The Effects Of Adenosine Receptors A1and A2A Mediated CAMP-PKA-CREB Signaling Pathway On The Activation Of HSC In Alcoholic Liver Fibrosis

Alcoholic liver disease (ALD) is a disease of the liver caused by long-term heavydrinking. Alcoholic liver fibrosis is an intermediate stage of ALD in the developingprogress. How to prevent and reverse its formation has become a hot academic attention.In the process of alcoholic liver fibrosis, ethanol can activate the hepatic stellate cells(hepatic stellate cell, HSC) through its metabolite acetaldehyde. The activated HSC isthe main source of extracellular matrix (ECM) during the liver fibrosis, and theactivation of HSC is a central part of the liver fibrosis caused by various factors.Therefore, inhibition of HSC activation and proliferation is the main strategy for thetreatment of liver fibrosis.In recent years, with the development of the adenosine receptor (adenosine receptors,ARs) subtype selective agonists and antagonists, researchers play more and moreattention on the in-depth research on the role of adenosine and its receptors, andgradually realize the important role of adenosine A1(adenosine A1receptor, A1R) andA2A receptors (adenosine both A2A receptor, A2AR) in the pathogenesis of alcoholicfatty liver and liver fibrosis. Chan, etc found that a large number of adenosine inducedby ethanol can promote the synthesis of collagen in rats and human HSC via activating the HSC adenosine A2AR. Peng has shown that a lack of A1R can reduce theoccurrence of alcoholic fatty liver in mice. Our previous researches suggest that caffeineas a non-selective adenosine receptor antagonist can significantly inhibit HSC T6activation induced by acetaldehyde, and evidently reduce the expression level ofTGF-beta1and CTGF in the HSC-T6. The mechanism may be related to theantagonism of adenosine A2AR-mediated signaling pathways.Thus, we speculate that both the adenosine A1R and A2AR can regulate the activationof HSC in the process of alcoholic liver fibrosis formation. But they may have differenteffects because of the opposite regulating effects on cAMP-PKA signaling pathway. Inorder to further clarify the effects of adenosine A1R and A2AR signaling pathways onregulating the proliferation of HSC, the cultured HSC was stimulated by differentconcentration of acetaldehyde at different times to prepare in-vitro HSC model ofalcoholic liver fibrosis. And on this basis, observe the expression level of HSC A1R andA2AR, and explain the effects of adenosine A1R and A2AR-mediated cAMP signalingpathways on regulating the activation and proliferation of HSC. The main content issummarized as follows:1Isolation, identification and culture of HSCHSCs were extracted and purified from normal male Sprague-Dawley rats (300-450g)in the liver with pronase and collagenase, followed by density gradient centrifugationover Nycodenz. Isolated HSCs were cultured in20%fetal bovine serum DMEM.2The expression level of A1R and A2AR mRNA and protein in HSC of alcoholicliver fibrosis The previous studies show that the effect of acetaldehyde was most obvious in200μMafter adding for48h. So we choose it to make an in-vitro model of rat alcoholic liverfibrosis HSC and set up a normal group. The expression levels of A1R and A2AR weredetected using qRT-PCR and Western Blot. The results show that the levels of A1R andA2AR expressed in the model group are obviously higher than the control group. Theexpression levels of A1R and A2AR in the model group are much higher compared withthe normal group.3The effect of adenosine A1receptor mediated cAMP signal pathway in HSC ofalcoholic liver fibrosisThe HSC were seeded in the plate and incubated for24hours. The HSC were dividedinto five groups: the control group, the model group, the PTX group (Adenosine A1receptor mediated Gi/o protein inhibitor), the NECA group (no-selective adenosinereceptor agonist) and the PTX+NECA group.200uM acetaldehyde was added into eachgroup for48h to establish ALD except the control group, then PTX was added into thePTX group and the PTX+NECA group for24h, NECA was added into the NECA groupand the PTX+NECA group for15min at last. The cAMP levels were measured with acAMP radio immunoassay kit according to the manufacturer’s instructions and theprotein expression levels of PKA and p-CREB were detected by Western Blot.Experiments were repeated at least three times. The results show that compared with thenormal group, the expression of cAMP and the key signal molecules, like PKA andp-CREB were significantly increased. And PTX can significantly enhance the effect ofNAEC. The results suggest that PTX as Gi/o protein coupled receptor inhibitor, canincrease the production of cAMP by