Caffeine Inhibits HSC-T6Cell Activation And Proliferation Induced By Acetaldehyde Via Adenosine A_2A Receptor Mediated CAMP/PKA/Src/ERKl/2/p38MAPK Signal Pathway

Alcoholic liver disease （ALD） is a disease of the liver caused by long-term heavydrinking[1]. In recent years,because of the rapid development of alcohol consumption,ALD has become a global public health problem.The ALD usually appears as fattyliver in the early,and then developed into alcoholic hepatitis, liver fibrosis andcirrhosis[2]. At present, alcoholic liver fibrosis is a turning point in the ALD process ofdevelopment.If we can removal the unfavorable factors that can reverse thepathological changes. Therefore, the study of its pathogenesis and therapeutic targetshas been the focus of attention of the academic circles.In the process of the development in alcoholic liver fibrosis, ethanol can throughmetabolizs toxicity products such as toxicity acetaldehyde and many kinds of factorsto activate hepatic stellate cells （HSC）^[3].Activation of HSC is the main source ofextracellular matrix （ECM） in the process of liver fibrosis, also is the center link ofliver fibrosis caused by various factors.Although the HSC has been widespreadconcern in the role of the pathogenesis of hepatic fibrosis and the transductionpathway of the HSC within relevant cytokines has also been extensively studied, butthere is no good way to reverse the development of alcoholic liver fibrosis because ofprocess of the ethanol induced is extremely complex and many affecting factors. In recent years, the adenosine A_2Areceptor has been more and more attention becauseof its complex biological role and the important regulatory function in a variety ofdiseases. Chan et al[4]show that the adenosine adenosine A_2Areceptor activation playsan important role of the HSC in the pathogenesis of liver fibrosis.There Is expected tobecome a novel target for prevention and treatment of liver fibrosis in2006.The study found that after ethanol-induced release a lot of adenosine can activativedthe adenosine A_2Areceptor and the promoted collagen synthesis of rat andhuman.Hashmi and Sohail^[5-6]also confirmed that adenosine can activated the A_2Areceptor induced liver fibrosis in HSC.Che etc^[7]also reported that the adenosine A_2Areceptor increased cAMP-PKA signaling pathway by its coupling of Gs protein, andthen induced Procollagen I and Procollagen III production of LX-2human liverthrough its downstream signaling pathway of PKA-Src-ERK1/2MAPK and p38MAPK in2007.Caffeine is the most widely daily diet and psychotropic drugs used in the world’sconsumer. Caffeine has a basic chemical structure was the active ingredient of themethylxanthines.In the body, caffeine can expressed pharmacological effects byadenosine A_2Areceptors and A1receptors^[8-10]. The relationship between caffeine andhealthy has been concern for scientists. In the traditional concepts, drinking caffeinewas bad for our health[11-12]. But in recent years, more and more interesting andprovocative epidemiological studies are being reported[13-16], that drinking caffeinecan significantly reduced the probability of suffer from chronic liver disease,including the risk of alcoholic liver fibrosis.In our prophase research，we determined that caffeine can inhibited the proliferationstimulated by acetaldehyde in HSC-T6^[17]. Caffeine can induce apoptosis, and itsmechanism may be raised with the TRAIL receptors: DR₄and DR₅^[18]. At present,the HSC in vitro culture often become an important study means of the liver fibrosis,and also has established several HSC to replace the former generation as theresearch target cells in HSC. The HSC-T6is the eternal life lines by SV40virus. TheHSC-T6can infinitely increase and reproduce,which is a good model research of liverfibrosis[19-20].In this study, the HSC-T6is induced by acetaldehyde.We can establish agood model research of liver fibrosis to explore how the caffeine inhibits HSC-T6cellactivation and proliferation induced by acetaldehyde via adenosine A_2A receptormediated cAMP/PKA/Src/ERK1/2/p38MAPK signal pathway.The main content issummarized as follows:1. Effects of different concentration of caffeine and adenosine receptors on theproliferation of HSC-T6stimulated by acetaldehyde at different timesThe proliferation of HSC-T6stimulated by acetaldehyde （200μM） was treated withdifferent doses of caffeine（0.5,1,2,4,8mM）,adenosine A_2Areceptor antagonistZM241385（1,10,100,1000,10000nM）,adenosine A_2Areceptor agonist CGS21680（1,10,100,1000,10000nM） pretreatment1h.To exclude the effect of caffeine,ZM241385, CGS21680on cell viability, the morphology and the proliferation of HSC-T6wasexamined after24h,48h,72h,treatment by microscope and MTT colorimetric assay,respectively.The results showed that inhibitory rate of caffeine （0.5,1,2,4,8mM）were3.56%,7.52%,18.02,25.15%,35.25%at24h; The inhibitory rate were12.05%,22.29%,51.01%,64.65%,67.36%at48h; The inhibitory rate were13.68%,16.27%,37.51%,54.51%,85.70%at72h. The inhibitory rate of caffeine （4mM） is mostobvious at48h. The inhibitory rate of