| Objective: Neuropathic pain is defined as pain caused by a lesion or disease of the somatosensory system.Neuropathic pain is one of the most common types of chronic pain.There are currently approximately 90 million neuropathic pain patients in China.However,due to the complex pathogenesis of neuropathic pain,there is still a lack of effective treatment methods.In recent years,clinical studies have confirmed that botulinum toxin type A(BTX-A)is effective in treating neuropathic pain with mild side effects,but its analgesic mechanism is still unclear.Studies have shown that the analgesic effect of BTX-A is mainly related to microglia.The activation of microglia is one of the necessary conditions for the formation of NP.The activation of microglia induces two functional phenotypes: pro-inflammatory(M1)phenotype and anti-inflammatory(M2)phenotype.The neuroinflammation mediated by M1 type microglia and the pro-inflammatory factors it releases subsequently plays a vital role in NP,while M2 type microglia antagonize the neuroinflammation mediated by M1 type microglia by releasing anti-inflammatory cytokines.The P2 X receptor family is a family of purinergic receptors mainly found on microglia.It is found that the P2X7 receptor induces the massive release of cellular inflammatory factors by mediating the activation of microglia,which promotes neuroinflammation.Participate in the occurrence and maintenance of neuropathic pain.The latest research shows that P2X7 receptor is involved in the regulation of microglia M1/M2 polarization,and BTX-A can inhibit the expression of P2X3,a member of the P2 X receptor family,suggesting that BTX-A may regulate microglia polarization through P2X7 receptor.In summary,we speculate that BTX-A may regulate the M1/M2 polarization of microglia by inhibiting the expression of P2X7 receptors to inhibit neuroinflammatory response,thereby alleviating neuropathic pain.This study aims to further clarify the mechanism of BTX-A’s analgesic effect and provide a basis for clinical application of BTX-A.Methods:1、Animal experiment.To study the effect of BTX-A on pain threshold,microglia polarization and P2X7 receptor in NP model rats.Experimental groups:Sprague-Dawley male rats were randomly divided into the following four groups:Sham,NP,BTX-A-10,and BTX-A-20 group.Except for the Sham group,neuropathic pain model induced by chronic compression injury(CCI)were established for the other three groups.Experiment contents:Observe the changes of MWT(mechanical withdrawal threshold)and TWL(thermal withdrawal latency)in each group of rats.Immunofluorescence was used to assess the expression levels of microglia marker Iba1,M1 marker CD68 and M2 marker CD206 in the spinal cord.Flow cytometry was used to assess the proportions of both M1 and M2 microglia in the spinal cord.Western blot was used to assess the protein level of P2X7.2、Cellular experiment1)In vitro experiments,observe the effects of BTX-A on microglia polarization and P2X7 receptors.Experimental groups: The rat microglial cell line(HAPI)were cultured in vitro and randomly divided into 3 groups: normal culture(Ctrl)group,LPS(100 ng/m L)group,and LPS+BTX-A(0.1 n M)group.Experiment contents:q RT-PCR was used to assess the m RNA levels of the M1 marker i NOS and the M2 marker Arg-1.ELISA was used to assess the levels of M1-related cytokine IL-6,TNF-α and M2-related cytokine IL-10.Western blot was used to assess the protein level of P2X7.2)In vitro experiments,observe whether BTX-A regulates the M1/M2 polarization of microglia by inhibiting the expression of P2X7 receptor.Experimental groups:(1)P2X7 overexpression vector and control vector were transfected into HAPI cells,and then treated with LPS and BTX-A.That is,it is divided into the following five groups: Ctrl,LPS,LPS+BTX-A,LPS+BTX-A+Vector,and LPS+BTX-A+P2X7 groups.(2)The P2X7 agonist Bz ATP(200 μM)and BTX-A were used to simultaneously treat HAPI cells.That is,it is divided into the following four groups: Ctrl,LPS,LPS+BTX-A,and LPS+BTX-A+Bz ATP groups.Experiment contents:Same as above.Results:1.The measurement results of MWT and TWL showed that,compared with the Sham group,the MWT and TWL values of the NP group decreased significantly(P<0.01).However,compared with the NP group,the MWT and TWL values of the BTX-A-10 and BTX-A-20 groups were significantly increased(P<0.01 or P<0.05).The results of immunofluorescence showed that compared with the Sham group,the number of Iba-1+ CD68+ cells(M1)in the NP group increased significantly(P<0.01);while the