Mechanism Of Mulberry Alkaloid On Liver Fibrosis Induced By CCl₄ Combined With A High-fat Diet

Liver fibrosis is a wound-healing process in liver with chronic injury and is characterized by the activation of liver stellate cells?HSC?and the excess production and deposition of extracellular matrix?ECM?components.It is the central link in the development of various chronic liver diseases to cirrhosis.There is substantial evidence that liver fibrosis is reversible.The present study will focus on natural products which display prevention and improvement effects on liver fibrosis.Mulberry alkaloid is the active ingredients of mulberry leaves.It was firstly used to ameliorate the experimental liver fibrosis mouse model and its mechanism of mulberry alkaloid on liver fibrosis in mice was revealed.Mice was injected intraperitoneally with 10%CCl₄ olive oil solution once every two days combined with a high-fat diet for 8 weeks to induce liver fibrosis.The mice in low,medium and high-dose groups were administered intragastrically with mulberry alkaloid at 50,100 or 200 mg/kg,and positive group were positive drug?100 mg/kg silibinin?after the liver fibrosis mouse model established.Accordingly,mice in the control and model group received an equal volume of distilled water.During intragastric administration,the study observed mice's general conditions and recorded body mass.Hematoxylin-eosin staining and Masson's trichrome staining were used for histological examination and the related indicators were measured.The experimental results are as follows:?1?Simultaneous administration of CCl₄ combined with a high-fat diet to cause mice to suffer mental deterioration,loss of appetite and significant decline in body mass.After intragastric administration for 45 days,the mental status of mice in all dose groups and the positive drug group were improved.The body mass of mice in all dose groups and the positive drug group were not significantly different with that of the model group?P>0.05?,but the liver coefficient of mice was significantly decreased in the high-dose group?P<0.05?and it was no obvious difference with the positive drug group.The HE and Masson staining showed,the all dose groups and the positive drug group had relatively clear hepatic lobules,neatly arranged liver cells,decreased collagen fibers,significantly reduced liver damage,and various degrees of amelioration on tissue fibrosis,compared to the model group.?2?Liver function-related indicators in each group of mice were measured.There was an abnormal change in plasma ALT,AST,AKP,TP,ALB,TBil,DBil,four indicators of liver fibrosis,and tissue HYP levels in liver fibrosis model mice.After intragastric administration,plasma biochemical indicators and collagen contents were ameliorated in all dose groups and positive drug groups.Compared to the model group,high-dose of mulberry alkaloid significantly reduced plasma ALT,AST and AKP by60.60%,54.85%,and 49.72%,respectively?P<0.05?;reduced HA,LN,PC-?,IV-C and liver tissue HYP by 12.01%,15.77%,17.55%,17.42%,and 28.81%,respectively?P<0.05?;increased TP and ALB by 26.50%and 27.75%,respectively?P<0.05?;decreased TBil and DBil by 35.56%and 74.50%,respectively?P<0.05?.The effects of high-dose of mulberry alkaloid on plasma ALT,AST,AKP,LN,PC-?,IV-C,TP,ALB,TBil,DBil and liver tissue HYP in liver fibrosis mice were equivalent to those of 100mg/kg silibinin under experimental conditions?P>0.05?.The results indicated that mulberry alkaloid can ameliorate liver fibrosis in mice.?3?The levels of blood lipids,antioxidant indicators and inflammatory factors in mice with liver fibrosis were measured.Liver fibrosis model mice had dyslipidemia,decreased antioxidant capacity and inflammatory response.After intragastric administration for 45 days,the above that of mice in all dose groups and positive drug group were ameliorated.High-dose of mulberry alkaloid significantly reduced plasma TC,TG,MDA by 28.67%,23.76%,and 33.60%,respectively?P<0.05?;increased GSH,SOD and T-AOC by 136.83%,45.14%,and 78.13%,respectively?P<0.05?,and reduced TNF-?,IL-6 and COX-2 by 9.96%,11.06%and 10.2%,respectively?P<0.05?.The effects of high-dose of mulberry alkaloid on TC,TG,HYP,MDA and GSH in liver fibrosis mice were equivalent to those of 100 mg/kg silibinin under experimental conditions?P>0.05?.The results indicated that mulberry alkaloid can ameliorate liver fibrosis mice by regulating the level of blood lipid,improving the anti-oxidative capacity and reducing inflammation.?4?The contents of?-SMA,Col?and Col?,and the relative expression of TGF-?1/Smads signal pathway relevant factors,MMP-13 and TIMP-1 mRNA in mice liver tissue were measured.CCl₄ combined with a high-fat diet stimulation activated HSC and led to ECM deposition in mice.The relative expression levels of TGF-?1,Smad2?Smad3?Smad4 and TIMP-1 mRNA in liver tissue were significantly increased in liver fibrosis model mice,while Smad7 and MMP-13 mRNA expression were decreased significantly?P<0.05?.Compared to the model group,high-dose of mulberry alkaloid reduced liver tissue?-SMA,Col?and Col?levels by 19.55%,19.93%and 13.84%,respectively?P<0.05?;decreased the relative expression of TGF-?1,Smad3,Smad4 and TIMP-1 mRNA by 59.04%,56.73%,50.70%and 49.09%,respectively?P<0.05?,and increased Smad7 and MMP-13 mRNA expression by 48.29%and 25.72%,respectively?P<0.05?.The effects of high-dose of mulberry alkaloid on?-SMA,Col?and Col?levels,and TGF-?1,Smad4 and MMP-13 mRNA expression were equivalent to those of 100 mg/kg of silibinin under experimental conditions?P>0.05?.The results indicated that mulberry alkaloid can ameliorate liver fibrosis by regulating the relative expression of TGF-?1/Smads signaling pathway relevant factors,MMP-13and TIMP-1 mRNA.On the one hand,mulberry alkaloid could improve antioxidant capacity and reduce inflammatory factors levels.One the other hand,it could regulate TGF-?1/Smads signaling pathways relevant factors,MMP-13 and TIMP-1 mRNA.These two aspects may be the reason for the reduction of HSC activation and ECM deposition in liver.In summary,mulberry alkaloid has the amelioration effect on liver fibrosis model mice induced by CCl₄ combined with a high-fat diet.