Effects Of Palmatine On P2X₇ Receptor Expression In Trigeminal Ganglion Of Trigeminal Neuralgia Rats And Its Mechanism Of Action

Objective:Trigeminal Neuralgia?TN?refers to severe,recurrent pain that occurs in the area of the trigeminal nerve,such as knife cutting,acupuncture,electric shock or burning.It most often occurs in the second and/or third trigeminal branches,with the right side affected slightly more frequently than the left.It has been reported that palmatine can reduce diabetic neuropathic pain and depression in rats by inhibiting the expression of P2X₇ receptor in the hippocampus,previous studies in our lab have also shown that palmatine can inhibit the expression of CGRP in trigeminal ganglion of rats with trigeminal neuralgia,but there has been no report on whether palmatine can affect the expression of P2X₇ receptor in trigeminal ganglion of trigeminal neuralgia rat.This experiment aims to observe the effect of palmatine treatment on P2X₇ receptor expression in trigeminal ganglion of trigeminal neuralgia rats,to understand the role and possible mechanism of palmatine in P2X₇-receptor-mediated trigeminal neuralgia,so as to provide experimental basis for finding new methods to treat trigeminal neuralgia in clinic.Methods:After buying healthy male Sprague-Dawley rats,they were randomly divided into six groups:sham control group?Sham?,sham treated with palmatine group?Sham+Palmatine?,TN model group?TN?,TN treated with palmatine group?TN+Palmatine?,TN treated with P2X₇shRNA group?TN+P2X₇shRNA?,TN treated with scrambleshRNA group?TN+scrambleshRNA?.The model of trigeminal neuralgia in rats was established by chronic constriction injury of the infraorbital nerve?CCI-ION?,rats in Sham+Palmatine group and TN+Palmatine group were intraperitoneally injected with palmatine solution on the first day after operation for14 consecutive days,rats in TN+P2X₇shRNA group and TN+scrambleshRNA group were injected with P2X₇shRNA and scrambleshRNA sublingual vein on the 10th day after operation.Mechanical withdrawal threshold?MWT?of rats in each group was measured by Mechanical pain test 1 week before modeling and 1,3,5,7,9,11,13and 15 days after operation.The right trigeminal ganglion of rats in each group was removed 15 days after operation,real-time fluorescence quantitative PCR?qPCR?was used to detect P2X₇mRNA expression changes in trigeminal ganglia of rats in each group.The expression changes of P2X₇ receptor and protein,p38 and p-p38 in trigeminal ganglion of rats in each group were detected by immunofluorescence and western blot.Changes of inflammatory cytokines IL-1?and TNF-?in serum of rats in each group were detected by ELISA.Results:Within 2 weeks after operation,compared with the Sham group,the mechanical pain-sensitivity threshold of the right face of the TN group was significantly reduced?p<0.01?,and that of the TN+Palmatine group and TN+P2X₇shRNA group was increased?p<0.01?.On day 15 after operation,q-PCR results showed that the expressions of P2X₇ receptor,IL-1?,and TNF-?in TGs were significantly increased in TN group compared with Sham group?p<0.05?,while the expressions of P2X₇receptor,IL-1?,and TNF-?in TN+Palmatine group and TN+P2X₇shRNA group were decreased in TGs compared with those in TN group?p<0.01?.Immunofluorescence results showed that P2X₇ was co-labeled with GFAP,and P2X₇receptor expression in TN group was significantly increased compared with Sham group?p<0.01?,after Palmatine or P2X₇shRNA treatment,P2X₇ receptor expression in trigeminal ganglion of TN+Palmatine group and TN+P2X₇shRNA group was significantly decreased compared with TN group?p<0.01?.Western blot results showed that P2X₇ receptor expression in TGs in TN group was significantly increased compared with Sham group?p<0.01?,while P2X₇ receptor expression in TN+Palmatine group and TN+P2X₇shRNA group was significantly decreased compared with that in TN group?p<0.01?.Phosphorylation in the p38 pathway showed that the treatment with palmatine and P2X₇shRNA could reduce the elevated p38 phosphorylation level in TGs in the TN group of rats.ELISA results showed that serum levels of inflammatory cytokines IL-1?and TNF-?in the TN group were significantly higher than those in the Sham group?p<0.05?,while serum levels of IL-1?and TNF-?decreased after treatment with palmatine and P2X₇shRNA?p<0.01?.Conclusion:P2X₇ receptor in trigeminal ganglion is involved in pathophysiological changes of cephalic and facial neuropathologic pain in rats with trigeminal neuralgia,by down-regulating the expression of P2X₇ receptor in TG of TN rats,palmatine can inhibit the phosphorylation of p38 signaling pathway and reduce the release of serum inflammatory cytokines IL-1?and TNF-?,thereby alleviating TN.