Effect Of Koumine Hydrochloride On Diabetic Neuropathic Pain And Its Mechanism Of Regulating Basolateral Amygdala Astrocytes

Diabetic neuropathic pain(DNP)is a common chronic complication of diabetes mellitus,which is characterized by persistent pain and difficult to treat.Current therapeutic strategies for DNP are mostly symptomatic therapies,which primarily concentrate on pain management.However,most analgesics were demonstrated to provide only partial pain relief and have multiple side effects,so it is urgent to develop new drug for the therapy of DNP.Koumine is an effective component with high efficiency and low toxicity extracted from the plant of Gelsemium elegans Benth.(G.elegans),studies have shown that it has the potential to relieve neuropathic pain,but its therapeutic effect on diabetic neuropathic pain remains to be further clarified and the mechanism remains to be explained.The role of astrocytes in neuropathic pain has caused the extensive concern in recent years.New evidence suggests that astrocytes are involved in the formation and development of peripheral and central neuropathic pain.After nerve injury,astrocytes become activated and release a flood of proinflammatory cytokines,which not only lead to further activation of the astrocytes themselves,but also promote the sensitization of neurons,resulting in chronic pain migration and long-lasting pain.Therefore,astrocytes may be a new target for the development of drugs for treating severe pain caused by neuropathic pain,including DNP.The amygdala is an important part of the limbic system,consisting of more than a dozen nuclei of different sizes,the more important ones including the central nucleus(Ce A)and basolateral amygdala(BLA).Currently,it is known that the BLA plays an important role in cognition,memory and attention control.Recent studies have further identified BLA as a key center for pain regulation,mainly involved in the modulation of pain-related emotions.In addition,BLA can not only passively receive multiple subcortical information,but also mediate the transmission of harmful information through mutual projection with other nuclei.Therefore,it is inferred that BLA plays a very significant role in the modulation of pain sensation and pain-related emotions.However,little is known about how BLA regulates pain,and whether there is a causal relationship between BLA astrocyte activation and DNP pathogenesis remains to be determined.Given all this,we establish DNP rats as the research object,comprehensive utilization of pharmacology,nerve morphology,cell and molecular biology,DREADDs and other multi-disciplinary technical means,to the BLA as the observation point,astrocyte activation as the breakthrough point,from the perspective of neuroinflammation,the mechanism of KM targeting BLA astrocytes alleviate DNP was systematically explored,which laid a theoretical and experimental basis for the development of original candidate new drugs of KM,and also provided new target and pathway for the research and development of novel drugs for the prevention and treatment of DNP.The main research contents are as follows:1 Effects of koumine on mechanical allodynia and pain-related emotions in DNP ratsEstablish the streptozocin(STZ)-induced DNP rats,and rats were randomly divided into control group,DNP group,koumine hydrochloride(KM)treated group(0.28,1.4 and 7.0 mg/kg)and gabapentin treated group(100 mg/kg)as positive control.From day 22 after STZ injection,intragastric(i.p.)administration of relevant drugs or solvent intervention,once a day for 7 days.The mechanical withdrawal threshold(WMT)and its behavior changes in open field(OF)and elevated plus maze(EPM)were used as indicators,the therapeutic effects of koumine on mechanical allodynia and pain-related emotions in DNP rats were observed.The results showed that koumine had significant effects on mechanical allodynia and negative emotion of DNP rats in a dosedependent manner.2 Relationship between the analgesic effect of koumine on DNP rats and its regulation of BLA astrocytes activationThe dynamic changes of BLA glial during the development of DNP were analyzed in DNP rats.Rats were randomly divided into control and DNP group.Brain tissues of rats were perfused after pain measurement at 3,7,14,21 and 28 days after modeling,respectively,and then slices were performed for immunofluorescence histochemistry staining.The results showed that BLA astrocytes were dynamically activated during the development of DNP,and were highly negatively correlated with changes in mechanical withdrawal threshold of DNP rats.The Pearson correlation coefficient was-0.693(P<0.001).It is suggested that the occurrence and development of DNP is closely related to the activation of BLA astrocytes.In order to observe the effect of koumine on the activation of BLA astrocytes in DNP rats,brain tissue was perfused after the behavioral experiment and immunofluorescence histochemistry staining of astrocyte marker GFAP was performed.The results showed that the activated astrocytes in the BLA area of DNP rats can be significantly inhibited by KM,and it was highly negatively correlated with the alleviating effect of koumine on DNP rats' allodynia,with a correlation coefficient of-0.782(P<0.001).These results suggest that inhibiting the activation of BLA astrocytes may be one of the analgesic mechanisms of koumine on diabetic neuropathic pain.3 Effects of DREADDs regulating BLA astrocytes on mechanical allodynia and pain-related emotions in ratsTo investigate the regulatory role of BLA astrocytes in DNP,we specifically inhibited or activated BLA astrocytes in DNP or naive rats by DREADDs.The results showed that inhibiting the activation of BLA astrocytes can dramatically improve the MWT,and effectively reverse the anxiety-depressive behavior caused by chronic pain in DNP rats.However,activation of BLA astrocytes in naive rats can cause mechanical allodynia in naive rats,but not enough to induce pain-related emotions.These results indicate that BLA astrocytes act a pivotal part in pain regulation.To further investigate whether specific inhibition of the