Exploring the Potential Mechanism of Zhizhu Decoction in the Treatment of Constipation-Predominant Irritable Bowel Syndrome Through Network Pharmacology and Experimental Validation.

aim of the studyZhizhu Decoction (ZZD), a classical traditional Chinese herbal formula composed of Atractylodis Macrocephalae Rhizoma and Aurantii Fructus Immaturus, has long been used to treat gastrointestinal disorders resembling constipation-predominant irritable bowel syndrome (IBS-C). This study aimed to elucidate the potential therapeutic mechanisms of ZZD in IBS-C by integrating network pharmacology, molecular docking, and in vivo experimental validation. MATERIALS AND

methodsActive compounds and targets of ZZD were identified using network pharmacology, followed by molecular docking to evaluate compound-target interactions. An IBS-C rat model was established to assess the therapeutic effects of ZZD. H&E and AB-PAS staining were used to observe histological lesions in colonic tissues. TUNEL staining was performed to detect apoptotic epithelial cells in colon sections. Immunohistochemistry (IHC) was used to assess the spatial distribution of TPH1 and tight junction proteins ZO-1 and Claudin-1 in colon tissues. RT-qPCR combined with western blotting was used to quantify the expression of ZO-1 and Claudin-1. Serum 5-HT concentrations were determined by ELISA, and liquid chromatography-mass spectrometry (LC-MS) was used to measure fecal short-chain fatty acid (SCFA) levels.

resultsNetwork pharmacology analysis: ZZD contains 23 bioactive compounds. This herbal formula mainly acts on core targets including AKT1, BCL-2, Caspase-3, and EGFR, with prominent enrichment of PI3K/AKT, apoptosis, TNF and NF-κB signaling. Molecular docking results further confirmed the favorable binding affinity of its main active ingredients (luteolin, naringenin, 3β-acetoxyatractylone, and 8β-ethoxyatractylenolide III) to these targets. In vivo experiments: In IBS-C model rats, ZZD intervention alleviated weight loss, elevated 24-h fecal output, fecal water content and Bristol stool scale scores, accelerated small intestinal carbon powder transit, and lowered visceral hypersensitivity as indicated by reduced AWR scores (P < 0.05), and it recovered goblet cell abundance, improved crypt architecture and thickened colonic mucus and muscular layers. ZZD markedly alleviated colonic injury and maintained intestinal barrier and tight junction integrity, as evidenced by upregulation of mRNA and protein levels of tight junction proteins ZO-1 and Claudin-1 (P < 0.05), as well as restoration of their continuous distribution in rat colonic tissues. ZZD suppressed epithelial apoptosis by downregulating Caspase-3 and upregulating BCL-2 and c-JUN (P < 0.05). In addition, IHC staining for TPH1, a specific marker of EC cells, revealed that ZZD treatment significantly increased the number of TPH1‑positive cells in the colonic epithelium (P < 0.0001), accompanied by elevated serum 5‑HT levels (P < 0.0001), suggesting recovery of the EC cell population and intestinal 5‑HT synthetic capacity. Moreover, ZZD increased the phosphorylation levels of colonic EGFR, PI3K and AKT (P < 0.05). It also significantly raised fecal SCFA levels (P < 0.05), implying improved gut microbial metabolism as a potential protective factor.

conclusionZZD exerts therapeutic effects against IBS-C that involve multiple mechanisms, including inhibition of intestinal epithelial apoptosis, preservation of tight-junction protein expression and distribution (ZO-1 and Claudin-1), restoration of EC cell integrity (as indicated by increased TPH1-positive cell abundance on IHC), and modulation of short-chain fatty acid metabolism. In addition, increased phosphorylation of EGFR, PI3K, and AKT was observed in association with these protective effects, suggesting potential involvement of this pathway in the underlying mechanisms.