| ObjectiveBy observing the protective effect of the method of shuganjianpi, huatanhuoxue effecting damaged cell model of nonalcoholic fatty liver disease (NAFLD) after builting NAFLD cell model successfully,we researched the effect of the method effecting the mitochondrial membrane liquidity, membrane potential level,energy metabolism, lipid peroxidation of liver cell on the NAFLD model cells,and discuss the protective effect of the method effecting the damaged liver cell.MethodsWe divided liver cells of cultivated immature rats into normal group and model group.Then we added DMEM-F12 culture solution containing 20% FBS to the normal group cells,and added 15μg/ml palmitic acid action to model group for 5 days.we counted the survival rate of liver cells with trypan blue staining,and observed the situation of cell attachment and morphological changes by inverted microscope.After establishing cell model of NAFLD successfully,we divided the model cells into three groups,including of cell model group of NAFLD,silymarin treatment group and Chinese medicine treatment group,and then intervended by the medicated serum of normal saline, silymarin and the shuganjianpi,huatanhuoxue herbs respectively.We detected liver cell activity,triglycerides(TG) level,Na+-K+-ATPase,Ca2+-ATPase,Cyto-Ox activity,SOD,MDA,NO,GSH activity in hepatocyte mitochondria with the methed of chemical colorimetry.we examined liver cell apoptosis rate by flow cytomety.we detected apoptosis related gene Bcl-2,Bax and the expression of Caspase-3 of liver cell mitochondria with Western Blot method.we inspected the mitochondrial membrane liquidity,and membrane potential level of liver cell mitochondria with fluorescence spectrophotometer.Results1. Effects of the medicated serum of Chinese Herbs on Survival rate,TG of NAFLD model cell.Compared with the normal group,cell survival of the model group was reduced significantly (P<0.05).Cell survival rates of traditional Chinese medicine group and silymarin group increased compared with the model group's significantly (P<0.01),and achieved the normal group level (P>0.05). Compared with the normal group,TG content of the model group increased significantly (P<0.01).TG levels of traditional Chinese medicine group and silymarin group were also less than the model group significantly (P<0.01). TG level of Traditional Chinese medicine group was less than silymarin group's (P<0.05).2. Effects of the medicated serum of Chinese Herbs on apoptosis rate,MPPT,Bcl-2,Bax,Caspase-3 of NAFLD model cell.Compared with the normal group, apoptosis rate and ODA540 quantity of the model group were increased significantly (P<0.01). And apoptosis rate and ODA54o value of the Chinese medicine group and silymarin group were less than the model group statistically(P<0.01 or P<0.05).Apoptosis rate of the traditional Chinese medicine group was less than silymarin group's (P<0.01), and ODA54o value of the traditional Chinese medicine group group reduced compared with the silymarin group(P<0.05). Compared with the normal group,Bcl-2 protein expression level of the model group decreased obviously(P<0.01).And compared with the model group,Bcl-2 protein expression level of the traditional Chinese medicine group and the silymarin group also rised(P<0.01).Bcl-2 protein expression level of the traditional Chinese medicine group was higher than the silymarin group's (P<0.05).Bax quantity and Caspase -3 protein expression of the model group was higher than the nomal group's(P<0.01).Compared with the model group, Bax quantity and Caspase-3 protein expression of the traditional Chinese medicine group and the silymarin group were also increased significantly (P<0.01).And the traditional Chinese medicine group's was less than the model group's (P<0.01).3. Effects of the medicated serum of Chinese Herbs on the mitochondrial membrane liquidity,and membrane potential level of liver cell mitochondria of the NAFLD model.Compared with the normal group,the mitochondrial membrane liquidity, and membrane potential level of NAFLD model cell were reduced signify-cantly(P<0.01).The mitochondrial membrane liquidity, and membrane poten-tial level of the traditional Chinese medicine group and the silymarin group were higher than the model group's (P<0.01 or P<0.05).The Chinese tradition-al medicine group's the mitochondrial membrane liquidity was increased com-pareing with the silymarin group(P<0.05).But the membrane potential level of the Chinese medicine group had no difference comparing with the silymarin group's(P>0.05).4.Effects of the medicated serum of Chinese Herbs on energy metabolism of liver cell mitochondria of the NAFLD model.Compared with the normal group ratio,the mitochondrial membrane liquidity,and membrane potential level of the model group were reduced signi-ficantly(P<0.01).The content of Na+-K+-ATPase,Ca2+-ATPase and Cyto-Ox activity of the traditional Chinese medicine group and the silymarin group were higher than the model group's(P<0.05).And the mitochondrial membrane liquidity of the traditional Chinese medicine group was higher than the sily-marin group's (P<0.05).But membrane potential level of the Chinese medicine group had no difference from the silymarin group's(P> 0.05).5.Effects of the medicated serum of Chinese Herbs on lipid peroxidation of liver cell mitochondria of the NAFLD model.Compared with the normal group,MDA content of liver cell mitochon-dria of the model group was increased significantly(P<0.01).MDA level of silymarin and Chinese medicine group were lower than the model group's(P< 0.01).But there was no significant difference between the traditional Chinese medicine group and the silymarin group (P>0.05).SOD, NO and GSH content of the model group were less than the normal group's significantly(P<0.01). Traditional Chinese medicine group can increase SOD, NO and GSH content compared with model group(P<0.01).NO and GSH levels of the traditional Chinese medicine group were higher than the silymarin group's (P<0.05).GSH level of the silymarin group had no difference from the model group (P>0.05).Conclusion1. Ganyupixu,tanyujiaozu is the basic pathogenesis of NAFLD since the beginning.2. The shuganjianpi,huatanhuoxue-herbs can reduce the TG level of the NAFLD in vitro model,increase the activity of the NAFLD model cells,and reduce the damage effectively.3. The shuganjianpi,huatanhuoxue herbs can reduce apoptosis rate of the NAFLD model cells by regulating the level of Bcl-2,Bax and Caspase-3 protein expression effectively.4. The shuganjianpi,huatanhuoxue herbs can improve cell level of energy metabolization obstacle by enhancing energy supplies of the NAFLD model cells,improve the level of the mitochondria lipid peroxidation by improving liver cell mitochondrial antioxidant levels,regulate the level of mitochondrial membrane liquidit and membrane potential of the NAFLD model cells effect-tively,which may be one of the mechanisms of Chinese Herbs effecting the NAFLD model cells. |
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