Study On Catestatin Involving In Neuropathic Pain Mediated By P2X₄ Receptor Of Dorsal Root Ganglia In Rats

Background and Objective:Neuropathic pain caused by nerve injury is a common and refractory disease.The molecular biological changes of primary sensory ganglia pay an important role in the pathogenesis of neuropathic pain.Adenosine triphosphate（ATP）can act on P2X₄ receptor of satellite glial cells（SGCs）in dorsal root ganglion（DRG）,which is closely related to neuropathic pain.Catestatin（CST）is a neuroendocrine peptide which is derived from the chromogranin A（CHGA）.Recently,the role of CST in acute and chronic pain has attracted much attention,but its exact mechanism of action remains unclear.This study was designed to investigate the effects and the possible mechanism of CST on P2X₄ receptor-mediated neuropathic pain in chronic constriction injury（CCI）rats by intrathecal CST-siRNA to down-regulate the expression of CST in CCI rats and by intrathecal CST in CCI rats,so as to provide a new way to alleviate or even cure neuropathic pain.Methods:Healthy and clean male Sprague-Dawley rats（180–220g）were randomly divided into divided into 7 groups:（1）Control（Ctrl,no operation）;（2）Sham operation group in which the sciatic nerve was separated without any ligation（Sham）;（3）CCI;（4）CCI treated with scrambled CST peptide（CCI+NC）;（5）CCI treated with CST（CCI+CST）;（6）CCI treated with CST-siRNA group（CCI+CST-siRNA）;（7）CCI treated with the scrambled siRNA group（CCI+NC-siRNA）.Each group contained six rats and all rats participated all procedures.According to different groups,CCI rats were modeled and postoperative administrated.The changes of the thermal withdrawal latency（TWL）and the mechanical withdrawal threshold（MWT）were observed by behavioral test.The expression of P2X₄ mRNA in the DRG of rats was detected by real-time PCR（qPCR）.The expression of the P2X₄ receptor protein level in the DRG of rats was assessed by western blot.The co-expression of P2X₄ receptor and GFAP in the DRG of rats was detected by double-labeled immunofluorescence.What’s more we tested whether the MAPK and NF-(B signal pathways were activated with western blot.Production of TNF-α,IL-1βin serum,and CST in DRG of rats were assessed by Enzyme linked immunosorbent assay（ELISA）.Statistical comparisons of the above results were made.Results:1.According to behavioral test,all CCI rats had lower MWT and TWL than Control group after the operation（p<0.01）.MWT and TWL for the Control and Sham groups did not differ across days（all p>0.05）.MWT and TWL did not significantly differ among the CCI,CCI+NC,CCI+NC-siRNA,CCI+CST,and CCI+CST-siRNA groups on Day 1,3,or 5（all p>0.05）.However,MWT and TWL were much lower in the CCI+CST group than that in the CCI group from Day 7through Day 11（all p<0.01）.In contrast,MWT and TWL were significantly greater in the CCI+CST-siRNA group than in the CCI+CST from Day 7 through Day 11（all p<0.01）.MWT and TWL did not significantly differ among the CCI,CCI+NC,and CCI+NC-siRNA groups at any time points（all p>0.05）.2.According to qPCR analysis,the expression of P2X₄ mRNA was significantly lower in the Control group than in the CCI group（p<0.01）.P2X₄ mRNA expression was significantly higher in the CCI+CST group than in the CCI group（p<0.01）.However,expression was much lower in the CCI+CST-siRNA group than in the CCI+CST group（p<0.01）.3.According to western blot,P2X₄ protein level was significantly higher in the CCI group than in the Control group（p<0.01）.P2X₄ protein level was significantly higher in the CCI+CST group than in the CCI group（p<0.01）and was significantly lower in the CCI+CST-siRNA group than in the CCI+CST group（p<0.01）.Phosphorylation level of p38 MAPK signaling was higher in the CCI group than in the Control group（p<0.01）.Additionally,phosphorylation was significantly higher in the CCI+CST group than in the CCI group（p<0.01）.Furthermore,phosphorylation level of the p38 MAPK signaling was lower in the CCI+CST-siRNA group than in the CCI+CST group（p<0.01）.ERK phosphorylation levels followed the same tendency.However,JNK phosphorylation level was higher in the CCI group than in the Control group（p<0.01）.JNK phosphorylation levels did not differ among the CCI and CCI+CST groups（p>0.05）,p-p65 levels showed the same tendency with p-JNK.4.According to double-labeled immunofluorescence analysis,co-expression of P2X₄ receptor and GFAP was higher in the CCI+CST group than in the CCI group.Co-expression was much lower in the CCI+CST-siRNA group than in the CCI+CST group.5.According to ELISA,the concentrations of TNF-α（pg/ml）,IL-1β（pg/ml）in serum,and CST（ng/ml）in DRG of rats in the CCI group were greater than that in the Control group（p<0.01）.In addition,TNF-α,IL-1βand CST levels were higher in the CCI+CST group than in the CCI group（p<0.01）.Conclusion:CST can aggravate neuropathic pain of CCI rats mediated by the P2X₄ receptor on the surface of SGCs in DRG.SGCs activation,p38 and ERK-MAPK signaling pathways may be involved in the pathophysiological process.