Medical Hemorrhoid Gel Ameliorates Croton Oil-Induced Hemorrhoids by Suppressing the NLRP3 Inflammasome Activation via NF-κB Signaling Pathway

Abstract

Background: Hemorrhoidal disease (HD) is characterized by the pathological dilation of anal vascular cushions, causing pain, itching and bleeding. Recent evidence links HD onset and progression to rectal inflammation. Medical Hemorrhoid Gel (MHG), a multi-component botanical preparation, has gained empirical validation for HD management. This study aims to systematically evaluate the safety, efficacy, and mechanism of action of MHG in treating HD.

Methods: The phytochemical composition of MHG was characterized using UPLC-QTOF-MS/MS and GC-MS/MS analyses. In vivo, the efficacy was assessed in a croton oil preparation (COP)‑induced HD rat model (n=10 per group) via anorectal coefficient (ARC) measurement, macroscopic severity score, Evans blue extravasation quantification, and H&E/PAS staining. Transcriptomic sequencing of anorectal tissues was integrated with experimental validation using ELISA, immunohistochemistry (IHC), and Western blotting to delineate molecular mechanism. Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison post hoc test (significance at p<0.05).

Results: MHG significantly reduced ARC, macroscopic severity score, and Evans blue extravasation, restored intestinal villus structure and goblet cell numbers, and alleviated inflammation. Acute toxicity tests showed that MHG did not cause anorectal abnormalities or systemic toxicity in rats. Transcriptomic analysis integrated with experimental validation suggested the therapeutic mechanism of MHG involves inflammation response and NF-κB pathway. Specifically, MHG suppressed the levels of the pro-inflammatory mediator TNF-α, while it enhanced the levels of the anti-inflammatory mediator IL-10. Mechanistic studies revealed that MHG inhibited NLRP3 inflammasome activation, reduced the phosphorylation level of p65 and enhanced IκBα expression. Phytochemical analysis identified 20 constituents that contribute to the bioactivity of MHG.

Conclusion: Our study substantiated that MHG exerts anti-hemorrhoidal effects through NLRP3 inflammasome suppression via NF-κB pathway regulation. This mechanistic insight provides scientific validation for clinical application of MHG in HD management.

Keywords: NK-κB signaling pathway; NLRP3 inflammasomes; hemorrhoidal disease; inflammation; medical hemorrhoid gel.

Figure 1

Flow chart of research scheme of MHG.

Figure 2

The anti-hemorrhoid effect of MHG. (A) Impact on anorectal coefficient. (B) Impact on macroscopic severity score. (C) Impact on Evans blue exudation. (D) Morphological observation of anorectal tissue. The mean ± SD is used to express the results.,***p < 0.001, vs the control group; ^##p < 0.01, ^###p < 0.001, vs the COP group (n = 10 per group).

Figure 3

The effect of MHG on histopathology of anorectal tissue. (A) H&E (magnification ×100) and (B) PAS (magnification ×100) staining analysis.

Figure 4

NF-κB mediated inflammatory response might be potential mechanism of MHG against HD (A) Volcano plot analysis of DEGs from the Control group and COP groups. (B) Volcano plot analysis of DEGs from the COP group and COP+MHG group. (C) Venn diagram illustrating DEGs downregulated in the Control vs COP comparison and upregulated in the COP vs COP+MHG comparison. (D) Venn diagram illustrating DEGs upregulated in the Control vs COP comparison and downregulated in the COP vs COP+MHG comparison. (E) The top 20 BP, CC and MF in GO enrichment analysis. (F) The top 15 key pathways in KEGG enrichment analysis (n = 3 per group).

Figure 5

MHG can inhibit the production of TNF-α and promote the production of IL-10 in HD rats. Concentrations of TNF-α (A) and IL-10 (B) in anorectal tissues were analyzed by using ELISA (n=8 per group). (C) The representative Western blotting bands of TNF-α and IL-10. Protein relative expression level of TNF-α (D) and IL-10 (E) (n = 3 per group). The mean ± SD is used to express the results. *p<0.05,***p < 0.001, vs the control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs the COP group.

Figure 6

MHG can inhibit the activation of NLRP3 inflammasomes in anorectal tissue. (A) The representative anorectal tissue sections were stained with IHC (magnification × 160). AOD value of NLRP3 (B), ASC (C), Caspase-1 (D) in anorectal tissue sections. (E) The representative Western blotting bands of NLRP3, ASC, Caspase-1, Pro-caspase-1 and IL-1β. Protein relative expression level of NLRP3 (F), ASC (G), Caspase-1 (H) and IL-1β (I). The mean ± SD is used to express the result. **p<0.01,***p < 0.001, vs the control group; #p < 0.05, ^##p < 0.01, ^###p < 0.001, vs the COP group (n = 3 per group).

Figure 7

MHG can modulate the NF-κB signaling pathway in anorectal tissue. (A) The representative anorectal tissue sections were stained with IHC (magnification ×160). AOD value of p65 (B), p-p65 (C) and IκBα (D) in anorectal tissue sections. (E) The representative Western blotting bands of p65, p-p65 and IκBα. Protein relative expression level of p-p65 (F) and of IκBα (G). The mean ± SD is used to express the results., **p<0.01,***p < 0.001, vs the control group; ^##p < 0.01, ^###p < 0.001, vs the COP group (n = 3 per group).

Figure 8

Phytochemical composition analysis of MHG. Total ion flow chromatograms of MHG sample analyzed by UPLC-QTOF-MS/MS in negative (A) and positive (B) ion modes. (C) Total ion chromatogram of MHG sample analyzed by GC-MS/MS.