Gegen Qinlian decoction (GQD) inhibits ulcerative colitis by modulating ferroptosis-dependent pathway in mice and organoids

Abstract

Background: Gegen Qinlian decoction (GQD) is a classic prescription for treating ulcerative colitis (UC) in traditional Chinese medicine. However, the therapeutic mechanism has not been fully clarified.

Purpose: In the present study, we aimed to evaluate the role of ferroptosis-mediated IEC death in UC treated mice with GQD by using DSS-induced a colitis mouse model and RSL3-induced ferroptosis in intestinal organoids.

Methods: The effects of GQD on DSS-treated colitis were examined via daily body weight, DAI, colon length, HE staining, PAS staining, ZO-1 and Occludin immunohistochemical staining. Ferroptosis was determined by analysis of iron load, MDA, GSH, mitochondrial morphology, and expression of ferroptosis-associated proteins (GPX4, SLC7A11 and ACSL4).

Results: In vivo, GQD administration reduced body weight loss and DAI scores, increased colon length, and improved intestinal histological characteristics and epithelial barrier dysfunction. GQD administration obviously improved the levels of ferroptosis markers (iron load, MDA, GSH, and mitochondrial morphology) and the expression of ferroptosis-associated proteins (GPX4, SLC7A11 and ACSL4). Consistent with in vivo results, GQD administration partially reversed the levels of mtROS, Fe2⁺ and MDA in intestinal organoids induced by RSL3, and notably improved morphological destruction, histological damage and epithelial barrier dysfunction in organoids.

Conclusions: In this study, we demonstrated that ferroptosis was triggered in DSS-induced experimental colitis and that GQD adiministration could protect against colonic damage and intestinal epithelial barrier dysfunction by inhibiting ferroptosis.

Keywords: Ferroptosis; Gegen Qinlian decoction; IEC death; Intestinal organoids; Ulcerative colitis.

Fig. 1

HPLC chromatograms of main ingredients in GQD

Fig. 2

GQD alleviated DSS-induced colitis. A The change in daily body weight of mice. B The daily DAI scores of mice. C–D Representative pictures of colon tissue and colon length in all groups. E Representative pictures of HE staining, scale bar = 100 μm. F Representative pictures of PAS staining, scale bar = 100 μm. G–H Immunohistochemistry staining of ZO-1 and Occludin, scale bar = 50 μm. Compared with control group, ^#P < 0.05, ^##P < 0.01; compared with DSS group, *P < 0.05, **P < 0.01

Fig. 3

GQD inhibited ferroptosis in DSS-induced colitic mice. A–C The level of Fe2⁺, GSH, and MDA in mouse colon tissue. D Electron microscope images of mouse colon tissue, enlarged image showing mitochondria, scale bar = 500 nm. E–H The protein levels of GPX4, SLC7A11 and ACSL4 in mouse colon tissue. Compared with control group, ^#P < 0.05, ^##P < 0.01; compared with DSS group, *P < 0.05, **P < 0.01

Fig. 4

RSL3 induced intestinal organoids injury and GQD treatment. A The morphological structure and FITC fluorescence intensity of intestinal organoids were induced at different concentrations of RSL3 for 24 h, scale bar = 200 μm. BFI is abbreviation for bright field imaging. B–C The viability of organoids at differen concentrations of RSL3 and GQD. Compared with control group, ^#P < 0.05, ^##P < 0.01; compared with DSS group, *P < 0.05, **P < 0.01

Fig. 5

GQD suppressed RSL3-induced intestinal organoids ferroptosis. A and C Mitochondria ROS level of intestinal organoids, scale bar = 200 μm. B and D Fe2⁺ level of intestinal organoids, scale bar = 100 μm. E GSH level of intestinal organoids. F MDA level of intestinal organoids. Compared with control group, ^#P < 0.05, ^##P < 0.01; compared with DSS group, *P < 0.05, **P < 0.01

Fig. 6

GQD treatment protect against colonic damage in intestinal organoids. A Morphological structure of intestinal organoids, scale bar = 20 μm. B The viability of intestinal organoids. C–D HE and PAS staining of intestinal organoids, scale bar = 20 μm. E Mount organoids immunofluorescence of ZO-1 and Occludin, scale bar = 50 μm. Compared with control group, ^#P < 0.05, ^##P < 0.01; compared with DSS group, *P < 0.05, **P < 0.01