Mechanism study of HuanglianKushen decoction intervention in DSS-induced ulcerative colitis mice based on integrative...

Ulcerative colitis (UC) is an inflammatory bowel disease characterized by variable extent colon and rectum inflammation. The pathogenesis is not yet clear, with no curative treatments available. HuanglianKushen decoction (HKD) is derived from Puji Fang (Prescriptions for Universal Relief) which records its effect for improving symptoms of acute severe UC. However, weak research on its efficacy and pharmacological mechanism limits its clinical application.

This study aims to establish the quality control method for the HKD aqueous extract and explore its mechanism of action on UC through intestinal microbiota and transcriptomics.

We employed high-performance liquid chromatography (HPLC) technology to develop the specific chromatogram of HKD aqueous extract. The UC model was induced by allowing free access to a solution of dextran sulfate sodium salt (DSS). Three doses of HKD were administered simultaneously for intervention. General signs were observed to score the disease activity index (DAI). The lengths of colon and small intestine tissues were detected and the colon tissues were subjected to histopathological examination. The weight indexes of spleen and thymus tissues was measured. The mRNA expression of inflammatory factors in colon tissues were assessed. After the efficacy was determined, the expression of key proteins for maintaining intestinal barrier integrity in colon tissues was detected and 16S rRNA sequencing was performed on the cecal contents. Meantime, high-throughput transcriptome sequencing of the colon tissues was performed to identify differentially expressed genes (DEGs) and the signaling pathways that regulated by HKD intervention. Based on the transcriptome results, the molecular mechanisms were further investigated by Western blotting (WB), Real-time quantitative PCR (RT-qPCR), and TdT-mediated dUTP nick-end labeling (TUNEL) staining of colonic tissue.

The HPLC specific chromatogram of HKD aqueous extract was established; 21 common peaks were identified and nine components were qualitatively determined. Pharmacodynamic studies have shown that HKD intervention improved the macroscopic pathological characterization of the UC mice and the pathological morphology of colon tissue. It also restored the immune organ indices and exerted anti-inflammatory effects. 16S rRNA sequencing results showed that HKD intervention improved the dysbiosis of gut microbiota. Colonic transcriptome sequencing results showed that the DEGs regulated after HKD intervention were primarily enriched in the IL-17 signaling pathway, Forkhead box protein O (FoxO) signaling pathway and p53 signaling pathway, etc. And the restoration of the mRNA expression of 14 DEGs in these three pathways after HKD intervention was validated.

This study preliminarily established a quality control method for HKD aqueous extract and provided a research basis for the pathogenesis of UC and the anti-UC mechanism of HKD by demonstrating that HKD improved the pathological characteristics and intestinal barrier damage, regulated the dysbiosis of the gut microbiota, and suppressed the apoptosis of IECs probably by inhibiting the apoptosis-related FoxO1 signaling pathway in UC mice.