the type and severityof infection [26] cTnI is a marker of myocardial injury withhigh sensitivity and specificity and is of great significance forthe diagnosis and risk stratification of myocardial infarctionIn recent years studies have found that cTnI is elevated inpatients with sepsis-related cardiomyopathy and its increaseoften reflects the severity of myocardial cell injury whichis closely related to disease severity and mortality [27] Theresults show that HQ (2000 mgkg) and adenosine (50 and100 mgkg) significantly reduced serum TNF-120572 IL-6 PCTand cTnI levels in septic rats Similarly HQ and adenosinehad a significant maleminusfemale difference in the regulation ofserum inflammatory factors in septic rats (P lt 005)

Mitogen-activated protein kinases (MAPKs) are serinethreonine-specific and are widely distributed in eukary-otic cells Typical MAPKs include c-Jun-N-terminal kinase(JNK) p38 kinases and extracellular signal transduction


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Figure 4 The antagonistic activity of ICI182780 against adenosine on the mitochondrial swelling in LPS-stimulated H9c2 cells as assessedby FCM H9c2 cells seeded on a 100 mm times 20 mm cell culture flask at a density of 2times105 cellsmL were treated with HQ (01 mgmL) andadenosine (10 120583M) for 24 h In another experiment the ER nonspecific antagonist Faslodex (ICI182780 1 120583M) was added 30 min prior toHQ (01 mgmL) and adenosine (10 120583M) to evaluate whether the observed effects elicited by HQ and adenosine were mediated by the ERCells in each group were collected 24 h later and mitochondrial swelling was assessed X plusmn SD n = 3 lowastlowastP lt 001 versus model group (-ICI)ampampP lt 005 versus each group


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Figure 5 The antagonistic activity of ICI182780 against adenosine on MMP (or ΔΨm) in LPS-stimulated H9c2 cells as assessed by FCMH9c2 cells seeded on a 100 mm times 20 mm cell culture flask at a density of 2times105 cellsmLwere treated with HQ (01 mgmL) and adenosine (10120583M) for 24 h In another experiment the ER nonspecific antagonist Faslodex (ICI182780 1 120583M) was added 30 min prior to HQ (01 mgmL)and adenosine (10 120583M) to evaluate whether the observed effects elicited by HQ and adenosine were mediated by the ER Cells in each groupwere collected 24 h later and ΔΨm was assessed

kinase (ERK) Serinethreonine-specific protein kinases canbe activated by extracellular signals or stimuli such as physicalstress proinflammatory cytokines growth factors and bac-terial complexes and this signal is subsequently amplified tothe nucleus by a phosphorylation cascadeThereforeMAPKsindirectly regulate the activity of transcription factors result-ing in altered expression of corresponding genes and reg-ulation of various important cell physiologicalpathologicalprocesses such as cell growth cell differentiation environ-mental stress adaptation and inflammatory response [2829] The results of the present study show that HQ (2000mgkg) and adenosine (50 and 100mgkg) could significantlyreduce the levels of p-JNK and p-P38 and increase the levelof p-ERK in septic rats Moreover the effect of adenosineon p-JNK levels also showed a significant maleminusfemaledifference (P lt 005) It can be speculated that adenosine

may play a protective role in septic rats through the JNKpathway

Gender differences can cause changes and differencesin the pharmacokinetics and pharmacodynamics of endoge-nous and exogenous compounds Female animals containhigher levels of estrogen which affects the bioavailabilitydistribution absorption metabolism and excretion of drugsIn order to rule out this interference we observed the effectsof adenosine onmale and female rats induced by LPS respec-tively Interestingly results showed that the therapeutic effectof adenosine in female septic rats was significantly superior tothat in male septic rats This might be associated with higherlevels of estrogen receptors in female mice adenosine alsohas estrogen-like activity [10] Subsequently the underlyingmechanisms of adenosine on the mitochondrial function ofH9c2 cells stimulated with LPSwere observed in the presence


