| ObjectiveIn this study,we intended to establish a model of non-alcoholic fatty liver cells induced by free fatty acids,and to investigate the therapeutic effect and therapeutic mechanism of sini powder,as well as the mechanism of glucocorticoid aggravating non-alcoholic fatty liver.Methods1.1 mM free fatty acid was used to induce LO2 cell steatosis,which was divided into four groups:blank control group,model group,sinisan decoction group and simvastatin group.FFA-induced nonalcoholic fatty liver model was established to determine the contents of intracellular triglyceride and total cholesterol,and to evaluate the pharmacodynamic effect of sinisan.The levels of IL-6,IL-18,TNF-and IL-1? in the supernatant were determined by ELISA.mRNA and protein expression levels of NLRP3,ASC and caspase-1 were detected by RT-PCR and Weston Blot.Furthermore,the therapeutic mechanism of sinisan in the treatment of nonalcoholic fatty liver disease through NLRP3/IL-1? pathway was explored.2.1mM free fatty acid was used to induce LO2 cell steatosis,which was divided into four groups:blank control group,FFA model group,GC+FFA composite model group,and sinisan decoction group.FFA-induced nonalcoholic fatty liver model was established to determine the contents of triglyceride and total cholesterol in each group,and to observe the accumulation of lipid in liver cells aggravated by glucocorticoids.The levels of IL-6,IL-18,TNF-? and IL-1? in the supernatant were determined by ELISA.mRNA and protein expression levels of NLRP3,ASC and caspase-1 were detected by RT-PCR and Weston Blot.To further verify the mechanism of glucocorticoids promoting nonalcoholic fatty liver disease through NLRP3/IL-1? pathway.Result1.Effect of sinitasan on FFA-induced LO2 cell fat change model:compared with the normal group,the contents of TC and TG in the model group increased significantly,and the contents of TC and TG in the simvastatin group,the high-dose sinitasan group and the medium-dose sinitasan group all decreased significantly compared with the model group,with statistically significant differences(P<0.05).However,there was no significant difference between the model group and the low-dose sinisan group.2.Establishment of LO2 cell fat model induced by GC+FFA and the effect of sini powder:the contents of IL-6,IL-18,TNF-?,and IL-1? in supernatant of each group were determined by ELISA.The results showed that compared with the blank group,the contents of IL-6,IL-18,TNF-?,and IL-1? in the supernatant of the two model groups were significantly increased.Compared with the model group,the contents of IL-6,IL-18,TNF-?,and IL-1? in the supernatant of GC+FFA were significantly increased.Compared with the FFA model group,the contents of IL-6,IL-18,TNF-?,and IL-1? in the sinuous powder group were significantly reduced.RT-PCR was used to detect the expression of NLRP3,caspase-1 and ASC mRNA in LO2 cells.The results showed that,compared with the control group,the expression levels of NLRP3,caspase-1 and ASC in the FFA model group and GC+FFA composite model group were significantly increased.Compared with the FFA model group,the expression levels of NLRP3,caspase-1 and ASC in the GC+FFA composite model group were significantly increased.The expression of NLRP3,caspase-1 and ASC mRNA were significantly decreased in the sinisan group compared with the FFA model group.The expressions of NLRP3,caspase-1 and ASC in LO2 cells were detected by Western Blot.The results showed that the protein expression levels of NLRP3,caspase-1 and ASC were significantly increased in the FFA model group and GC+FFA composite model group compared with the control group.The protein expression levels of NLRP3,caspase-1 and ASC in the GC+FFA composite model group were significantly increased compared with the FFA composite model group.Compared with the FFA model group,the protein expressions of NLRP3,caspase-1 and ASC mRNA in the sinisan group were significantly reduced,with statistically significant differences(P<0.01).3.Mechanism of intervention of sini powder on NLRP3/il-1 in LO2 cell fat model:the contents of IL-6,IL-18,TNF-?,and IL-1? in supernatant were detected by ELISA.Compared with the blank group,the contents of IL-6,IL-18,TNF-?,and IL-1? in supernatant of model group were significantly increased.Compared with the model group,the contents of IL-6,IL-18,TNF-?,and IL-1? in the sinisan group were significantly reduced,with statistically significant differences(P<0.01).Compared with simvastatin group,there was no significant difference in the expression level of each index in the supernatant between sinisan group and simvastatin group.The expressions of NLRP3,caspase-1 and ASC in LO2 cells were detected by Western Blot.The results showed that the expression levels of NLRP3,caspase-1 and ASC in the model group were significantly increased compared with the control group,and the differences were statistically significant(P<0.01).There was no significant difference between sinisan decoction group and simvastatin positive group.Protein expressions of NLRP3,caspase-1 and ASC were significantly decreased in both groups compared with the model group.RT-PCR was used to detect the expression of NLRP3,caspase-1 and ASC mRNA in LO2 cells.The results showed that compared with the control group,the expression levels of NLRP3,caspase-1 and ASC in the model group were significantly increased,and the differences were statistically significant(P<0.01).The expression levels of NLRP3,caspase-1 and ASC mRNA in the sinisan positive group were significantly lower than that in the model group(P<0.05).Conclusion1.Sinisan can reduce the secretion of triglyceride and total cholesterol in steatosis cells2.By upregulating the gene expression of NLRP3/IL-1?pathway,glucocorticoids can increase the inflammatory response of cells,strengthen the insulin resistance of cells,and thus aggravate the inflammation of non-alcoholic fatty liver.3.Sinisan decoction may reduce the secretion of IL-6,IL-18,TNF-?and other inflammatory factors by down-regulating the gene expression of NLRP3/IL-1?pathway,thereby reducing the inflammatory response of cells and improving the insulin resistance of cells,thus achieving the therapeutic effect of non-alcoholic fatty liver disease. |
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