Study On The Effect And Mechanism Of Cordyceps Mycelium Active Component C02 On Nonalcoholic Steatohepatitis

Objective:1.Induce normal mice to establish a non-alcoholic steatohepatitis（NASH）animal model through a methionine-choline deficiency（MCD）diet and observe the best modelling cycle.Using this model to evaluate the effect of the active component of Cordyceps mycelium C02 on NASH Intervention;2.Through the Western diet（WD）combined with CCl₄ to induce normal mice to establish NASH model and observe the best modelling cycle.Using this model to evaluate the intervention effect of the active component C02 of Cordyceps mycelium on NASH,and to clarify its possible mechanism.Method:1.The mice model of Nash was induced by MCD diet,and set the dynamic observation groups for 4,6,and 8 weeks respectively.According to the serum liver function（ALT,AST activity）,liver tissue triglyceride（TG）content,liver tissue pathology（HE,Red O,Sirius red staining）,SAF score and other tests to determine the best model cycle.The NASH model of mice is established by the above optimal modeling cycle.After the treatment of C02 intervention,serum and liver tissue would be collected to evaluate the effect of C02 on the mice model of NASH induced by MCD diet.2.WD/CCl₄ was used to establish NASH model,and set dynamic observation groups for 8,12,and 16 weeks respectively.The optimal model period was determined by the serum liver function（ALT,AST activity）,liver tissue triglyceride（TG）content,liver tissue pathology（HE,Red O,Sirius red staining）,SAF score and hydroxyproline（Hyp）content.The NASH model of mice with the optimal model period above would be used.After the treatment of C02 intervention,serum and liver tissue would be collected.The above indexes and the gene,protein and immunohistochemical expression levels of liver lipid metabolism,fibrosis,and antioxidant related indexes were detected to evaluate the efficacy of C02 on WD/CCl₄ induced NASH model in mice,and to preliminarily explore its possible mechanism.Result:1.The MCD diet model can simulate the pathological characteristics of human NASH（such as elevated serum ALT and AST levels,liver tissue TG,steatosis,ballooning degeneration of hepatocytes,inflammatory cell infiltration and perisinusoidal fibrosis）.According to the dynamic observation of pathological changes in mice,it was found that the body weight and wet liver weight of mice in each period were significantly lower than that of the MCS group at the same period（P<0.001）,the serum ALT and AST activities and the content of TG in liver tissue were significantly increased（P<0.05,P<0.01,P<0.001）;In the MCD 4-week group,hepatocytes swelled and round,fatty vacuoles were formed,inflammatory cell infiltration and mild perisinus fibrosis were visible;In the MCD 6-week group,fatty vacuoles,ballooning degeneration of hepatocytes,and focal infiltration of inflammatory cells coexisted in the liver lobules,and short fibrous septa were formed;In the MCD 8-week group,the degree of liver fibrosis increased compared with that of 6 weeks,but the balloon like changes in the liver lobules reduced,and the weight of the mice was reduced by more than 50%compared with before modeling.Therefore,the 6-week diet model was the best,which is used to further verify the effect of C02 in prevention and treatment of NASH.2.C02 can significantly improve NASH in mice induced by MCD diet.Compared with the MCD group,20 mg/kg C02 and 40 mg/kg C02 can significantly reduce serum ALT and AST activities（P<0.05,P<0.01）,and 40 mg/kg C02 can significantly reduce liver tissue TG content（P<0.001）.The results of pathological staining and SAF scores of liver tissues showed that the C02 medication groups could improve liver steatosis,ballooning,and inflammation in mice induced by MCD diet,and reduce the degree of liver fibrosis.The SAF score decreased by 2.5 and 2.6 points respectively.3.WD/CCl₄ can induce liver steatosis,inflammation and fibrosis in the mice,which can simulate the pathological characteristics of human NASH.According to the dynamic observation of pathological changes in mice,it was found that the activities of serum ALT and the content of TG in liver tissue of mice were significantly higher than those in the ND/Oil group in the same period（P<0.05,P<0.01,P<0.001）.The results of pathological staining of liver tissue and SAF score showed that there were obvious fatty changes in the liver lobules of mice in the WD/CCl₄-8w group,with