Study On Protective Effect And Protective Mechanism Of The Roots Of Indigofera Stachyoides On Acute Liver Injury In Mice

Objective:To study the protective effects of the roots of Indigofera stachyoides on three models of acute liver injury in mice Methods:1)Acute toxicity study of the roots of Indigofera stachyoides:24 male KM mice were random Ly divided into 4 groups with 6 mice in each group.The roots of Indigofera stachyoides200,400,800,and 1600 mg·kg^-1groups were given subcutaneous 0.1 m L·10 g^-1doses,fasting and irritating to water,and the blood was decapitated on the following day and separated.Mouse liver.The activity of serum alanine aminotransferase?ALT?in mice was measured,and the death of mice and the pathological changes of liver in mice were observed.2)Effect of the roots of Indigofera stachyoides on liver of normal mice:30 male KM mice were randomLy divided into normal control group,low dose of the roots of Indigofera stachyoides group(200 mg·kg^-1),high dose of the roots of Indigofera stachyoides(400 mg·kg^-1),10 mice in each group,low dose group was injected subcutaneously with a certain dose of 200 mg·kg^-1the roots of Indigofera stachyoides aqueous solution,and high dose group was injected subcutaneously with a certain dose of 400 mg·kg^-1the roots of Indigofera stachyoides aqueous solution for4 consecutive days,fasting can not help but water,the next day morning decapitation blood,and liver separation.Serum ALT activity was measured in mice;Real-time PCR was used to detect CYP1A2,CYP2E1 and CYP3A11 gene levels in liver tissue.3)Protective effects of the roots of Indigofera stachyoides on carbon tetrachloride?CCl₄?-induced acute liver injury in mice:Fifty male KM mice were randomLy divided into 5 groups with 10 rats in each group as normal controls.The model group,low dose group of the roots of Indigofera stachyoides(200 mg·kg^-1),high dose group of the roots of Indigofera stachyoides(400 mg·kg^-1),and biphenyl diester group(200mg·kg^-1).The roots of Indigofera stachyoides low dose group and high dose group were administered subcutaneously for 4 consecutive days.The bifendate group was intragastrically administered for 7 consecutive days.Twenty-four hours after the last administration,0.1%CCl₄-vegetable oil(10 mL·kg^-1)was intraperitoneally injected,and water was fasted.16 hours later,the blood was decapitated and the mouse liver tissue was isolated.The activities of ALT and AST in mice serum,and the contents of glutathione?GSH?and malondialdehyde?MDA?in liver tissue were measured.Observe the pathological changes of liver tissue,Realtime-PCR detection of liver-related gene metallothionein 1 MT-1,nuclear factor-associated factor Nrf-2,HO-1,Nqo1,inflammatory factors Mip2,IL-1beta and endoplasmic reticulum Stress genes Gadd45 and Gadd153.4)Protective effects of the roots of Indigofera stachyoides on acute liver injury induced by acetaminophen?APAP?in mice:Male KM mice were randomLy divided into normal control group,model group,bicyclol group,and the roots of Indigofera stachyoides low-dose group(200mg·kg^-1)and high-dose ginseng group(400 mg·kg^-1),10 in each group,the roots of Indigofera stachyoides low-dose group and high-dose group were injected subcutaneously respectively,continuous 4 day.The rats in the bicyclol group were intragastrically administered for 4 consecutive days.Eight hours after the last administration,the acute liver injury model was established by intraperitoneal injection of 300 mg·kg^-1APAP aqueous solution in each group except for the normal control group.Can not help but water,after 16 h,take blood and seperate serum,and dissect liver.The activity of alanine ALT and aspartate AST in serum and the content of reductive GSH and MDA in liver tissue were measured.Observe the pathological changes of liver tissue.Realtime-PCR was used to detect the expression level of Egr-1 and TNF-?genes in liver tissue.5)Protective effects of the roots of Indigofera stachyoides on D-galactosamine?D-Gal?-induced acute liver injury in mice:Male KM mice were randomLy divided into normal control group,model group,bicyclol group,and the roots of Indigofera stachyoides low and high Dose groups,10 in each group,and the roots of Indigofera stachyoides low-dose group and high-dose group were administered subcutaneously for 4 consecutive days.Bicyclol was intragastrically administered for 4 consecutive days.Eight hours after the last administration,D-GalN(800 mg·kg^-1)was intraperitoneally injected.Water was fasted,and blood was decapitated after 16 h,and the mice were isolated.Liver tissue.The activity of alanine aminotransferase ALT and AST in serum and the content of reductive GSH and MDA in liver tissue were measured.Observe changes in liver pathology.Results 1)the roots of Indigofera stachyoides was observed for 7 days after a single dose of 200mg·kg^-1,400 mg·kg^-1,800 mg·kg^-1,1600 mg·kg^-11 in a gradient dose.No deaths occurred.The roots of Indigofera stachyoides 200 mg·kg^-1,400 mg·kg^-1,800 mg·kg^-1groups had no significant effect on the liver,and the roots of Indigofera stachyoides1600 mg·kg^-11 could increase ALT activity in mice.2)After continuous administration of the roots of Indigofera stachyoides for 4 days,there was no effect on the liver of normal mice and the expression of P450 enzymes CYP1A2,CYP2E1 and CYP3A11was inhibited.3)The roots of Indigofera stachyoides low-dose group and high-dose group can reduce the serum ALT,AST activity in CCl₄ mice,reduce the liver MDA content,increase the GSH content and significantly improve the degree of hepatocyte necrosis,Realtime-PCR results showed that increased expression of MT-1 and Nrf2further induced the expression of Nqo1,Gclc,and HO-1,which in turn inhibited the expression of the inflammatory factors Mip2 and IL-1?,the endoplasmic reticulum stress genes Gadd45 and Gadd153,and protected the role of the liver.4)The roots of Indigofera stachyoides low-dose group and high-dose group did not cause obvious liver damage to normal mice and could reduce the serum ALT,AST activity in APAP model mice and significantly improve the degree of hepatocyte necrosis,inhibit Egr-1,TNF-?gene expression level.The roots of Indigofera stachyoides low dose group and high dose group can reduce the serum ALT,AST activity in D-Gal model mice,reduce the MDA content in liver tissue of mice and significantly improve the degree of hepatocyte necrosis.Conclusion:The roots of Indigofera stachyoides has no obvious hepatotoxicity and has protective effects on CCl₄,APAP and D-Gal-induced acute liver injury in mice.The protective mechanisms may be related to the following two pathways:1)the pathways that induce the metallothionein MT-1 and Nrf2 antioxidant damage;2)the inhibition of P450 enzymes CYP1A2,CYP2E1,and CYP3A11expression;3)downregulation of the inflammatory factors related genes Expression.