| ObjectiveIn recent years,the prevalence of non-alcoholic steatohepatitis(NASH)has increased rapidly,and 20% of NASH patients will develop to cirrhosis.Studies have shown that NLRP3 inflammasome are related to inflammation and induce NASH via releasing inflammatory factors.Quzhi Ruangan Fang(QZRGF)is an effective experience recipe from Professor Su Lian,a famous Chinese medicine of Yunnan Province and expert in liver disease.Early clinical and experimental studies have shown that QZRGF could reduce lipid deposition and prevent and treat NASH.However,it is still unclear whether the action mechanism of QZRGF for the prevention and treatment of NASH is related to the inhibition of NLRP3 inflammasome and related factors.The study was designed to observe the effect and potential mechanism of the QZRGF on the prevention and treatment of NASH from the perspective of the NLRP3 inflammasome,in order to provide a theoretical basis for the clinical application of QZRGF,and enrich the scientific connotation of tranditional Chinese medicine(TCM)in the treatment of NASH.Methods 1.According to the random number table,SD male rats were randomly divided into normal group,model group,QZRGF low-dose group,QZRGF medium-dose group,QZRGF highdose group,and the simvastatin group.The NASH rat model was replicated,and drug intervention was given at the same time.The low,medium and high dose of QZRGF were 7.11g/kg/d,14.22g/kg/d and 28.44g/kg/d and the dose of simvastatin was 1.8mg/kg/d,the normal group and the model group were given 2ml of normal saline.Samples were collected and indices were tested at week 8 and 14 respectively.2.The therapeutic effect of QZRGF on NASH was observed.The samples collected at the 8th and 14 th weeks were examined respectively.The pathological morphology of the liver in each group was observed by HE staining and oil red O staining.Rat serum liver function indexes(ALT,AST)were detected by colorimetry.Serum lipids(TC,TG,LDL-C,HDL-C)were detected by enzyme method.Rat liver fat(TC,TG)was detected by GPO-PAP method.3.The effect of QZRGF on the expression of NLRP3 inflammasome and related factors was observed.The samples collected at 14 th week were tested.The expression of NLRP3 mRNA,ASC mRNA and Caspase-1 mRNA in the livers of rats in all groups was detected by realtime fluorescence quantitative PCR(RT-qPCR)method.The expression of NLRP3 and Caspase-1 protein in the liver of all groups of rats was detected by immunohistochemical two-step method.The content of inflammatory factors IL-1β and IL-18 in the liver of all groups of rats was detected by enzyme-linked immunosorbent assay(Elisa).Results 1.QZRGF alleviated the pathological changes in the liver of NASH rats.The results of HE staining found that at 8th week,compared with the control group,the model group had a disordered liver cell arrangement,unclear hepatic cords,a large number of fat vacuoles,or mild cytoplasmic changes,indicating the presence of NAFL model.At the 14 th week,the liver lipid deposition area of the model group increased,lipid vacuoles increased,obvious cytoplasmic loosening,ballooning and a small amount of inflammatory cell infiltration,indicating that the NASH model was successfully replicated.While the liver of rats in all QZRGF group and simvastatin group showed reduced fat vacuoles,cytoplasmic loosening,ballooning and inflammatory cell infiltration,especially in the QZRGF high-dose groups and simvastatin group.The results of Oil Red O also showed that a large number of round orange-red lipid droplet vacuoles appeared in the liver of the model group at 8th and 14 th.But in the all QZRGF groups and the simvastatin group,the number of orange-red lipid drops decreased.2.QZRGF decreased the rat liver index,blood lipid index,liver lipid index and improved liver function.At 8th and 14 th week,compared with the control group,the liver index,blood lipid index(TC,TG,LDL-C),liver fat(TC,TG),liver function(ALT,AST)of the model group were significant Increased(P<0.05).Compared with the model group,these indicators were significantly reduced in the QZRGF medium-dose and high-dose groups,and simvastatin group(P<0.05),but the blood lipid and liver lipid TG of the QZRGF low-dose group showed a downward trend(P>0.05).In addition,HDL-C increased in all QZRGF groups and simvastatin group(P<0.05).3.QZRGF reduced the expression of NLRP3 mRNA,ASC mRNA and Caspase-1 mRNA in the liver of NASH rats.At week 14,compared with the control group,the relative expression levels of NLRP3 mRNA,ASC mRNA,and Caspase-1 mRNA in the liver of the model group were all increased(P<0.05).Compared with the model group,the relative expression level of NLRP3 mRNA in all QZRGF groups and simvastatin group decreased(P<0.01).The relative expression levels of ASCmRNA and Caspase-1 mRNA in QZRGF medium-dose and high-dose groups decreased(P<0.01).The relative expression of ASCmRNA and Caspase-1 mRNA in the QZRGF low-dose and simvastatin group showed a downward trend(P>0.05).4.QZRGF reduced the expression of NLRP3 and Caspase-1 protein in NASH rat liver.At 14 th week,compared with the control group,the cytoplasmic cytoplasmic NLRP3 protein and Caspase-1 protein positive expression in the model group increased significantly,and the IOD / AREA value also increased significantly(P<0.05).Compared with the model group,the positive expression of NLRP3 protein in hepatocytes of all QZRGF groups and simvastatin group was significantly reduced,and the IOD/AREA value was also significantly reduced(P<0.01).The positive expression of Caspase-1 protein in liver cells of QZRGF medium-dose and high-dose groups was significantly reduced,and the IOD/AREA value was also significantly reduced(P<0.05).The positive expression of Caspase-1 protein in QZRGF low-dose group and simvastatin group showed a downward trend(P> 0.05).5.QZRGF reduced the expression of IL-1β and IL-18 in the liver of NASH rats.At 14 th week,compared with the normal group,the relative expression levels of IL-1β and IL-18 in the rat liver of the model group were increased(P<0.01).Compared with the model group,the relative expression of IL-1β in QZRGF medium-dose group,high-dose groups and simvastatin group were reduced(P<0.01),and the relative expression of IL-1β in QZRGF low-dose group showed a downward trend(P>0.05);The relative expression of IL-18 in the QZRGF high-dose group was reduced(P<0.01),while the relative expression of IL-18 in the QZRGF low-dose group and simvastatin group showed a downward trend(P>0.05).Conclusion 1.QZRGF can prevent and cure NASH by reducing liver fatty degeneration and inflammation in rats,lowering liver index,blood lipid and liver fat levels in rats,and improving liver function.2.The mechanism of action of QZRGF in the prevention and treatment of NASH may be related to the down-regulation of the expression of NLRP3 inflammasome and related factors. |
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