Mechanistic Study Of Curcutin-activated Nrf2 System And Its Relation To Alleviating Mitochondrial Damage And Insulin Resistance

ObjectiveMitochondrial dysfunction which is induced by oxidative stress involves in insulin resistance(IR).The mechanism of curcumin(Cur)for alleviating mitochondrial oxidative stress and IR by activating Nrf2 system has not been clearly elucidated.The purpose of our study was to investigate the mechanism of Nrf2 for sensitizing insulin sensitivity,the target of this action and related signaling on whether and how curcumin could relieve the mitochondrial damage and IR.The study will elucidate the pharmacological basis for curcumin translation and thus lay the theoretical foundation for the prevention and treatment of diabetes mellitus.MethodsFirst of all,we observed the effect of Cur intervention in the IR model of C57BL/6J mice induced by high fat diet,and discussed the effective target for Cur protected mitochondria and reversed IR.In order to deeply understand the aforementioned effect of Nrf2 antioxidant system,the HepG2 IR model was induced by palmitic acid treatment combined with the Nrf2 inhibitor retinoic acid(RA)treatment to observe the effect of Cur.Since mitochondrial morphological regulation is closely related to its function the mitochondrial splitting inhibitor Mdivi-1 was also treated,and we explored whether Cur could play a role in the regulation of mitochondrial function,activation of the Nrf2 system and insulin signaling as well via modulating mitochondrial morphology.Since the mitochondrial redox balance was closely related to the calcium homeostasis,we observed the effects of Cur and Nrf2 on the mitochondrial calcium content and used mitochondrial calcium channel inhibitor to further explore its regulation on insulin signaling.The experiments are as follows:The animal IR model were induced by using Male C57BL/6J mice to feed 60%high fat diet and according to the results of intraperitoneal glucose tolerance test(IPGTT)to judge whether the IR model was established successfully.HepG2 cells were chosen as the cellular IR model which was induced by the treatment of 600μM saturated fatty palmitate acid(PA).HFD-fed mice were treated with curcumin after the IR model wasestablished.Curcumin was given by oral gavage daily at the dose of 50mg/kg BW for 2weeks.Furthermore,1μM Nrf2 inhibitor retinoic acid(RA),50μM mitochondrial cleavage inhibitor Mdivi-1,30μM ruthenium red(RuR)and 10μM Curcumin were treated to HepG2 IR model.Then we detected mitochondrial membrane potential,ROS,calcium content and WB was employed to analyze protein content.WB was employed to analysis the changes of mitochondrial cleavage and fusion proteins(Drp1,Mfn2 and OPA1)and related regulatory factors(Nrfl,mtTFA,TFB1M and TFB21M)in C57BL/6J mice.The content of ATP and the activity of ATPase(Na+K+-ATPase,Mg2+-ATPase,Ca2+-ATPase,Ca2+Mg2+-ATPase)in the liver of mice were measured by phosphomolybdate colorimetric method to evaluate the effect of curcumin on mitochondrial energy metabolism in liver of mice.The content of malonaldchyde(MDA)indicated for mitochondrial lipid peroxidation was determined by TAB combined techniques.The mitochondrial membrane potential(MMP)was measured by using JC-1 kit.The mitochondria ROS were detected by staining with MitoSOXTM in HepG2 cells.At the same time,WB employed to analysis the PKBSer473 phosphorylation,expression of GRP78,IκBα and related mitochondrial protein as well as regulatory factors after cells were treated with RA,Mdivi-1 and RuR.ResultsMitochondrial fusion proteins were inhibited(decreased Mfn2,OPA1 fusion proteins),increased expression of mitogen-regulated protein(increased Drp1 protein)and downregulated transcriptional regulatory proteins(Nrfl,mtTFA,TFB1M and TFB2M),both in high-fat diet-induced IR model and PA-induced cell IR model.Curcumin can significantly attenuated or even reversed these effects caused by high fat.In addition,Cur could markedtly increased the ATP content in the liver tissue of IR mice and enhance the activity of Na+ K+-ATPase,Mg2+-ATPase,Ca2+-ATPase,Ca2+ Mg2+-ATPase.In addition,Cur can partially antagonize PA-induced decrease of mitochondrial ROS content and function damage of mitochondria,that is decreased;mitochondrial membrane potential.When the cells were treated with Nrf2 inhibitor,the above-mentioned improvements by Cur were significantly inhibited.Thus,we can recognize that Cur regulates mitochondrial function via activating of Nrf2.Further studies have shown that after PA-induced IR cells were treated with either mitochondrial cleavage agents Mdivi-1 or Cur,both of them can up-regulate mitochondrial synthetic and transcriptional regulatory proteins,increase mitochondrial fusion protein exprssion,and inhibit mitochondrial cleavage regulatory protein.Finally,since curcumin reduces mitochondrial calcium overload in the IR cells.In order to further evaluate the efficacy of curcumin,we used IR cells and treated them with mitochondrial calcium channel inhibitor,and found that mitochondrial calcium overload of IR cells was inhibited,.This inhibition can alsp play a role in inhibition of inflammation,endoplasmic reticulum stress,while activating Nrf2 System and enhancing insulin signaling.ConclusionAll of the experiments in vitro and vivo shows that Curcumin can promote up-regulation of mitochondrial synthesis and transcriptional regulatory proteins,enhance mitochondrial fusion protein and inhibit the expression of mitochondrial biogenesis-regulating proteins,and these mitochondrial regulations are related to the effects of Cur on its improvement of Nrf2 antioxidant function and regulation of insulin signaling.Therefore,curcumin can activate the Nrf2 system by which to attenuate the mitochondrial oxidative stress,modulate the morphology and function of mitochondria..These effects are the important mechanisms in improving insulin resistance,and curcumin interacts with the mitochondrial oxidative stress and inhibies calcium overloading-induced activation of inflammation and endoplasmic reticulum stress through activating Nrf2 system function,and thus improve insulin sensitivity.