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Figure 6 The antagonistic activity of ICI182780 against adenosine on ROS production in LPS-stimulated H9c2 cells as assessed by FCMH9c2 cells seeded on a 100 mm times 20 mm cell culture flask at a density of 2times105 cellsmLwere treated with HQ (01 mgmL) and adenosine (10120583M) for 24 h In another experiment the ER nonspecific antagonist Faslodex (ICI182780 1 120583M) was added 30 min prior to HQ (01 mgmL)and adenosine (10 120583M) to evaluate whether the observed effects elicited by HQ and adenosine were mediated by the ER Cells in each groupwere collected 24 h later and ROS levels were assessed X plusmn SD n = 3 lowastlowastP lt 001 versus model group (-ICI) ampampP lt 005 versus each group


and absence of the estrogen receptor-specific antagonistICI182780

In recent years mitochondrial dysfunction has beenrecognized as one of the major factors in the pathogen-esis of sepsis The reported mechanism includes excessiveROS production during sepsis which causes oxidative stressinjury and results in oxidative damage to the mitochondrialrespiratory chain and insufficient ATP production therebyimpairing mitochondrial membrane potential and ultimatelyleading to mitochondrial permeability transition mitochon-drial energetic dysfunction and organelle swelling Our FCMresults show that HQ (01mgmL) and adenosine (10 120583M)could significantly reduce ROS levels and mitochondrialmembrane potential and inhibit the increase in mitochon-drial swelling in H9c2 cells induced by LPS (20 120583gmL 24 h)This effect could be blocked by the specific estrogen receptorantagonist ICI182780 suggesting that the protective effect ofadenosine in H9c2 cells is mediated by the ER

Previous studies have shown that the condition of sepsisis closely related to gender In comparison with male septicanimals the prognosis is better for females which may beexplained by the fact that female animals have more anti-inflammatory factors such as interleukin 10 (IL-10) anddrugs with estrogen-like activity can protect the immunesystem of mice with blood loss reducing the occurrenceof immunosuppression This indicates that there may be aconnection between estrogen-like activity and sepsis symp-toms Accordingly previous studies in our laboratory haveshown that adenosine has estrogen-like activity and that itsfunction is mediated by estrogen receptorsThe present studydemonstrates that there was a maleminusfemale difference in thetherapeutic effect of HQ and adenosine in septic rats andthat the improvement in mitochondrial function in H9c2cells elicited by HQ and adenosine could be antagonized byICI182780 It is reasonable to suggest therefore that HQ andadenosine executes its protective function via the ERpathway

In our study HQ is a positive control drug HQ hasestrogen-like activity which was used for treatment ofmyocardial injury such as sepsis diabetes and hypertensionResults of animal experiments and cell experiments alsoshowed that HQattenuated LPS-induced cardiac dysfunctionby inhibiting mitochondrial function via the ER pathwayMoreover adenosine has the same molecular mechanism asHQ

5 Conclusion

Medium and high doses of adenosine could significantlypromote cardiac function (LVEF and LVFS) and reduceinflammatory factors (TNF-120572 IL-6 PCT and cTnI) andp-JNK levels in septic rats with an obvious maleminusfemaledifference The results of experiments using ICI182780 toantagonize the effect of adenosine on LPS-stimulated H9c2cells show that adenosine could significantly inhibit theincreases in ROS levels mitochondrial membrane poten-tial and mitochondrial swelling and that this effect couldbe blocked by the estrogen receptor-specific antagonistICI182780 In conclusion adenosine attenuated LPS-induced

cardiac dysfunction by inhibiting mitochondrial function viathe ER pathway

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Mengnan Zeng and Xiaoke Zheng designed and performedthe experiments and analyzed the raw data Beibei ZhangBenke Li Yuxuan Kan and Shengchao Wang assisted withthe experiments Weisheng Feng supervised the project

Acknowledgments

Our work was supported by the Central GovernmentGuide Local Science and Technology Development Funds(14104349) and the study on the key technology for qualitycontrol and the key characteristics of Rehmannia glutinosaDioscorea oppositaThunb and Achyranthes bidentata Blumefrom Henan Province (171100310500) Thanks for Meng Lisupervised the project

References

[1] R P Dellinger M M Levy and A Rhodes ldquoSurviving sepsiscampaign international guidelines for management of severesepsis and septic shock 2012rdquo Critical Care Medicine vol 42no 1 pp 580ndash637 2014

[2] C Fleischmann D O Thomas M Hartmann et al ldquoHospitalincidence and mortality rates of sepsisrdquo Deutsches ArzteblattInternational vol 113 no 10 pp 159ndash166 2016

