改良芍药甘草汤调控lncRNA H19/miR-185-5p/Tph1轴干预脊髓损伤后肌痉挛的机制研究OA

Objective To investigate the biological mechanisms underlying the therapeutic effect of modified Shaoyao Gancao decoction(芍药甘草汤)in alleviating muscle spasms following spinal cord injury.Methods ①Extract and modify the active components from the original Shaoyao Gancao decoction(芍药甘草汤),determined their concentrations using chro-matography-mass spectrometry(HPLC-MS),and compared the differences between the two formulations.②Forty SD rats were randomly divided into four groups:normal control group,model group,treatment group,and positive control group,with 10 rats in each group.With the exception of the normal control group,all other rats were subjected to a spinal cord injury model via se-mi-transverse sectioning of the T9-T10segments.Three days post-surgery,the normal control group and the model group received oral administration of deionized water;the drug-treated group received oral administration of modified Shaoyao Gancao decoc-tion(芍药甘草汤),Shaoyao∶Gancao=3∶1,3.3 g of crude drugs per kg body weight;the positive control group received oral ad-ministration of Standard Shaoyao Gancao Decoction(Shaoyao∶Gancao=1∶1,3.3 g of crude drugs per kg body weight).The total treatment duration was 2 weeks.The drug efficacy was evaluated using the BBB scale(Basso,Beattie & Bresnahanlocomotor rating scale,BBB scale),and 5-hydroxytryptamine(5-HT)levels in spinal cord tissue were measured.The expression of long noncoding RNA H19(lncRNA H19,hereinafter referred to as H19)and microRNA 185-5p(miR-185-5p)in spinal cord tis-sues was examined using polymerase chain reaction(RT-qPCR)in both the model group and the treatment group.Furthermore,a dual-fluorescein enzyme reporter assay was employed to verify that H19 regulates the expression of miR-185-5p and that miR-185-5p,in turn,regulates the expression of tryptophan hydroxylase 1(Tph1).③In vitro culture of spinal cord nerve cells was conducted,where modified Shaoyao Gancao decoction(芍药甘草汤),H19inhibitor,and miR-185-5p inhibitor were adminis-tered to investigate the effects of these agents as well as H19 and miR-185-5p on Tph 1 expression.④Twelve SD rats were ran-domly divided into four groups:the model group,the drug administration group,the H19 inhibitor group,and the positive con-trol group,with three rats in each group.Rats in the H19 inhibitor group received intravenous injection of H19 inhibitor at a dose of 50 mg·kg-1 via the tail vein,while all other groups were treated as described in step ①.The expression levels of 5-HT were determined using enzyme-linked immunosorbent assay(ELISA).Results ①The overall ion current profile showed no sig-nificant differences;however,in the modified Shaoyao Gancao decoction(芍药甘草汤),compounds related to Paeonia exhib-ited higher responses,while those related to Glycyrrhiza demonstrated reduced responses.②At 2 weeks post-administration,the BBB score was 10.000±1.291 in the drug-treated group and 9.667±1.633 in the positive control group,both significantly higher than those in the model group 3.286±0.756(P＜0.01).Additionally,5-HT expression levels were elevated in the drug-treated group(7.097±1.414)pg·mL-1 compared to the model group(5.144±0.904)pg·mL-1(P＜0.01).RT-qPCR and Western blot results demonstrated increased expression of H19 and Tph1 in spinal cord tissue following administration,while miR-185-5p expression was downregulated.Dual-fluorescein reporter gene experiments further confirmed that H19 regulates miR-185-5p expression,which in turn modulates Tph1 expression.③In the cultured model group,Tph1 expression in spinal cord cells was lower than that in the normal control group,whereas it increased in the drug-treated group.In the H19 inhibitor group,Tph1 expression was reduced compared to both the normal control group and the drug-treated group;however,it was elevated in the miR-185-5p inhibitor group compared to both the model group and the H19 inhibitor group.④Further experiments demonstrat-ed that 5-HT exhibited high expression levels in the spinal cord tissues of rats in the drug-treated group,and the H19 inhibitor could reverse this phenomenon.Conclusion Shaoyao Gancao decoction(芍药甘草汤)exerts a positive effect on spinal cord repair in rats with spinal cord injury by modulating the H19/miR-185-5p/Tph1 axis,thereby promoting the synthesis of the neurotransmitter 5-HT.