Effect Of CBS On P2X₃ Receptor In Dorsal Root Ganglion Of Rats With Chronic Constriction Injury Of Sciatic Nerve

Objective: To investigate the role of Cystathionine-β-Synthetase(CBS)in neuralgia caused by chronic constriction injury(CCI)in rat sciatic nerve and its regulation mechanism for P2X3 receptor in dorsal root ganglion in neuropathic pain.Methods:Thirty-six SPF healthy male Sprague-Dawley rats(180~200g,5-6 weeks),in which intrathecal catheters were successfully implanted,were divided into 3 groups(n =12):(group Sham),chronic constriction injury(group CCI)and CBS inhibitor O-(carboxymethyl)hydroxylamine hemihydrochloride(group CCI+AOAA).3 days after intrathecal catheters were successfully implanted,the sciatic nerve was only exposed but not occluded in group Sham,and the group CCI and the group CCI+AOAA were exposed to the right sciatic nerve trunk behind the femur,and were loosely ligated 4 places with a chrome gut(4/0)at intervals of 1 mm.Intrathecal injection was performed once a day for14 consecutive days starting from 1st day after operation.AOAA(10ug/kg)10 μl and normal saline 10 μl were intrathecally injected in group CCI+AOAA,and normal saline 20μl was intrathecally injected in group Sham and group CCI.Thermal withdrawal latency and mechanical withdrawal threshold were measured 30 min after intrathecal injection of AOAA and saline at day 1 before surgery,1,3,7,10,and 14 days after surgery.Six rats in each group were decapitated,and the L4-L6 dorsal root ganglia on the right side of the spinal cord were taken at 7d and 14 d.Western blot was used to detect the expression of CBS and P2X3 receptors,and the distribution of CBS and P2X3 receptors were observed by double immunofluorescence labeling.Results: 1 The rats developed chronic neuralgia such as dorsiflexion,squatting,and sling,and no autophagy or motor dysfunction,on the 1st postoperative day in the group CCI.The above-mentioned chronic neuralgia performance was observed to be reduced in the group CCI+AOAA.The above neuropathic manifestations were not observed in the group Sham.2 Compared with 1st day before operation,the group CCI showed thermal pain and mechanical allodynia at 1st day after operation,and TWL and MWT decreased significantly(P<0.05);Compared with group Sham,TWL and MWT decreased significantly at each time point in group CCI(P<0.05);Compared with CCI group,TCL and MWT increased at each time point in group CCI+AOAA(P<0.05).3 Compared with group Sham,CBS and P2X3 receptor expression were significantly up-regulated at the 7th and 14 th day after operation in group CCI(P<0.05);Compared with group CCI,CBS and P2X3 receptor expression were down-regulated at the 7th and 14 th day after operation in group CCI+AOAA(P<0.05).4 Immunofluorescence double-labeling staining revealed that CBS and P2X3 receptors were co-expressed on spinal DRG neurons.Conclusion: 1 Both CBS and P2X3 receptors are involved in the regulation of neuropathic pain allergy caused by chronic constriction injury of the sciatic nerve.2 The CBS-H2 S signaling pathway plays an important role in the rat model of sciatic nerve constriction injury.Endogenous hydrogen sulfide synthase CBS may participate in the development and maintenance of neuropathic pain by up-regulating the expression of P2X3 receptor in the dorsal root ganglia of CCI rats.