[3] J N Pulido B Afessa M Masaki et al ldquoClinical spectrumfrequency and significance of myocardial dysfunction in severesepsis and septic shockrdquoMayo Clinic Proceedings vol 87 no 7pp 620ndash628 2012

[4] N Varga J C Ruiz-Rodrıguez and R Ferrer ldquoMelatonin andmitochondrial dysfunction are key players in the pathophysiol-ogy of sepsisrdquo Enfermedades Infecciosas y Microbiologıa Clınicavol 36 no 9 pp 535ndash538 2018

[5] S Li H Wu D Han et al ldquoA Novel Mechanism of Mesenchy-mal Stromal Cell-Mediated Protection against Sepsis Restrict-ing Inflammasome Activation in Macrophages by IncreasingMitophagy and Decreasing Mitochondrial ROSrdquo OxidativeMedicine and Cellular Longevity vol 2018 Article ID 353760915 pages 2018

[6] J Lyu G Zheng Z Chen et al ldquoSepsis-induced brain mito-chondrial dysfunction is associated with altered mitochondrialSrc and PTP1B levelsrdquo Brain Research vol 1620 pp 130ndash1382015

[7] B-G Han J-W Cho Y D Cho et al ldquoCrystal structure ofhuman transglutaminase 2 in complex with adenosine triphos-phaterdquo International Journal of Biological Macromolecules vol47 no 2 pp 108ndash115 2010


[8] W Geldenhuys A Hanif J Yun and M Nayeem ldquoExploringAdenosine Receptor Ligands Potential Role in the Treatment ofCardiovascular DiseasesrdquoMolecules vol 22 no 6 p 917 2017

[9] M Peleli and M Carlstrom ldquoAdenosine signaling in diabetesmellitus and associated cardiovascular and renal complica-tionsrdquoMolecular Aspects of Medicine vol 55 pp 62ndash74 2017

[10] M Zeng L Zhang M Li et al ldquoEstrogenic Effects of theExtracts from the Chinese Yam (Dioscorea opposite Thunb)and Its Effective Compounds in Vitro and in Vivordquo Moleculesvol 23 no 11 pp 330ndash343 2018

[11] E J Filardo ldquoA role for G-protein coupled estrogen recep-tor (GPER) in estrogen-induced carcinogenesis Dysregulatedglandular homeostasis survival and metastasisrdquoThe Journal ofSteroid Biochemistry and Molecular Biology vol 176 pp 38ndash482018

[12] P Habib D Dreymueller B Rosing et al ldquoEstrogen serumconcentration affects blood immune cell composition andpolarization in human females under controlled ovarian stim-ulationrdquo The Journal of Steroid Biochemistry and MolecularBiology vol 178 pp 340ndash347 2018

[13] T-C Chao H-H Chao M-F Chen J A Greager and RJ Walter ldquoFemale sex hormones modulate the function ofLPS-treated macrophagesrdquo American Journal of ReproductiveImmunology vol 44 no 5 pp 310ndash318 2000

[14] Y Tien J Lin C Lai et al ldquoCarthamus tinctorius L preventsLPS-induced TNF120572 signaling activation and cell apoptosisthrough JNK12ndashNF120581B pathway inhibition in H9c2 cardiomy-oblast cellsrdquo Journal of Ethnopharmacology vol 130 no 3 pp505ndash513 2010

[15] Z Liang B Du L Xie et al ldquoPulverization Using Liquid Nitro-gen Significantly Improves Physical Properties of Powder andExtractionYieldof Polysaccharides ofAstragalusmongholicusrdquoInternational Journal of Food Engineering vol 13 no 2 pp 164ndash175 2017

[16] L Zhou and X Mei ldquoReview of study on peripheral nerveregeneration promoted by traditional chinese medicinesrdquoZhongguo Zhongyao Zazhi vol 36 no 1 pp 77ndash80 2011

[17] H Tian J Lu H He et al ldquoThe effect of Astragalus as anadjuvant treatment in type 2 diabetes mellitus A (preliminary)meta-analysisrdquo Journal of Ethnopharmacology vol 191 pp 206ndash215 2016

[18] G Zheng J Lyu S Liu et al ldquoSilencing of uncoupling protein 2by small interfering RNAaggravatesmitochondrial dysfunctionin cardiomyocytes under septic conditionsrdquo International Jour-nal of MolecularMedicine vol 35 no 6 pp 1525ndash1536 2015

