Anti-diabetic Neuropathic Pain Effect Of Koumine And The Mechanism Involved In Inhibiting Microglia M1 Polarization In The Spinal Cord Of Rats

In recent decades,the prevalence of diabetes in China and even in the world has rapidly increased.Diabetes and its complications are among the top causes of death currently,and they pose a serious threat to human health.Diabetic neuropathic pain(DNP),a chronic pain,is one of the serious complications of diabetes.However,current medical methods are not only difficult to cure for diabetic pain,but also have a lot of side effects.Therefore,it is urgent to develop new therapeutic drugs with less side effects.Komine(KM)is the highest content of indole alkaloids in the medicinal plant Gelsemium elegans.Our research group has found that KM has a highly effective and low-toxic anti-DNP effect.In order to further explore the target and route of action of the anti-DNP of KM,the present study examined the anti-DNP effect of KM and the mechanism involved in inhibiting spinal microglia M1 polarization in the rat model of DNP induced by streptozotocin(STZ);then to explain the molecular mechanism of KM inhibiting spinal microglia M1 polarization,and to clarify the molecular pathways and key effector molecules.The main research contents are as follows:In this study,a single intraperitoneal injection of STZ(70 mg/kg)was used to establish a model of typeⅠDNP in Sprague-Dawley rats.It was found that intragastric administration of KM can significantly reduce mechanical hyperalgesia in diabetic rats,suggesting that KMhas a significant anti-DNP effect and is dose-dependent.To further investigate whether the effect of KM on rat DNP is related to microglia IRF8,microglia activation,and M1 polarization,wedetected the expression levels of IRF8,iba-1(microglia activation markers)and M1 polarization markers(CD86,CD68,TNF-α,and IL-1β)m RNA and proteinby RT-q PCR and western blot.The results showed that KM can inhibite microglia M1 polarizationby down-regulating the expression of IRF8,then reduces production of proinflammatory cytokines such as CD86,CD68,TNF-α and IL-1β,thereby suppressing DNP.Jagged 1 is one of the important ligands of the Notch receptor,and activates the Notch signal pathway.In order to explore whether the anti-DNP effect of KM is regulated by Notch signal pathway,the present studyobserved the effects of intrathecal injection Jagged 1 on mechanical withdrawal threshold of KM on STZ-induced DNP rats.The results showed that KM could inhibit DNP,whereas the activation of Notch signaling pathway by Jagged 1 reversed the anti-DNP effect of KM,which increased the hyperalgesia,suggesting that the anti-DNP effect of KM may be regulated through the Notch signaling pathway.On this basis,wedetected the expression levels of IRF8,iba-1,CD86,CD68,TNF-α,and IL-1β by RT-q PCR and western blot.The results observed that,compared with STZ+PBS+KM group,the STZ+Jagged 1+KM group IRF8,iba-1,CD86,CD68,TNF-α and IL-1β expression was significantly increased,indicating that i.t.Jagged 1 can reverse the effect of KM,which increases the expression of IRF8 in spinal microglia and activates microglia M1 polarization,which increases the release of proinflammatory cytokines and exacerbates hyperalgesia.In summary,the results of this study suggest that KM has a significant analgesic effect on diabetic neuropathic pain in rats;KM can down-regulate IRF8 expression through Notch signaling pathway,and then inhibitspinal microglia M1 polarization,thereby reduce the expression of proinflammatory cytokines and exerting anti-diabetic neuropathic pain.