| Sciatica is a common neuropathic pain in clinic,which refers to the syndrome of pain along the sciatic nerve pathway and its distribution.The prevalence of sciatica is very high and most patients have a long course of disease,which seriously affects the quality of life of patients.Mongolian medicine ascribes sciatica to "Baimai disease of the lower limbs".It is considered that”Sangen disorders make Heyi excess,which is fight with blood and stroke into Baimai of lower limbs,then disorder Baimai function of lower limbs" is the basic etiology and pathogenesis of the disease.Living and environmental factors such as lying in a cool and humid place in a long time,rain,wind or sudden temperature drop,imbalance of cold and warm,as well as trauma damage,lumbar deformation,and adjacent lesions are external causes.Therefore,the main therapeutic principle of Mongolian medicine is to improve the circulation of Heyi and blood,regulate the body hormone,dredge the Baimai,and relieve pain.The patients are usually given Mongolian medicine or traditional external therapy according to the differentiation of symptoms and signs,and combinat with diet and daily life,so as to achieve good clinical effect.Among them,Mongolian Medicine Warm Acupuncture has a significant analgesic effect in the treatment of sciatica,and it often has a significant effect on those who have no effect of commonly used western painkillers.A large number of modern studies in the early stage of our team have confirmed that Mongolian Medicine Warm Acupuncture can regulate the neural-endocrine-immune network of the body through its acupuncture effect,warm effect and acupoint specific stimulation,so as to play its multiple functions of warming the meridians,harmonizing the Heyi and blood,anti-inflammatory,analgesic,sedative and hypnotic,enhancing immunity,and achieve the purpose of regulating the body and treating diseases.However,the cellular and molecular mechanism of its analgesic effect on sciatica is not clear.Modern medicine considers that the pathogenesis of neuropathic pain is very complex,and there are great limitations in the understanding of it so far,so it is difficult to carry out effective treatment for the cause.At present,western medicine mainly use analgesics or nerve modulation methods such as transcutaneous nerve stimulation and spinal cord electrical stimulation in clinic,but the treatment effect is limited.Therefore,the research on the pathogenesis and treatment of neuropathic pain is always the focus and hot spot.At present,peripheral sensitization and central sensitization play an important role in the production and maintenance of pathological pain.When peripheral nerve is injured and inflamed,inflammatory cells and nerve endings release a series of inflammatory pain causing substances to extracellular fluid,which can make local microenvironment form"inflammatory soup".These inflammatory pain causing substances can directly or indirectly cause the excitement of nociceptors,reduce the excitation threshold and sensitize them,that is to say,peripheral sensitization.When the nerve is damaged,pain will continue to exist.This kind of nociceptive stimulation can cause the excitability of spinal dorsal horn neurons to increase continuously and then plastic changes,that is,central sensitization.A large number of studies have confirmed that many protein kinases can activate and maintain central sensitization.Extracellular signal regulated kinase(ERK)is one of the main members of MAPK family,which can be activated by neurotrophic factors and other biological stimuli ERK/MAPK signaling pathway plays an important role in the regulation of pain sensitization.Therefore,from a holistic perspective,this study mainly studied the effect of Mongolian Medicine Warm Acupuncture on the content of inflammatory pain factors in peripheral nerves and the activity of ERK/MAPK signal pathway in spinal dorsal horn neurons of CCI model rats,in order to explain the analgesic mechanism of Mongolian Medicine Warm Acupuncture on CCI model rats at the cellular and molecular levelObjectiveIn this study,the analgesic effect of Mongolian Medicine Warm Acupuncture on CCI model rats was taken as the starting point,and various advanced techniques of modern molecular biology were used to study the correlation between the analgesic effect of Mongolian Medicine Warm Acupuncture and the changes of inflammatory pain factors in peripheral nerves and ERK/MAPK signal pathway activity in spinal dorsal horn.The analgesic effect and mechanism of Mongolian Medicine Warm Acupuncture on CCI model rats were