[19] Y Liu and Z Lin ldquoPathogenesis of sepsis-induced myocardialdysfunctionrdquo Zhonghua Wei Zhong Bing Ji Jiu Yi Xue vol 30no 4 pp 374ndash376 2018

[20] G SinghG SinghR Bhatti et al ldquoIndolyl-isoxazolidines atten-uate LPS-stimulated pro-inflammatory cytokines and increasesurvival in a mouse model of sepsis Identification of potentleadrdquo European Journal of Medicinal Chemistry vol 153 pp 56ndash64 2018

[21] M M Markiewski R A DeAngelis and J D Lambris ldquoCom-plexity of complement activation in sepsisrdquo Journal of Cellularand Molecular Medicine vol 12 no 6a pp 2245ndash2254 2008

[22] X Li R Luo R Jiang et al ldquoThe role of the Hsp90Aktpathway in myocardial calpain-induced caspase-3 activationand apoptosis during sepsisrdquo BMC Cardiovascular Disordersvol 13 no 1 pp 8ndash13 2013

[23] H Yorulmaz E Ozkok G Ates A Aksu N Balkıs and STamer ldquoGhrelin Impact on Muscle Energy Metabolism in Sep-sisrdquo International Journal of Peptide Research and Therapeuticsvol 24 no 2 pp 259ndash264 2018

[24] E Y Birati H Mathelier and M Molina ldquoComparison ofCauses of Death After Heart Transplantation in Patients WithLeft Ventricular Ejection Fractions =35 Versus gt35rdquo Ameri-can Journal of Cardiology vol 117 no 8 pp 1322ndash1326 2018

[25] S K Vanaja A J Russo B Behl et al ldquoBacterial OuterMembrane Vesicles Mediate Cytosolic Localization of LPS andCaspase-11 Activationrdquo Cell vol 165 no 5 pp 1106ndash1119 2016

[26] J Szederjesi E Almasy A Lazar A Hutanu I Badea andA Georgescu ldquoAn Evaluation of Serum Procalcitonin and C-Reactive Protein Levels as Diagnostic and Prognostic Biomark-ers of Severe Sepsisrdquo The Journal of Critical Care Medicine vol1 no 4 pp 147ndash153 2015

[27] R Haines S Crichton J Wilson D Treacher and M Oster-mann ldquoCardiac biomarkers are associatedwithmaximum stageof acute kidney injury in critically ill patients A prospectiveanalysisrdquo Critical Care vol 21 no 1 2017

[28] J N Sanchez T Wang and M S Cohen ldquoBRAF and MEKInhibitors Use and Resistance in BRAF-Mutated CancersrdquoDrugs vol 78 no 5 pp 549ndash566 2018

[29] L XingHNi andYWang ldquoQuercitrin attenuates osteoporosisin ovariectomized rats by regulating mitogen-activated proteinkinase (MAPK) signaling pathwaysrdquo Biomedicine amp Pharma-cotherapy vol 89 pp 1136ndash1141 2017

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


normal sinus rhythm of patients with paroxysmal supraven-tricular tachycardia (PSVT) (with or without preexcitationsyndrome) [8 9] Whether adenosine can contribute toprotection of cardiomyopathy induced by LPS has not beenreported In our previous studies it has been reported thatadenosine has estrogen-like effects mediated by the estrogenreceptor (ER) [10] Other studies have shown that drugswith estrogen-like activity can protect the immune system ofmice with traumatic blood loss and reduce the occurrence ofimmunosuppression [11 12] Furthermore estradiol (E2) hasbeen demonstrated to modulate the function of LPS-treatedmacrophages and H9c2 cells [13 14] suggesting a possiblelink between estrogen-like activity and sepsis Neverthelessit remains to be clarified whether the estrogen-like effects ofadenosine can contribute to protection of cardiac dysfunctionassociated with LPS-induced sepsis This study was designedto examine the effects of adenosine on LPS-induced cardiacanomalies and the underlying mechanisms involved

Astragali Radix (named Huang Qi in China called HQ inthis study) is a traditional Chinesemedicine that has the effectof supplementing qi mainly containing astragalus saponinssucrose astragalus polysaccharides amino acids seleniumzinc copper and so on [15] Researches have reported thatHQ have many kinds of pharmacological activities such asstrong heart diuresis and antithrombotic fight free radicaldamage protect myocardium and enhance the immunefunction [16] HQ is used for treatment of myocardial injurysuch as sepsis diabetes and hypertension [17] In additionHQ has estrogen-like activity In our study HQ is a positivecontrol drug

