The Expression And Role Of AQP9 In Models Of Cultured Steatosis Hepatocytes And The Role Of P38 MAPK Signal Transduction Pathway In The Expression Of AQP9 In NAFLD.

Objective: To explore the expression and role of AQP9 and its mRNA in the models of cultured steatosis hepatocytes. And to investigate the role of p38 Mitogen-activted protein kinase(MAPK) signal transduction pathway in the expression of AQP9 in nonancoholic fatty liver disease(NAFLD).So as to further explore the possible mechanism of AQP9 in NAFLD.Methods:L-02 cell strain hepatocytes(normal adult human hepatocytes) underwent subcultures with 1640 medium contining 10% bovine calf serum.Investigate the localization of AQP9 in position of L-02 cell strain by Immunocytochemistry(ICC) . Steatosis models of hepatocytes was established by adding oleic acid with suitable concentration by MTT to the growing L-02 cell strain.Also add the SB203580(the selective inhibitor of p38 MAPK) into steatosis models of hepatocytes.So the cultures were divided into the control group(Group C,cultured with normal medium),the model group(Group M,cultured with the normal medium +oleic acid),the blockage group (Group B,cultured with the normal medium +oleic acid +blocking agent SB203580). The accumulation of lipid droplets in hepatocytes were observed by light microscopy after oil red staining . The contents of triglyceride ,free fatty acid(FFA),glycerol in hepatocytes were measured by analyzed kit respectively.The chang of the expression of AQP9 mRNA was measured by RT-PCR and the protein expression of AQP9 ,total-p38 MAPK , phospho-p38 MAPK protein were measured by Western blot.Results:The model of oleic acid-induced steatosis of L-02 strain hepatocytes was established successfully.No red lipid droplet was observed in hepatocytes which distributed densely ,with clear nuclei in the control group by optical microscopy after oil red-O straining.A small mumber of steatotic hepatocytes were found in the model group only 24h after culture and many orange-red lipid droplets were found in the cytoplasm 72h after culture,with some lipid droplets fused into larger ones.By using the method of immuneocytochemistry ,the positive products of AQP9 were aboundently-expressed on the memebrane of normal L-02 strian of hepatocyte,not including cytoplasm and nucleus. The content of TG,FFA,glycerol were increased in group M and B,compared with that in group C(P<0.01),with highest level detected in group M. The expression of AQP9 mRNA coincided with protein were both upregulated in group M and group B,compared with that in group C(P<0.01),with lower expression in group B compared with that in group M(P<0.05).The expression of t-p38 MAPK(total p38 Mitogen-activted protein kinase) protein in group M and group B had no significant differences ,compared with that in group C(P>0.05). Compared with that in group C, the expression of p-p38 MAPK(phospho-p38 Mitogen-activted protein kinase) protein was upregulated in group M,but had no significant difference in group B(P>0.05). compared with that in group M,the expression of p-p38 MAPK in group B was much lower(P<0.01).Conclusion:A model of steatosis of human hepatocyte can be established successfully by adding oleic acid to L-02 strain of hepatocyte.In the model of steatosis of hepatocyte ,the expression of AQP9 was upregulated gruandually with the fatty degeneration of hepatocyte aggravated.After blocked the p38 MAPK signal transduction pathway,the expression of AQP9 downregulated, compared with the control group,and also fatty degeneration of hepatocyte relived.So it proves that AQP9 might take a part in the course of fat metalism and fatty degeneration of hepatocyte,and p38 MAPK singal transduction pathway might regulate the expression of AQP9 in the model of steatosis of hepatocyte.