The Role And Related Mechanism Of Zhen Bao Pill In Regeneration And Treatment Of Spinal Cord Injury

BackgroundSpinal Cord Injury(SCI)is a common clinical condition with high morbidity and lack of effective treatment in the long-term.This project is based on years of experience in clinical treatment of Treasure Pills and previous experimental studies found that Jumbo Pills containing nacre,peony,musk,and antelope have a good therapeutic effect on spinal cord injury(SCI).However,the complex mechanism of repairing spinal cord injury is still not clear.The various complex molecular mechanisms in spinal cord injury are still not very clear.Further studies and research are needed.This article starts with Zhenbao Pill and uses a rat model of spinal cord injury for research,In order to get the role of Zhenbao Pill in the regeneration of spinal cord injury and related mechanisms.Part1 Zhenbao Pill reduces the percentage of Treg cells byinducing HSP27 expressionObjectiveThis study attempts to investigate the role of CD4+CD25+Foxp3+ regulatory T cells(Tregs)in the mechanism of the action of Zhenbao pill,and further explore the relationship between Tregs and HSP27 expression in repair mechanism.MethodTreatment of human peripheral blood mononuclear cells(PBMCs)with different concentrations(0,0.5,2.5,5,10 mg/m L)of Zhenbao pill,flow cytometry was used to detect the expression of thespecific factors CD4+CD25+Foxp3+ in Tregs,and detection of Tregs related regulatory factor TGF-P contentwas performed with ELISA assay.The relationship between miR-214 and HSP27 was assessed by Luciferase assay,and the level of miR-214 was detected by qPCR.The expression of HSP27 was examined with qPCR and western blotting.RNA interference technology and gene recombination were used to inhibit and up-regulate the expression of HSP27.ResultsZhenbao pill with 11.61 mg/m L of IC50 for Tregs can significantly inhibit the differentiation into Tregs in human PBMCs and up-regulate by more than 1-fold of HSP27 expression.Essentially,it enhanced the expression of HSP27 by inhibiting mi R-214 expression(50%).Inhibition of HSP27 expression,followed by the differentiation into Tregs,was promoted in human PBMCs.When the HSP27 expression was up-regulated,the differentiation into Tregs was decreased by 30%.It indicated that the expression of HSP27 regulated the differentiation into Tregs.Inhibition of HSP27 expression and Zhenbao pill treatment,the differentiation into Tregs was decreased but remained at a higher level than that of the group was only treated with pill.Under the action of Zhenbao pill,the expression of HSP27 was not completely interfered,and its expression level was still increased.ConclusionIn the process of repairing the SCI,Zhenbao pill inhibits reduces numbers of Treg lymphocytes as well as TGF-βlevels by inducing HSP27 expression.Part2 Zhenbao pill protects against acute spinal cord injuryvia miR-146a-5p regulating the expression of GPR17ObjectiveThe aim of the present study was to observe the effect of zhenbao pill on the motor function of acute spinal cord injury(ASCI)rats and the molecular mechanisms involving miR-146a-5p and G-protein-coupled receptor 17(GPR17).MethodASCI rat model was established by modiied Allen method,and then the rats were divided into three groups.SH-SY5Y cells were cultured overnight in hypoxia condition and transfected with mi R-146a-5p mimic or miR-146a-5p inhibitor.The hind limb motor function of the rats was evaluated by Basso,Beattie,Bresnahan(BBB)scoring system.Quantitative real-time PCR(qRT-PCR)and Western blot were usedto detect the expression of miR-146a-5p,GPR17,inducible nitric oxide synthase(i NOS),interleukin 1β(IL-1β),and tumor necrosis factor a(TNF-a).Neuronal apoptosis was measured using low cytometry assay.Luciferase reporter assay was performed to determine the regulation of miR-146a-5p on GPR17.ResultsZhenbao pill could enhance hind limb motor function and attenuate the inlammatory response caused by ASCI.Moreover,zhenbao pill increased the level of miR-146a-5p and decreased GPR17 expression in vivo and in vitro.Bioinformatics software predicted that GPR17 3-UTR had a binding site with mi R-146a-5p.Luciferase reporter assay showed that miR-146a-5p had a negative regulatory effect on GPR17 expression.Knockdown of miR-146a-5p could reverse the effect of zhenbao pill on the up-regulation of GPR17 induced by hypoxia,reversed the inhibitory effect of zhen bao pill on the cell apoptosis induced by hypoxia and the recovery of zhenbao pill on hind limb motor function in ASCI rats.ConclusionZhenbao pill could inhibit neuronal apoptosis by regulating miR-146a-5p/GPR17 expression,and then promoting the recovery of spinal cord function.