Effect Of Co-culture Of Adipocytes And Hepatocytes On The Expression Levels Of AQP9 MRNA And Protein In Hepatocytes And Its Mechanism

Objective This study established one model that co-culture of hepatocytes and mature human adipocytes,to observe the effect of mature adipocytes on the expression levels of AQP9 in HepG2 cells,and to explore its possible mechanism.Methods Firstly,Culturing human preadipocytes and promoting them differentiation to fully mature cells,co-culture HepG2 cells with human preadipocytes and mature human adipocytes for 48 hours,and labeled as control group and experimental group,respectively.The co-cultured HepG2 cells were stained with oil red o and the contents of triglycerides were detected.The changes in PI3K-Akt signaling pathway and expression levels of AQP9 m RNA and protein were detected by real-time fluorescence-based quantitative PCR and Western blotting,respectively.Subsequently,the experimental group was co-cultured with 100ng/ml PI3K-Akt pathway agonist,recombinant human insulin-like growth factor-1(IGF-1),labeled as experimental group+IGF-1 group.WB verified the activation of PI3K-Akt pathway.Meanwhile,q RT-PCR and WB detected the expression of AQP9.Secondly,HepG2 cells were infected with recombinant lentivirus LV-AQP9 or vector lentivirus LV-PWPI by recombinant lentivirus transfection technology.They were labeled as HepG2-AQP9 group andHepG2-PWPI group respectively.Laser confocal detection of transfection efficiency,RT-q PCR and WB were used to detect the changes of AQP9 expression level after virus transfection.Afterwards,the HepG2-AQP9 cells with stable over-expression of AQP9 as well as the HepG2-PWPI cells were separately co-cultured with mature human adipocytes for 48 hours,and labeled as HepG2-AQP9 co-culture group and HepG2-PWPI co-culture group respectively.The co-cultured HepG2-PWPI and HepG2-AQP9 cells were stained with oil red o and the contents of triglycerides were detected.Finally,IGF-1 was added to the HepG2-AQP9co-culture group and recorded as the HepG2-AQP9 co-culture+IGF-1group.Oil red o staining and the contents of triglycerides were detected.Meanwhile,WB verified the activation of PI3K-Akt pathway,and q RT-PCR and WB detected the expression of AQP9.Results Compared with the control group,the intracellular lipid droplets increased significantly in the experimental group,and the contents of triglycerides increased significantly(P=0.006),suggesting that co-culture of adipocytes can induce adipose degeneration of HepG2 cells.The results of q RT-PCR and WB indicated that the expression levels of AQP9 m RNA and protein in the experimental group were significantly higher than those in the control group(P values were 0.005 and 0.000 respectively),while WB results showed that the expression level of p-Akt protein in the experimental group was significantly lower than that in the control group(P=0.000),the change of total Akt protein was insignificant,and p-Akt/total Akt was significantly lower than that in the control group(P=0.001),suggesting that co-culture of adipocytes could inhibit PI3K-Akt signaling pathway and up-regulation of AQP9 expression in HepG2 cells.WB results showed that the expression level of p-Akt protein in the experimental group+IGF-1 group was significantly higher than that in theexperimental group(P=0.007),the change of total Akt protein was not obvious,p-Akt/total Akt was significantly higher than that in the control group(P=0.000).Meanwhile,RT-q PCR and WB results indicated that the expression levels of AQP9 m RNA and protein in the experimental group+IGF-1 group was significantly lower than that in the experimental group(P value was 0.000,0.005 respectively),suggesting that co-culture of hepatocytes and mature human adipocytes may regulate the expression of AQP9 by PI3K-Akt pathway.Laser confocal analysis showed that the transfection rate was more than 90%.RT-q PCR and WB results indicated that the expression levels of AQP9 m RNA and protein in HepG2-AQP9 group were significantly higher than those in HepG2-PWPI group(P values were 0.002 and 0.000 respectively),suggesting that the stable overexpression of AQP9 cell line was successfully constructed.Compared with HepG2-PWPI co-culture group and HepG2-AQP9 co-culture+IGF-1group,the intracellular lipid droplets in HepG2-AQP9 co-culture group increased significantly,and the intracellular triglyceride content increased significantly(P value was 0.005),suggesting that the increased expression of AQP9 could promote the steatosis of HepG2 co-cultured with adipocytes.WB results showed that compared with HepG2-AQP9co-culture group,the expression levels of p-Akt protein and p-Akt/total Akt in HepG2-AQP9 co-culture+IGF-1 group increased significantly(P=0.000),while the expression levels of AQP9 m RNA and protein decreased significantly(P values were 0.000 and 0.001,respectively).Conclusion Co-culture of adipocytes can induce steatosis of HepG2 cells,and may play an importent role during the steatosis of HepG2 cells by inhibiting PI3K-Akt signaling pathway and up-regulating the expression of AQP9.