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. 2024 May 21:15:1405084.
doi: 10.3389/fimmu.2024.1405084. eCollection 2024.

Cynaroside ameliorates methotrexate-induced enteritis in rats through inhibiting NLRP3 inflammasome activation

Wuying Lang #  1   2   3 , Xin Wen #  1   4 , Shuangqi Zhang  1 , Xuhua Liang  1   2   3 , Lin Chen  1   3 , Dezhu Zhang  5 , Ruina Zhou  4 , Ihsan Ali  6 , Xuansheng Hu  1 , Haihua Zhang  7 , Min Cheng  1   2
Affiliations

Cynaroside ameliorates methotrexate-induced enteritis in rats through inhibiting NLRP3 inflammasome activation

Wuying Lang et al. Front Immunol. .

Abstract

Introduction: Cynaroside exhibits various biological properties, including anti-inflammatory, antiviral, antitumor, and cardioprotective effects. However, its involvement in methotrexate (MTX)-induced intestinal inflammation remains inadequately understood. Thus, we investigated the impact of cynaroside on MTX-induced intestinal inflammation and its potential mechanisms.

Methods: To assess the protective potential of cynaroside against intestinal inflammation, Sprague-Dawley rats were subjected to a regimen of 7 mg/kg MTX for 3 days, followed by treatment with cynaroside at varying doses (10, 20, or 40 mg/kg). Histopathological evaluations were conducted alongside measurements of inflammatory mediators to elucidate the involvement of the NLRP3 inflammasome in alleviating intestinal inflammation.

Results: Administration of 7 mg/kg MTX resulted in decreased daily food intake, increased weight loss, and elevated disease activity index in rats. Conversely, treatment with cynaroside at 20 or 40 mg/kg ameliorated the reductions in body weight and daily food intake and suppressed the MTX-induced elevation in the disease activity index. Notably, cynaroside administration at 20 or 40 mg/kg attenuated inflammatory cell infiltration, augmented goblet cell numbers and lowered serum levels of tumor necrosis factor-α, interleukin (IL)-1β, and IL-18, as well as the CD68-positive cell rate in the intestines of MTX-induced rats. Furthermore, cynaroside downregulated the expression levels of NLRP3, cleaved caspase 1, and cleaved IL-1β in MTX-induced rats.

Discussion: Collectively, our findings indicated that cymaroside alleviates intestinal inflammatory injury by inhibiting the activation of NLRP3 inflammasome in MTX-induced rats.

Keywords: NLRP3; cleaved IL-1β; cleaved caspase 1; cynaroside; intestinal inflammation; methotrexate.

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Conflict of interest statement

Author WL, XL and MC were employed by the company Shaanxi Qinling Industrial Technology Research Institute of Special Biological Resources Co. Ltd. Author DZ was employed by the company Shaanxi Panlong Pharmaceutical Group Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Cynaroside alleviates the decline in growth performance in MTX-induced rats. (A) Final body weight in the different treatment groups. (B) Daily food intake in the different treatment groups. (C) DAI in the different treatment groups. Data are expressed as the mean ± SD. n = 6. ##P < 0.01, compared with the control group. **P < 0.01, compared with the MTX group.
Figure 2
Figure 2
Effect of cynaroside on intestinal pathological alterations in MTX-induced rats. (A) Representative small intestine tissue of histopathological H&E staining (200x). (B) Statistical analysis of the inflammation score. Data are expressed as the mean ± SD. n = 6. ##P < 0.01, compared with the control group. **P < 0.01, compared with the MTX group. Scale bars represent 100 μm.
Figure 3
Figure 3
Effect of cynaroside on the small intestinal goblet cells in MTX-induced rats. (A) Goblet cells were assessed by PAS staining (200x). (B) Statistical analysis of the number of goblet cells. Data are expressed as the mean ± SD. n = 6. ##P < 0.01, compared with the control group. **P < 0.01, compared with the MTX group. Scale bars represent 100 μm.
Figure 4
Figure 4
Effect of cynaroside on serum pro-inflammatory factors levels in MTX-induced rats. Serum levels of (A) TNF-α, (B) IL-1β, and (C) IL-18 as assessed using by ELISA. Data are expressed as the mean ± SD. n = 6. ##P < 0.01, compared with the control group. **P < 0.01, compared with the MTX group.
Figure 5
Figure 5
CD68-positive cell rate in the small intestine. (A) Immunofluorescence analysis of CD68-positive cells in the different treatment groups (200x). (B) CD68-positive rate in the different treatment groups. Data are expressed as the mean ± SD. n = 6. ##P < 0.01, compared with the control group. **P < 0.01, compared with the MTX group. Scale bars represent 100 μm.
Figure 6
Figure 6
Effect of cynaroside on the NLRP3 inflammasome in MTX-induced rats. (A) the expression of NLRP3, cleaved caspase 1, cleaved IL-1β as measured by western blot. GAPDH was used as a control. (B) NLRP3 immunofluorescence (200x). (C) Immunofluorescence cumulative optical density (IOD) of NLRP3. Data are expressed as the mean ± SE. n = 3. #P < 0.05, ##P < 0.01 compared with the control group. *P < 0.05, **P < 0.01, compared with the MTX group. Scale bars represent 100 μm.
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