2 Materials and Methods

21 Plant Material and Reagents Adenosine and LPS werepurchased from Sigma (St Louis MO USA) HuangqiZhusheye (referred to HQ positive control group in thisstudy) was purchased from Zbd Pharmaceutical Co Ltd(HeiLongJiang China) Dulbeccorsquos modified Eagle medium(DMEM Gibco Pittsburgh PA USA) heat-inactivatedfetal calf serum (HyClone Logan UT USA) methyl thi-azolyl tetrazolium (MTT) and dimethyl sulfoxide (DMSO)(Amresco Seattle WA USA) ICI182780 (Tocris BristolUK) and Salirasib (FST APExBIO Amazon USA) Amicroplate reader was also used (Bio-Rad Hercules CAUSA) were used

22 Cell Culture and Treatment H9c2 cells for in vitro exper-iments were purchased from ATCC (Rockville MD USA)and cultured in DMEM supplemented with 50 unitsmLpenicillin 50 120583gmL streptomycin and 10 heat-inactivatedfetal calf serum at 37∘C in a humidified incubator with 5CO2(Thermo Scientific Waltham MA USA) There were

four experimental groups in the present study control model(LPS 20 120583gmL) HQ (01 mgmL LPS 20 120583gmL positivecontrol) and adenosine (10120583M LPS 20 120583gmL) groups H9c2cells were seeded on a 100 mm times 20 mm cell culture flaskat a density of 2times105 cellsmL Following treatment withHQ and adenosine for 24 h the cells were collected For

antagonism the estrogen antagonist Faslodex (ICI182780 1120583M ER nonspecific) was added 30 min prior to treatmentwith HQ and adenosine to evaluate whether the observedeffects were mediated by the ER Cells from each groupwere used for mitochondrial swelling membrane potential(ΔΨm) and ROS detection assays

23 Evaluation of the Degree of Mitochondrial Swelling [18]To determine the large amplitude swelling of H9c2 cells theisolation of the mitochondria and the cytosol was performedusing a Cell Mitochondria Isolation kit (SM0020 BeijingSolarbio Science amp Technology Co Ltd China) BrieflyH9c2 cells in each group were incubated in ice-cold mito-chondrial lysis buffer for 20 min The cell suspension wastransferred to a glass homogenizer and homogenized for 20strokes The homogenate was subjected to centrifugation at800 x g for 15 min at 4∘C to remove the nuclei and unbrokencells The supernatant was collected and centrifuged againat 12000 x g for 15 min at 4∘C to obtain the mitochondrialfraction Samples of mitochondria were dissolved in bufferand subjected to flow cytometry (FCM BD FACS Aria IIIBD Biosciences USA)

24 Evaluation of Mitochondrial Membrane Potential(ΔΨm) [18] ΔΨm was assessed using FCM with 551015840661015840-tetrachloro-111015840331015840-tetraethylbenzimidazole-carbocyanideiodine staining (JC-1 GK3610 Genview Beijing China)H9c2 cells in each group were stained with JC-1 for 20min at 37∘C and subjected to FCM JC-1 (red fluorescence)represented normal cells and monomeric JC-1 (greenfluorescence) represented cells in which ΔΨmwas increased

25 Determination of Intracellular ROS Levels The produc-tion of ROS in H9C2 cells was fluorometrically monitoredusing the nonfluorescent probe 2101584071015840-dichlorofluoresceindiacetate (DCFH-DA) (CA1410 Beijing Solarbio Science ampTechnology Co Ltd China) DCFH-DA passively diffusesinto cells and is deacetylated becoming a fluorescent com-pound 2101584071015840-dichlorofluorescein (DCFH) DCFH reacts withROS to form the fluorescent product DCF which is trappedinside the cells Cells in each group cultured on 6-well disheswere trypsinized and collected by centrifugation DCFH-DA diluted in DMEM to a final concentration of 1 120583Mwas added to the H9c2 cells and incubated at 37∘C for20 min Subsequently H9c2 cells were washed three timeswith PBS and DCF fluorescence was measured by FCMMean fluorescence intensity values represented the levels ofintracellular ROS

