The Research On The Cross-talks Between Peripheral TRPV1 And P2X₃ And Intervention Effect Of Electroacupuncture In Rats With Different Pathological Pain

Objective To investigate the cross-talks between transient receptor potential vanilloid 1(TRPV1)and P2X₃at the periphery of rats with different pathological pain models and to investigate the intervention effect of electroacupuncture(EA)on the cross-talks between peripheral TRPV1 and P2X₃under conditions of different pathological pain.Methods Part I:Healthy male Sprague Dawley(SD)rats were subcutaneously injected with complete freund's adjuvant(CFA)in the right hind paw to establish a model of inflammatory pain and selective ligation of the sciatic nerve branches to established a model of neuropathic pain.The mechanical paw withdrawal thresholds(MPWTs)of the ipsilateral hind paw were measured at 1 d before and 1 d after modeling.Different doses of TRPV1/P2X₃receptor agonists(capsaicin/?,?-me ATP)or inhibitors(capsazepine/TNP-ATP)were injected on the dorsal foot at 3rd day after establishment of different pathological pain.Spontaneous pain behaviors(spontaneous flinching times and the spontaneous licking time)were observed within 20 min after administration and the dosage gradients of capsaicin,capsazepine,?,?-me ATP and TNP-ATP were screened.Then,according to different dosing regimens,agonists and/or inhibitors were subcutaneously administered to rats to observe the effects of TRPV1 on spontaneous pain induced by P2X₃and the effects of P2X₃on spontaneous pain induced by TRPV1 under different pathological conditions.Whole-cell patch clamp electrophysiology was used to observe the response of TRPV1 and P2X₃channels to capsaicin and?,?-me ATP in the ipsilateral DRG of rats under different pathological conditions and the cross-talk between TRPV1 and P2X₃.The part II:Healthy male SD rats were selected and the inflammatory pain model and neuropathic pain model were established by the same method.Partial rats of the CFA model and the SNI model were divided into groups according to randomized methods,CFA+?,?-me ATP+capsaicin group,CFA+EA+?,?-me ATP+capsaicin group,CFA+capsaicin+?,?-me ATP group,CFA+EA+capsaicin+?,?-me ATP group and SNI+?,?-me ATP+capsaicin group,SNI+EA+?,?-me ATP+capsaicin group,SNI+capsaicin+?,?-me ATP group,SNI+EA+capsaicin+?,?-me ATP group,and 6 in each group.The other part of rats in the CFA model and SNI model were completely randomized divided into Control group,Model group,Sham EA group,EA group and Sham SNI group,SNI group,Sham EA group,EA group,and 6 in each group.One day after the modeling,rats in the EA group began to be intervened with EA.The bilateral points like“Zusanli”and“Kunlun”of rats were chose.The stimulation frequencies of EA were 100 Hz(CFA model)and 2 Hz(SNI model),respectively.Other stimulation parameters were 1m A for15min,and then the intensity was raised to 2m A,totally 30 min,once every day.The MPWTs of the ipsilateral hind paw were measured 1 day before modeling,1 day after modeling before the EA intervention,1 day after modeling after the EA intervention,and 3 days after the EA intervention.At 3 days after modeling,the rats were sacrificed and their ipsilateral side L4-L6DRG was harvested.The effect of EA on the cross-talk between TRPV1 and P2X₃under different pathological conditions was observed by observing spontaneous pain behaviors.The co-IP assay was used to detect the cross-talk between TRPV1 and P2X₃in the ipsilateral DRG under the condition of pathological pain and EA treatment.Results Part I:(1)Changes in pain threshold:Before modeling,there was no significant difference in the basic MPWTs between rats in each group;after the modeling,the MPWTs of the two model rats were significantly decreased(P<0.01).(2)Change of spontaneous pain behaviors:observing spontaneous pain behaviors,capsaicin(CFA model:30?g,SNI model:3?g),capsazepine(CFA model:113?g,SNI model:400?g),?,?-me ATP(CFA model:50nmol,SNI model:250 nmol),TNP-ATP(CFA model:50 nmol,SNI model:250 nmol)were sequentially screened.It can be seen that under the CFA model,capsazepine can significantly antagonize spontaneous pain behaviors induced by?,?-me ATP during the first 2 min(P<0.05),and?,?-me ATP can increase spontaneous painful behavior induced by capsaicin during the first 2 min(P<0.05).However,in the SNI model,capsaicin significantly increased spontaneous pain behaviors induced by?,?-me ATP during 3rd 2 min(P<0.05)and 8th 2 min(P<0.05),but both?,?-me ATP and TNP-ATP can increase spontaneous pain behaviors induced by capsaicin.(3)Electrophysiological changes of whole-cell patch clamp:Compared with the normal group,the model induced by CFA can significantly increase the response of TRPV1 to capsaicin(P<0.05)but cannot interfere with the response of P2X₃to?,?-me ATP,and the SNI model can significantly reduce response of TRPV1 to capsaicin(P<0.01)and increased response of P2X₃to?,?-me ATP(P<0.01).Under model of CFA and SNI,the response of both TRPV1 or P2X₃to capsaicin or?,?-me ATP could be inhibited by intervention of?,?-me ATP or capsaicin.Part II:(1)Changes in pain threshold:Before modeling,there was no significant difference in the basic MPWTs between rats in each group;1d after modeling,the MPWTs in the model group of 2 models decreased significantly(P<0.01)and maintained at a lower level until the 3rd day(P<0.01).In the CFA model,compared with model group,the MPWTs of rats in the EA group increased significantly after EA treatment on 1d(P<0.01).After 3 days'EA treatment,compared with model group,the MPWTs in the EA group maintained at a higher level(P<0.01).In the SNI model,compared with model group,MPWTs of the EA group were significantly increased after 3 day's EA treatment(P<0.05).(2)Changes of spontaneous pain behaviors:Under the condition of CFA model,EA can reduce both the enhancement effect of TRPV1 on the spontaneous pain behaviors induced by P2X₃and the enhancement effect of P2X₃on the spontaneous pain behaviors induced by TRPV1.Under the condition of SNI model,EA is also alleviating EA can reduce both both the enhancement effect of TRPV1 on the spontaneous pain induced by P2X₃and the enhancement effect of P2X₃on the spontaneous pain induced by TRPV1.(3)co-IP assay:After precipitation of TRPV1,the P2X₃expressions of the model(Model,SNI)and EA groups were positive in both two models compared with the control group and sham SNI group;when P2X₃was precipitated,compared with control group and sham SNI group,the TRPV1 expressions of the model(Model,SNI)and EA groups were also positive in both two models.Conclusions There were differences in the cross-talks between TRPV1 and P2X₃at peripheral afferent terminals under different pathological conditions,but there was a mutual inhibitory effect between TRPV1 and P2X₃in DRG neurons.There were differences in the cross-talks of TRPV1 and P2X₃at peripheral afferent terminals and in the DRG neurons under the same pathological condition.EA has a good analgesic effect on different pathological pain and can inhibit the enhancement of the cross-talks between TRPV1 and P2X₃at peripheral afferent terminals on spontaneous pain behaviors in rats with different pathological pain.There were cross-talks between TRPV1 and P2X₃in DRG under different pathological pain,and EA can not eliminate the cross-talks between the them.