Role Of STAT3 Acetylation In The Regulation Of Glutamate Induced Neuronal Cells Injury By Interleukin-6

Objective:To investigate the protective effect of interleukin 6（IL-6）on glutamate（Glu）-induced neuronal damage and observe the regulatory effect of signal transducer and activator of transcription 3（STAT3）acetylation modification in this process,and the relationship with phosphorylation modification.Methods:1.The effect of IL-6 on apoptosis induced by glutamate:SH-SY5Y cells were designed in Control group,IL-6 group,Glu group and IL-6+Glu group.The apoptotic expression levels of each groups were determined by western blot（WB）.2.The effect of IL-6 on glutamate-induced calcium overload:SH-SY5Y cells were designed in Control group,calcium free group and IL-6 group.The intracellular calcium(Ca²⁺)levels were detected by dynamic calcium imaging system（PTI）.3.Regulation of STAT3 acetylation and phosphorylation on IL-6 resistance to glutamate induced cell injury and their relationship:（1）The expression of STAT3 acetylated and phosphorylated proteins was detected by WB.（2）The effect of CREB binding protein（CBP）on STAT3 acetylation:Immunofluorescence and WB were used to observe the expression of CBP in cytoplasm and nucleus;The relationship between CBP and STAT3acetylation was detected by Co-immunoprecipitation（CO-IP）.（3）The effect of inhibiting STAT3 acetylation on cell injury:（1）The effect of resveratrol（Rev）on STAT3 acetylation and apoptotic,experiments were designed in Control group,Glu group,IL-6+Glu group and IL-6+Glu+Rev group,the expression of STAT3 acetylation and apoptotic protein were detected by WB.（2）The effect of Rev on calcium overload:The changes of intracellular Ca²⁺level in IL-6 group and IL-6+Rev group were detected by PTI.（4）The relationship between STAT3 acetylation and phosphorylation:（1）Rev inhibits STAT3 acetylation,phosphorylation of STAT3 was detected by WB.（2）CBP silences lentivirus transfected cells,the changes of STAT3 acetylation and phosphorylation were detected by WB.（3）Stattic inhibits STAT3phosphorylation,phosphorylation and acetylation of STAT3 were detected by WB.Results:1.The effect of IL-6 on cell apoptosis induced by glutamate:the results of WB showed that compared with Control group,Glu group increased the expression of Bax and decreased the expression of Bcl-2（all P<0.05）,suggesting that glutamate can induce apoptosis;Compared with Glu group,IL-6+Glu group decreased the expression of Bax and increased the expression of Bcl-2（all P<0.05）,suggesting that IL-6 inhibits glutamate induced apoptosis.2.The effect of IL-6 on glutamate-induced calcium overload:the results of PTI showed that compared with Control group,intracellular Ca²⁺was significantly decreased in IL-6 group（P<0.05）,suggesting that IL-6 can reduce the calcium overload induced by glutamate;Glutamate did not induce the increase of intracellular Ca²⁺in the calcium free group,suggesting that the calcium overload induced by glutamate was caused by external calcium influx.3.Regulation of STAT3 acetylation and phosphorylation on IL-6 resistance to glutamate induced cell injury and their relationship:（1）STAT3 acetylation and phosphorylation modification:the results of western blot showed that compared with Control group and Glu group,the expression levels of Ac-STAT3 and p-STAT3 were significantly increased in IL-6 group and IL-6+Glu group（all P<0.05）,suggesting that IL-6 can activate the acetylation and phosphorylation of STAT3.（2）The effect of CBP on STAT3 acetylation:（1）The results of western blot showed that compared with Control group and Glu group,the expression levels of CBP were significantly increased in IL-6 group and IL-6+Glu group（P<0.05）,suggesting that IL-6can increase CBP expression.（2）The results of immunofluorescence and western blot showed that compared with Control group,the expression levels of cytoplasmic CBP were significantly increased in IL-6 group（P<0.05）,suggesting that IL-6 can induce CBP expression in cytoplasm.（3）The results of Co-IP showed that CBP can specifically co-precipitate with Ac-STAT3 and STAT3,suggesting that CBP,Ac-STAT3 and STAT3bind to each other.（3）The effect of inhibiting STAT3 acetylation on cell damage:（1）The results of western blot showed that compared with IL-6+Glu group,the expression levels of Ac-STAT3 were decreased in Rev+IL-6+Glu group（P<0.05）,the acetylation of STAT3could be inhibited by Rev.（2）Compared with IL-6+Glu group,Bax expression in IL-6+Glu+Rev group was significantly increased,and Bcl-2 expression was decreased（all P<0.05）,it was confirmed that Rev blocked the anti apoptotic effect of IL-6 while blocking the acetylation of STAT3,which indicated that the acetylation of STAT3 was involved in the anti apoptotic effect of IL-6.（3）The results of PTI showed that compared with IL-6group,intracellular Ca²⁺was significantly increased in Rev+IL-6 group（P<0.05）,it was confirmed that Rev blocked the effect of IL-6 on calcium overload while blocking the acetylation of STAT3,which further indicated that STAT3 acetylation was involved in the anti calcium overload effect of IL-6.（4）The relationship between STAT3 acetylation and phosphorylation:（1）The results of western blot showed that compared with IL-6+Glu group,the expression levels of p-STAT3 decreased in Rev+IL-6+Glu group（P<0.05）,indicating that while inhibiting STAT3 acetylation,it inhibit STAT3 phosphorylation at the same time.（2）Compared with NC group and NC+IL-6 group,the expression levels of Ac-STAT3 decreased in CBPsh RNA group and CBPsh RNA+IL-6 group（all P<0.05）,indicating that silencing CBP inhibit STAT3 acetylation;Compared with NC group and NC+IL-6 group,the expression levels of p-STAT3 decreased in CBPsh RNA group and CBPsh RNA+IL-6 group（all P<0.05）.The results proved that silencing CBP inhibited STAT3 acetylation and inhibited STAT3 phosphorylation at the same time.（3）Compared with IL-6+Glu,the expression levels of p-STAT3 decreased in Stattic+IL-6+Glu group（P<0.05）,indicating that the phosphorylation of STAT3 be inhibited by Stattic;Compared with IL-6+Glu group,the expression levels of Ac-STAT3 decreased in Stattic+IL-6+Glu group（P<0.05）,indicating that while inhibiting phosphorylation of STAT3,it can inhibit the acetylation.Conclusions:1.IL-6 protects nerve cells by reducing cell apoptosis and reducing intracellular calcium overload,and resists excitatory neurotoxicity induced by glutamate;2.The acetylation modification of STAT3 participates in the neuroprotection of IL-6,and it is interdependent with the phosphorylation modification,and both are indispensable.