Adenosine Receptor A1 Mediates Activation Of HSCs Via PI Signaling Pathway In Alcoholic Liver Fibrosis

Alcoholic liver disease is one kind of liver disease which caused by long-term heavy drinking. It has a large spectrum of injury ranging from fatty liver to cirrhosis even liver cancer. Alcoholic liver fibrosis is an important part of this progress and has become a hot academic spot. In the process of alcoholic liver fibrosis, acetaldehyde, which is the metabolite of ethanol in liver can activate hepatic stellate cells. The activated hepatic stellate cell can produce extracellular matrix and that’s the central part of liver fibrosis. Therefore, inhibit the activation of hepatic stellate cells is the main target for the treatment of liver fibrosis.With the development of study adenosine and adenosine receptors, people realize adenosine receptors play an important role of liver disease. Our previous researches suggest that caffeine as a non-selective adenosine receptor antagonist can protect against alcohol-induced liver fibrosis in rat model and inhibit activation of hepatic stellate cell induced by acetaldehyde. We also found that adenosine A2 A receptor can mediate activation of hepatic stellate cell via c AMP-PKA-CREB signaling pathway.Thus, we speculate that adenosine A1 receptor also plays an important role in alcoholic liver fibrosis and it can mediate activation of hepatic stellate cell via PI signaling pathway. In order to clarify this, we use acetaldehyde to prepare HSC-T6 cell line to establish alcoholic liver fibrosis cell model. On this basis, we try to explain the effects of adenosine A1 receptor-mediated PI signaling pathway on regulating the activation and proliferation of hepatic stellate cell.The main content is as follows:1. The expression of m RNA and protein level of A1 R in alcoholic liver fibrosis HSC model.We use acetaldehyde to prepare HSC-T6 cell line to establish alcoholic liver fibrosis cell model, then detected the expression of m RNA and protein level of A1 R by Real Time PCR and Western blot. The results shows that the m RNA and protein level of A1 R in model group are higher than the normal group.2. Silencing A1 R by RNAi significantly inhibited the HSC proliferation.siRNA targeting A1 R was designed and synthesized according to its mRNA. The si RNA was transfected into rat HSC-T6 cells by liposome LipofectamineTM2000. The HSC were divided into four groups: the control group, the model group,the A1 R si RNA group and the scramble si RNA group. HSC cell proliferation was measured by MTT. The m RNA level of A1 R, α-SMA, Collagen I in the supernatant of the cell culture were measured by Quantitative Real-Time PCR. The protein level of A1 R, α-SMA, Collagen I were measured by Western blot. A1 R si RNA effectively inhibit the cell proliferation, and it also significantly decreases the levels of A1 R, α-SMA, Collagen I, suggesting that A1 R may be potential target genes in the alcoholic liver fibrosis.3. The effect of PI signaling pathway in alcoholic liver fibrosis HSC model.Rat hepatic stellate cell-T6 line(HSC-T6) incubated for 24 hours. The HSC were divided into five groups: the control group, the model group, the UTP group(precursor of IP3), the NECA group(non-selective adenosine receptor agonist) and the UTP+NECA group. 200μM acetaldehyde was added into each group for 24 h to establish alcoholic liver fibrosis model except the control group, then UTP and NECA were added into each group respectively. The IP3 levels were measured with an Elisa kit, the protein expression level of PLC was detected by western blot and [Ca2+] concentration was monitored by confocal laser scanning microscope(CLSM). The results show that compared with the normal group, the expression of IP3 and the key signal molecules, like PLC and [Ca2+] were significantly increased. And UTP can significantly enhance the effect of NECA. The results prompt the presence of PI signaling pathway in HSC.4. The effect of adenosine A1 receptor mediated PI signaling pathway in alcoholic liver fibrosis HSC model.Rat hepatic stellate cell-T6 line(HSC-T6) incubated for 24 hours. The HSC were divided into five groups: the control group, the model group, the CPA group(adenosine A1 R agonist), the DPCPX group(adenosine A1 R antagonist) and the CPA+DPCPX group. 200μM acetaldehyde was added into each group for 24 h to establish alcoholic liver fibrosis model except the control group, then CPA and DPCPX were added into each group respectively. The expression levels of α-SMA and Collagen I were detected by real-time quantitative PCR and western blot. The IP3 and DAG levels were measured with Elisa kits, the protein expression level of PLC and PKC was detected by western blot and [Ca2+] concentration was monitored by confocal laser scanning microscope(CLSM). The results show that the levels of α-SMA, Collagen I, IP3, DAG, PLC, PKC and [Ca2+] concentration in model group were enhanced obviously after 48 hours while significantly increased after CPA treatment but reduced after DPCPX treatment. However, the effect above in the CPA+DPCPX combination group was obviously reversed. The results indicate that the A1R-PI signaling pathway has a certain role during the proliferation of acetaldehyde-induced HSC.5. Cross-talk between PLC-PKC signal pathway and c AMP-PKA signal pathwayRat hepatic stellate cell-T6 line(HSC-T6) incubated for 24 hours. The HSC were divided into four groups: the control group, the model group, the PMA group(PKC agonist) and the SC-3088 group(PKC antagonist). 200μM acetaldehyde was added into each group for 24 h to establish alcoholic liver fibrosis model except the control group, then PMA and SC-3088 were added into each group respectively. The expression levels of α-SMA and Collagen I were detected by real-time quantitative PCR and western blot. The c AMP level were measured with Elisa kits, the protein expression level of PKA was detected by western blot. The results show that the levels of α-SMA, Collagen I, c AMP and PKA concentration in model group were enhanced obviously after 48 hours while significantly increased after PMA treatment but reduced after SC-3088 treatment. Then we divided the HSC into four groups: the control group, the model group, the FSK group(PKA agonist) and the H89 group(PKA antagonist). 200μM acetaldehyde was added into each group for 24 h to establish alcoholic liver fibrosis model except the control group, then FSK and H89 were added into each group respectively. The expression levels of α-SMA and Collagen I were detected by real-time quantitative PCR and western blot. The protein expression level of PLC and PKC were detected by western blot. The results show that the levels of α-SMA, Collagen I, PLC and PKC concentration in model group were enhanced obviously after 48 hours while significantly increased after FSK treatment but reduced after H89 treatment. The results indicate that there have cross-talk between PLC-PKC signaling pathway and c AMP-PKA signaling pathway in acetaldehyde-induced HSC.To sum up, the expression levels of A1 R in HSC of alcoholic liver fibrosis are much higher than the normal HSC. Silencing A1 R by RNAi can significantly inhibited the HSC proliferation, A1R-PI signaling pathway has a certain role during the proliferation of acetaldehyde-induced HSC and have a cross-talk with c AMP-PKA signaling pathway.