The Intervention Of Major Alkaloids In Sophorae Tonkinensis On Immune Liver Injury In Mice

Objective: To study the active components of the main alkaloids of Sophorae Tonkinensis on immunological liver injury in mice by pharmacological methods,then to investigate the effects of major alkaloids on NK cells and its surface-activated receptor NKG2 D by molecular biology,in order to investigate the effects of the main alkaloids of the roots of Sophorae Tonkinensis by inhibiting the activation of NK cells and thus interfering with immune liver injury.Methods: The levels of ALT and AST in serum were measured by automatic biochemical analyzer,combine with the result of liver injury,which was detected by HE staining,to screening out the active components of arbutin and Sophora alkaloids from the interaction of sorghum root.The effects of Oxymatrine and Sophocarpine on the immunological liver injury in mice were further confirmed by changes of ALB,CHE and TP,hepatocyte apoptosis,immunoglobulin level and inflammatory factors.The number of NK cells and NK cell surface receptors NKG2 D and CRACC were detected by flow cytometry.Western blot was used to detect NKG2 A,NKG2D,DAP10,DAP12.Immunoblotting and immunohistochemistry were used to detect PI3 K,AKT and ERK.And Q-PCR detect the mRNA level of NKG2 A,NKG2D,DAP10,DAP12,PI3 K,AKT,ERK,ZAP70 and Syk,in order to investigate the mechanism of Oxymatrine and Sophocarpine intervening in immunological liver injury.Result:(1)Screening active components of anti-immune liver injury Sophorae TonkinensisTaking the model of poly I: C/D-GaIN-induced injury in mice as research object,serum ALT,AST and pathological state of mice were taken as indicators to screen for alkaloids having inhibition on immunological liver injury.We have found that OMT and Sophocarpine in Sophorae Tonkinensis can dramatically decreased the serum levels of ALT and AST(p<0.05 or p<0.01)causing by poly I: C/D-GaIN-induced injury in mice,and the effect of high-dose group was superior to low-dose group,which shows that OMT and Sophocarpine can obviously alleviate immunological liver injury induced by poly I: C/D-GaIN(PD).The result of hisopathology shows that high-dose groups of OMT and Sophocarpine can dramatically reduce the immunological liver injury causing by poly I: C/DGaIN,though pretreatment by low-dose groups can slightly reduce inflammatory cell infiltration and injury of liver tissue,while matrine did not have these tervention.Above all,OMT and Sophocarpine have prevention of poly I: C/D-GaIN-induced immunological liver injury in mice.Result:(2)Research of the mechanisms of inhibition of Oxymatrine on immunological liver injuryBy measuring the serum level of TP、ALB and CHE.The result showed that TP、ALB and CHE in PD group were decreased compared with the control group(p<0.01);while in high-dose and lowdose groups TP、ALB and CHE rised slightly(p<0.05 or p<0.01),and the effect of high-dose group was superior to low-dose group.The result of tunel staining showed that hepatocyte apoptosis were reduced in high/low-dose group compared with PD group,and the effect of high-dose group were better than low-dose group.Using Procartaplex multiplex detection to research level of TNF-α、IL-18 and concentration of lgG,lgM,lgA,we have discovered that they all have been decreased(p<0.01 or p<0.05)after administration.Considering the variations of mRNA levels of INF-γ and IL-12 in hepatic tissue of mice,which reveals that OMT have prevention on poly I: C/D-Ga IN(PD)-induced immunological liver injury.We found that the proportion of NK cells in the liver of PD group was higher than control group(p<0.01)by using flow cytometry analysis.Comparing with PD group proportion of NK cells in liver of High/Low-dose group was lower,but higher control group(p<0.05 or p<0.01).In PD group,the proportion of NK cells in spleen was lower than control group(p<0.05).Meanwhile,after OMT treatment,the number of NK cells in spleen was higher than PD group and lower than control group(p<0.05).Considering papers and the experiment results,it revealed that NK cells transfer from spleen into liver,causing immunological liver injury,and OMT can suppressed the accumulation of NK cells in liver which could attenuate immunological liver injury.By using flow cytometry analysis we found that the expression of NKG2 D and CRACC was higer in PD group than that in the control group.After drug administration,OMT group can ouviously down-regulate the expression of NKG2 D,but the effect on CRACC was not patent.Besides,we have found that the expression of NKG2 D the NK cells surface receptor in PD group was higher than control group(p<0.05;p<0.01)by Western blot and Q-PCR.After drug administration,OMT group can obviously down-regulate the expression of NKG2D(p<0.05;p<0.01).The effect of OMT on NKG2 A was not obvious.Conclusion: OMT might down-regulate the expression of NKG2 D to attenuate immunological liver injury.By using Q-PCR,after treated with poly I: C/D-GaIN we have discovered that mRNA level of DAP10,AKT and PI3 K all increased(p<0.05、p<0.01).After drug administration the expression of NKG2 D decreased and mRNA levels of DAP10,AKT and PI3 K all have decreased(p<0.05;p<0.01).By Western blot and immunohistochemical we can know that after treated with poly I: C/D-GaIN the expression of the adaptor of NKG2 D and its downstream proteins all increase(p<0.05;p<0.01).After drug administration the expression of DAP10 and its downstream proteins decreased(p<0.05;p<0.01).The results reveal that OMT may inhibit the expression of NKG2 D to affect downstream proteins of DAP10 which can inhibit the activity of NK cells.(3)Research of mechanism of modulating effect of Sophocarpine on immunological liver injuryWe establish a model of immunological liver injury by poly I: C/D-Ga IN to investigate the effect of Sophocarpine on immunological liver injury.We found that Sophocarpine can increase proportion of ALB、CHE and TP(p<0.05;p<0.01),decrease the mRNA level of TNF-α,IL-12 and INF-γ(p<0.05;p<0.01).Considering the result of Tunel staining,we found that Sophocarpine have hepato-protection against immunological liver injury.The results of flow cytometry analysis to determinie the number of NK cells in liver showed that proportion of NK cells in lymphocytes is higher than Control group,but decreased after drug administration.Besides,the expression of NK cell surface activated receptor NKG2 D is lower than PD group.DAP12 is the adaptor of NKG2 D,which is closely interrelated with activation of NK cells.By determined with Western Blot and Q-PCR,we have found that the expression of DAP12、ZAP70 and Syk all higher than control group(p<0.05;p<0.01).After pretreatment with Sophocarpine which were all decreased(p<0.05;p<0.01).The results demonstrated that Sophocarpine can inhibit activation of NK cells by down-regulating NKG2D-DAP12 signaling pathways to attenuate liver damage.Conclusion: This study shows that Oxymatrine and Sophocarpine could significantly relieve the liver injury in mice,and its mechanism is related to the inhibition of NK cell activity by drugs.