Protective Effect And Underlying Mechanism Of Daidzein On Acute Liver Injury In Mice

Part Ⅰ:Pretreatment with daidzein ameliorated concanavalin A-induced liver injury in miceObjective:To investigate the protective effects of daidzein in ConA-induced acute liver injury,and the underlying mechanism of daidzein on ConA-induced liver injury.Methods:ConA was dissolved in a saline solution and injected intravenously at 15 mg/kg to induce liver injury.Daidzein(200 mg/kg/d,dissolved in saline solution when it was used)was administered intraperitoneally to mice for 3 d before ConA injection.One hour after the last daidzein dose,the mice were treated with ConA.The mice were divided randomly into four groups:(1)Control group:mice were treated with saline solution only;(2)Daid group:mice were treated with daidzein only;(3)ConA group:mice were treated with ConA at a dose of 15 mg/kg;(4)ConA+Daid group:mice were treated with daidzein(200mg/kg/d)three days before ConA injection.All mice were sacrificed at 12 h after ConA injection by anesthesia with 1%pentobarbital(10 ml/kg),and the serum and liver tissues were harvested and stored at-80℃.For the survival assay,an approximate lethal dose of ConA(25/mg/kg)was administered,the survival rate of each group was observed within72 hours.12 hours after ConA(15mg/kg)injection,serum samples and liver tissues were harvested,the levels of ALT,AST were detected and liver tissues were stained with H&E and TUNEL to evaluate the degree of liver injury and the number of apoptotic cells,respectively.The expression of TNF-α,IL-1βand IL-6 in liver tissue were measured by real-time PCR,and the levels of TNF-α,IL-1βand IL-6 in serum were measured using ELISA kits according to the manufacturer’s instructions.To detect the oxidative stress induced by ConA,Dihydroethidium(DHE)fluorescence was used to test ROS levels and the SOD,GPX,and MDA levels in liver tissues were measured by SOD detection kits,GPX detection kits,and MDA detection kits,according to the manufacturer’s instructions.To investigate the possible mechanism of daidzein alleviating ConA induced oxidative stress and hepatocytes apoptosis,the expression of p-Akt,p-GSK3βand its downstream Nrf2,HO-1 and NQO1 were also analyzed by western blot.The hepatic mononuclear cells(MNs)and splenocytes were isolated from liver and spleen respectively,and the percentage of CD4~+T lymphocytes and activated CD4~+T lymphocytes in liver and spleen were detected by flow cytometry.in vitro study,AML-12 cells were pretreated with daidzein with or without LY294002(20μm)and then treated with ConA for 12 h.The viability and apoptosis of AML-12 cells after 12 h of ConA treatment were determined using a CCK-8assay and Annexin V-FITC/PI apoptosis detection kit.Results:The survival rate of mice injected with lethal dose of ConA reached 40%within72 hours.Pretreatment with daidzein showed a significant increase in survival,and 100%of the mice were still alive.In ConA treated mice,the levels of ALT,AST in serum were obviously elevated.However,pretreatment with daidzein efficiently decreased the levels of ALT,AST induced by ConA injection.H&E staining showed extensive hepatocyte necrosis and inflammatory cells infiltration after ConA injection.Strikingly,these pathological alterations were markedly improved by daidzein pretreatment.TUNEL staining exhibited plenty of apoptotic hepatocytes after ConA stimulation,daidzein pretreatment could significantly decreased the number of apoptotic hepatocytes in ConA challenged mice.Besides,the levels of TNF-α,IL-1βand IL-6 in liver and serum were significantly increased in serum and liver tissues by ConA challenge,but which could be diminished by daidzein pretreatment.After ConA stimulation,the levels of ROS and SOD,GPX in liver tissues were significantly increased,while daidzein pretreatment distinctly alleviated oxidative stress injury induced by ConA.Western blot showed that pretreatment with daidzein significantly prevented the decrease of intrahepatic protein levels of phosphorylated Akt(p-Akt),phosphorylated GSK3β(p-GSK3β),nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),and NOQ1(NAD(P)H quinone dehydrogenase 1)in response to ConA administration.Besides,after ConA stimulation,CD4~+T lymphocytes in liver tissues and spleen were both activated,while daidzein pretreatment had no effect on the activation of CD4~+T lymphocytes.In vitro study,daidzein pretreatment prevented ConA-induced murine hepatocyte death.This effect was partly diminished by