26 Animals Thepresent studywas conducted in accordancewith theRegulations of Experimental AnimalAdministrationissued by the State Committee of Science and Technologyof the Peoplersquos Republic of China Male (6 weeks old 200plusmn 20 g) and female (6 weeks old 180 plusmn 20 g) Wistar ratswere obtained from Beijing Vital River Laboratory AnimalTechnology Co Ltd (Ethical approval reference numberSCXK2016-0011) All rats were housed in cages on a 12 h12h light-dark schedule at a controlled temperature (22∘C)


amp

0

20

40

60

80

100


LVEF

()

Adenosine

amp amp amp

femalemale

lowastlowast


lowastlowast

lowast

(a)

0

10

20

30

40

50

60

70

80


LVFS

()

Adenosine

ampamp

amp

femalemale

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)







3 Results




0

100

200

300

400

500

600


TNF-

(p

gm

L)

Adenosine

ampamp

amp

femalemale

lowastlowast

lowastlowast


lowast

(a)

0

100

200

300

400

500

600

700


IL-6

(pg

mL)

Adenosine

amp amp amp

femalemale

lowastlowast


lowast

(b)

0

200

400

600

800

1000

1200

1400


PCT

(ng

mL)

Adenosine

amp amp

amp

femalemale



lowast

(c)

0

5

10

15

20

25

30

35


CTN

I (ng

mL)

Adenosinefemalemale

lowastlowast


lowast

(d)







Con M HQ

Low

Mid

dle

Hig

h

0

2

4

6

8

10

12p-

ERK

12

(Uni

tsm

L)

Adenosine

femalemale

lowastlowast

lowastlowast

lowast

lowast

(a)

Con M HQ

Low

Mid

dle

Hig

h

0

20

40

60

80

100

120

p-JN

K1

2 (U

nits

mL)

Adenosine

ampamp

amp

femalemale

lowastlowast

lowast

lowast

(b)

Con M HQ

Low

Mid

dle

Hig

h

050

100150200250300350400

p-p3

8 (U

nits

mL)

Adenosine

amp amp

femalemale

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)




4 Discussion






Con

350300250200150100

50

50

250200150100500

150

100

50

0

Cou

nt

300

250

200

150

100

50

010

20

30

40

50

60

0

Cou

nt

Cou

nt

10

20

30

40

50

60

0

Cou

ntC

ount

Cou

nt

Cou

ntC

ount

SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000) 25020015010050

SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

353025201510

50

3530

4540

25201510

50

25

75

100

125

0

M

HQ

Ad

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

FSC

SSC

minusICI+ICI

ampampampamp

lowastlowast

lowastlowast

lowastlowast



Con

M

HQ

Ad

888

436

748

708

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

881

449

569

521

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

PE-A






Con

M

HQ

Con+ICI

M+ICI

HQ+ICI

Ad Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

ROS

(MFI

)

minusICI+ICI

ampampampamp

105

104

103

102

50Cou

nt

25

75

100

125

0

50Cou

nt25

75

100

125

0

50Cou

nt

25

75

100

125

0

50Cou

nt

25

75100125

0

50Cou

nt25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75

100

125

0

lowastlowast

lowastlowast

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A







5 Conclusion



Data Availability






Acknowledgments


References



































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















amp

0

20

40

60

80

100


LVEF

()

Adenosine

amp amp amp

femalemale

lowastlowast


lowastlowast

lowast

(a)

0

10

20

30

40

50

60

70

80


LVFS

()

Adenosine

ampamp

amp

femalemale

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)







3 Results




0

100

200

300

400

500

600


TNF-

(p

gm

L)

Adenosine

ampamp

amp

femalemale

lowastlowast

lowastlowast


lowast

(a)

0

100

200

300

400

500

600

700


IL-6

(pg

mL)

Adenosine

amp amp amp

femalemale

lowastlowast


lowast

(b)

0

200

400

600

800

1000

1200

1400


PCT

(ng

mL)

Adenosine

amp amp

amp

femalemale



lowast

(c)

0

5

10

15

20

25

30

35


CTN

I (ng

mL)

Adenosinefemalemale

lowastlowast


lowast

(d)







Con M HQ

Low

Mid

dle

Hig

h

0

2

4

6

8

10

12p-

ERK

12

(Uni

tsm

L)

