Effect And Mechanisms Of Paeoniflorin On Inflammation In Diabetic Liver Injury

Background and objective:Diabetic liver injury（DLI）is a common complication and damages the quality of life of diabetic patients.When present in a hyperglycemic environment for a long time,it can cause a disorder of metabolism in the liver,lead to liver inflammation and even liver fibrosis as the disease progresses.Therefore,it is of great significance to explore the pathogenesis of diabetic liver injury and find an effective therapeutic target.Paeoniflorin（PF）,the main active component of total glycoside of paeony（TGP）,has shown anti-inflammatory,antioxidant and immunomodulatory effects in many diseases.In recent years,studies have confirmed that paeoniflorin has a potential therapeutic effect on a variety of liver diseases.However,it is still unclear whether PF has a protective effect on liver injury in type 2 diabetes mellitus.We further investigated the protective effect and mechanism of paeoniflorin on liver injury in type 2 diabetes mellitus,searching for potential therapeutic targets for diabetic liver injury.Methods:In vivo study: In this study,db/db mice were used as a type 2 diabetic mouse model,and they received gavage doses of PF（25,50 and 100 mg/kg）for 12 weeks.The db/m mice were used as control group.The animals were divided into six groups: db/m group,db/m + paeoniflorin group,db/db group,db/db + paeoniflorin group（25,50,100mg/kg）.Serum ALT,AST,TG,TC and FFA were measured after 12 weeks.HE staining,oil red O staining and transmission electron microscopy were used to observe the pathological changes of liver.The expression levels of IL-1β,IL-18 and TNF-α were detected using Western blot,Real-time PCR and immunohistochemistry.We also detected the expression of F4/80 in mouse liver by immunohistochemistry.The expression of TXNIP was also detected by Western blot and immunohistochemistry.The expression of NLRP3,ASC and Caspase-1 were detected by Western blot.In vitro study: Mouse liver parenchymal cell line AML12 was selected for the experiment.The toxic dose and the optimal concentration of the drug were examined using MTT assay,and the time of high glucose stimulation was screened using Western blot and Real-time PCR.Experimental groups: control group,control + paeoniflorin group,mannitol group,high glucose group,high glucose + paeoniflorin（4,8,16μmmol/L）group.We treated AML12 cells induced by HG with paeoniflorin.The effect of paeoniflorin on the expression of inflammatory cytokines（IL-1β,IL-18 and TNF-α）was detected by Western blot and Real-time PCR.The expression of NLRP3,ASC and caspase-1 were analyzed by Western blot.We analyzed the effect of paeoniflorin on the expression of TXNIP by Western blot and immunofluorescence.Through molecular docking experiments and cellular thermal shift assay（CETSA）,we studied the binding ability of paeoniflorin to TXNIP in AML12 cells.The binding ability of TXNIP and NLRP3 was tested by CO-IP experiment.After we knock out TXNIP in AML 12 cells with TXNIP si RNA,the inflammatory response of hepatocytes were detected using Western blot and Real-time PCR.Results:In vivo study: We found that paeoniflorin significantly reversed abnormal liver function and liver pathology in db/db mice.Western blot,immunohistochemistry and Real-time PCR demonstrated that paeoniflorin significantly inhibited the expression of proinflammatory cytokines（IL-1β,IL-18 and TNF-α）in the liver.Furthermore,we found that expression of TXNIP in the liver of diabetic mice was increased,while the expression of TXNIP was inhibited by paeoniflorin.Western blot showed that the activation of NLRP3 inflammasome was increased in db/db mice,while it was significantly inhibited after paeoniflorin treatment.In vitro study: The optimal concentrations of paeoniflorin were 4,8 and 16 μmmol/L according to the results of MTT assay.We found that the inflammatory response was most pronounced when the AML12 cells were stimulated with high glucose for 24 hours.Western blot and Real-time PCR demonstrated that paeoniflorin decreased the expression of proinflammatory cytokine（Il-1β,Il-18 and TNF-α）in mouse AML12 cells induced by high glucose.Western blot showed that the expressions of TXNIP,NLRP3,ASC,caspase-1 were increased in the HG group,while paeoniflorin decreased the expression of these proteins.Immunofluorescence also showed that paeoniflorin decreased the expression of TXNIP.We also found that TXNIP may be a target of paeoniflorin through molecular docking experiments and CETSA.CO-IP experiment showed that paeoniflorin treatment reduced the binding ability of TXNIP to NLRP3.After TXNIP was silenced using si RNA in AML12 cells,paeoniflorin could not further reverse hepatocytes injury induced by high glucose.Conclusion:（1）In vivo experiments,paeoniflorin reversed abnormal liver function and inhibited inflammatory response of liver.（2）In vitro experiments,paeoniflorin inhibited HG-induced hepatocyte injury and inflammatory response.（3）Paeoniflorin attenuated diabetic liver injury by targeting TXNIP to inhibit the activation of NLRP3 inflammasome.