Antispasmodic effects and potential mechanism of the traditional chinese medicine Shaoyao–Gancao decoction on intestinal spasm in rats

• SGD ameliorates intestinal spasmodic disorders through inhibiting smooth muscle contraction. • SGD has multi-component-and multi-target characteristics in the treatment of anti-intestinal spasm. • SGD inhibits intestinal smooth muscle contraction through calcium signaling pathway. Shaoyao–Gancao decoction (SGD) is a classic Chinese herbal formula extensively used to treat various spastic diseases in clinical settings. SGD exerts a significant antispasmodic effect with high economic value. However, the biological activity and the potential mechanisms of SGD against the intestinal spasm remain elusive. This study aimed to scientifically evaluate the antispasmodic effect of SGD on inhibiting the intestinal spasm in rats and to elucidate the multi-component and multi-targets mechanism of SGD to treat gastrointestinal spasms. In vitro and In vivo experiments were designed and conducted to verify that SGD had intestinal antispasmodic and relaxed intestinal smooth-muscle activity. In addition, the expression levels of l -type voltage-gated Ca 2+ channel 1.2 (Cav1.2), inositol 1,4,5-trisphosphate receptor (IP 3 R), calcium/calmodulin dependent proteinase Ⅱ (CaMKΠ), myosin light chain kinase (MLCK), and ryanodine receptor (RyR2) in intestinal tissue were analyzed by Western blotting (WB). Moreover, network pharmacology analysis was performed to predict the key antispasmodic active ingredients of SGD, which were further subjected to in silico studies for molecular docking. In vitro experiments revealed that SGD had a concentration-dependent relaxant effect on the spontaneous and Ach (1 mM)-induced spastic contraction. The application of a potassium channel blocker (TEA) did not affect the SGD-mediated anti-contraction activity, wheras SGD significantly inhibited the enhancement of intestinal smooth-muscle contraction induced by Bayky8644 (an l -type calcium-channel activator). Similarly, the attenuation of contraction induced by SGD can be reversed by Baky8644. In addition, the traditional Chinese medicine-based network pharmacology study revealed that SGD exerted the anti-intestinal spasm effect by acting on multiple pathways. The main active components of SGD, liquiritigenin and benzoylpaeoniflorin, had stronger binding to the voltage-gated calcium channel β2a (VGCC), calcium/calmodulin-dependent protein kinase IIB (CAMK2B), and myosin light-chain kinase-1 (MLCK-1), thereby exerting calcium channel blocker activity. Moreover, SGD exhibited antiperistaltic effects as demonstrated by In vivo studies. Western blotting studies further revealed that the antispasmodic mechanism of SGD involved regulating the target proteins of calcium that mediated signalling by increasing the level of Cav1.2, IP 3 R, and CaMKΠ and decreasing the level of RyR2. SGD exerted a significant inhibitory effect on intestinal smooth-muscle contraction, probably by altering the expression of the regulatory genes involved in the calcium-mediated smooth contraction process. Multi-component-and multi-target characteristics of SGD for treating anti-intestinal spasm were demonstrated. Overall, this study provided a foundation for the quality control, product development, and clinical application of further classic formula-development research.