Protective Effect Of Diammonium Glycyrrhizinate On Acute Liver Injury Induced By Acetaminophen In Mice

Objective:To explore the protective effect of diammonium glycyrrhizinate(DG)on acute liver injury induced by acetaminophen(APAP)in mice,the effects of inflammatory response and cell apoptosis,and to preliminarily explore its possible mechanism,so as to provide a basis for basic research on the clinical application of DG.Method:1.Model establishment and drug administration intervention: In this study,50 healthy adult SPF male ICR mice were used to establish an acute liver injury model.They were adaptive fed for 5 days and randomly divided into 5 groups with 10 mice in each group,which were divided into normal control group,APAP model group,glutathione(GSH)positive control group,DG low-dose group and DG high-dose group.The normal control group was given the same amount of normal saline intragastrically and intraperitoneally.APAP group was given a one-time gavage of 300mg/ k GAPAP solution,and the same amount of normal saline was injected intraperitoneally every day.GSH group was given300mg/ KGAPAP solution by gavage once,and 400mg/kg glutathione was intraperitoneally injected once a day for 5 consecutive days.The low-dose and high-dose DG groups were given 300mg/ k GAPAP solution by one-time gavage,respectively.After gavage in each group,low-dose and high-dose DG(50 and200mg/kg)were given intraperitoneally once a day,respectively,for consecutive5 days.2.Mental activity,diet,water intake,urine volume and other conditions of the experimental mice were observed every day,and the changes in body weight were recorded.After the end of the experiment,blood samples were collected from the eyes,and serum samples were collected from the mice for viscera sampling.Liver tissue samples were collected from each group,fixed and sliced to observe and compare the pathological morphological changes of each tissue.3.The levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH)in serum samples of mice were detected by enzyme plate analyzer.4.Serum levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in mice were determined by enzyme-linked immunosorbent assay(ELISA).5.Fluorescence microscope was used to observe the liver tissue where the fluorescence was detected,and the proteins such as IκB-α,NF-κB p65,Bcl-2and Bax were qualitatively,locatively and semi-quantitatively analyzed.6.The m RNA expression levels of IκB-α,NF-κB p65,Bcl-2 and Bax were detected by real-time quantitative PCR.Results:1.DG can improve the physical status of mice with acute liver injury.2.Effects of DG on body weight and liver index of APAP-induced acute liver injury mice: Compared with the control group,the body weight of APAP-induced acute liver injury mice showed a gradually decreasing trend.Compared with the control group,after APAP-induced acute liver injury,liver enlargement tended to occur in APAP-model group,GSH group and low and high dose DG intervention group.Among them,the liver weight and liver index of mice in APAP model group were significantly higher than those in the control group,GSH and low and high dose DG intervention groups,with statistical significance(P < 0.01).3.Effects of DG on serum ALT,AST and LDH in APAP-induced acute liver injury mice: Compared with normal control group,the contents of ALT,AST and LDH in serum in APAP model group were significantly increased(P<0.01);Compared with APAP model group,the contents of ALT,AST and LDH in serum of mice in GSH and DG intervention groups were significantly decreased(P<0.01),which had statistical significance.4.Influence of DG on liver histomorphology of APAP-induced acute liver injury mice: HE staining results showed that,compared with the model group,the liver tissue damage of APAP-exposed mice was alleviated to some extent after GSH and DG intervention.5.Effect of DG on serum inflammatory factors in APAP-induced acute liver injury mice: Compared with the control group,the levels of serum IL-1β,TNF-αand IL-6 in model group were significantly increased(P<0.01);Compared with model group,the levels of IL-1β,TNF-α and IL-6 in serum of mice were significantly decreased after GSH and DG intervention(P<0.01),with statistical significance.6.Effects of DG on protein levels of IκB-α,NF-κBp65,Bcl-2 and Bax in liver tissue of mice with acute liver injury induced by APAP:Compared with the control group,the number of IκB-α and Bcl-2 positive protein cells in liver cells of model group was significantly decreased(P<0.01),while the number of NF-κB p65 and Bax positive protein cells was increased(P<0.01).Compared with model group,after GSH and DG intervention,the number of endogenous IκB-α and Bcl-2 positive protein cells in mouse liver increased(P<0.01),while the number of NF-κB p65 and Bax positive protein cells decreased(P<0.05 or P<0.01),with statistical significance.7.Effects of DG on m RNA expression levels of IκB-α,NF-κB p65,Bcl-2and Bax in liver tissues of mice with acute liver injury induced by APAP:Compared with normal control group,the expressions of IκB-α and Bcl-2m RNA in APAP model group were down-regulated(P<0.01),while the expressions of NF-κB p65 and Bax m RNA were significantly up-regulated(P<0.01).Compared with model group,the expressions of endogenous IκB-α and Bcl-2 m RNA in liver tissues of mice treated with GSH and DG were increased(P<0.05 or P<0.01),while the expressions of NF-κB p65 and Bax m RNA were significantly decreased(P<0.01),with statistical significance.Conclusion:DG can improve the general signs of APAP-induced acute liver injury mice,improve the liver function indexes and LDH to a certain extent,relieve the pathological damage of liver tissue,reduce the activity of serum inflammatory factors of liver injury mice to a certain extent,reduce the inflammatory response,regulate the apoptotic factors.It is speculated that the mechanism of DG against acute liver injury may be related to the protection of liver function,inhibition of inflammatory response and apoptosis.