The Effects Of Adenosine Receptor A1 And A2A On Activation Of Hepatic Stellate Cells In Alcoholic Liver Fibrosis And The Interaction Between Adenosine Receptor A1 And A2A

Long-term excessive drinking is the most important cause of liver disease alcoholic (ALD), and alcoholic liver fibrosis is the intermediate stage and the essential pathological process of ALD progression. In the process of alcoholic liver fibrosis, ethanol can activate the hepatic stellate cells (HSCs) through its intermediate metabolite acetaldehyde. The preliminary studies have confirmed that 200 μM acetaldehyde stimulated in vitro HSC cells for 48 hours can be successfully established alcoholic liver fibrosis model. Adenosine is an endogenous purine nucleotide, widely distributed in the body, and most of the adenosine receptors (ARs) play an important role in corresponding physiological on the cell membrane. ARs belong to the superfamily of G- protein coupled receptors, which can be divided into four subtypes, namely A1R, A2AR, A2BR and A3R. A1 receptors and A2A receptors are highly expressed receptors, and are the two most important types between them. In recent years, with the development of selective agonists and antagonists of A1R and A2AR, there are more and more researches on the role of adenosine and its receptors. However, there is little research on the role of A1R and A2AR in alcoholic liver fibrosis and the interaction between them. Therefore, this study selected in vitro HSC cell as the research object, using 200 μM acetaldehyde in vitro HSC stimulated cells for 48 hours to establish alcoholic liver fibrosis model, in the model were overexpressed A1R and A2AR, effects of A1R and A2AR in alcoholic liver fibrosis play and their interaction.Objective:Through the construction of eukaryotic recombinant plasmid pEGFP-C2-A1R and pEGFP-C2-A2AR, were transfected into HSC-T6 cells, the establishment of AIR and A2AR in HSC expression model, to observe the AIR and A2AR mRNA and its protein expression in HSC. Experimental determination and monitoring related gene mRNA and protein expression levels by MTT and cell cycle, detection of AIR and A2AR respectively the influence on the expression of rat alcoholic liver fibrosis HSC activation and proliferation of and by immuno coprecipitation technique combined with Western blot method to study the AIR and A2AR in alcoholic liver fibrosis HSC cells between the two interactions.Methods:Extraction of AIR and A2AR gene from rat brain tissue, and then HindⅢ, KpnI of the digested and digested vector pEGFP-C2. The digested products were connected method and transformed into competent Escherichia coli DH5a connection according to conventional, plasmid pEGFP-C2-AlR and pEGFP-C2-A2AR eukaryotic expression. After the successful construction of pEGFP-C2-A1R and pEGFP-C2-A2AR recombinant plasmid by enzyme cutting and sequencing by liposome mediated guide HSC cells transfected with and under the inverted fluorescence microscope to observe the expression of green fluorescent protein, expression of AIR and A2AR mRNA and protein were detected by RT-PCR and Western blot, to identify the expression model is established successfully. Then referring to the early research results using 200μM acetaldehyde stimulated cells for 48 hours to establish alcoholic liver fibrosis model. After overexpression of AIR and A2AR respectively, RT-PCR and Western blot were used to detect the expression changes of AIR, A2AR, a-SMA, collagen I and cyclin-D1. At the same time, the MTT assay and cell cycle assay to detect HSC activation and proliferation. PEGFP-C2-A1R and pEGFP-C2-A2AR eukaryotic expression plasmids were co-transfected into HSC cells, and the interaction between AIR and A2AR was detected by immunoprecipitation.Results:1. Successfully constructed the recombinant plasmid:after double enzyme digestion and identification, the expression of green fluorescent protein was observed under the fluorescence inverted microscope. RT-PCR and Blot Western method can be used to detect the expression of and mRNA in A2AR and AIR respectively.2. Effects of AIR and A2AR over expression respectively on HSC activation and proliferation:After the overexpression of AIR and A2AR respectively, a-SMA, collagen I and cyclin-D1 expression were significantly up-regulated with 200 μM acetaldehyde stimulated in vitro HSC cells for 48 hours. MTT results showed that the model group can obviously promote the HSC proliferation and over expression group promoting effect is more obvious. And cell cycle assay results showed that model group, the distribution of the increase in the proportion of S phase cells, to reduce the proportion of G0/G1 phase cells. Compared with model group, the proportion of the cells in S phase was significantly increased, and the proportion of cells in G0/G1 phase was significantly decreased. This suggests that AIR and A2AR can promote the activation and proliferation of HSC cells respectively.3. Interaction between AIR and A2AR protein:using Western blot method combined with co precipitation technique, choosing the last time on the washing of the supernatant as a negative control to eliminate the nonspecific protein and for the purpose of residual interference. The results showed that the corresponding target protein could be detected in the immune complex, the bands in the Western blot and HSC cell line were consistent with the position of the bands in the cell line and negative control group did not detect the stripe, indicating that there was an interaction between A1R and A2AR.Conclusion:AIR and A2AR can promote the activation and proliferation of HSC cells respectively and there is an interaction between AIR andA2AR.