an inhibitor of the phosphatidylinositol 3-kinase(PI3K)/Akt pathway.Conclusion:Daidzein pretreatment protects against ConA-induced liver injury,which might be through inhibiting the oxidative stress response and hepatocyte apoptosis by regulating the PI3K/Akt/GSK3βsignaling pathway.Part Ⅱ: Daidzein preconditioning attenuates hepatic ischemia reperfusion injury via HMGB1-induced TLR4/MyD88/NF-κB signaling pathwayObjective: Daidzein is a soybean isoflavone and have been reported to have protective effect on cerebral and myocardial ischemia reperfusion injury.However,whether daidzein preconditioning could also reduce hepatic ischemia-reperfusion injury(HIRI)is unknown.The present study aimed to investigate the protective role of daidzein in liver I/R injury.Methods: Animal model of segmental(70%)hepatic warm ischemia was performed to induce acute liver injury.The mice were randomly to one of four groups:(1)Sham group: The surgery of laparotomy was performed without HIRI.(2)H/R group: The surgery of HIRI was performed.(3)H/R + Daid group: Mice were treated with daidzein(200 mg/kg/d)via intraperitoneal injection for 3d before the HIRI surgery performed.(4)H/R + Daid + r HMGB1 group: Mice were treated with daidzein(200 mg/kg/d)via intraperitoneal injection for 3d before the HIRI surgery performed,20 μg of r HMGB1 was administered immediately after reperfusion.All mice were killed 6h after reperfusion and all serum and liver tissues are harvested and stored in-80℃ for further analysis.Serum samples were collected to detect the levels of ALT,AST.Liver tissues were harvested and stained with H&E and TUNEL to evaluate the degree of liver injury and the number of apoptotic cells,respectively.Immunohistochemistry and ELISA was performed to detect the translocation of High-mobility group box 1(HMGB1).The expression of TNF-α,IL-1β,IL-6 and Bax,Bcl-2 in liver tissue were measured by real-time PCR.Western blots were used to examine the expression of Bax,Bcl-2 and HMGB1 to evaluate apoptosis and the release of HMGB1 in liver.To investigate the protective effects of daidzein on the I/Rinduced liver injury,TLR4/MyD88/NF-κB signal pathway was detected and the expression of TLR4,MyD88 and p-NF-κB in liver were measured by western blot.In vitro study,AML-12 cells were pretreated with daidzein(80u M)and then treated with Cocl2(300u M)for 24 h.To observed the nucleocytoplasmic shuttling of HMGB1 in hepatocytes,immunofluorescent staining and western blot for HMGB1 expression in AML-12 cells was performed.Results: The level of ALT and AST were obviously increased 6h after reperfusion,conversely,there were significantly reduced in daidzein pretreatment group.In addition,H&E staining showed that the structure of liver tissues was entirely maintained in the sham group,whereas a disordered lobular structure,marked hepatocyte degeneration or necrosis,and inflammatory cell infiltration were observed in the H/R group.However,daidzein pretreatment distinctly ameliorated all the pathological features presented in the H/R group.TUNEL staining exhibited plenty of apoptotic hepatocytes after HIRI,daidzein pretreatment obviously reduced Bax expression and upregulated the expression of Bcl-2,and the number of TUNEL-positive cells were also declined.RT-PCR showed a significant increase in the expression of proinflammatory cytokine(TNF-α,IL-1β and IL-6)in the H/R group,while daidzein pretreatment can inhibit inflammatory response after HIRI.To evaluate the nucleocytoplasmic shuttling of HMGB1 during HIRI,immunohistochemical staining and western blot for HMGB1 expression in liver tissues were performed.As expected,the translocation of HMGB1 during HIRI was significantly decreased by daidzein pretreatment,and the expression of TLR4,MyD88 and p-NF-κB were also declined in the daidzein pretreatment group.To further determine whether the protective effects of daidzein on HIRI are correlated with HMGB1 inhibition,we administered 20 ug of r HMGB1 to daidzein-treated mice immediately after reperfusion.As compared to the daidzein pretreatment group,the mice received r HMGB1 exhibited significant increases in ALT,AST,and the neutrophil infiltration and hepatocytes apoptosis were also increased.In vitro study,daidzein pretreatment prevented Cocl2-induced murine hepatocyte death,and the nucleocytoplasmic shuttling of HMGB1 in AML-12 cells were also inhibited by daidzein pretreatment.Conclusion: Daidzein pretreatment alleviated HIRI via HMGB1-induced TLR4/MyD88/NF-κB signaling pathway.