Oxymatrine Attenuates Ulcerative Colitis through Inhibiting Pyroptosis Mediated by the NLRP3 Inflammasome

Abstract

Ulcerative colitis (UC) is difficult to cure and easy to relapse, leading to poor quality of life for patients. Oxymatrine (OMT) is one of the main alkaloids of Sophora flavescens Aiton, which has many effects, such as anti-inflammation, anti-oxidative stress, and immunosuppression. This study aimed to investigate whether OMT could attenuate ulcerative colitis by inhibiting the NOD-like receptor family pyrin domain containing three (NLRP3) inflammasome-mediated pyroptosis. In this study, the UC rat models were established by 2,4,6-Trinitrobenzenesulfonic acid (TNBS) in vivo, while RAW264.7 cells and peritoneal macrophages were stimulated with Lipopolysaccharides/Adenosine Triphosphate (LPS/ATP) in vitro to simulate pyroptosis models, and Western blotting (WB) and other detection techniques were applied to analyze proteins involved in the NLRP3 inflammasome pathway. Our results showed that OMT alleviated colitis ulcers and pathological damage in the TNBS-induced UC rats and exhibited an inhibitory effect on pyroptosis at the early stage of UC. In the model group, the pyroptosis reached the peak at 24 h after modeling with the contents of active-cysteine-aspartic proteases-1 (caspase-1), Gasdermin D (GSDMD)-N, and cleaved-interleukin-1 beta (IL-1β) to the highest expression level. Meanwhile, we found that OMT (80 mg kg^-1) remarkably decreased the expression levels of NLRP3, active-caspase-1, and cleaved-IL-1β at 24 h in the lesion tissue from UC rats. Further experiments on cells demonstrated that OMT at concentrations of 100 and 250 μM significantly inhibited cell death caused by NLRP3 inflammasome activation (p < 0.05), downregulated caspase-1, GSDMD, and decreased the levels of active-caspase-1, GSDMD-N, cleaved-IL-1β in RAW326.7 cells, and peritoneal macrophages. In summary, these results indicated that OMT could attenuate ulcerative colitis through inhibiting pyroptosis mediated by the NLRP3 inflammasome. The inhibition of the NLRP3 inflammasome may be a potential strategy for UC.

Keywords: GSDMD; NLRP3 inflammasome; oxymatrine; pyroptosis; ulcerative colitis.

Figure 1

OMT alleviates symptoms and histopathological damage in TNBS-induced UC. (A) The DAI score was calculated by combining fecal properties, body weight changes, and fecal occult blood in rats. Model, TNBS-induced UC; OMT, oxymatrine treatment; 5-ASA, 5-aminosalicylic acid treatment. (B) The CMDI, which integrated the adhesion status and the severity of mucosal lesions, was used to assess the symptoms of ulcerative colitis in rats. (C) Representative anatomical images of colon in each treatment group on the sixth day of TNBS-induced UC. (D) Representative H&E staining of colonic lesions (scale bar: 100 μm). (E) RHI score was calculated according to the H&E staining results. Data are shown as mean ± SE. Compared to the model group, * p < 0.05, ** p < 0.01, *** p < 0.001; Compared to the control group, ### p < 0.001.

Figure 2

Changes in NLRP3 signaling pathway proteins in rats at different time points and the effects of OMT on early stage of UC after TNBS injection. (A) The protein expression levels of NLRP3 signaling pathway were detected at different time points after TNBS injection with the lesion tissues from UC rats. (B) The effects of 80 mg kg⁻¹ OMT on the activation of NLRP3 signaling pathway at the early stage of UC were evaluated by measuring the expression levels of NLRP3, caspase-1, active-caspase-1, IL-1β, and cleaved-IL-1β at 8 h and 24 h after TNBS injection.

Figure 3

Effects of OMT on LPS/ATP-induced NLRP3-mediated pyroptosis in primary cultured rat peritoneal macrophages. (A) Representative images of PI staining of rat peritoneal macrophages (scale bar: 100 μm). Model, LPS/ATP stimulation with 3 mM ATP for 30 min after 10 μg mL⁻¹ LPS pretreatment for 12 h; OMT, oxymatrine pretreatment for 1 h before LPS/ATP stimulation. (B) Percentage of PI-positive cells from primary peritoneal macrophage cultures (n = 4). Data are shown as mean ± SE. Compared to the model group, * p < 0.05, ** p < 0.01. Compared to the control group, ### p < 0.001. (C) Western blot analysis of the proteins of NLRP3 signaling pathway with the cultured rat peritoneal macrophage lysates.

Figure 4

Effects of OMT on LPS/ATP-induced pyroptosis in RAW264.7 cells. (A) Representative images of the morphological changes in RAW264.7 cells in bright field under the LPS/ATP stimulation in the absence or presence of OMT (scale bar: 100 μm). Model, the LPS/ATP-stimulation. (B) Detection of the LDH release to determine the OMT effects on the LPS/ATP-induced cell damage. Data are shown as mean ± SE. Compared to the model group, * p < 0.05, ** p < 0.01. Compared to the control group, ### p < 0.001. (C) The changes in NLRP3 signaling-related protein levels in the RAW264.7 cell lysates under different conditions.

Figure 5

OMT attenuates UC through inhibiting pyroptosis mediated by the NLRP3 inflammasome. The blue arrow represents the drug treatment, the black arrow represents the protein formation process, and the red arrow represents the protein content decline.