explained at the cellular and molecular level.At the same time,it provides a simple,safe,efFective,non-invasive and non drug treatment for patients with chronic pathological pain such as sciatica.MethodsExperiment 1,behavioral experiment:50 SPF male SD rats were randomly divided into five groups:sham operation group,CCI model group,Mongolian Medicine Warm Acupuncture group,acupuncture group and Western medicine group,with 10 rats in each group.The neuropathic pain model of chronic compression injury(CCI)caused by sciatic nerve ligation was established by Bennett and Xie.The treatment was started 15 days after the model was made,and the course of treatment was 14 days.Conduct behavioral tests at different time points,which including general condition observation,ambulatory score,mechanical withdraw threshold and thermal withdraw latency.In the second experiment,the changes of inflammatory pain mediators such as PGE2 and SP,and inflammatory cytokines such as IL-6 and TNF-? were detected by enzyme-linked immunosorbent assay(ELISA)after the treatmentExperiment 3,molecular biology experiment:30 SPF male SD rats were randomly divided into 6 groups:sham operation group,CCI model group,U0126 inhibitor group,DMSO group,Mongolian Medicine Warm Acupuncture group and U0126+Mongolian Medicine Warm Acupuncture group,with 5 rats in each group.After treatment,3 rats in each group were randomly selected to detect the expression of BDNF,RAS,c-raf,MEK,ERK1/2,p-ERK1/2,CREB and p-CREB in spinal dorsal horn neurons by Western blot,and the expression of TRK2 mRNA,ERK1/2 mRNA and CREB mRNA in spinal dorsal horn neurons by real-time PCR.Results1.Behavioral experiment results:?changes of body weight:There was no significant difference in the weight of rats in each group before modeling(P>0.05);the weight gain of rats in CCI model group was not significant at each time point from modeling to sampling;after treatment,the body weight of rats in Warm Acupuncture group,acupuncture group and drug group was significantly higher than that in CCI model group(P<0.05),and there was no significant difference with sham operation group(P>0.05).?changes of ambulatory score After the CCI model,the ambulatory score of rats increased gradually and reached the peak value 15 days after the model was established;after treatment,the score of rats in the Warm Acupuncture group decreased significantly compared with that in the CCI group(P<0.05);the change trend of the ambulatory score in the acupuncture group and the drug group was almost the same as that in the Warm Acupuncture group,and there was no difference between the three groups at each time point(P>0.05).?changes of mechanical withdraw threshold(MWT)and thermal withdraw latency(TWL):MWT and TWL in CCI model group decreased significantly from the 4th day after model establishment,reached the lowest value at the 15th day after model establishment,and were still significantly lower than those in sham operation group before material collection(P<0.05);after intervention,MWT and TWL in Warm Acupuncture group,acupuncture group and drug group were significantly higher than those in CCI model group(P<0.05),but there was no significant difference between sham operation group(P>0.05).2.The contents of PGE2,SP,IL-6 and TNF-? in the serum of CCI model rats were significantly higher than those of sham operation group(P<0.05).After intervention,the contents of PGE2,SP,IL-6 and TNF-? in serum of Warm Acupuncture group,acupuncture group and drug group were significantly lower than those in CCI model group(P<0.05).There was no significant difference in the above indexes between the Warm Acupuncture group and drug group(p>0.05),indicating that Warm Acupuncture Therapy and ibuprofen treatment had similar effects on inflammatory media.The contents of PGE2,IL-6 and TNF-? in acupuncture group were significantly different from those in Warm Acupuncture group and drug group(P<0.05).3.Expression of BDNF and its receptor trk2 mRNA:The expression of BDNF protein and its receptor trk2 mRNA in spinal dorsal horn of CCI model group were significantly higher than that of sham operation group(P<0.05).Compared with the CCI model group,the BDNF protein expression and trk2 mRNA expression in the spinal dorsal horn of rats in the Warm Acupuncture group and the Warm Acupuncture+U0126 group were significantly reduced(P<0.05),and there was no significant difference in the U0126 inhibitor group and the DMSO group(P>0.05).4.Results of ERK1/2 signal pathway:The expression of Ras,C-Raf,MEK,p-ERK1/2 and ERK2 mRNA in spinal dorsal horn of CCI model group and DMSO group