Study On Analgesic Mechanism Of Tetrahydropalmatine Based On Gene Sequencing

Background and Purpose:Neuropathic pain(NP)treatment remains a challenge because the pathomechanism is not yet fully understood.Because of low treatment efficacy,there is an important unmet need in neuropathic pain patients,and the development of a more effective pharmacotherapy is urgently required.Traditional Chinese medicine treatment has the advantages of multi-target,multi-channel and weak side effects,which needs our efforts to develop.Tetrahydropalmatine(THP)is the main effective component from the tuber of Corydalis yanhusuo(W.T.Wang),a plant in the poppy family.THP has the effects of analgesia and sedation,reduce addiction,anti arrhythmia,delay myocardial remodeling,improve hemodynamics and reduce blood lipid.Neuroinflammation induced by oxidative stress-mediated plays an important role in NP.Ca2+ channel(Cacng1)subunit is a membrane protein that participates in calcium transport and affects NP.In this study,we aimed to investigate the protective properties of THP on a spared nerve injury(SNI)model of neuropathic pain in mice in in vivo and also in in vitro experiments.Thought and Method:(1)Firstly,this study used the spared nerve injury(SNI)model to give THP treatment.Through the pain behavior experiment of different groups of mice,the differences between mechanical pain and cold pain in mice were detected,and whether THP participated in the analgesic effect of SNI model was judged from the behavior.The genes affecting THP in neuropathic pain were analyzed by gene sequencing of dorsal root ganglion(DRG)in model group and THP treatment group.H&E staining showed the effect of THP on the inflammatory response of SNI model.Detection of the expression of iNOS and IL-1β in DRG and sciatic nerve(SN)by qRT-PCR.Griess detected NO levels in DRG,SN and serum.The expression of related proteins was detected by Western blot.(2)After using iNOS inhibitor,the differences of mechanical pain and cold pain in mice were detected.The binding of THP to P3 8,P65 and iNOS was detected by molecular docking between protein and active components.ELISA detected IL-1β and Griess detected the level of NO in serum of each group.The expression of related proteins was detected by Western blot.(3)In order to determine the molecular mechanism of THP against NP,CCK-8 experiment was used to explore the cytotoxicity of THP in nerve cells.In order to further study the mechanism of THP,the related proteins in the models of sodium nitroprusside(SNP)-induced neuro-2a(N2a)cells,LPS-induced BV2 cells,and LTA-induced astrocytes were detected by Western blot and immunofluorescence.(4)qRT-PCR and Western blot were used to detect the expression of Cacngl in DRG and SN after THP treated SNI model.After N2a cells overexpressed Cacngl plasmid,the effect of THP on Cacng1 expression was detected by Western blot,the distribution and concentration of Ca2+ in each group were detected by calcium ion fluorescence probe(Fura-2AM),and the content of Ca2+ in cell lysate and culture supernatant were detected by calcium content detection kit.Results:(1)Compared with the SNI groups,THP decreased mechanical hyperalgesia and cold allodynia compared with the SNI group.A microarray was applied to analyze differentially expressed of mRNA among different groups,and THP noticeably changed the expression of MAPK-related proteins compared with the SNI groups.H&E staining showed that the THP changed the inflammation after the spared nerve injury.Compared with SNI group,the expression of NO in DRG、SN and serum decreased after THP treatment.Further results indicated that THP suppressed the expression of inducible nitric oxide synthase(iNOS),P38MAPKs and p65 phosphorylation in sciatic nerve.(2)Compared with the model group,the mechanical hyperalgesia and cold hyperalgesia of mice treated with iNOS inhibitor decreased.The serum levels of the pro-inflammatory cytokines IL-1β were significantly higher in the SNI group as compared to the THP group.(3)To identify the molecular mechanism of the antineuropathic activity of THP,sodium nitroprusside(SNP)-induced neuro-2a(N2a)cells,LPS-induced BV2 cells,and LTA-induced astrocytes were further investigated in signaling pathways.In vitro experiments indicated that THP suppressed the expression of IL-1β,iNOS,phosphorylated P38MAPKs,and P65,which were assayed using western blotting,and immunofluorescence.(4)The expression of Cacngl was increased in SNI model,and THP could inhibit the expression of Cacngl.In vitro,after overexpression of Cacngl,THP could inhibit the expression of Cacng1.THP can inhibit the increased concentration and distribution of Ca2+ in cells after Cacngl overexpression.Conclusion:This study found that tetrahydropalmatine(THP)has analgesic effect in neuropathic pain;THP may alleviate neuroinflammation through P38MAPK/NF-κB/iNOS pathway and NO expression;THP may also regulate the expression of Cacngl in neuropathic pain,affect the transport of Ca2+,and alleviate neuropathic pain.