Study On The Active Ingredients And Its Mechanisms Of Albiziae Flos In Attenuating Acetaminophen-induced Acute Liver Injury

Objective: To explore the active ingredients and its mechanisms of Albiziae flos in attenuating APAP-induced acute liver injury,and to provide scientific basis for its development and application in the treatment of APAPinduced liver injury.Methods: Using human normal hepatocytes L-02 cells as subjects,MTT assay was used to detect the effects of different extracts of Albiziae flos on the viability of L-02 cells,and effective extracts were screened.LC-MS analysis was used to identify the chemical ingredients in the effective extracts,select the main ingredients for active screening,and determine the effective ingredient in conjunction with relevant literature.The APAP-induced acute liver injury model was established using BALB/c mice.Mice were given different concentrations of active ingredient by gavage,and the effects of active ingredient on liver appearance and viscera coefficient were observed.Hematoxylin-eosin(H&E)staining is used to determine the pathophysiological changes of liver.Biochemical kits are used to detect the levels of ALT,AST,and LDH in mouse serum,and contents of SOD,GSH,GSH-Px,CAT,and MDA in mouse liver tissue homogenate.Immunohistochemical staining was used to detect changes in reactive oxygen species(ROS)and F4/80 in mouse liver.Enzyme-linked immunosorbent assay(ELISA)was used to determine the levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in mouse serum.L-02 cells were used to establish an APAP-induced cell injury model,and the blank group,drug group,model group and treatment group were set up.Flow cytometry was used to determine the changes in intracellular ROS levels.Biochemical kits were used to measure NADH dehydrogenase activity.MTT was used to detect the effect of cell survival rate in the blank group and treated group with APAP or APAP and NADH dehydrogenase inhibitor rotenone.Western blot was used to detect the expression of NADH dehydrogenase in L-02 cells.Results: The MTT method showed that ethyl acetate extract was the effective part of Albiziae flos to reduce APAP-induced liver injury.When the ethyl acetate extract concentration reached 80 μg/m L,the cell survival rate increased to 94.86 ± 1.21%.The LC-MS results showed that the chemical ingredients of the ethyl acetate extract were mainly flavonoid glycosides,including myricetin,isoquercitrin,cynaroside,guajavarin,quercitrin,and afzelin.Quercitrin(Que)was the main chemical ingredient,accounting for more than 90% of the total content of ethyl acetate extract.Activity screening showed that APAP(10 m M)reduced the cell survival rate to 66.26 ± 1.54%,while after Que pretreatment,the cell survival rate significantly increased(P <0.01).When Que concentration reached 80 m M,the cell survival rate increased to 96.03 ± 1.26%.The results of the whole animal experiment showed that,compared with the control group,the liver organ coefficient of the model group(APAP)was significantly increased(P <0.01),the liver cells showed extensive coagulative necrosis,and the levels of ALT,AST and LDH were significantly increased(P <0.01),indicating that the modeling was successful;compared with the APAP group,the liver tissue cell structure of the mice pretreated with Que at various concentrations tended to be normal,and the liver organ coefficient of the mice in the medium and high dose groups was significantly decreased(P <0.01),Que at various concentrations significantly reduced ALT,AST and LDH levels in the serum of the mice(P <0.05 or P <0.01);compared with the control group,the levels of ALT,AST,and LDH in the low and medium dose groups and the level of ALT in the high dose group were significantly different(P <0.01),while the levels of AST and LDH in the high dose group were not significantly different.Compared with the control group,F4/80 in liver tissue of the APAP group significantly increased(P <0.01),the levels of IL-6 and TNF-α in serum were greatly increased,and there were significant differences(P <0.01);Que pretreatment in each group could significantly alleviate the increase of F4/80 caused by APAP(P <0.01),and significantly reduced the levels of IL-6 and TNF-α(P <0.01);compared with the control group,there were significant differences in F4/80,IL-6 and TNF-α in the low and medium dose groups(P <0.01),but there was no significant difference in F4/80,IL-6 and TNF-α in the high dose group.Compared with the control group,the APAP group’s liver ROS levels increased significantly(P <0.01),SOD,GSH,GSH-Px and CAT levels in liver tissue homogenate significantly decreased(P <0.01),and MDA content increased significantly(P <0.01);compared with the APAP group,the ROS levels of the quercitrin groups were significantly reduced(P <0.01),and the levels of SOD,GSH,GSH-Px and CAT in mouse liver tissues were significantly increased(P <0.05 or P <0.01),showing a dose-dependent effect,the medium and high dose groups can significantly reduce MDA levels(P <0.05 or P <0.01);compared with the control group,there were significant differences in ROS and CAT in each dose group and GSH and MDA in the low and medium dose groups and SOD and GSH-Px in the low dose group(P <0.05 or P <0.01),and there was no significant difference in SOD,GSH-Px in the middle and high dose groups and GSH and MDA in the high dose group.Flow cytometry results showed that compared with the control group,ROS in the APAP group was significantly increased(P <0.01);compared with the APAP group,ROS level in L-02 cells in the treatment group(APAP + Que)was significantly decreased(P <0.01);compared with the control group,ROS in the APAP + Que group had no significant difference.NADH dehydrogenase activity assay showed that compared with the control group,the activity of NADH dehydrogenase in the APAP group was significantly decreased(P <0.01);compared with the APAP group,the NADH dehydrogenase activity of the APAP + Que group was increased significantly(P <0.05);compared with the control group,the activity of NADH dehydrogenase in the APAP + Que group had no significant difference.MTT method was used to detect the survival rate of cells in each group,compared with APAP treatment,the combined treatment of APAP and NADH dehydrogenase inhibitor rotenone significantly reduced the survival of the Que group cells(P <0.01).Western blot shows that compared with the control group,the expression of NADH dehydrogenase protein in L-02 cells in the APAP group was significantly reduced(P <0.01);after quercitrin pretreatment,APAP + Que significantly increased the content of NADH dehydrogenase in injured cells(P <0.01).Conclusion: 1.The ethyl acetate extract of Albiziae flos can effectively reduce APAP-induced acute liver injury,and its effective chemical ingredient is quercitrin.2.Quercitrin from Albiziae flos can effectively reduce APAPinduced liver injury in mice,and can reduce inflammatory factor levels and oxidative stress in liver tissue of mice.3.Quercitrin up-regulates the expression of mitochondrial NADH dehydrogenase,increases the activity of NADH dehydrogenase,reduces the production of intracellular ROS,avoids the cells from oxidative stress,and finally reduces the cell damage caused by APAP.In this study,quercitrin in Albiziae flos was identified as the effective ingredient to reduce APAP-induced acute liver injury,and its mechanism was further elucidated.