[Modified Shaoyao Gancao decoction()alleviates post-spinal cord injury muscle spasticity by regulating the lncRNA...

To investigate the biological mechanisms underlying the therapeutic effect of modified Shaoyao Gancao decoction () in alleviating muscle spasms following spinal cord injury.

①Extract and modify the active components from the original Shaoyao Gancao decoction (), determined their concentrations using chromatography-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 semi-transverse sectioning of the T₉-T₁₀ segments. 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 decoction (), Shaoyao∶Gancao=3∶1, 3.3 g of crude drugs per kg body weight;the positive control group received oral administration 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 tissues 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 (), H19 inhibitor, and miR-185-5p inhibitor were administered to investigate the effects of these agents as well as H19 and miR-185-5p on Tph1 expression.④Twelve SD rats were randomly divided into four groups:the model group, the drug administration group, the H19 inhibitor group, and the positive control 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).

①The overall ion current profile showed no significant differences;however, in the modified Shaoyao Gancao decoction (), compounds related to Paeonia exhibited 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 demonstrated 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.

Shaoyao Gancao decoction () 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.