were significantly higher than that of sham operation group(all P<0.05),but the expression of ERK1/2 and ERK1 mRNA had no significant difference(P>0.05).After intervention,the expression of Ras,C-Raf,MEK and p-ERK1/2 in spinal dorsal horn neurons in Warm Acupuncture group was significantly lower than that in CCI group(all P<0.05),and the relative expression of ERK2 mRNA had no significant change(P>0.05).The expression of MEK and p-ERK1/2 protein in U0126 inhibitor group was significantly lower than that in CCI model group(all P<0.05),and there was no significant difference in the expression of ras,C-Raf,ERK1/2 and ERK1/2 mRNA(all P>0.05).Compared with CCI model group,the expression of Ras,C-Raf and MEK protein in spinal dorsal horn neurons of rats in Warm Acupuncture+U0126 inhibitor group decreased significantly(all P<0.05),and the expression of ERK1/2,p-ERK1/2 and ERK1/2 mRNA had no significant difference(P>0.05).In the comparison between the three treatment groups,Compared with Warm Acupuncture group and U0126 group,the expressions of Ras and c-raf in spinal dorsal horn neurons in the two groups were significantly different(all p<0.05),and the expression of MEK,ERK1/2,p-ERK1/2 and ERK1/2 mRNA had no significant difference(all P>0.05),indicating that Mongolian Medicine Warm Acupuncture and U0126 inhibitor both can effectively inhibit the activity of ERK signaling pathway.Compared with Warm Acupuncture group and Warm Acupuncture+U0126 group,the expression of Ras,MEK and p-ERK1/2 in spinal dorsal horn neurons in the two groups were significantly different(all p<0.05),and the expression of other indexes were not significantly different(P>0.05),which indicated that the inhibitory effects of Mongolian Medicine Warm Acupuncture and U0126 inhibitor on the activity of ERK signaling pathway are not superimposed.5.Expression of CREB,p-CREB and CREB mRNA:The expression of p-CREB protein in spinal dorsal horn neurons of CCI model group and DMSO group was significantly higher than that of sham operation group(P<0.05),but the expression of CREB and CREB mRNA of the two groups was no significant difference with sham operation group(P>0.05).After intervention treatment,the expression of p-CREB protein in spinal dorsal horn neurons of Warm Acupuncture group,acupuncture group and drug group were significantly lower than that of the CCI group(all P<0.05).Among the three treatment groups,there was no significant difference in the expression of p-CREB protein in spinal dorsal horn neurons of rats in the Warm Acupuncture group and the U0126 group(p>0.05),indicating that both Mongolian Medicine Warm Acupuncture and U0126 inhibitor could inhibit the activity of ERK1/2 signaling pathway,thereby reducing the phosphorylation of its downstream protein CREB.Compared with the Warm Acupuncture group and U0126 group,the expression of p-CREB in the Warm Acupuncture+U0126 group was more significantly decreased than that in the two groups(p<0.05),indicating that Mongolian Medicine Warm Acupuncture could not only directly interfere with the activation of ERK1/2 signaling pathway downstream protein CREB,but also play a synergistic effect with the combined application of U0126 inhibitor.Conclusion1.Mongolian Medicine Warm Acupuncture therapy can effectively reduce the spontaneous pain behavior of CCI model rats,increase the pain threshold of mechanical stimulation,and reduce the state of thermal radiation pain sensitivity.so,Mongolian Medicine Warm Acupuncture has obvious analgesic effect2.Mongolian Medicine Warm Acupuncture can significantly reduce the contents of PGE2,P,IL-6 and TNF-? in the serum of CCI model rats.Therefore,the anti-inflammatory and analgesic effects of Mongolian Medicine Warm Acupuncture on CCI model rats may be related to the reduction of peripheral neuroinflammatory response and peripheral sensitization factors3.Mongolian Medicine Warm Acupuncture can significantly reduce the expression of BDNF,RAS,c-raf,MEK,p-ERK1/2 and p-CREB in ERK1/2 signal pathway of spinal dorsal horn in CCI model rats.Therefore,Mongolian Medicine Warm Acupuncture may reduce the plasticity of spinal dorsal horn by inhibiting the ERK/MAPK signal pathway activity,so as to play an analgesic role in Neuropathic pain.4.Mongolian Medicine Warm Acupuncture can achieve good analgesic effect by improving the circulation of Heyi blood,and repairing the function of Baimai in lower limbs of CCI model rats.At the same time,it has the characteristics and advantages of small toxic,few selected acupoint,safety and effectiveness. |
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