Adenosine

femalemale

lowastlowast

lowastlowast

lowast

lowast

(a)

Con M HQ

Low

Mid

dle

Hig

h

0

20

40

60

80

100

120

p-JN

K1

2 (U

nits

mL)

Adenosine

ampamp

amp

femalemale

lowastlowast

lowast

lowast

(b)

Con M HQ

Low

Mid

dle

Hig

h

050

100150200250300350400

p-p3

8 (U

nits

mL)

Adenosine

amp amp

femalemale

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)




4 Discussion






Con

350300250200150100

50

50

250200150100500

150

100

50

0

Cou

nt

300

250

200

150

100

50

010

20

30

40

50

60

0

Cou

nt

Cou

nt

10

20

30

40

50

60

0

Cou

ntC

ount

Cou

nt

Cou

ntC

ount

SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000) 25020015010050

SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

353025201510

50

3530

4540

25201510

50

25

75

100

125

0

M

HQ

Ad

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

FSC

SSC

minusICI+ICI

ampampampamp

lowastlowast

lowastlowast

lowastlowast



Con

M

HQ

Ad

888

436

748

708

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

881

449

569

521

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

PE-A






Con

M

HQ

Con+ICI

M+ICI

HQ+ICI

Ad Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

ROS

(MFI

)

minusICI+ICI

ampampampamp

105

104

103

102

50Cou

nt

25

75

100

125

0

50Cou

nt25

75

100

125

0

50Cou

nt

25

75

100

125

0

50Cou

nt

25

75100125

0

50Cou

nt25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75

100

125

0

lowastlowast

lowastlowast

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A







5 Conclusion



Data Availability






Acknowledgments


References



































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

100

200

300

400

500

600


TNF-

(p

gm

L)

Adenosine

ampamp

amp

femalemale

lowastlowast

lowastlowast


lowast

(a)

0

100

200

300

400

500

600

700


IL-6

(pg

mL)

Adenosine

amp amp amp

femalemale

lowastlowast


lowast

(b)

0

200

400

600

800

1000

1200

1400


PCT

(ng

mL)

Adenosine

amp amp

amp

femalemale



lowast

(c)

0

5

10

15

20

25

30

35


CTN

I (ng

mL)

Adenosinefemalemale

lowastlowast


lowast

(d)







Con M HQ

Low

Mid

dle

Hig

h

0

2

4

6

8

10

12p-

ERK

12

(Uni

tsm

L)

Adenosine

femalemale

lowastlowast

lowastlowast

lowast

lowast

(a)

Con M HQ

Low

Mid

dle

Hig

h

0

20

40

60

80

100

120

p-JN

K1

2 (U

nits

mL)

Adenosine

ampamp

amp

femalemale

lowastlowast

lowast

lowast

(b)

Con M HQ

Low

Mid

dle

Hig

h

050

100150200250300350400

p-p3

8 (U

nits

mL)

Adenosine

amp amp

femalemale

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)




4 Discussion






Con

350300250200150100

50

50

250200150100500

150

100

50

0

Cou

nt

300

250

200

150

100

50

010

20

30

40

50

60

0

Cou

nt

Cou

nt

10

20

30

40

50

60

0

Cou

ntC

ount

Cou

nt

Cou

ntC

ount

SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000) 25020015010050

SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

353025201510

50

3530

4540

25201510

50

25

75

100

125

0

M

HQ

Ad

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

FSC

SSC

minusICI+ICI

ampampampamp

lowastlowast

lowastlowast

lowastlowast



Con

M

HQ

Ad

888

436

748

708

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

881

449

569

521

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

PE-A






Con

M

HQ

Con+ICI

M+ICI

HQ+ICI

Ad Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

ROS

(MFI

)

minusICI+ICI

ampampampamp

105

104

103

102

50Cou

nt

25

75

100

125

0

50Cou

nt25

75

100

125

0

50Cou

nt

25

75

100

125

0

50Cou

nt

25

75100125

0

50Cou

nt25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75

100

125

0

lowastlowast

lowastlowast

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A







5 Conclusion



Data Availability






Acknowledgments


References



































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Con M HQ

Low

Mid

dle

Hig

h

0

2

4

6

8

10

12p-

ERK

12

(Uni

tsm

L)

Adenosine

femalemale

lowastlowast

lowastlowast

lowast

lowast

(a)

