Kaempferol in Jichuan decoction alleviates slow-transit constipation by attenuating oxidative stress-induced interstitial cells of Cajal apoptosis: A network pharmacological study

Background: Slow transit constipation (STC), a common intestinal disorder, has a continuously rising incidence. The classical Chinese herbal formula Jichuan decoction (JCD) has been clinically applied for STC, yet the pharmacological mechanisms remain unclear. Kaempferol (Kae), as a potential active constituent of JCD, possesses antioxidant, anti-inflammatory, and gut-regulating capabilities. Investigating the role of Kae in STC treatment may provide a mechanistic basis for the application of JCD.

Methods: Network pharmacology was employed to screen the active constituents of JCD and their potential targets in STC. A protein-protein interaction network and a "herb compound-target-disease" network were constructed for the overlapping targets. GO and KEGG enrichment analyses were conducted. An STC model was established in mice by administering oral loperamide (LOP) for 7 consecutive days. The model mice were treated with 50 mg/kg/d Kae for a 14-day treatment cycle. Body weight, fecal count, and fecal water content were measured. Histopathological analyses included H&E, PAS and AB-PAS staining. Interstitial cells of Cajal (ICCs) were labelled with c-Kit, and Apoptosis was detected by TUNEL. The expression of water metabolism regulatory factors, AQP4 and AQP8, was assessed by Western blot (WB). Oxidative stress was gauged by MDA assays. Molecular docking and cellular thermal shift assays verified the interaction between Kae and GSTP1. Primary ICCs from STC mice were isolated. GSTP1 expression was determined by WB. Cell viability and Apoptosis were assessed by CCK-8 and flow cytometry. ICC oxidative stress was evaluated through Reactive Oxygen Species, malondialdehyde and superoxide dismutase levels detected by fluorescent probes and kits.

Results: Network pharmacological analyses identified 127 potential targets of JCD for STC, among which the top 5 hub genes were predominantly associated with oxidative stress and Apoptosis. In vivo models confirmed that Kae alleviated the LOP-induced constipation, evidenced by shortened fecal transit time, increased fecal pellet number, and higher fecal water content. Kae also attenuated the Apoptosis of ICC cells in the mouse intestines. Findings also revealed that Kae attenuated oxidative stress and Apoptosis in ICCs by binding to and stabilizing GSTP1.

Conclusion: Active constituent Kae in JCD attenuates LOP-induced intestinal dysmotility in mice by suppressing oxidative stress-driven ICC Apoptosis targeting GSTP1. This study provides experimental evidence for GSTP1 as a potential target for the treatment of STC.