number of Iba-1+ CD206+ cells(M2)had no statistically significant difference(P>0.05).Compared with the NP group,the number of Iba-1+CD68+ cells(M1)in the BTX-A-10 and BTX-A-20 groups was significantly reduced(P<0.01 or P<0.05),while the number of Iba-1+CD206+ cells(M2)was significantly increased(P<0.01).The results of flow cytometry showed that compared with the Sham group,the number of M1 type microglia in the NP group was significantly increased(P<0.01),and the difference in the number of M2 type microglia was not statistically significant(P>0.05).Compared with the NP group,the number of M1 type microglia in the BTX-A-10 and BTX-A-20 groups was significantly reduced(P<0.01),and the number of M2 type microglia was significantly increased(P<0.01 or P<0.05).The results of Western blot showed that compared with the Sham group,the expression of P2X7 protein in the NP group was significantly increased(P<0.01);compared with the NP group,the expression of P2X7 protein in the BTX-A-10 and BTX-A-20 groups was significantly reduced(P<0.01).2.The results of q RT-PCR and ELISA showed that compared with the Ctrl group,the expression of i NOS and M1-related cytokines(TNF-α and IL-6)in the LPS group increased significantly(P<0.01).At the same time,there was no statistically significant difference in the expression of Arg-1 and M2 type microglia-related cytokines(IL-10)(P>0.05).Compared with the LPS group,the expression of i NOS and M1-related cytokines in the LPS+BTX-A group was significantly reduced(P<0.01 or P<0.05),and the expression of Arg-1 and M2-type microglia-related cytokines was significantly increased(P<0.01).The results of Western blot showed that compared with the Ctrl group,the expression of P2X7 protein in the LPS group was significantly increased(P<0.01);compared with the LPS group,the expression of P2X7 protein in the LPS+BTX-A group was significantly reduced(P<0.01)).3.1)The results of Western blot showed that compared with the Ctrl group,the expression of P2X7 protein in the LPS group was significantly increased(P<0.01);compared with the LPS group,the expression of P2X7 protein in the LPS+BTX-A group was significantly reduced(P<0.01).Importantly,compared with the LPS+BTX-A+Vector group,the expression of P2X7 protein in the LPS+BTXA+P2X7 group was significantly increased(P<0.01).The results of q RT-PCR and ELISA showed that compared with the Ctrl group,the expression of M1 markers(i NOS,TNF-α and IL-6)in the LPS group was significantly up-regulated(P<0.01),and M2 markers(Arg-1,IL-10)expression difference was not statistically significant(P>0.05).Compared with the LPS group,the expression of M1 markers in the LPS+BTX-A group was significantly down-regulated(P<0.01 or P<0.05),while the expression of M2 markers was significantly up-regulated(P<0.01).Compared with the LPS+BTX-A+Vector group,the expression of M1 markers in the LPS+BTXA+P2X7 group was significantly up-regulated(P<0.01),and the expression of M2 polarization markers was significantly down-regulated(P<0.01).2)The results of Western blot showed that compared with the Ctrl group,the expression of P2X7 protein in the LPS group was significantly increased(P<0.01);compared with the LPS group,the expression of P2X7 protein in the LPS+BTX-A group was significantly reduced(P<0.01).Importantly,compared with the LPS+BTX-A group,the expression of P2X7 protein in the LPS+BTX-A+Bz ATP group was significantly increased(P<0.05).The results of q RT-PCR and ELISA showed that compared with the Ctrl group,the expression of M1 markers in the LPS group was significantly up-regulated(P<0.01),and M2 markers expression difference was not statistically significant(P>0.05).Compared with the LPS group,the expression of M1 markers in the LPS+BTX-A group was significantly downregulated(P<0.01),while the expression of M2 markers was significantly up-regulated(P<0.01).Compared with the LPS+BTX-A group,the expression of M1 markers in the LPS+BTX-A+Bz ATP group was significantly up-regulated(P<0.05),and the expression of M2 markers was significantly down-regulated(P<0.05).Conclusion: BTX-A has a good analgesic effect on neuropathic pain.Its analgesic mechanism may be related to the neuroprotection.In other words,it promotes the polarization of microglia M2 and inhibit the polarization of microglia M1 by inhibiting the expression of P2X7 receptor,thereby inhibiting the release of pro-inflammatory factors TNF-α and IL-6 and promoting the release of antiinflammatory factor IL-10. |
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