ZM241385（1,10,100,1000,10000nM） were10.25%,10.85%,12.85%,30.88%,23.87%at24h; The inhibitory rate were13.58%,14.81%,16.41%,40.98%,29.51%at48h; The inhibitory rate of ZM241385（1uM）foris most obvious at48h. The inhibitory rate of CGS21680（1,10,100,1000,10000nM）,the inhibitory rates were-6.79%,-7.89%,-25.88%,-34.7%,-16.1%at24h; Theinhibition rates were-14.33%-16.33%-29.12%-42.34%,-19.1%at48h; Theinhibitory rate of CGS21680（1uM） proliferation is most obvious at48h. The results showed that proliferation of HSC-T6was inhibited by caffeine and ZM241385.CGS21680can further promote HSC-T6proliferation, and show some concentration andtime dependent.2. Caffeine inhibits HSC-T6cell activation and proliferation induced byacetaldehyde via adenosine A_2AreceptorIn the above study on the basis of the experimental, the experimental set up thenormal group （conventional culture）, model group and the adenosine receptor （AR）regulator group.We use caffeine （4mM）, ZM241385（1uM）, CGS21680（1uM）,caffeine+CGS21680,ZM241385+CGS21680and HSC-T6common foster, then addto200uM acetaldehyd （per12h, supplement1） after1h, continue foster for48h. Totreatment the adenosine A_2Areceptor mRNA expression by fluorescence quantitativepolymerase chain reaction （Real-time PCR） assay; To detect the alpha-smooth muscleactin （α-SMA） content in the HSC-T6by immunocytochemistry assay; To detect theprocollagen I,Procollagen III, transforming growth factor-β1（TGF-β1）, connectivetissue growth factor （CTGF） mRNA expression by reverse transcriptase PCR（RT-PCR） assay; To detect the CTGF protein expression by Western blot method inHSC-T6.The results show that acetaldehyde（200uM） can significantly enhanced theexpression of adenosine A_2Areceptor,α-SMA, Procollagen I, Procollagen III, TGF-β1and CTGF; Compared with model group, caffeine and ZM241385were significantlyreduced the expression of adenosine A_2Areceptor,α-SMA, Procollagen I, ProcollagenIII, TGF-β1and CTGF in HSC-T6, CGS21680has a further role. Likewise, the A_2Areceptor antagonist ZM241385or caffeine completely abrogated the effect ofCGS21680.The results show that caffeine can significantly reduce the activation ofHSC-T6by acetaldehyde-induced,and significantly inhibited the expression levels ofadenosine A_2Areceptor, Procollagen I, Procollagen III,TGF-β1and CTGF. Themechanism that may be blocking the adenosine A_2Areceptor-mediated signalingpathway. 3. Caffeine inhibits HSC-T6cell activation and proliferation induced by Acetaldehyde via adenosine A_2Areceptor mediated cAMP/PKA/Src/ERK1/2/p38MAPKsignal pathwayAs mentioned above experimental groups, To measured the cAMP levels in HSC-T6by radioimmunoassay;To detected the PKA mRNA expression in HSC-T6byRT-PCR assay;To detected the P-Src, P-ERK1/2, P-of p38expression in HSC-T6byWestern blot assay. The results showed that acetaldehyde（200uM） can significantlyenhanced cAMP,PKA, P-Src, P-ERK1/2and P-p38expression at48h; Compared withmodel group, caffeine and ZM241385were significantly lower the cAMP,PKA,P-Src, the P-ERK1/2and P-p38expression, but CGS21680has a further role.With thedrug alone group, caffeine+CGS21680and ZM241385+CGS21680combinationgroup were significantly reversed the above effects. The results show that Caffeineinhibits HSC-T6cell activation and proliferation induced by Acetaldehyde viaadenosine A_2Areceptor mediated cAMP/PKA/Src/ERK1/2/p38MAPKsignal pathway.On this basis, we use of caffeine （4mM）,PKA blocker:H89+caffeine （20Îœm+4mM）,Src blocker:PP2+caffeine （20μM+4mM）, ERK1/2blocker: U0126+caffeine （20μM+4mM）, P-p38blocker: SB202190+caffeine （20μM+4mM） co-cultured with HSC-T6,then to join the acetaldehyde （200uM）stimulation （every12h, supplement1） culturedfor48h;At the same time,we set the normal group and model group. To detected theProcollagen I, Procollagen III,TGF-β1and CTGF mRNA expression by RT-PCRassay. Treatment of the cells with caffeine plus H89, PP2, U0126significantlyattenuated procollagen I mRNA up-regulation by acetaldehyde but SB202190did not.In contrast, only SB202190completely abolished acetaldehyde-induced procollagenIII mRNA expression. In a similar fashion, inhibition of PKA, src kinase, and p38MAPK reversed the effect of CTGF and TGF-β1mRNA expression. Then todetected the P-Src, P-ERK1/2, P-p38expression in HSC-T6by Western blotassay.The results show that inhibitors of p38MAPK and erk1/2plus caffeine activation did not prevent phosphorylation of src,although an src kinase inhibitorblocked activation of src by acetaldehyde.Acetaldehyde stimulates erk1/2phosphorylation,which is blocked by inhibitors of PKA, src kinase, and erk1/2pluscaffeine. In contrast, the p38MAPK inhibitor did not interfere with any of thesesignaling events. Acetaldehyde also stimulated the phosphorylation of p38MAPK,which was blocked by SB202190but not by the inhibitors of PKA, src, anderk1/2plus caffeine kinase. Thus, We finally deciphered that caffeine may be dividedthrough cAMP/PKA/Src/ERK1/2pathway and the p38MAPK pathway inhibitivedthe proliferation and activation in HSC-T6induced by acetaldehyde and the sequenceof signaling most consistent with our results is PKA–src–erk1/2.