Neutrophil Granule Protein Proteinase 3 Mediates The Mechanism Of NASH Liver Fibrosis And The Intervention Study Of Total Glycosides Of Cirsium Japokicum

Background Nonalcoholic fatty liver disease(NAFLD)includes simple fatty liver disease(NAFL)and nonalcoholic steatohepatitis(NASH).Compared with NAFL,patients with NASH have a significantly increased risk of developing cirrhosis and hepatocellular carcinoma.The pathogenesis of NAFLD/NASH has not been fully elucidated.There are currently no approved drugs targeting to NASH.Protease 3(PR3)is a serine protease released mainly by neutrophil estrogenic granules,which has the effect of inducing infiltration of proinflammatory immune cell and increasing inflammation.Previous clinical studies reported that the level of PR3 in peripheral blood in obese NASH patients was significantly increased.Animal experiments showed that the inhibition of PR3 significantly reduced fasting blood glucose and hepatocyte lipid overcollection in mice.At present,whether PR3 plays a pathophysiological function in the progression of NASH and its potential mechanisms have not been clarified.The total glycosides of Cirsium japonicum(CJTG)is the main flavonoid chemical components in Chinese medicine Cirsium japonicum Fisch.ex DC,which mainly contains pectolinarin and linarin.Pectolinarin and linarin.have antioxidant,anti-inflammatory,anti-diabetic and hepato-protective effects.However,there are no relevant studies on whether CJTG have an intervention effect on NASH liver fibrosis.This project focuses on PR3 and researches the effects and mechanism of CJTG on NASH.It has certain scientific significance for discovering potential target molecules and effective drugs(including active ingredients of traditional Chinese medicine)that intervene in NASH.Objectives1.Based on the research strategy of gene function deletion,PR3 gene knockout mice were used to reveal the pathophysiological function and mechanism of PR3 in NASH liver fibrosis.2.To investigate the effect and mechanism of PR3 inhibitor Elafin blocking PR3 intervention in NASH liver fibrosis mice.3.To study the intervention effect of CJTG on the liver of PR3 and in NASH mice.MethodsPart Ⅰ Based on the PR3 gene deletion strategy,the pathophysiological function and mechanism of PR3 in NASH liver fibrosis were studied Under the background of C57BL/6J,10-week-old PR3 knockout(KO)and wild-type(WT)control mice were fed a high-fat diet(CDAHFD),which was made of L-amino acid rodent diets with 60 kcal% fat with low methionine and no added choline,for 10 weeks to establish NASH and liver fibrosis model.Histopathological features of NASH and its liver fibrosis and hepatocyte damage levels were comprehensively analyzed by H&E staining,Masson staining,Sirius red staining,immunohistochemical staining and western blotting of α-SMA,hepatic fibrosis-related factor m RNA level detection,and serum ALT and AST biochemical detection of liver tissue.Part Ⅱ Using adeno-associated virus(AAV),the intervention effect and mechanism of liver overexpression of Elafin in NASH and hepatic fibrosis mice were studied Elafin is an endogenous inhibitor of PR3.In this project,AAV(AAV-Elafin)expressing Elafin and its control AAV(AAV-Vehicle)were constructed with AAV9 serotype,which is high liver affinity.The 8-week-old C57BL/6J male mice were injected with AAV(1.8E11～2.1E11 vg/mice)to induce liver overexpression of Elafin.NASH liver fibrosis is induced using the same method as Part Ⅰ.After 12 weeks of diet induction,the above experimental methods were used to detect NASH histopathological features,liver fibrosis and liver damage.Part Ⅲ Intervention effect of CJTG in NASH liver fibrosis mice10-week-old male mice were randomly divided into standard diet(SC)group,CDAHFD-Vehicle group,and CDAHFD-CJTG group.The CDAHFD-Vehicle group and CDAHFD-CJTG group were induced NASH liver fibrosis by feeding CDAHFD for 10 weeks,and gastric therapy with CJTG suspension or distilled water were given at sixth week(once daily for35 consecutive days).The intervention of CJTG on liver PR3 level and NASH was evaluated by PR3 immunohistochemical staining,H&E staining,small animal nuclear magnetic body component analysis and serum ALT and AST biochemical detection.Results1.Deletion of PR3 gene significantly reduced NASH and liver fibrosis in miceCompared with the SC group,the histopathological characteristics and collagen deposition of NASH in the liver of CDAHFD-fed PR3 KO mice were significantly reduced.The α-SMA expression levels and the m RNA levels of liver fibrosis-related factors α-SMA,Col1a1,TGF-β1,Timp1 and Lox in the liver of PR3 KO mice as the same as the serum AST and ALT levels were significantly reduced.These results suggest that PR3 gene deletion can significantly reduce NASH and liver fibrosis in mice.2.Liver overexpression of Elafin significantly improves NASH and hepatic fibrosis in micePCR results showed that AAV-Elafin successfully induced Elafin overexpression in the liver of mice.The results of NASH and liver fiber phenotype analysis showed that the ballooning changes of hepatocytes,inflammatory infiltration and collagen deposition in liver of AAV-Elafin mice were significantly reduced.The expression levels of liver fibrosisrelated proteins α-SMA and the m RNA levels of related factors Col1a1,TGF-β1,Timp1 and Lox were significantly reduced,and the levels of serum liver function detection index AST and ALT were significantly reduced.The above results suggest that hepatic overexpression of Elafin significantly improves NASH and liver fibrosis in mice.3.CJTG significantly reduced the level of PR3 in NASH mice and also improved NASH and fat content in miceThe results of qualitative analysis by HPLC suggest that CJTG contains pectolinarin and linarin.Compared with NASH mice administered to the control,the levels of PR3,inflammatory infiltration in the livers of NASH mice,which treated with CJTG,were significantly reduced.The results of serum AST and ALT showed that the serum ALT level of mice treated with NASH was 1.4 times higher than that of mice treated with CJTG.The results of small animal NMR body component analysis showed a significant reduction in fat content in mice treated with CJTG.Conclusions(1)PR3 gene deletion significantly improved the liver histopathology of NASH mice,and alleviated NASH liver fibrosis and liver injury.(2)Liver overexpression of Elafin improved liver histopathology in NASH mice,and alleviated NASH liver fibrosis and liver injury.(3)CJTG inhibited the expression of PR3 in the liver,improved the inflammation of liver lobules in NASH mice,and significantly reduced NASH,liver damage and fat content.In summary,this topic reveals that PR3 plays an important pathophysiological function in promoting NASH and liver fibrosis,and liver overexpression of AAV-induced PR3 endogenous inhibitor Elafin can significantly improve NASH and liver fibrosis in mice.The Chinese herbal extract CJTG significantly reduced the level of PR3 and the fat content and improved NASH in mice.Therefore,PR3 can be used as a potential target in the development of drugs,which can cure NASH and liver fibrosis.The results of this project support further research on the role of CJTG in the intervention of NASH.