Experimental Study Of The Effects Of NAFL On Hepatic Fibrosis In Rats

Objective:To explore the direct activation of NAFL steatosis on stellate cell(HSC)by establishment of rat NAFL in vivo and hepatocyte steatosis and mixed culture model in vitro,and reveal the non-inflammatory mechanism of NAFL fibrosis.Methods:In vivo experiment: 1.Forty Sprague-Dawley(SD)rats were randomly grouped into model(n = 20)and control(n = 20).Model rats were fed with high-fat diet(HFD)and chow diet for 20 weeks to produce NAFL,and the control rats received chow diet.The body weight and body length were measured weekly.After intraperitoneal injection of 3% pentobarbital sodium(30 mg/kg),the liver was extracted,fixed in 10% neutral formalin solution,and the middle lobes were harvested.Steatosis,collagen deposition and fibrosis,TGF-beta1,PDGF and ?-SMA expression were detected by HE,masson and immunohistochemistry(SP)staining respectively.DN-3A microscopic image system was used for semi-quantitative analysis.In vitro experiment: the human hepatocyte L-02 and stellate cell LX-2 were used and the RPMI 1640 with 50% fetal bovine serum was applied to L-02 to establish steatosis cytological model.Hepatocyte-stellate cell interaction was simulated by L-02 and LX-2 co-culture,and the separate culture and supernatant culture were set as control.The steatosis,the expression of ?-SMA,collagen type ? and type ? were detected by O il red O staining,immunofluorescence and western blot(WB)respectively,and the morphology of L-02,LX-2 and steatosis was observed under microscope and measured with Image J software.All data were processed and statistically analyzed with IBM SSPS software.Results:In vivo experiment: 1.Compared with the control,the liver of model rats showed significant steatosis(P < 0.001).According to the degree of lipid deposition,the steatosis was classified into mild(> 20%)(26.23 ± 7.40)%,moderate(> 30%)(33.54 ± 4.22)%,severe(> 40%)(43.69 ± 9.13)%,and extremely severe(> 50%)(54.12 ± 6.79)%.The differences were statistically significant(P < 0.05).2.In the control group,only a small amount of collagen deposited in the portal area,and lack in the lobule.While the model rats showed that lots of collagen deposited around the hepatocytes,and was consistent with the degree of steatosis.There is no obvious difference in the portal area between two groups.3.In the control group,only a small amount of lymphocyte in the portal area,and lack in the lobule.While a small number of spotty necrosis and inflammatory cell infiltration can be noticed in the model group,but the expression of PDGF and TGF-beta 1 was negative in the two groups.In vitro experiment: 1.RPMI 1640 medium containing 50% serum could induce steatosis of L-02 cells,and oil red O staining showed a lot of lipid droplets.2.L-02/LX-2 co-culture with 10:1 inoculation density could induce the positive expression of LX-2 ?-SMA,and the expression of type ? and type ? collagen increased(P < 0.01).Conclusions:NAFL showed significant collagen deposition around hepatocytes,while inflammatory cell infiltration was mild,the ?-SMA was positively expressed and the fibrotic factor was negatively expressed,suggested that there may be a way of the fatty changed hepatocyte to activate stel ate cel s to promote liver fibrosis.