The Effects Of Insulin And Ly294002 Inhibitors Of PI3K On The Regulations/expression Of Aquaporin9 In Nomal Liver Cells

Objective To explor the expression of AQP9 after insulin effect on the membrane of normal human hepatocyes。And to investigate the role of PI3K/AKT signal transduction pathway in the regulation of AQP9 expression。Methods L-02 cell strain hepatocytes ( normal adult human hepatocytes)underwent subculture with 1640 medium contining 10% bovine serum。Investigate the localization of AQP9 in position of L-02 cell strain by Immunocytochemistry(ICC)。Establish the insulin stimulates hepatocytes model of concentration gradient:harvest the L-02 cell strain hepatocytes after incubating with 100nM/L, 250 nM/L, 500 nM/L insulin concentrations respectively;Establish the time gradient :harvest the L-02 cell strain hepatocytes at 3h,6h,12h,24h after incubating with insulin;Establish the drug stimulates hepatocytes model :Add the optimal concentration insulin stimulates the L-02 cell strain hepatocytes that incubated with the LY294002(the selective inhibitor of PI3K),A-443654 (the selective inhibitor of AKT),SB2030580(the selective inhibitor of MAPK)。The change of the expression of AQP9 mRNA was measured by RT-PCR and the protein expression of AQP9 was measured by Western blot.Results: By using the method of Immunocytochemistry ,the positive product of AQP9 were aboundently-expressed on the memebrane of normal L-02 strain of hepatocyte,not including cytoplasm and nucleus. The insulin stimulates hepatocytes model of concentration and time gradient was established successfully.The expression of AQP9 began to reduce from 100nM/L insulin,especially at 500nM/L insulin (p < 0.01 ); and the expression of AQP9 began to reduce from 3 hours of insulin stimulation(p<0.05); Incubated with the selective inhibitor of PI3K and AKT ,the inhibitory effects of insulin on AQP9expression is decreased(p<0.05);but it does not change significant by blocking the MAPK signaling pathway.Conclusion: The expression of AQP9 on the memebrane of normal L-02 is time and concentration dependence after incubating with insulin,that is the higher the concentration of insulin and the role of the longer,the more obviously inhibited the expression of AQP9。Incubated with the selective inhibitor of PI3K and AKT ,the inhibitory effects of insulin on AQP9expression is decreased;but it does not change significant by blocking the MAPK signaling pathway. As for that, the PI3K/akt signal transduction pathwsys was involved in the swap mechanism of insulin on AQP9.