| 【Research Purpose】In this study,we established a rat model of nonalcoholic steatohepatitis(NASH)to observe the therapeutic effect of Ginkgolide B(GB)on NASH rats and to explore whether the mechanism of GB is related to the regulation of CB2/NF-κB signal pathway to reduce inflammation in vivo.【Research Methods】1.The therapeutic effect of GB on NASH rats.The experimental animals were SD rats,and 64 rats of SPF grade.Then the rats were divided into normal group(n=12)and model group(n=52).NASH model was established by feeding rats with high-fat and high-sugar diet.After 12 weeks of feeding,two rats in each group were selected to verify the NASH model.After successful modeling,the rats were randomly divided into low,middle and high dose groups of GB(GB-D,GB-Z,GB-G)and fenofibrate(FNBT)group,with 10 rats in each group.Each dose of GB group was treated by intragastric administration of 0.5,1.0 and 2.0mg/kg/d GB suspension respectively.Fenofibrate group was given 18mg/kg/d fenofibrate suspension by intragastric administration.The rats in the normal group and the model group were given normal saline intragastric administration respectively.After 8 weeks of treatment,the rats were anesthetized and the blood and liver of the rats were collected.The changes of liver index were observed by measuring the body weight and liver weight of rats.Hepatic steatosis,inflammation and fibrosis were observed by HE staining,oil red O staining and Masson staining.Biochemical kit was used to detect the changes of liver function indexes(AST,ALT)in rat serum.The lipid metabolism indexes such as total cholesterol(TC,TG,HDLC,LDL-C)in serum and liver were detected.To observe the therapeutic effect of GB on NASH.2.GB regulates CB2/NFκB signal pathway to reduce inflammation and slow down the progression of NASH to liver fibrosis.ELISA method was used to detect the inflammatory factors such as IL-1β,IL-6 and TNF-α in serum and liver.HA,LN,PC Ⅲ and Ⅳ-C in serum of rats in each group were detected by ELISA method.WB and q PCR were used to detect the expression of CB2,IKKβ,NF-κB protein and m RNA in liver tissue of rats in each group.To study the anti-inflammatory mechanism of GB in NASH rats.【Research Result】1.The therapeutic effect of GB on liver of NASH rats.The NASH rat model was successfully established by high-fat and high-sugar diet for12 weeks.The results of liver index showed that the body weight and liver index in the model group were significantly higher than those in the normal group(P<0.05),while those in the GB treatment group and fenofibrate group were significantly lower than those in the model group(P<0.05).The results of pathological examination showed that compared with the normal group,the HE staining images of the liver of the model group showed disordered arrangement of hepatic cords,fat deformation of hepatocytes,balloon degeneration,loose cytoplasm,inflammatory cell infiltration,connective tissue hyperplasia and so on.Compared with the model group,the treatment groups of GB reduced the fat vacuoles and inflammatory infiltration in liver tissue in a dose-dependent manner.Compared with the normal group,a large number of orange lipid droplets could be seen by oil red O staining in the model group.Compared with the model group,the lipid droplets in GB treatment group and fenofibrate group decreased significantly,and the therapeutic effect became more obvious with the increase of GB dose.The detection of serum liver function showed that the serum liver function indexes AST and ALT in the model group were significantly higher than those in the normal group(P<0.05).Compared with the model group,the levels of AST and ALT in GB treatment group and fenofibrate group decreased significantly(P<0.05).The results of serum and liver lipid metabolism indexes showed that the levels of TC,TG and LDL-C in liver and serum in model group were significantly higher than those in normal group(P<0.05),while the level of HDL-C in model group was significantly lower than that in normal group(P<0.05).Compared with the model group,the levels of TC,TG and LDL-C in serum and liver in all GB treatment groups and fenofibrate group decreased(P<0.05),while the levels of HDL-C in GB-G group and fenofibrate group increased(P<0.05).Although the level of HDL-C in GB-D group increased to some extent,there was no significant difference compared with model group(P>0.05).2.GB regulates GB/NF-κB signal pathway to reduce hepatitis and slow down the progression of NASH to liver fibrosis.2.1 GB attenuates liver inflammation and slows down the progress of NASH to liver fibrosisThe results of inflammatory factors detected by ELISA showed that the levels of inflammatory factors IL-1β,IL-6 and TNF-α in liver and serum in model group were significantly higher than those in normal group(P<0.05).Compared with the model group,the levels of IL-1β,IL-6 and TNF-α in serum and liver of rats in GB treatment group and fenofibrate group decreased significantly(P<0.05).The results of Masson staining showed that compared with the model group,the fibrous connective tissue of each treatment group was improved in varying degrees,but there were still a large number of fat vacuoles of different sizes,and a small amount of collagen fibers in fenofibrate group and GB-G group.The results of ELISA showed that the serum levels of HA,LN,PC Ⅲ and IV-C in the model group were significantly higher than those in the normal group(P<0.05).Compared with the model group,each dose of GB treatment group and fenofibrate group could significantly reduce the levels of serum HA,LN,PC Ⅲ and Ⅳ-C in rats(P<0.05).It is suggested that GB can reduce the inflammatory reaction in NASH rats and slow down the progression of NASH to liver fibrosis.2.2 GB regulates CB2/NF-κB signal pathway.The results of WB and q PCR showed that the expression of CB2 protein and m RNA in the liver of the model group was lower than that of the normal group(P<0.05).Compared with the model group,the expression levels of CB2 protein and m RNA in the liver of GB treatment groups and fenofibrate group increased(P<0.05).Compared with the normal group,the expression levels of IKKβ and NF-κB protein and m RNA in the liver of the model group were significantly increased.(P<0.05)Compared with the model group,the expression levels of IKKβ,NF-κB p65 protein and m RNA in each GB treatment group and fenofibrate group were significantly decreased(P<0.05).It is suggested that GB can regulate the expression of CB2/NF-κB signal pathway and reduce liver inflammation.【Conclusion】1.GB can improve the liver fat degeneration and inflammatory reaction of NASH rats induced by high-fat and high-sugar diet,protect liver function and slow down the development of NASH to liver fibrosis,and has therapeutic effect on NASH rats.2.The mechanism of GB treatment on NASH rats may be to protect liver function and reduce liver inflammation by regulating CB2/NF-κB signal pathway in the liver of NASH rats.GB up-regulated the expression of CB2 protein and m RNA,down-regulated the expression of IKKβ and NF-κB protein and m RNA,decreased the levels of IL-1β,IL-6and TNF-α,and then decreased the levels of HA,LN,PC Ⅲ and IV-C,and slowed down the progression of NASH to liver fibrosis. |
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