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

Objective: To observe the protective effect of interleukin-6(IL-6)on glutamate induced apoptosis of SH-SY5 Y cells,and further explore the effect of Signal transduction and activeator of transcription 3(STAT3)acetylation in the progress.Methods:(1)SH-SY5 Y cell line was selected in this study.SH-SY5 Y cells were randomly divided into the following four groups: Control group,IL-6 group(10ng/mL),Glu group(20mmol/L),and IL-6+Glu group.Each group was tested as follows:(1)Cell activity was detected by CCK8 assay.(2)Lactate dehydrogenase(LDH)activity was detected by LDH release assay.(3)Cell morphology were observed by inverted microscope.(4)The apoptosis level of SH-SY5 Y cells was detected by flow cytometry.(5)Real-time PCR and Western Blot were used to detect the expression levels of apoptosis-related genes and proteins in SH-SY5 Y cells,including mRNA and protein expression levels of anti-apoptotic member Bcl-xl,pro-apoptotic component Bax and key apoptotic enzyme cysteinyl aspatate-specific proteinase3(caspase3).(6)The levels of STAT3 lysine 685 acetylation were quantified by Western Blot assay.(2)Further to determine the role of STAT3 acetylation in IL-6 resistance to glutamate induced SH-SY5 Y cells apoptosis,resveratrol(Rev)(12.5μmol/L),which is an activator of SIRT1,was used to inhibit STAT3 acetylation.The inhibition efficiency of resveratrol were detected by Western Blot.Real-time PCR and Western Blot were used to detect the expression changes of Bcl-xl,Bax and caspase3 genes and proteins after deacetylation.Results:(1)Results of cell damage and apoptosis(1)Compared with the Control group,the cell viability rate of the Glu group(65.13±3.52% vs.100±0%)was significantly reduced.Compared with the Glu group,the cell viability rate of the IL-6+Glu group(75.76±3.82% vs.65.13±3.52%)increased significantly.There was no significant difference between the IL-6 group and the Control group.(2)Compared with the Controlgroup,the LDH activity of the Glu group(39.15±5.56 U/L vs.14.24±4.03 U/L)was significantly increased.Compared with the Glu group,the LDH activity of the IL-6+Glu(13.13±4.71 U/L vs.39.15±5.56 U/L)group was significantly decreased.There was no significant difference between the IL-6 group and the Control group.(3)Morphological observation: the cell morphology in the Control group was long spindle,and the cell processes were obvious.The cells in the Glu group became round,shrivelled,clustered,and the cell processes were reduced or disappeared.The rounded and wrinkled cells in the IL-6+Glu group were less than that in the Glu group,and the cell processes were more than that in the Glu group.The cell morphology of IL-6 group was the same as that of Control group.(4)Flow flow apoptosis detection: compared with the Control group,the apoptosis rate of the Glu group(30.28±8.1% vs.7.19±1.44%)was significantly higher.Compared with the Glu group,the apoptosis rate of the IL-6+Glu group(19.83±5.84%vs.30.28±8.1%)significantly decreased.There was no significant difference between the IL-6 group and the Control group.(5)Detection of apoptosis-related genes and proteins:compared with the Control group,the expression of pro-apoptosis-related genes and proteins(Bax mRNA and protein and caspase-3 mRNA and protein-activated fragment(cleaved caspase-3))was significantly increased in the Glu group,and the anti-apoptotic Bcl-xl mRNA and protein expression were significantly decreased in the Glu group.Compared with the Glu group,the expression of pro-apoptotic genes and proteins(Bax mRNA and protein,caspase3 mRNA and cleaved-caspase3 protein)in the IL-6+Glu group were significantly decreased,and the anti-apoptotic Bcl-xl mRNA and protein expression were significantly increased in the IL-6+Glu group.There was no significant difference between the IL-6 group and the Control group.(6)The acetylation levels of STAT3 Lys685 in the IL-6 group and the IL-6+Glu group were significantly higher than those in the Control group and the Glu group.There was no significant difference in the acetylation level between the IL-6 group and the IL-6+Glu group.There was no significant difference between the Control group and the Glu group.(2)Rev inhibits the expression of STAT3 Lys685 acetylated protein.Rev inhibits the effect of IL-6 against the expression of glutamate induced pro-apoptotic genes and proteins(BaxmRNA and proteins,caspase-3 mRNA and cleaved caspase-3 protein).Rev inhibited the effect of IL-6 against the expression of glutamate-induced anti-apoptotic Bcl-xl mRNA and protein.Conlusion:(1)IL-6 protects SH-SY5 Y cells from glutamate-induced apoptosis.(2)IL-6 increases the acetylation of STAT3 Lys685 in SH-SY5 Y cells.(3)IL-6 regulates glutamate-induced SH-SY5 Y cells apoptosis via STAT3 acetylation.