Mechanism Of The Protective Effect Of Neferine On APAP-induced Liver Injury In Mice

Background:Drug-induced liver injury(DILI)refers to liver damage caused by an allergic reaction caused by the drug itself or its metabolites or drugs during drug use,also known as drug-induced liver disease(DILD).Clinically,it can be manifested as various acute and chronic liver diseases.In severe cases,it may be life-threatening and requires active rescue treatment.As a clinically widely used antipyretic analgesic,acute liver injury caused by acetaminophen(APAP)occupies a considerable proportion of drug-induced liver damage,and currently severe liver caused by excessive use and abuse of APAP Toxic problems have drawn increasing attention.The mechanism of APAP-induced liver injury is very complex.Many intracellular and extracellular events are involved in its pathophysiological processes,including APAP metabolism,mitochondrial oxidative stress,endoplasmic reticulum stress,autophagy,aseptic inflammation,and microcirculatory dysfunction.And liver regeneration.Neferine(Nef)is a water-insoluble total alkali extract of the natural medicine lotus seed.It is a bisbenzylisoquinoline alkaloid with anti-inflammatory,anti-fibrotic,vasodilator,blood pressure lowering and platelet inhibiting.Aggregation,anti-arrhythmia and anti-tumor effects.Based on previous studies,we found that neferine has protective effects on liver of rats with hepatic fibrosis.However,whether Nef intervention has protective effect on liver injury induced by APAP has not been studied.Therefore,this study aims to explore whether Nef plays a role in APAP liver injury and studies related mechanisms.Method:(1)APAP lethal dose experiment40 male C57BL/6 mice were randomly divided into the following 4 groups: 1APAP+NS group,NS gavage 0.4ml once daily,n=10;2APAP+Nef(5)group,Nef suspension 5mg/kg Oral administration of 0.4ml,once daily,n=10;3APAP+Nef(10)group,Nef suspension10mg/kg gavage 0.4ml,once daily,n=10;4APAP+Nef(20)group,Nef Suspension20mg/kg gavage 0.4ml,once a day,n=10.Nef suspension was continuously administeredto each group of mice for 14 days.After the last gavage for 0.5 hours,mice in each group were intraperitoneally injected with 2.5% APAP solution 600 mg/kg.(2)APAP sublethal dose experimentThirty male C57BL/6 mice were randomly divided into the following 5 groups: 1 saline control group(NS control): NS gavage 0.4 ml once daily,n=6;2APAP+NS group,NS gavage 0.4 ml Once daily,n=6;3APAP+Nef(5)group,Nef suspension 5mg/kg gavage0.4ml,once daily,n=6;4APAP+Nef(10)group,Nef suspension 10 mg /kg gavage 0.4ml,once a day,n = 6;5APAP + Nef(20)group,Nef suspension 20 mg / kg gavage 0.4ml,once a day,n = 6.The mice were continuously intragastrically administered with Nef suspension for 14 days.After the last gavage for 0.5 hours,except for the NS control group,the other groups of mice were intraperitoneally injected with 2.5% APAP solution 400 mg/kg.Results:(1)Nef increased the 5-day survival rate of mice with APAP-induced liver injury.The5-day survival rate of APAP+ Nef(20)group was higher than that of APAP+NS group(P<0.05).(2)Nef reduced serum transaminase levels in mice with APAP-induced liver injury.Compared with APAP+NS group,ALT and AST levels were lower in APAP+Nef(10)group and APAP+Nef(20)group,and APAP+Nef(20)The group was lower than the APAP+Nef(10)group,and the difference was statistically significant(P<0.05).(3)Nef attenuated histopathological changes in mice with APAP-induced liver injury.HE staining showed hepatic lobular necrosis and fat infiltration in APAP+Nef(10)group and APAP+Nef(20)group compared with APAP+NS group.The degree of lymphocyte infiltration was significantly reduced,and in the APAP+Nef(20)group,the above improvement was more significant than the APAP+Nef(10)group,and the histological scores were statistically significant(P<0.05).(4)Nef inhibited the expression of inflammatory factors in mice with liver injury induced by APAP.Compared with APAP+NS group,the expression of TNF-α and IL-6 decreased in APAP+Nef(10)group and APAP+Nef(20)group.The APAP+Nef(20)group was lower than the APAP+Nef(10)group,and the difference was statistically significant(P<0.05).(5)Nef inhibited apoptosis of hepatocytes in mice with liver injury induced by APAP.Compared with APAP+NS group,the percentage of hepatocyte apoptosis decreased in APAP+Nef(10)group and APAP+Nef(20)group,and APAP+Nef The(20)group was lower than the APAP+Nef(10)group,and the percentage of apoptotic cells was statistically significant(P<0.05).(6)Nef reduced the level of oxidative stress in mice with liver injury induced by APAP.Nef can reduce the production of ROS in the body.The difference was statistically significant(P<0.05).Nef reduced the damage of MDA on the cells of the body.The difference wasstatistically significant.(P<0.05);Nef increased the body’s ability to resist oxygen free radicals,and the expression levels of SOD,GSH and GSH-px increased,and the difference was statistically significant(P<0.05).(7)Nef can up-regulate Nrf2/HO-1/NQO1 pathway protein expression,compared with APAP+NS group,protein expression of Nrf2,HO-1,NQO1 in APAP+Nef(10)group and APAP+Nef(20)group The amount of AAP+Nef(20)group was significantly higher than that of APAP+Nef(10)group,and the difference was statistically significant(P<0.05).Conclusions:The results suggest that Nef plays a prophylactic and therapeutic role in APAP-induced hepatotoxic injury,probably due to Nef’s powerful antioxidant capacity,blocking ROS progression,inhibiting oxidative stress and lipid peroxidation,and upregulating Nrf2/ HO-1/NQO1 anti-oxidation pathway protein expression,reduce hepatocyte necrosis,reduce the expression of inflammatory factors,thereby playing a protective role of Nef on acetaminophen-induced liver injury.This study can provide a new strategy for the treatment of APAP liver injury and provide a theoretical basis for the application of Nef in APAP-induced liver injury.