activation of BLA astrocytes affected the activity of BLA local neurons,the expression of neuronal activation marker c-Fos in rat BLA region was detected by immunofluorescence histochemical method.The results showed that c-Fos was greatly expressed in BLA of DNP rats,regulation of the activity of BLA astrocytes could affect the expression of cFos,and most c-Fos were co-labeled with excitatory neuronal marker Ca MKII,and only a few were co-labeled with GAD67(an inhibitory neuronal marker),suggesting that BLA astrocytes can regulate the excitatory neurons in BLA region.In order to investigate whether the analgesic effect of astrocytes in BLA region was realized by inhibiting the activity of BLA excitatory neurons,a mixed virus that activates astrocytes and inhibits the activity of excitatory neurons was injected into the BLA area of naive rats,it was observed that after CNO administration,the MWT of the BLA astrocyte-activated rats was significantly reduced,but there was no significant change in the BLA astrocyte-activated rats while the excitatory neuron activity was inhibited,suggesting that BLA astrocytes may regulate pain behavior by regulating the activity of BLA excitatory neurons.In order to study how BLA astrocytes affect the activity of their local excitatory neurons,enzyme linked immunosorbent assay(ELISA)was used to determine tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?)and the expression of chemokines CXCL1 and MCP-1 in the BLA region.The results showed that inhibition of BLA astrocyte can alleviate mechanical allodynia in rats with DNP coincident with the reduce of TNF-?,IL-1?,CXCL1 and MCP-1.On the contrary,after activation of BLA astrocytes it can induce mechanical allodynia in naive rats and is accompanied by increased expression of TNF-?,IL-1? and CXCL1 and MCP-1.In summary,these results suggest that BLA astrocytes are one of the crucial mediators of DNP,the regulatory effect may be mediated by the release of inflammatory mediators and the activation of the local excitatory neurons.4 BLA astrocytes mediate the effect of koumine in the treatment of diabetic neuropathic painUsing the combination of DREADDs and KM to further confirm that BLA astrocytes are the target cells of koumine to exert its analgesic effect.Rats were divided into control group,DNP group,DNP rats transfected with gfa ABC1D-M4-EGFP group and its KM treated group.On the 21 st day after modeling,the above-mentioned transfected with gfa ABC1D-M4-EGFP and group with KM treated were given 1.0mg/kg CNO intraperitoneally(i.p.),and 1 h later 7.0 mg/kg KM or saline were given intragastrically(i.g.).After intragastric administration for 1 h,the MWT of each group was measured and once a day for 7 days,then the synergistic effect of koumine on the analgesic effect of CNO-activated receptor was analyzed.The results showed that continuous specific inhibition of the activation of BLA astrocytes could significantly alleviate the mechanical allodynia in DNP rats.After the combined application of KM,the mechanical allodynia relief effect had no significant change compared with that of the single application.Furthermore,DREADDs was used to continuously activate the activation of astrocytes in BLA to simulate chronic neuropathic pain in naive rats.Rats were set up as control group,DNP group,transfected with gfa ABC1D-M3-EGFP and its KM treated group.21 days after virus injection,the gfa ABC1D-M3-EGFP transfected group and its KM treated group were i.p.0.5 mg/kg CNO,1 hour later KM(7.0 mg/kg)or saline was administered i.g.,and after intragastric administration for 1 h the MWT of each group was measured,once a day for 7 days,then the antagonistic effect of koumine on mechanical allodynia induced by CNO activated receptors was analyzed.The results demonstrated that rats with long-term activation of BLA astrocytes showed persistent mechanical allodynia and pain-related emotions like DNP rats.Under the effect of KM,the mechanical allodynia was gradually relieved,and the anxiety behavior was significantly improved.In conclusion,it is suggested that BLA astrocytes may be one of the important targets for koumine in treatment of diabetic neuropathic pain.5 Effect of koumine on the inflammatory mediators produced and released by activated astrocytesIn order to observe the effect of koumine on the inflammatory mediators released by activated astrocytes,the tissues of BLA region in each group were collected after the behavioral experiment,and the expression levels of TNF-?,IL-1?,CXCL1 and MCP-1 were determined by enzyme linked immunosorbent assay(ELISA).Our results indicated that the levels of tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?),CXCL1 and MCP-1 were significantly increased in BLA of DNP rats,and koumine could dose-dependently decrease the levels of inflammatory mediators in BLA region.At the culture cell level,lipopolysaccharides(LPS)was used to induce the activation of C6 cells.The experiment was divided into control,model,and model cells treated with different concentrations of KM(25 ?M,50 ?M,100 ?M and 200 ?M).The cell supernatants of each group were collected and ELISA was used to detect the level of TNF-?,IL-1?,CXCL1 and MCP-1 in each group.The results showed that LPS induced neuroinflammation in C6 cells and increased the expression of inflammatory mediators.After KM pretreatment,the level of TNF-?,IL-1?,CXCL1 and MCP-1 were decreased in a concentration-dependent manner,suggesting that koumine could effectively inhibit the production and release of inflammatory mediators by C6 cells.In conclusion,the results suggest that the activation of astrocytes in the BLA plays an important role in the development of diabetic neuropathic pain.Activation of astrocytes in the BLA may be one of the key factors leading to diabetic neuropathic pain.Koumine has significant analgesic effect and anti-anxiety effect on diabetic neuropathic pain,which is related to its inhibition of the activation of BLA astrocytes and the formation and release of inflammatory mediators,which may be one of the important mechanisms for its treatment effect of diabetic neuropathic pain.