Con M HQ

Low

Mid

dle

Hig

h

0

20

40

60

80

100

120

p-JN

K1

2 (U

nits

mL)

Adenosine

ampamp

amp

femalemale

lowastlowast

lowast

lowast

(b)

Con M HQ

Low

Mid

dle

Hig

h

050

100150200250300350400

p-p3

8 (U

nits

mL)

Adenosine

amp amp

femalemale

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)




4 Discussion






Con

350300250200150100

50

50

250200150100500

150

100

50

0

Cou

nt

300

250

200

150

100

50

010

20

30

40

50

60

0

Cou

nt

Cou

nt

10

20

30

40

50

60

0

Cou

ntC

ount

Cou

nt

Cou

ntC

ount

SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000) 25020015010050

SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

353025201510

50

3530

4540

25201510

50

25

75

100

125

0

M

HQ

Ad

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

FSC

SSC

minusICI+ICI

ampampampamp

lowastlowast

lowastlowast

lowastlowast



Con

M

HQ

Ad

888

436

748

708

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

881

449

569

521

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

PE-A






Con

M

HQ

Con+ICI

M+ICI

HQ+ICI

Ad Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

ROS

(MFI

)

minusICI+ICI

ampampampamp

105

104

103

102

50Cou

nt

25

75

100

125

0

50Cou

nt25

75

100

125

0

50Cou

nt

25

75

100

125

0

50Cou

nt

25

75100125

0

50Cou

nt25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75

100

125

0

lowastlowast

lowastlowast

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A







5 Conclusion



Data Availability






Acknowledgments


References



































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Con

350300250200150100

50

50

250200150100500

150

100

50

0

Cou

nt

300

250

200

150

100

50

010

20

30

40

50

60

0

Cou

nt

Cou

nt

10

20

30

40

50

60

0

Cou

ntC

ount

Cou

nt

Cou

ntC

ount

SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000) 25020015010050

SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

25020015010050SSC-A (times 1000)

353025201510

50

3530

4540

25201510

50

25

75

100

125

0

M

HQ

Ad

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

FSC

SSC

minusICI+ICI

ampampampamp

lowastlowast

lowastlowast

lowastlowast



Con

M

HQ

Ad

888

436

748

708

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

881

449

569

521

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

PE-A






Con

M

HQ

Con+ICI

M+ICI

HQ+ICI

Ad Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

ROS

(MFI

)

minusICI+ICI

ampampampamp

105

104

103

102

50Cou

nt

25

75

100

125

0

50Cou

nt25

75

100

125

0

50Cou

nt

25

75

100

125

0

50Cou

nt

25

75100125

0

50Cou

nt25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75

100

125

0

lowastlowast

lowastlowast

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A







5 Conclusion



Data Availability






Acknowledgments


References



































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Con

M

HQ

Ad

888

436

748

708

Con+ICI

M+ICI

HQ+ICI

Ad+ICI

881

449

569

521

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

FITC-A

105

104

103

102

105

104

103

102

PE-A

FITC-A

105

104

103

102

PE-A






Con

M

HQ

Con+ICI

M+ICI

HQ+ICI

Ad Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

ROS

(MFI

)

minusICI+ICI

ampampampamp

105

104

103

102

50Cou

nt

25

75

100

125

0

50Cou

nt25

75

100

125

0

50Cou

nt

25

75

100

125

0

50Cou

nt

25

75100125

0

50Cou

nt25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75

100

125

0

lowastlowast

lowastlowast

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A







5 Conclusion



Data Availability






Acknowledgments


References



































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Con

M

HQ

Con+ICI

M+ICI

HQ+ICI

Ad Ad+ICI

0

10

20

30

40

50

60

Con M HQ Ad

ROS

(MFI

)

minusICI+ICI

ampampampamp

105

104

103

102

50Cou

nt

25

75

100

125

0

50Cou

nt25

75

100

125

0

50Cou

nt

25

75

100

125

0

50Cou

nt

25

75100125

0

50Cou

nt25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75100125

0

50Cou

nt

25

75

100

125

0

lowastlowast

lowastlowast

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A

105

104

103

102

FITC-A10

510

410

310

2

FITC-A







5 Conclusion



Data Availability






Acknowledgments


References



































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























5 Conclusion



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



















adenosine attenuates lps-induced cardiac dysfunction by...

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