| ObjectivesTo observe the protective effect of quercetin on immunological liver injury induced by triptolide,and to explore the relevant mechanisms.Methods1.Protective effects of quercetin on triptolide induced liver injury.Fifty C57BL/6J mice were randomly divided into five groups: control group,model group,(20、50、80)mg/kg Quercetin pre-treatment group.Animals were treated with different dose of quercetin once daily for 10 days.Mice in control group and TP group were given 5‰CMC-Na instead.At the end of the experiment,triptolide(500 μg/kg)was given intragastric to the mice except control group.22 hours later the level of serum ALT,AST level were detected.The contents of GSH,SOD and MDA in liver tissue homogenates were measured through commercial kits.HE staining was performed to observe pathologic change of liver.2.The variety of the balance of Th17/Treg cells after immunological liver injury and quercetin application and the effect of TLR4-MyD88-NF-κB and Tim3 signal pathway involved.To explore the variety of the balance of Th17/Treg cells,Th17 and Treg related cytokine IL-17 and IL-10 were detected by ELISA and RT-PCR,ROR-γt and Foxp3 were checked by immunohistochemistry and RT-PCR.To investigate whether TLR4-MyD88-NF-κB and Tim3 signaling was involved in the protective action of QE,the expression level of TLR4,MyD88,NF-κB and Tim3 were measured by Western blot.3.Effect of TAK-242 on the variety of the balance of Th17/Treg cells.Thirty C57BL/6J mice were randomly divided into three groups: control group,model group and TAK-242 group(1mg/kg).Triptolide(500 μg/kg)was given intragastric to the mice except control group.An hour before inducing liver injury,mice were injected TAK-242 intraperitoneally.22 hours later the level of serum ALT,AST level were detected.The contents of GSH,SOD and MDA in liver tissue homogenates were measured through commercial kits.HE staining was performed to observe pathologic change of liver.Th17 and Treg related cytokine IL-17 and IL-10 were detected by ELISA and RT-PCR,ROR-γt and Foxp3 were checked by immunohistochemistry and RT-PCR.To investigate whether TLR4-MyD88-NF-κB and Tim3 signaling was involved in the protective action of QE,the expression level of TLR4,MyD88,NF-κB and Tim3 were measured by Western blot.Results1.In TP-treated mice,activities of ALT,AST and MDA significantly increased as compared with those of the controls,and the formation of GSH and SOD was dramatically decreased.The histopathological evaluation of liver sections in TP treatment group showed extensive changes in liver morphology,including extensive hepatocellular necrosis and inflammatory cellular infiltrations.Administration of QE for 10 consecutive days markedly decreased the level of ALT,AST and MDA,elevated the level of SOD and GSH,especially at the higher dose.These data suggest that QE could protect against TP induced liver injury.2.Administration with QE visibly inhibited IL-17 secretion and promoted IL-10 secretion.In comparison with the control,the expression level of ROR-γt dramatically increased and Foxp3 significantly decreased in the model group.Immunohistochemistry results showed that QE markedly suppressed ROR-γt and elevated Foxp3 expression in the liver tissues of TP-induced mice.Compared with the control,the expression levels of TLR4,MyD88 and NF-κB observably increased while Tim-3 was slightly reduced in TP-treated mice.However,treatment with high dose of QE markedly suppressed this elevation and the expression of Tim-3 was significantly increased.3.Administration with TAK-242,the level of ALT,AST in serum and MDA,IL-17,TLR4,MyD88 and NF-κB were observably decreased,the level of SOD,GSH,IL-10 and Tim-3 were dramatically elevated.Immunohistochemistry results showed that TAK-242 markedly suppressed ROR-γt and elevated Foxp3 expression in the liver tissues.The histopathological evaluation of liver sections in TAK-242 treatment group showed that there is little hepatocellular necrosis and inflammatory cellular infiltrations.Conclusions1.Quercetin could protect liver injury induced by TP.2.The inhibition of TP induced liver injury by Quercetin is associated with its ability to shift Th17/Treg imbalance to Treg dominance through Tim3 and TLR4-MyD88-NF-κB signaling pathway.3.Blocking TLR4 signal pathway could protect TP induced liver injury by regulating Th17/Treg imbalance. |
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