inhibiting Gi/o-AC-cAMP channel andstrengthen the effect of nonselective AR agonist. To further clarify the role of adenosine A1R and A1R-mediated cAMP signalingpathway in the HSC model of alcoholic liver fibrosis, respectively cultivate HSC withadenosine A1R agonist CPA, A1R antagonist DPCPX or CPA+DPCPX for48hours.The expression levels of a-SMA, Collagen I and Collagen III were detected by real-timequantitative PCR and Western blot. The results show that the levels of a-SMA, CollagenI, Collagen III in model group were enhanced obviously after48h while significantlyreduced after DPCPX treatment. And CPA can further increase the expression levels ofa-SMA, Collagen I and Collagen III. However, the effect above in the CPA+DPCPXcombination group was obviously reversed. It indicates that acetaldehyde can promotethe proliferation of HSC via activating the acetaldehyde A1R.Further study suggests the levels of cAMP, PKA and p-CREB in the model group weredecreased after CPA treatment obviously. And DPCPX can further increase theexpression levels of cAMP, PKA and p-CREB in the model group. At the same time,the effect above in the CPA+DPCPX combination group was significantly reversed.The results showed that the A1R-Gi/o-AC-cAMP-PKA-CREB signaling pathway has acertain role within the activation and proliferation of acetaldehyde-induced HSC.4The effect of adenosine A2A receptor mediated cAMP signal pathway in HSC ofalcoholic liver fibrosisThe HSC were seeded in the plate and incubated for24hours. The HSC were dividedinto five groups: the control group, the model group, the Rolipram group, the NECAgroup and the NECA+Rolipram group.200uM acetaldehyde was added into each groupfor48h to establish ALD except the control group, then Rolipram was added into theRolipram group and the NECA+Rolipram group for15min, NECA was added into theNECA group and the NECA+Rolipram group for10min at last. The levels of cAMP were measured with a cAMP radio immunoassay kit according to the manufacturer’sinstructions and the protein expression levels of PKA and p-CREB were detected byWestern Blot. Experiments were repeated at least three times. The results show thatcompared with the normal group, the expression of cAMP and the key signal molecules,like PKA and p-CREB were significantly increased. Rolipram and NECA cansignificantly increase the level of cAMP, PKA and p-CREB.in the model group. Thecombination of the Rolipram can and NECA can further improve the effects. The levelof cAMP can be increased in two ways: First, as the antagonists of ARs; Second,inhibition of the intracellular phosphodiesterase activity. The results indicate thatRolipram as phosphodiesterase inhibitors (IV) significantly increase level of cAMP, andNECA as non-selective AR agonist also significantly increase level of cAMP, promptthe presence of AR-Gs-AC-cAMP signaling pathways in HSC.To further clarify the role of adenosine A1R and A1R-mediated cAMP signalingpathway in the HSC model of alcoholic liver fibrosis, respectively cultivate HSC withadenosine A2AR agonist CGS21680, adenosine A2AR antagonist ZM241385, orCGS21680+ZM241385for48hours. The expression levels of a-SMA, Collagen I andCollagen III were measured by real-time quantitative PCR and Western blot. The resultssuggest that the levels of a-SMA, Collagen I, Collagen III in model group wereenhanced obviously after acetaldehyde treatment for48hours while significantlyreduced after ZM241385treatment. And CGS21680can further increase the expressionlevels of a-SMA, Collagen I and Collagen III. However, the effect above in theZM241385+CGS21680combination group was obviously reversed. The results indicatethat acetaldehyde can promote the proliferation of HSC via activating the acetaldehydeA2AR and the effect can be inhibited by the antagonist of A2AR. Further study shows that the protein expression levels of cAMP, PKA and p-CREB inthe HSC of model group were significantly reduced after the ZM241385treatment.However the levels were futher increased after the treatment of CGS21680. However,the effect above in the ZM241385+CGS21680combination group was obviouslyreversed. The result indicates that the A2AR-Gs-AC-cAMP-PKA-CREB signalingpathway has a certain role during the proliferation of acetaldehyde-induced HSC.To sum up, the expression levels of adenosine A1R and A2AR in HSC of alcoholic liverfibrosis are much higher than the normal HSC. Adenosine A1R and A2AR were jointlyparticipate in regulating the activation of HSC proliferation in alcoholic liver fibrosis.Although both of the Adenosine A1R and A2AR antagonists have significant inhibitoryeffect in alcoholic liver fibrosis, they have the opposite effects on cAMP-PKA-CREBsignaling pathway.