Study On The Role Of Catestatin In P2X₄ Receptor-mediated Activation Of Primary Cultured Glial Cells From Rat

Background and Objective:Under pathological conditions,glial cells（satellite glial cells,microglial cells,oligodendrocytes）rapidly changed from the static form to the activated form.To explore the activation of glial cells and its possible mechanism is of great research value in understanding the pathophysiological processes of nervous system diseases.Adenosine triphosphate（ATP）can act on dorsal root ganglion（DRG）satellite glial cells（SGCs）and spinal cord microglia（MG）P2X₄receptor-mediated gel Activation of stromal cells.The neuroendocrine polypeptide catestatin（CST）,which is an inflammatory mediator,is closely related to the pathogenesis of cardiovascular diseases,and its role in the activation of glial cells with inflammatory factors has not been reported.This study aimed to observe the role of CST in P2X₄receptor-mediated activation of dorsal root ganglion SGCs and spinal cord MG activation and its possible mechanism.Methods:The research object of this subject is primary cultured rat dorsal root ganglion SGCs and spinal cord MG.CCK8,q PCR（Quantitative real-time PCR,q PCR）and Western Blot method were used to detect the effects of various concentrations of ATP and CST on the activity of dorsal root ganglion SGCs;The expression of P2X₄receptor in primary cultured dorsal root ganglion SGCs and spinal cord MG were detected by immunofluorescence double labeling method;The expressions of P2X₄mRNA and ERK/P-ERK receptor protein in primary cultured dorsal root ganglion SGCs and spinal cord MG were detected by q PCR and Western Blot,respectively;The kit was used to detect the content of TNF-αand IL-1βin primary cultured dorsal root ganglion SGCs and spinal cord MG supernatant.Results:1.Detection of DRG SGCs and spinal cord MG specific markers GFAP/CD11b by immunofluorescence method,the results showed that the purity of primary cultured SGCs/MG was above 95%and 90%,which were in full compliance Requirements for later experiments.2.Effect of ATP/CST on the survival of primary cultured dorsal root ganglion SGCs:CCK8 method was used to detect the effect of different concentrations of ATP/CST on the survival of primary cultured dorsal root ganglion SGCs,suggesting that CST may enhance ATP-induced dorsal root ganglion SGCs activation.3.Real-time quantitative PCR to detect the effect of ATP on P2X₄mRNA levels:q PCR method was used to detect the relative expression of P2X₄mRNA in primary cultured dorsal root ganglion SGCs in each group.The results showed that when the concentration of ATP treatment was 0.1m M,the upward trend of P2X₄mRNA expression was the most obvious.Western blot detection of ATP on P2X₄receptor protein expression results were similar to real-time quantitative PCR results.4.The effect of CST on the primary cultured dorsal root ganglion SGCs and spinal cord MG morphology:inverted microscope observation of normal dorsal root ganglion SGCs/spinal cord MG added ATP,cell morphological changes were enhanced.5.Real-time quantitative PCR to detect the effect of CST on P2X₄mRNA levels:The results showed that compared with the control group,P2X₄mRNA expression in the ATP group was significantly increased;P2X₄mRNA expression in the ATP+CST group was significantly higher than that in the ATP group.The above results indicate that CST can upregulate the P2X₄mRNA levels in primary cultured dorsal root ganglion SGCs/spinal cord MG.6.Western blot analysis of the effect of CST on P2X₄receptor protein expression:The results showed that:compared with the control group,the expression of P2X₄receptor protein in the ATP group was significantly increased;compared with the ATP group,the P2X₄protein in the ATP+CST group The expression level was significantly higher than that in the ATP group.The above results indicate that CST can enhance the expression of P2X₄receptor in dorsal root ganglion SGCs/spinal cord MG induced by ATP.7.Co-expression of GFAP and P2X₄receptors in primary cultured dorsal root ganglion SGCs and CD11b and P2X₄receptors in spinal cord MG were detected by immunofluorescence double labeling method,the results showed that the coexpression of P2X₄receptor and GFAP/CD11b in glial cells of ATP group was higher than that of control group,the ATP+CST group was higher than the ATP group.The results suggest that CST can increase the co-expression of dorsal root ganglion SGCs and spinal cord MG P2X₄and GFAP/CD11b.8.The role of ERK1/2 signaling pathway in CST enhancing P2X₄-mediated activation of primary cultured dorsal root ganglion SGCs/spinal cord MG:The ratio of the integrated optical density of p-ERK1/2 and ERK1/2 in the dorsal root ganglion SGCs and spinal cord MG in the ATP group was higher than that in the Control group,compared with the ATP group,the ATP+CST group,the integrated optical density ratios of p-ERK1/2 and ERK1/2 in the ATP group treated with CST were significantly increased,compared with the control group,the statistical data of the ATP group were significantly different.The results show that the enhanced effect of CST on P2X₄receptor-mediated dorsal root ganglion SGCs and spinal cord MG activation may be related to ERK1/2 phosphorylation9.ELISA was used to detect the content of TNF-αand IL-1βin the primary cultured dorsal root ganglion SGCs and spinal cord MG supernatant:statistical analysis showed that the expression of TNF-αin the ATP group was higher than that in the control group,compared with the ATP group,the expression level of TNF-αin the ATP+CST group was higher than that in the ATP group;the detection results of IL-1βshowed the same trend.These results indicate that CST can increase the expression of TNF-αand IL-1βin activated dorsal root ganglion SGCs and spinal cord MG.Conclusion:CST can enhance the P2X₄receptor-mediated activation of primary cultured dorsal root ganglion SGCs and spinal cord MG,and the ERK-MAPK signaling pathway may be involved in its activation process.