Zhenbao Pill reduces Treg cell proportion in acute spinal cord injury rats by regulating TUG1/miR-214/HSP27 axis.

Background: Acute spinal cord injury (SCI) is one of the weakest pathologies that seriously affect the quality of life of patients. Objective: To study the mechanism of how Zhenbao Pill reduces Treg cell proportion and improves acute SCI. Methods: A rat SCI model was established. Flow cytometry analysis was performed to determine the Treg cell proportion. RNA immunoprecipitation (RIP) and RNA pull-down were applied in confirming taurine up-regulated gene 1 (TUG1) and miR-214 binding. Intrathecal injection of TUG1 siRNA was also conducted to determine the effect of TUG1 in vivoResults: Zhenbao Pill promoted the expression of TUG1 and heat shock protein 27 (HSP27) protein, and reduced the expression of miR-214 and forkhead box protein p3 (Foxp3) as well as Treg cell proportion in a concentration-dependent manner in SCI rats or in vitro cultured CD4+ T cells. Knockdown of TUG1 reversed the high protein expression of HSP27 and the inhibition of Treg cell proportion as well as Foxp3 protein induced by Zhenbao Pill, and miR-214 inhibitor canceled the TUG1 knockdown effect. Further, miR-214 mimic reversed the inhibition of Treg cell proportion and Foxp3 protein expression by Zhenbao Pill, which was abolished by the overexpression of HSP27. The mechanism was validated in animal experiments. Conclusion: Zhenbao Pill regulated TUG1/miR-214/HSP27 signaling pathway to reduce Treg cell proportion and thus relieve acute SCI.

Zhenbao pill protects against acute spinal cord injury via miR-146a-5p regulating the expression of GPR17.

The 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). ASCI rat model was established by modified Allen method, and then the rats were divided into three groups. SH-SY5Y cells were cultured overnight in hypoxia condition and transfected with miR-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 used to detect the expression of miR-146a-5p, GPR17, inducible nitric oxide synthase (iNOS), interleukin 1ß (IL-1ß), and tumor necrosis factor α (TNF-α). Neuronal apoptosis was measured using flow cytometry assay. Luciferase reporter assay was performed to determine the regulation of miR-146a-5p on GPR17. Zhenbao pill could enhance hind limb motor function and attenuate the inflammatory 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 miR-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 zhenbao pill on the cell apoptosis induced by hypoxia and the recovery of zhenbao pill on hind limb motor function in ASCI rats. Zhenbao pill could inhibit neuronal apoptosis by regulating miR-146a-5p/GPR17 expression, and then promoting the recovery of spinal cord function.

Zhenbao Pill reduces the percentage of Treg cells by inducing HSP27 expression.

CONTEXT: Zhenbao pill containing Nacre, Safflower, Musk and Cornu Bubahas has been proved to have a good therapeutic effect on the repair of spinal cord injury (SCI). However, its complex mechanism of repairing SCI is not yet known. OBJECTIVE: This 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. MATERIALS AND METHODS: Treatment of human peripheral blood mononuclear cells (PBMCs) with different concentrations (0, 0.5, 2.5, 5, 10mg/mL) of Zhenbao pill, flow cytometry was used to detect the expression of the specific factors CD4+CD25+Foxp3+ in Tregs, and detection of Tregs related regulatory factor TGF-ß content was 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. RESULTS: Zhenbao pill with 11.61mg/mL 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 miR-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. CONCLUSIONS: In the process of repairing the SCI, Zhenbao pill inhibits reduces numbers of Treg lymphocytes as well as TGF-ß levels by inducing HSP27 expression.