Based On Microglia α7nAchR Mediated BDNF/TrkB Signaling Pathway Is Involved In The Molecular Mechanism Of Trifluoro-Icaritin Intervention In Neuropathic Pain

Background: Neuropathic pain is one of the most stubborn and intractable diseases,which seriously affects the quality of life of patients and makes them miserable.In addition,neuropathic pain is also accompanied by anxiety,depression,and social barriers,which will reduce patients’ enjoyment of life and make them suffer.Reduce the ability to work,and in severe cases,suicidal tendencies can occur,which can be life-threatening.The complex pathogenesis of neuropathic pain increases the difficulty of treatment.Numerous studies have confirmed that there are complex interactions between microglia and neurons,affecting the transmission of pain signals.Activation of α7 nicotinic acetylcholine receptor(α7n ACh R)inhibits the activation of microglia,promotes the transition of M1 type to M2 type microglia,releases anti-inflammatory mediators,and exhibits inhibition of neuroinflammation effect.Trifluoro-icaritin(ICTF),a derivative of icaritin(ICT),has antiinflammatory and neuroprotective effects.Studies have shown that ICTF has analgesic and anti-inflammatory effects on chronic inflammatory pain.However,whether ICTF has analgesic and anti-inflammatory effects on neuropathic pain in chronic pain needs further exploration.Therefore,the purpose of this experiment was to investigate the effect of ICTF on the α7n ACh R-BDNF/Trk B/KCC2 signaling pathway,inflammatory factors and microglia in SNI-induced neuropathic pain rats at the spinal cord level,in order to further reveal the analgesic,anti-inflammatory and molecular effects of ICTF.The mechanism provides experimental basis.Objective:1.To observe the effect of ICTF on mechanical hyperalgesia,thermal hyperalgesia,gait,rotarod,open field and elevated plus maze in rats with SNI-induced neuropathic pain.2.To observe the effect of ICTF on the relationship between α7n ACh R and microglia and their polarization state in spinal cord of rats with SNI-induced neuropathic pain,as well as α7n ACh R,BDNF,Trk B,KCC2,IL-1β,IL-10,CD11 b,CD40,CD206 molecular effects on protein expression levels.3.To observe intrathecal administration of α7n ACh R antagonist α-Bgtx on the mechanical hyperalgesia and the protein expression of α7n ACh R,BDNF,Trk B,KCC2,IL-1β,IL-10,CD11 b,CD40,CD206 molecules in the spinal cord of rats with ICTFinduced neuropathic pain induced by SNI level of influence.4.To observe the effect of intrathecal administration of exogenous BDNF on the mechanical hyperalgesia and protein expression levels of BDNF,Trk B,and KCC2 molecules in the spinal cord of rats with ICTF-induced neuropathic pain induced by SNI.Methods:1.Preparation of SNI-induced neuropathic pain rats according to literature reports.2.The mechanical and thermal hyperalgesia of rats with SNI-induced neuropathic pain were detected by dynamic plantar tactile tester and plantar tester,respectively,and the effect of ICTF on the nociceptive behavior of rats with SNI-induced neuropathic pain was observed,and screen the optimal dose for ICTF therapy.3.Cat Walk gait behavior was used to detect the effect of ICTF on gait parameters in rats with SNI-induced neuropathic pain.4.The effect of ICTF on the overall motor ability of rats with SNI-induced neuropathic pain was detected by rotarod test.5.Apply correlation to analyze the correlation between mechanical hyperalgesia,gait strength parameters and rotarod speed.6.Using open field test and elevated plus maze test to detect the effect of ICTF on anxiety in rats with SNI-induced neuropathic pain.7.The effect of ICTF on the protein expression levels of α7n ACh R,BDNF,Trk B,KCC2 IL-1β,IL-10,CD11 b,CD40 and CD206 in the spinal cord of SNI rats was detected by Western blot.8.The effect of ICTF on α7n ACh R and microglia in spinal cord of SNI rats was detected by immunofluorescence.9.The effect of intrathecal injection of α-Bgtx,an antagonist of α7n ACh R,on mechanical hyperalgesia in SNI rats with ICTF intervention was detected by dynamic plantar tactile measuring instrument.10.The