Scrophularia Ningpoensis Improves Insulin Resistance Through Regulation Of The AMPK-NLRP3 Inflammasome Axis

Background: Insulin resistance（IR）is the pathological basis behind the onset and development of Type 2 diabetes mellitus（T2DM）.Effectively improving IR is the key to treatment of T2 DM.Accumulating evidence demonstrates that disturbed insulin signaling caused by NLRP3 inflammasome-mediated metaflammation forms the pivotal etiology linked to insulin resistance.Scrophularia plant Scrophularia ningpoensis Hemsl is a traditional Chinese medicine.Traditional Chinese medicine theory holds that Scrophularia ningpoensis can nourish yin and clear heat,and can be used for the treatment of "diabetes" unilaterally or in combination.Recent studies have shown that the aqueous extracts of Scrophularia ningpoensis（AESN）and polysaccharide components have therapeutic effects on experimental diabetic animal models,but their mechanism and pharmacodynamic material basis are unknown at present.Objective: To observe the improving effect of AESN on IR,and to reveal whether its improving effect is related to the activation of AMPK and the inhibition of NLRP3 inflammasome activation.Method:1.AESN was prepared and characterized by HPLC.The contents of harpagoside,anglo glycoside C,harpagoside and cinnamic acid in the samples were detected.2.Db/db mice were randomly divided into five groups,and treated by gavage with saline,AESN（50,100 and 200 mg/kg）or metformin（300 mg/kg）for 8 weeks,respectively.Wild type（WT）C57BLKS mice were treated with saline by gavage.Body weight and blood glucose were monitored weekly.Insulin and glucose tolerance tests were performed at 7 and 8 weeks after treatment,respectively.Then mice were sacrificed under anesthesia,and blood samples were collected.The serum was separated by centrifugation and frozen for the determination of TC,NEFA,TG,ALT,HDL-C,AST,LDL-C and insulin contents.The liver,adipose and pancreas were collected for histopathological analysis.The contents of TC,TG and IL-1β in liver tissue were detected after homogenization.The levels of Akt,p-Akt,NLRP3,caspase-1,IL-1β,AMPK,pAMPK,GSK3β and p-GSK3β were detected by Western blotting.3.Mouse primary hepatic parenchymal cells or Hep G2 cells were cultured in a high glucose and high insulin environment to induce the establishment of IR cell model.The effects of AESN on the viability,lipid accumulation,gluconeogenesis,expression and phosphorylation of NLRP3 inflammatory related protein and insulin conduction related signal protein in primary mouse hepatocytes were observed;Effects of AESN on Hep G2 cell viability,gluconeogenesis and the expression of NLRP3 inflammatory related protein and insulin conduction related signal protein;Compound C was used to observe the effect of AESN on the above effects.Result:1.The contents of harpagide,angloride C,harpagoside and cinnamic acid in AESN were 0.37%,0.42%,0.17% and 0.07%,respectively.2.AESN reduced blood glucose and improve IR in db/db mice,but has no significant effect on body weight.Biochemical analysis showed that AESN reduced serum TC,TG,LDL-C,NEFA,ALT and AST levels,and increase HDL-C levels.Biochemical analysis of liver tissue showed that AESN decreased the contents of TC,TG and IL-1β in liver tissue.Histopathological analysis showed that AESN alleviated the accumulation of liver lipid,reduced the size of adipocytes and relieved the abnormal morphology of islets.Western blot results showed that AESN activated AMPK,inhibited NLRP3 inflammasome activation and improved insulin sensitivity.3.Under normal culture condition,AESN incubation increased AMPK phosphorylation and inhibited glucose output,but had no significant effect on cell viability.Under the condition of high glucose plus insulin exposure,AESN can protect the impaired AMPK activity,inhibit the NLRP3 inflammasome activation,improve insulin signal transduction and reduce lipid accumulation.Inhibition of AMPK by Compound C could block the effect of AESN.Conclusion: AESN improves insulin sensitivity via AMPK-mediated NLRP3 inflammasome inhibition.