Basing Electroacupuncture And Polymethoxyflavones On The Alternatively Spliced Variants Of OPRM1:the Efficacy And Mechanism Of Analgesia

Background/AimFor decades,multiple clinical and experimental studies demonstrated that electroacupuncture and herbs were widely used in the treatment of various damage and disease-evoked pain.Previous investigation showed that electroacupuncture could promote the release of endogenous opioid peptide(EOP)and the analgesic effect was mediated by opioid receptors in peripheric and central nerve system(CNS)or some inflammatory mediators.Also,other investigations found that ?-opioids,morphine as a representative,could give rise to the differential expressions of the alternatively spliced variant mRNAs of OPRM1 in brain regions.The present study used wild type C57BL/6 mice(WT)and the same strain mE7b mice(total ?-opioid receptors gene knockout),concentrating on OPRM1 in selective regions of CNS,trying to search the relationship between electroacupuncture,herbs and OPRM1,aiming to propose the analgesic potency and mechanism of electroacupuncture and herbs.MethodSince the schemes of EA to alleviate pain are different,papers referred to this region were mostly gathered and analyzed,confirming the option of acupoints and specific operational details,for instance,specification of stainless steel needle,frequency of HANS apparatus,depth of needle stimulation,intensity of electricity and latency of stimulation.There are many analgesic effects in traditional Chinese medicines,including Yuanhu(Corydalis yanhusuo),Angelica dahurica,toad venom and so on.According to the different mechanisms of action,this study selected polymethoxyflavones(extracted from citrus)which mainly act on opioid receptors to carry out experiments.First,we establish a mice pain model.After the model was established successfully,electro-acupuncture stimulation and drug treatment were given to detect the changes of pain behavior,record and analyze the data,and observe the differences between WT and mE7b mice under the same conditions.To observe the effect of electroacupuncture and drugs on the expression of mu opioid receptor gene in different brain regions of mice,we used qPCR.After confirming that electro-acupuncture and drugs exert their effects through opioid receptors,local(peripheral)or ventricular(central)injection of beta-FNA,a mu-opioid receptor antagonist,was given to WT mice,followed by electro-acupuncture stimulation and drug treatment.The behavioral differences between peripheral and central use of beta-FNA were compared,and the main targets of electro-acupuncture and traditional Chinese medicine analgesia were explored.At last,Western Blot and immunohistochemical techniques were used to analyze the changes of ERK1/2 and c-Fos in pain after electroacupuncture and TCM treatment.Result1.Electro-acupuncture and polymethoxyflavone can improve formalin-induced acute pain,especially second-phase pain,and also improve the response of CFA-induced pain model rats to thermal and mechanical stimulation within 24 hours.2.After intraventricular injection of beta-FNA,the analgesic effect of electro-acupuncture was significantly reduced,while that of local injection of beta-FNA had little effect,but decreased only at 4 hours.In contrast,intraventricular injection of beta-FNA could significantly reduce the analgesic effect of polymethoxyflavones,while local injection of beta-FNA had no effect on the analgesic effect of polymethoxyflavones.3.Electro-acupuncture and polymethoxyflavone can regulate the expression of OPRM1 in pain-related brain areas,especially electro-acupuncture.4.The expression of p-ERK in the brain region of pain model mice induced by CFA was significantly increased.Electro-acupuncture treatment could reduce the phosphorylation level of ERK in WT mice,which was close to normal level.For mE7b mice,electro-acupuncture decreased the phosphorylation level of THA region but had no effect on RVM region.At the same time,the expression of c-Fos in the brain region of pain model mice induced by CFA also increased significantly.In WT mice,electro-acupuncture can reduce this effect,except for THA region,but for mE7b mice,electro-acupuncture can only reduce the expression of c-Fos in RVM region.5.Different from electro-acupuncture,polymethoxyflavone can reduce the phosphorylation of ERK in WT mice,but it can not reduce the phosphorylation of ERK in mE7b mice.Meanwhile,polymethoxyflavone can not reduce the expression of c-Fos in the brain of mE7b mice.Conclusion:1.Both electroacupuncture and polymethoxyflavone can reduce formalin-induced biphasic pain and CFA-induced persistent pain,but the analgesic effect decreases after mu-opioid receptor gene knockout,especially in the polymethoxyflavone treatment group.2.Electro-acupuncture mainly acts through central opioid receptors,while polymethoxyflavone completely acts through central opioid receptors.3.It is known that EA can produce endogenous opioid peptides.Our results show that EA also participates in the analgesic process of EA by regulating the expression level of OPRM1 gene and its splicing isomers,while polymethoxyflavone acts as an opioid drug and has no effect on OPRM1.4.The phosphorylation of ERK and the expression of c-Fos were significantly up-regulated in the brain of CFA-induced pain model mice,suggesting that the up-regulation of these two indicators could represent the pain state of mice,while the down-regulation of p-ERK and c-Fos by electro-acupuncture and polymethoxyflavone could occur in the brain of most WT mice,but not in mE7b mice,suggesting that Electro-acupuncture and polymethoxyflavones can exert analgesic effect by decreasing the phosphorylation level of ERK and the expression level of c-Fos,which requires the participation of mu-opioid.