effect of intrathecal injection of exogenous BDNF on the mechanical hyperalgesia of ICTF-intervention SNI rats was detected by dynamic plantar tactile measuring instrument.11.Western blot was used to detect the effect of α7n ACh R antagonist α-Bgtx on the protein expression levels of α7n ACh R,BDNF,Trk B,KCC2,IL-1β,IL-10,CD11 b,CD40and CD206 in the spinal cord of SNI rats after ICTF intervention.12.Western blot was used to detect the effect of exogenous BDNF on the protein expression levels of BDNF,Trk B,and KCC2 molecules in the spinal cord of ICTFintervention SNI rats.Results:1.ICTF(5 mg/kg)can relieve the mechanical hyperalgesia in rats with SNI-induced neuropathic pain,but is insensitive to thermal hyperalgesia.To explore whether ICTF has analgesic effect on SNI induced neuropathic pain rats?Based on the literature reports,we set the ICTF as 0.5 mg / kg,1.5 mg / kg,5 mg / kg and15 mg / kg dose groups.The results showed that the mechanical pain sensitivity of SNI rats decreased significantly with sham group;Compared with SNI group,after 21 consecutive days of ICTF treatment,5 mg / kg ICTF significantly increased the mechanical pain sensitivity threshold;Compared with mechanical pain sensitivity,SNI rats are not sensitive to thermal pain sensitivity.ICTF(5 mg/kg)can effectively improve the gait parameters of rats with SNI-induced neuropathic pain.2.ICTF(5 mg/kg)can effectively improve gait parameters in rats with SNI-induced neuropathic painTo explore whether ICTF can improve gait in rats with SNI-induced neuropathic pain?We used the Catwalk gait analysis system for testing,and the results showed that,compared with Sham,Max Contact Max Intensity,Maximum Intensity,Print area and standing time of the left hind paw in the SNI group significantly decreased,while the left hind paw swing time decreased significantly in the SNI group.Significantly increased;compared with the SNI group,Max Contact Max Intensity,Maximum Intensity,Print area and standing time of the left hind paw maximal contact time of SNI rats on the 21 st day after ICTF treatment significantly increased,while the left hind paw swing time decreased significantly.3.ICTF(5 mg/kg)can effectively improve overall motor balance ability induced by SNI.Through gait behavior,we found that ICTF can improve local motor impairment induced by SNI rats,then,ICTF can improve the overall motor ability of SNI-induced neuropathic pain rats? The results of the rotarod test showed that compared with the Sham group,the rotational speed of the rotarod and the dwell time of the rotarod in the SNI rats were significantly decreased;on the 21 st day after ICTF treatment,the rotational speed of the rotarod and the dwell time of the rotarod in the SNI rats increased significantly.4.Correlation analysis of mechanical hyperalgesia,gait intensity parameters and rotational speed of the rotarod.To further explore whether mechanical allodynia,gait intensity parameters and rotarod speed are related to each other,we performed a correlation analysis.The results showed that the mechanical pain sensitivity,the maximum intensity of the maximum contact time and the rotational speed of the rotator were positively correlated with each other,and the difference was statistically significant.5.ICTF(5 mg/kg)can effectively relieve SNI-induced anxiety.The results of the open field test showed that compared with Sham,the total movement distance and the stay time in the central area of the rats in the SNI group were shortened,while the residence time in the peripheral area was increased.time increases,while the dwell time in the peripheral zone decreases.The results of the elevated plus maze test showed that compared with Sham,SNI rats significantly decreased the number of open-arm stays,the open-arm dwell time,the percentage of open-arm dwell times,and the percentage of open-arm dwell time;SNI increased significantly on the 21 st day after ICTF treatment.The number of times the rats stayed in the open arms,the time of the open arms,the percentage of the times of the open arms,and the percentage of the time of the open arms increased