severe vacuolation,and focal hepatocyte necrosis in the lobules,accompanied by inflammatory cell infiltration,it can be seen that the fibrous interval is formed,most of the fibrosis stages are in the F2 stage,and the SAF score is 8.4;In the WD/CCl₄-12w group,fatty degeneration,ballooning of hepatocytes,and inflammatory cell infiltration coexisted in the liver lobules of mice,accompanied by liver fibrosis,and more than half of the fibrosis stages reached stage F3,with a SAF score of 9.3;In the WD/CCl₄-16w group,a large number of inflammatory cell infiltration and hepatic tissue necrosis appeared in the liver tissue of mice,fat vacuoles and ballooning of hepatocytes were reduced compared with the previous ones,but the degree of liver fibrosis was aggravated,and the fibrosis stage was F3-F4 stage,SAF score 9.6 points.According to the degree and time of the model,12 weeks is the best model-building cycle,which can be used to further verify the effect of C02 in preventing and treating NASH,and to explore its related mechanism.4.C02 can significantly improve WD/CCl₄ induced NASH in mice.Compared with the WD/CCl₄ group,the C02 medication groups can significantly reduce the serum ALT and AST levels in mice（P<0.05,P<0.01）,20 mg/kg C02 and 40 mg/kg C02 can significantly reduce the liver tissue TG content（P<0.05）;The results of pathological staining of liver tissue and SAF score in mice showed that each C02 medication group can improve liver steatosis,hepatocyte ballooning,liver inflammation and fibrosis,and compared with the WD/CCl₄ group,the SAF score decreased by 1.9,2.4,and 2.9 points;C02 can reduce the expression of liver tissue fibrosis-related indicators.The results of immunohistochemistry,RT-PCR and Western-blot show that C02 significantly reduce the expression levels of Col-I andα-SMA（P<0.05,P<0.01,P<0.001）;C02 can improve the oxidation state of liver tissue in NASH mice,20 mg/kg C02 and 40 mg/kg C02 can significantly reduce the NO content in liver tissue（P<0.01）,and 40 mg/kg C02 can significantly reduce the MDA content in liver tissue（P<0.01）,and C02 can significantly increase liver tissue SOD and GSH-Px activity（P<0.05,P<0.01,P<0.001）;C02 can inhibit the expression of inflammatory factors in liver tissue,RT-PCR results show that each administration group of C02 can significantly reduce the expression levels of F4/80and TNF-αm RNA（P<0.01,P<0.001）,20 mg/kg C02 and 40 mg/kg C02 can significantly reduce the expression level of TGF-βand CCL2 m RNA（P<0.05,P<0.01,P<0.001）.Western-Blot results show that each administration group of C02 can significantly reduce the expression level of TGF-βprotein in the liver tissue of mice（P<0.01）.The immunohistochemical staining results of liver tissue show that C02 can reduce the expression of F4/80 in liver tissue of mice;C02 can regulate the expression of liver tissue lipid metabolism-related factors,RT-PCR results show that the expression levels of SCD1,ACOX1,and LXRαm RNA in the liver of mice in each CO2 administration group are significantly reduced（P<0.01,P<0.001）,20 mg/kg C02 and 40 mg/kg C02 can significantly reduce the expression level of PLIN2 m RNA（P<0.01,P<0.001）.Western-Blot results show that 40 mg/kg C02 can significantly reduce the protein expression of SREBP1c（P<0.01）.5.Through the detection of liver tissue samples,it is found that C02 can significantly inhibit the expression of keap1,increase the expression of Nrf2 and its downstream antioxidant genes HO-1,NQO1,GSTs,and increase the expression of PPARαand PPARγin liver.RT-PCR results show that 20 mg/kg C02 and 40 mg/kg C02can significantly reduce the expression level of keap1 m RNA（P<0.01,P<0.001）,and 40mg/kg C02 can significantly increase the expression level of Nrf2,HO-1,and GSTs m RNA（P<0.05,P<0.01）.Western-Blot results show that 40 mg/kg C02 can significantly increase the protein expression levels of Nrf2,HO-1,and NQO1;RT-PCR and Western-Blot results show that 40 mg/kg C02 can significantly increase the expression levels of PPARγ,PPARαm RNA and protein（P<0.05,P<0.01,P<0.001）.Conclusion:1.Both MCD diet model and WD/CCl₄ compound model can induce the NASH pathological characteristics in mice,and C02 can effectively improve the degree of liver steatosis,inflammation and fibrosis induced by these two models.2.C02 has significant anti-oxidant,lipid metabolism,anti-inflammatory and anti-fibrosis,and its mechanism may be related to the regulation of keap1/Nrf2/HO-1signaling pathway.