The Inhibition Effect And Mechanism Of Puerarin On Neuropathic Pain

Background:neuropathic pain(NP)is a common chronic pain.In clinical practice,many patients suffer from spontaneous pain,abnormal pain,induced,hyperalgesia and hyperalgesia.It seriously affects the survival and life of the patients.However,there is no effective treatment for this chronic neuralgia.Therefore,our study on the pathogenesis of neuropathic pain aims at obtaining more effective treatment measures,improving the life of the patients and reducing the cost of medical treatment which is very significant.Transient response pathway(TRP)plays an important role in neuropathic pain.In addition,the inflammatory response to the activation of glial cells in the spinal cord and the proinflammatory cytokines IL-1 beta,IL-6 and tumor necrosis factor(TNF-alpha),which are produced by peripheral nerve injury,play an important role in the sensitization of the central nervous system.In recent years,the studies on analgesics of Chinese medicine has been widely reported.Many Chinese herbs have significant effects on inhibiting neuropathic pain,but the mechanism is unknown.The aim of this study is to using the pSNL neuropathic pain model to explore the exact analgesic effect of puerarin on neuropathic pain rats,futher,we want to explore the pain related signaling pathway and the specific analgesic molecules mechanism of puerarin,so as to provide a theoretical basis and a new strategy for the prevention and control of neuropathic pain.The first part The analgesic effect of Puerarin on pSNL neuropathic pain rats and the effects of Puerarin on locomotor activity in rats.Objective:To explore the analgesic effect of Puerarin on pSNL neuropathic pain rats.Methods:40 adult male SD rats,SPF,weighing 180-210g,were randomly divided into control(n=10,group C),model(n=10,group M),low dose of puerarin(n=10,L group,30mg/kg),and high dose of puerarin(n=10,group H,60mg/kg).In the three groups outside the control group,peripheral nerve pain models were established by Partial Sciatic Nerve Ligation(pSNL).After operation,normal saline was injected into the control group and model group for 7 consecutive days.The other two groups were given the corresponding dose of Puerarin:the rats’ mechanical pain threshold(MWT)and the latent period(PWL)were measured before and 1-7 days after the operation.The rats’ weight was recorded during the period of the operation,and the rats’ autonomic activity was detected on the 0,1,3,7 day of the experiment and the cold tolerance threshold of rats was detected by the cold acetone test.At the end of behavioral experiments,rats were killed and the spinal cord of L4-L6 segment dorsal root ganglion and lumbar enlargement segment were taken for subsequent detection.Results:compared with group C,the MWT and PWL of rats in group M were significantly decreased(P<0.05),these results suggested that the model had been established successfully.As compared with the M group,MWT and PWL increased significantly in L and H groups(P<0.05),and the increase in L groups was more significant,suggesting that puerarin could alleviate the mechanical and thermal pain sensitivity of rats.In group M,the locomotor activity in rats decreased significantly,while in L and H groups,the locomotor activity in group M was improved(P<0.05).Conclusion:Puerarin can improve the mechanical pain sensitivity,cold pain sensitivity and thermal pain sensitivity of pSNL neuropathic pain rats,and can also improve the ability of autonomic activity in rats.The second part The effect of Puerarin on the expression of TRPV1/TRPA1 protein in rats with neuropathic pain.Objective:To explore the effect of puerarin on TRPV1/TRPA1 protein in rats with neuropathic pain.Methods:The dorsal root ganglion of L4-L6 segments of rats in each group was taken after the final behavioral experiment.The expression of TRPV1/TRPA1 protein and mRNA in the dorsal root ganglion of rats was detected by Westernblot and q-PCR.Results:The content of TRPV1 and TRPA1 mRNA in group M increased significantly compared with group C(P<0.05).Compared with the M group,the expression of TRPV1 and TRPA1mRNA in L and H groups was obviously reduced(P<0.05).Compared with the C group,the expression of TRPV1 and TRPA1 protein in the M group increased significantly(P<0.05),and the expression of TRPV1 and TRPA1 proteins in the L and H groups also decreased significantly compared with the M group.Conclusion:Puerarin can improve the expression of TRPA1/TRPV1 protein and mRNA in rats with neuropathic pain.The third part The effect of Puerarin on the activation of glial cells and inflammatory factors in spinal cord of rats with neuropathic pain.Objective:To investigate the effect of puerarin on the activation of microglia and astrocytes in the spinal cord of rats with neuropathic pain and the effects of puerarin on the inflammatory factors in the spinal cord.Methods:After the final test,the spinal cord was fully exposed,and the spinal cord was removed and placed in 4%polyformaldehyde to fix 6-10h(or 4℃refrigerator overnight),and then the tissue was transferred to the dehydrated base of 30%sucrose prepared by PBS(4 ℃ refrigerator,24 h).After the tissue was deposited,the tissue was embedded with OCT embedding agent and stored in the-80 ℃refrigerator.The spinal cord used to detect inflammatory factors was frozen directly after-80 C refrigerator was removed then cut into frozen sections with a thickness of 4μm before dyeing.The content of the activation markers Iba-1 of spinal microglia and the activation marker GFAP of astrocytes were detected by immunofluorescence,and the content of IL-1 beta,IL-6 and TNF-alpha in the spinal cord was detected by Elisa.Results:compared with the C group,the expression of astrocyte activation marker GFAP protein and microglia Iba-1 protein in the spinal dorsal horn of the M group increased significantly,while the L and H groups were significantly lower than those in the M group(P<0.05).It suggested that puerarin can inhibit the activation of astrocytes and microglia in the dorsal horn of spinal cord which were activated by pSNL.Compared with group C,the contents of IL-1 beta,IL-6 and TNF-alpha in the spinal cord of M group increased significantly,both in L group and H group.