significantly.6.Intrathecal administration of α-Bgtx and exogenous BDNF both reversed the relieving effects of ICTF on mechanical hyperalgesia in rats with neuropathic pain.To explore the effects of intrathecal administration of α-Bgtx and exogenous BDNF on the mechanical hyperalgesia of ICTF-treated SNI rats,we started intrathecal injection of α-Bgtx and α-Bgtx on the 14 th and 17 th day of ICTF treatment in SNI rats,respectively.Exogenous BDNF,it was found that compared with the SNI+ICTF+PBS group,the mechanical allodynia threshold of SNI rats given α-Bgtx and exogenous BDNF was significantly reduced.7.ICTF may play a therapeutic role through α7n ACh R-mediated BDNF/Trk B/KCC2 signaling pathway.To explore whether ICTF plays a therapeutic role through α7n ACh R-mediated BDNF/Trk B/KCC2 signaling pathway? Western blot and immunofluorescence results showed that compared with the Sham group,the protein expression and average immunofluorescence intensity of α7n ACh R in the spinal cord of SNI rats were decreased.The intensity increased;compared with the Sham group,the protein expressions of BDNF and Trk B in the spinal cord of the SNI rats increased,and the protein expression of KCC decreased,and on the 21 st day after ICTF treatment,the protein expressions of BDNF and Trk B in the spinal cord of the SNI rats decreased,and the protein expression of KCC rise.Next,in order to verify whether the BDNF/Trk B/KCC2 signaling pathway is involved in the therapeutic effect of ICTF,exogenous BDNF was intrathecally administered.Western blot results showed that compared with the SNI+ICTF+PBS group,the BDNF in the SNI+ICTF+BDNF group The expression of Trk B and Trk B were increased,and the expression of KCC protein was decreased.To further explore whether ICTF exerts a therapeutic effect by regulating theα7n ACh R-mediated BDNF/Trk B/KCC2 signaling pathway,intrathecal intervention on the function of α7n ACh R,Western blot and immunofluorescence results showed that compared with the SNI+ICTF+PBS group,SNI The expression of α7n ACh R and KCC2 decreased in the +ICTF+α-Bgtx group,while the expression of BDNF and Trk B increased.8.ICTF(5 mg/kg ICTF)may change the polarization state of microglia by upregulating α7n ACh R and release anti-inflammatory factors to play an anti-inflammatory effect.To explore whether ICTF exerts anti-inflammatory effects through microglia? We detected inflammatory factors by Western blot,and the results showed that compared with the Sham group,the expression levels of CD11 b and CD40 proteins in microglia of SNI rats were increased,while the expression levels of CD206 white were decreased;in addition,compared with the Sham group,the The expression level of IL-1β protein in SNI rats increased,while the protein expression level of IL-10 decreased.On the 21 st day after ICTF treatment,the protein expression level of IL-1β in SNI rats decreased,while the protein expression level of IL-10 decreased.Next,in order to test the hypothesis that ICTF may change the polarization state of microglia by up-regulating α7n ACh R and release antiinflammatory factors to play an anti-inflammatory role,we intervened α7n ACh R and administered the α7n ACh R antagonist α-Bgtx intrathecally.The results of Western blot Compared with the SNI+ICTF+PBS group,the protein expression levels of CD11 b and CD40 in the SNI+ICTF+α-Bgtx group were increased,while the expression level of CD206 was decreased;IL-10 Protein expression levels decreased.Conclusion:1.ICTF has analgesic and anti-inflammatory effects on SNI-induced neuropathic pain in rats.2.ICTF can improve the local movement disorder and improve the overall movement ability of SNI rats.3.The analgesic and anti-inflammatory effects of ICTF on SNI rats may,on the one hand,inhibit BDNF/Trk B signaling by up-regulating α7n ACh R in microglia,and upregulate KCC2 expression pathway,thereby alleviating mechanical hyperalgesia;On the one hand,it may inhibit the activation of microglia by activating the α7n ACh R of microglia,causing microglia to tend to M2 type and release anti-inflammatory factors,thereby exerting anti-inflammatory effects.