The Effects Of Caffeine On Signal Pathways Of CAMP And PI In HSC Of Alcoholic Liver Fibrosis In Rats

Alcoholic liver fibrosis（ALF） is a form of alcoholic liver disease（ALD） in thedeveloping progress. The ALD has symptoms such as alcoholic fatty liver, alcoholichepatitis and so on in the prophase, and even develop to ALF even alcholic cirrohosis,result to the necrosis of hepatic cells widely, and form the liver failure finally.ALF is adisease which is characterized by extracellular matrix （ECM） hyperplasia, and ALF isthe key link of ALD. If we can cure ALF effectively, the ALD will be blocked orpostponed to be liver cirrohosis, but there isn’t ideal clinical drug to prevent and cureALF high effectively at present.Caffeine is the dominant components of coffee and tea. which are the usual drink inlife, serving drinks containing caffeine is widely spread in the world. In rencent years,more and more studies reported that having drink which containing caffeine will reducethe proability of suffering from the liver disease, but the mechanism of it has not yetbeen elaborated completely. Our group has confirmed that caffeine can prevent and curethe rats of ALF in early research of histology, but the mechanism of caffeine has beenunknown. Basing on a review of relevant literatures, in this research, at first weestablished the alcohol-induced hepatic fibrosis model in rats by increasing concen- tration of alcohol twice a day for12weeks. On this basis, we isolated the primary HSCsby collagenase type IV, in situ perfusion. And we detect the content of cyclic adenosinemonophosphate（cAMP）, protein kinase A and the cAMP response element bondingprotein（CREB）, to clear the mechanism of caffeine protect the ALF, and on this basis,we stimulated the HSC-T6by acetaldehyde to establish the alcohol-induced liverfibrosis model in vitro, and we detect key material such as Phosphatidase C（PLC）,diacylglycerol （DAG） and protein kinase C（PKC） in PI pathway, then explored thetherapeutic and prevention effects of low concentration of caffeine on alcohol-inducedliver fibrosis and the effects on proliferation of HSC-T6stimulated by acetaldehyde andits partial mechanisms. The main contents summarized as follows:1.The effects of caffeine on HSC isolated from the rats of liver fibrosis incAMP-PKA-CREB signal pathwayThe rats were fed by alcohol twice everyday for12weeks, the liver fibrosis modelwas established,the rats were divieded into6groups randomly:normol, model,caffeine（5,10,20mg/kg） and the positive control （colchicine）, the caffeine was also fedto rats everyday as the dose above, at the12thweekend, we killed the rats and isolatedthe HSCs from the rats by collagenase IV. We we observe that the HSCs’auto-fluorescence at the328nm of flurescence microscope, and count the cells by trypanblue excluding test, and detect the expression of α-SMA by immunocytochemistry andthe cell cycle by FCM, the cAMP content by¹²⁵I-cAMP radioimmunoassa.The mRNAexpression of α-SMA, type I and III procollegan, PKA and CREB were measured byRT-PCR.It indicates that HSCs have spontaneous blue-green fluorescence in model. Theamount and activity of HSC in model was more than normal. Compared with thenormal,the cells in S and G2/M phases were more, the cAMP was more, and there ismuch α-SMA, type I and III procollegan, PKA and CREB in the model. Compared withthe model, caffeine（10,20mg/kg） can block the cell cycle at G2/M phase and decreasethe cAMP content, and inhibits the mRNA expression of type I and III procollegan, α-SMA, PKA and CREB markedly. The results showed that caffeine can inhibit theproliferation and activation of HSC isolated from the rats of liver fibrosis.Themechanism may be involved with caffeine inhibits the cAMP-PKA-CREB signalpathway in HSC.2. The effect of caffeine on HSC-T6stimulated by acetaldehyde in PLC-DAG-PKC signal pathway.The HSC-T6pretreatmented with different doses of caffeine were stimulated by200μmol/L acetaldehyde.In the time of24,48,72hours,we use MTT to explore theproliferation of HSC-T6. The mRNA expression of PLC were measured by RT-PCR, thecontent of DAG by liquid scintiloscope and the activity of PKC in HSC-T6by cell PKCkinase activity spectrometry quantitative kit, The results showed acetaldehyde canactivate and proliferate the HSC, the that caffeine can inhibit the proliferation ofHSC-T6stimulated by acetaldehyde and the50%inhibiting concentration （IC50） were22.512,4.553,2.021mmol/L respectively. Compared with the control, the expression ofPLC and the content of cAMP was more, and the activity was stronger, caffeine（1,2,4mmol/L） can down-regulate the expression of PLC, and reduce the content of DAG, anddecrease the activity of PKC.The research above showed caffeine could inhibitproliferation of HSC-T6stimulated by acetaldehyde.The mechanism may be involvedwith caffeine inhibits the PLC-DAG-PKC pathway, The research provided a promisingapproach to using caffeine as an anti-alcoholic liver fibrosis drug and providing a newidea for evaluation, and screening effective treatment of alcoholic liver fibrosis.