Protective effect of San Huang Pill and its bioactive compounds against ulcerative colitis in Drosophila via modulation of JAK/STAT, apoptosis, Toll, and Nrf2/Keap1 pathways

Abstract

Ethnopharmacological relevance: San Huang Pill (SHP) is a prescription in Dunhuang Ancient Medical Prescription, which has the efficacy of heat-clearing and dampness-drying, and is a traditional formula for the treatment of gastrointestinal diseases. However, its efficacy and mechanism in treating ulcerative colitis (UC) are still unclear.

Aim of the study: To investigate the protective effects of SHP and its bioactive compounds against Dextran Sulfate Sodium (DSS)-induced intestinal damage using the Drosophila melanogaster model, and to detect the molecular mechanism of SHP in the treatment of UC.

Methods: Survival rate, locomotion, feeding, and excretion were used to explore the anti-inflammatory effects of SHP. The pharmacotoxicity of SHP was measured using developmental analysis. Intestinal integrity, intestinal length, intestinal acid-base homeostasis, and Tepan blue assay were used to analyze the protective effect of SHP against DSS-induced intestinal damage. The molecular mechanism of SHP was detected using DHE staining, immunofluorescence, real-time PCR, 16 S rRNA gene sequencing, and network pharmacology analysis. Survival rate, intestinal length, and integrity analysis were used to detect the protective effect of bioactive compounds of SHP against intestinal damage.

Results: SHP supplementation significantly increased the survival rate, restored locomotion, increased metabolic rate, maintained intestinal morphological integrity and intestinal homeostasis, protected intestinal epithelial cells, and alleviated intestinal oxidative damage in adult flies under DSS stimulation. Besides, administration of SHP had no toxic effect on flies. Moreover, SHP supplementation remarkably decreased the expression levels of genes related to JAK/STAT, apoptosis, and Toll signaling pathways, increased the gene expressions of the Nrf2/Keap1 pathway, and also reduced the relative abundance of harmful bacteria in DSS-treated flies. Additionally, the ingredients in SHP (palmatine, berberine, baicalein, wogonin, rhein, and aloeemodin) had protection against DSS-induced intestinal injury, such as prolonging survival rate, increasing intestinal length, and maintaining intestinal barrier integrity.

Conclusion: SHP had a strong anti-inflammatory function, and remarkably alleviated DSS-induced intestinal morphological damage and intestinal homeostatic imbalance in adult flies by regulating JAK/STAT, apoptosis, Toll and Nrf2/Keap1 signaling pathways, and also gut microbial homeostasis. This suggests that SHP may be a potential complementary and alternative medicine herb therapy for UC, which provides a basis for modern pharmacodynamic evaluation of other prescriptions in Dunhuang ancient medical prescription.

Keywords: Bioactive compounds; Drosophila melanogaster; Dunhuang ancient medical prescription; San huang pill; Signaling pathway; Ulcerative colitis.