Study On The Mechanism Of Shenge Decotion In Treating NASH By Reducing Hepatic Oxidative Stress Response And Regulating Lipid Matabolism

Purpose:To study the related mechanisms of Shenge Decotion in the treatment of NASH model mice with high-fat diet by reducing liver oxidative stress,inflammatory response and regulating lipid metabolism.Methods:1.Grouping: 76 male C57BL/6J mice were adapted for feeding for one week.,then they were divided into Normal group,Model group,Shenge Decotion low-dose group,Shenge Decotion middle-dose group,Shenge Decotion high-dose group,Yishanfu group,and Fenofibrate group by body weight.Both Normal and the Model group have 13 mice,and and 10 in the remaining medication groups.2.Modeling and identification: The Normal group was given a normal diet,and the other groups were given a high-fat diet.At 12 weeks,3 mice of both Normal group and Model group were executed for model identification by gross observation,blood lipids,liver lipid content,serum transaminase detection and histological staining,and the follow-up diet as before.3.Administration method: After 12 weeks,the Normal group was treated with distilled water,and the mice in the low-dose,middle-dose and high-dose groups of Shenge Decotion were gavaged with Shenge Decotion in 7.875g/kg/d,15.75g/kg/d(human equivalent dose),31.5g/kg/d,respectively.Yishanfu and Fenofibrate were intragastrically administered according to human equivalent dose(0.2052g/kg/d,0.03g/kg/d).The gastric volume of mice was adjusted in proportion of 0.1ml/10 g weight,once a day,all mice were sacrificed after 4 weeks of drug treatment.4.Detection methods for each index(1)The effect of Shenge Decotion on the weight,liver index,liver lipid deposition and metabolism in NASH model mice:Weigh the body,liver and spleen of each mouse,then calculate the organ index;The lipid deposition of liver tissue was observed by Oil red O staining;The levels of TG,TC,LDL-c,HDL-c,Glu,UA,Cr in serum and TG?TC contents in liver homogenate supernatant were detected by biochemical method.The expression levels of fatty acid oxidation genes CPT1 and PPAR-? were detected by Real-Time PCR.(2)Anti-inflammatory and hepatoprotective effects of Shenge Decotion on NASH model mice:The expression levels of serum ALT and AST were detected by biochemical method,serum TNF-? levels were detected by ELISA,and the expression levels of liver tissue TNF-?,CD68,F4/80 were detected by Real-Time PCR.Observe the inflammatory infiltration by HE staining of liver tissue;(3)Inhibition effect of Shenge Decotion on liver oxidative stress in NASH model mice:Biochemical method for detecting MDA content in liver homogenate supernatant,serum Adiponectin level were detected by ELISA,the expression levels of Adipo R1,Adipo R2,SIRT1,PGC1? and NRF2 m RNA were detected by Real-Time PCR.The expression levels of related proteins in adiponectin signaling pathways such as Adiopo R1,p-AMPK/AMPK,SIRT1,SIRT3,PGC1?,NRF2 were detected by Western Blot.Results:1.NASH model identification:Compared with the normal group,the body weight and liver weight of the model group increased significantly(P<0.01).Serum ALT,AST and liver tissue TG levels increased significantly(P<0.05).HE staining of liver tissue showed hepatic lobular structure disorder,mixed steatosis(predominately small vesicles and a little large vesicles),inflammatory cell infiltration in the Hepatic lobule,mildly balloon-like changes,the NAS score is 4.67 ± 1.53.Oil red staining showed a large number of orange-red bulk lipid droplets.2.The efficacy and mechanism of Shenge Decotion in the treatment of NASH mouse model:(1)Shenge Decotion can reduce the body,liver weight and liver lipid deposition,regulate metabolism of NASH model mice:Compared with the normal group,the mice in the model group had increased liver volume,blunt edge,yellowish color,texture like muddy and greasy,and a large amountof fat deposition in the viscera;body weight and liver index increased significantly(P<0.05);Oil red staining showed a large number of lipid droplets;serum TG,TC,LDL-c,FBG,Cr,liver tissue TG,TC have increased to different degrees(P<0.05);The expression levels of liver PPAR-? and CPT1 m RNA was lower significantly(P < 0.01).Compared with the model group,the liver color in each drug group was reddish and oily,the body weight of the mice in drug groups was significantly reduced(P<0.0001);Except for Shenge Decotion low-dose group and the fenofibine group,the liver indexes of other groups were significantly decreased(P<0.01);Oil red O staining of liver tissue showed that the number of lipid droplets decreased in each drug group;Except for Yishanfu group,each group could significantly reduce serum TG and liver TG level(P<0.05);Except for the low and middle-dose groups of Shenge Decotion,the other groups could significantly reduce serum TC levels(P<0.001);Except for the low-dose group of Shenge Decotion,the serum LDL-c levels in other drug group were decreased significantly(P<0.05).The m RNA expression levels of fatty acid oxidation genes PPAR-? and CPT1 were significantly up-regulated except for the low-dose group of Shenge Decotion(P<0.01).(2)Shenge Decotion can alleviate inflammatory damage in NASH model mice:Compared with the normal group,hepatic HE staining of the model group showed hepatic cell steatosis,inflammatory cell infiltration in the lobule,hepatocyte swelling and looseness in the whole field;NAS score was 5.3±1.15;serum ALT,AST,TNF-?levels And the expression levels of TNF-?,CD68 and F4/80 m RNA in liver tissue increased significantly(P<0.01).Compared with the model group,each drug group can reduce the degree of hepatic steatosis and inflammatory cell infiltration in the lobular area,and reduce the NAS score.Except for the low-dose group of Shenge Decotion,other groups can significantly reduce the ALT level.(P<0.0001);Except for Yishanfu group,each group can significantly reduce serum AST level(P<0.05);Each group can significantly reduce serum TNF-? level and hepatic m RNA expression levels of CD68 and F4/80,which are the macrophage surface markers(P<0.01).Except for the low-dose group of Shenge Decotion,the levels of TNF-? m RNA in the liver tissues of the mice in each group were significantly decreased(P<0.05).(3)Shenge Decotion can alleviate liver oxidative stress in NASH model mice:Compared with the normal group,MDA of liver oxidative stress products in the model group was significantly increased(P<0.0001);Serum adiponectin and liver adiponectin receptor levels were significantly decreased(P<0.01),The expression levels of regulating oxidative stress reaction-related genes such as p-AMPK,SIRT1,SIRT3,PGC1?,and NRF2 were significantly down-regulated(P<0.01);Compared with the model group,each drug group significantly reduced the MDA level of liver tissue(P<0.0001);Except for the low-dose group of Shenge Decotion,other group could significantly up-regulate serum adiponectin,Adipo R1,Adiopo R2,SIRT1.PGC1?,SIRT3,and NRF2 expression levels(P<0.01).Conclusions:1.Shenge Decotion 's weight loss and lipid-lowering effect on NASH model mice are related to increasing the expression levels of fatty acid oxidation genes PPAR-? and CPT1;2.The anti-inflammatory and hepatoprotective effects of Shenge Decotion on NASH model mice are related to reducing the number of KC cells in liver tissue and decreasing the expression level of TNF-?.3.Shenge Decotion inhibited the liver oxidative stress response in NASH model mice is related to activating APN signaling pathway.