| PurposesPain is an unpleasant sensory and emotional experience that can often be accompanied by tissue damage.Acute pain has a protective effect on the body,while chronic pain has an adverse effect on the body.Drugs targeting a single target are often less effective because of the complex mechanisms of chronic pain,and most of the current analgesics prone to incomplete analgesia,tolerance and side effects.Electroacupuncture(EA)has been widely used in the clinical treatment of pain.Compared with the drugs,EA is safe,effective and has fewer side effects.The World Health Organization(WHO)lists chronic pain in sports system,postoperative pain,rheumatoid arthritis,trigeminal neuralgia and other pain as acupuncture indications.Although clinical trials have confirmed the efficacy of EA analgesia,the mechanism is still unclear.In this study,it was found that EA could down-regulate the expression of Nav 1.7 in Dorsal Root Ganglion(DRG)to alleviate inflammatory pain in a rat model of plantar inflammatory pain induced by Complete Freund’ s Adjuvant(CFA).Therefore,the analgesic mechanism of EA in the CFA-induced chronic inflammatory pain model is explored,which provides a theoretical basis for the clinical treatment of inflammatory pain and EA analgesia.Methods1.Establishment of rat model of plantar inflammatory pain50 u L CFA was injected into the left hind limb plantar of rats to establish an animal model,and 0.9%normal saline was injected into the plantar of rats in control group after anesthesia.Changes of paw withdrawal mechanical threshold(PWT)and paw withdrawal thermal latency(PWL)were detected after injection of CFA or normal saline to verify the success of model.2.EA therapyEA therapy was started 1 d after injection of CFA.EA stimulated the left Huantiao(GB30)and Zusanli(ST36)of the rats once every two days until 14 days after injection of CFA.Changes of PWT and PWL in rats treated with EA were detected to verify whether EA was effective in analgesia.3.Detection of depression statusSucrose preference test and forced swimming test were performed to detect whether the rats were depressed or not.4.Detection of proteins and moleculesThe concentration of serum corticosterone of rats was detected by Elisa and the mRNA and protein expressions of Nav1.7 and Glucocorticoid receptor(GR)in DRG were detected by Western blot and qRT-PCR on the 14 day after CFA injection.5.Observation of the effect of GR expression on pain behavior and Nav1.7 expression in rats7 days after CFA injection,GR antagonist RU38486 was injected into the subarachnoid for 5 consecutive days.The pain behavior and expression of Nav1.7 protein in rats were detected 7 days after CFA injection,GR sense oligonucleotide(hereinafter referred to as GR sense)or antisense oligonucleotide(hereinafter referred to as GR antisense)was injected intrathecally and the pain behavior was detected in rats.6.Detection of the co-labeling of Nav1.7 and GR in DRG by Immunofluorescence.7.Observation of the effect of GR expression on anti-nociceptive action of EA7 days after the injection of CFA,two group of rats was intrathecally injected with GR sense or GR antisense respectively on the basis of EA treatment,and the other group was only treated with EA.The PWT and PWL and expression of Nav1.7 was compared among three groups of rats.Results1.PWT and PWL of rats were decreased after CFA injection.Compared with the control group,the ipsilateral PWT and PWL in the model group and the EA group were significantly reduced 1 day after CFA injection(P<0.001),indicating the model was successfully made.14 days after injection,PWT and PWL in the model group were lower than those in the control group(P<0.001).2.EA relieved inflammatory pain in rats.Compared with the model group,the PWT and PWL of rats in the EA group were significantly improved 7 days after CFA injection(f<0.001),and were still higher than those in the model group 14 days after CFA injection(P<0.001).3.EA relieved depression in rats.The sucrose preference rate of the model group was significantly lower than that of the control group(P<0.001),and significantly increased after EA treatment(P<0.01).In the forced swimming experiment,the immobility time of the model group was significantly increased compared with the control group(P<0.01),and decreased after electroacupuncture treatment(P<0.05).4.EA reduced the expression of Nav1.7 and GR in DRG of rats,and decreased the concentration of corticosterone in serum of rats.14 days after CFA injection,the expression of Nav1.7 protein and mRNA in DRG of rats in the model group were increased(P<0.01,P<0.001).And the expression of GR mRNA and protein in nucleus were increased in the model group(P<0.001),while there was no statistically significant difference in GR protein in cytoplasm among the three groups(P>0.05).The concentration of corticosterone in serum of rats in the model group was significantly increased(P<0.001).The expression levels of Nav1.7 and GR protein and mRNA were significantly decreased after EA treatment(P<0.05,P<0.01,P<0.001,P<0.01),as well as the concentration of corticosterone in serum(P<0.01).5.Intrathecal injection of GR antagonist relieved mechanical and thermal hyperalgesia induced by CFA,and reversed the up-regulation of Nav1.7.Mechanical and thermal hyperalgesia of rats were aggravated after intrathecal injection of GR sense and alleviated after injection of GR antisense.PWT and PWL in rats were increased after intrathecal injection of GR antagonist RU38486(P<0.05,P<0.01),and Nav1.7 expression was decreased(P<0.01).PWT and PWL of the rats were increased after intrathecal injection of GR antisense(P<0.001,P<0.01),and decreased after injection of GR sense(P<0.05,P<0.01).6.Immunofluorescence results showed that Nav1.7 and GR were co-labeled in DRG.7.Intrathecal injection of GR antisense reduced the expression of Nav1.7 protein and enhanced the analgesic effect of EA,while injection of GR sense increased the expression of Nav1.7 and blocked the analgesic effect of EA.On the basis of EA treatment,GR sense/antisense was injected intrathecally respectively 7 days after CFA injection,and PWT and PWL were tested.The results showed that PWT and PWL were significantly lower in the EA plus GR sense group compared with the EA group(P<0.001).PWT and PWL in rats treated with EA plus GR antisense were higher than those in EA group(P<0.01).Western blot results showed that the expression of Nav1.7 protein increased in DRG of rats in EA plus GR sense group compared with the EA group(P<0.001),and decreased in DRG of rats in EA plus GR antisense group(P<0.05).ConclusionIn the rat model of plantar inflammatory pain,GR activation promotes the expression of Nav1.7 protein in DRG.EA reduced the expression of Nav1.7 protein by inhibiting the transcriptional activity of GR in DRG,thus alleviating inflammatory pain and depression in rats.Silencing GR reverses the high expression of Nav1.7 caused by CFA and enhances the analgesic effect of EA.In summary,GR may be an important target for EA to participate in analgesia of inflammatory pain.This study provides a theoretical basis for a more comprehensive explanation of the mechanism of EA analgesia,and provides new ideas for the treatment of chronic pain. |
PDF Full Text Request