An’s anorectal fumigation lotion for nuclear factor kappa-B pathway-targeted treatment of inflammatory mixed hemorrhoids

doi:10.19852/j.cnki.jtcm.2025.04.008

Abstract

Abstract:

OBJECTIVE: To investigate the mechanism by which An’s anorectal fumigation lotion (AAFL) treats inflammatory mixed hemorrhoids.

METHODS: Eighty Sprague-Dawley rats, with an equal number of males and females, were randomly assigned to the following four groups: control, model, AAFL, and positive groups. Following hemorrhoid induction, hemorrhoidal tissues were collected from the rats for analysis. Pathological alterations in these tissues were examined via hematoxylin-eosin staining. Immun-ohistochemistry was used to detect inflammatory markers. The ultrastructural pathological changes in these tissues were observed by transmission electron microscopy. Reverse transcription-polymerase chain reaction and Western blotting were used to analyze the gene and protein expression of nuclear factor kappa-B (NF-κB) p65, inhibitor of kappa-B (IκB), inhibitor of NF-κB kinase (IκK-β), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α).

RESULTS: Compared with the control group, the rats in each treatment group showed general improvements in hemorrhoidal tissue pathology. The AAFL group showed increased IκB expression and decreased IL-1β, IL-6, TNF-α, NF-κB, p65, and IκK-β expressions.

CONCLUSION: AAFL can decrease the production of inflammatory markers by targeting the NF-κB pathway, resulting in improved pathological conditions in mixed hemorrhoids. Our findings will aid in the treatment of mixed hemorrhoids.

Key words: hemorrhoids, NF-kapa B, inflammatory factors, An’s anorectal fumigation lotion

FENG Dayong, AN Ayue, FENG Yuening, WANG Chunhui, BAI Zhiyong, WANG Qian. An’s anorectal fumigation lotion for nuclear factor kappa-B pathway-targeted treatment of inflammatory mixed hemorrhoids[J]. Journal of Traditional Chinese Medicine, 2025, 45(4): 777-785.

Figures/Tables 4

Figure 1 Effect of AAFL on the morphology of hemorrhoid tissues after 7 d treatment HE and immunohistochemistry staining were performed. A: HE stains of hemorrhoid tissues (Scale bar: 200 μm); B: immunohistochemistry stains of IL-1β of hemorrhoid tissues (Scale bar: 200 μm); C: immunohistochemistry stains of IL-6 of hemorrhoid tissues (Scale bar: 200 μm); D: immunohistochemistry stains of TNF-α of hemorrhoid tissues (Scale bar: 200 μm); A1, B1, C1, D1: Control group; A2, B2, C2, D2: Model group; A3, B3, C3, D3: AAFL group; A4, B4, C4, D4: Positive group. Control group: normal rats; saline-soaked cotton balls applied rectally,10 min, twice daily for 7 d; Model group: hemorrhoid-induced rats; saline-treated cotton balls applied rectally,10 min, twice daily for 7 d; AAFL group: hemorrhoid-induced rats; treated with 37 ℃ AAFL granule solution (200 g/1000 mL),10 min, twice daily for 7 d; Positive group: hemorrhoid-induced rats; treated with 37 ℃ potassium permanganate (1:5000) 10 min, twice daily for 7 d. AAFL: An’s anorectal fumigation lotion; HE: hematoxylin-eosin; IL-1β: interleukin-1 beta; IL-6: interleukin-6; TNF-α: tumor necrosis factor-alpha.

Figure 2 Effect of AAFL on the morphology of hemorrhoid tissues after 14 d treatment HE and Immunohistochemistry staining were performed. A: HE stains of hemorrhoid tissues (Scale bar: 200 μm),B: immunohistochemistry stains of IL-1β of hemorrhoid tissues (Scale bar: 200 μm); C: immunohistochemistry stains of IL-6 of hemorrhoid tissues (Scale bar: 200 μm); D: immunohistochemistry stains of TNF-α of hemorrhoid tissues (Scale bar: 200 μm); A1, B1, C1, D1: Control group; A2, B2, C2, D2: Model group; A3, B3, C3, D3: AAFL group; A4, B4, C4, D4: Positive group. Control group: Normal rats; saline-soaked cotton balls applied rectally,10 min, twice daily for 14 d; Model group: Hemorrhoid-induced rats; saline-treated cotton balls applied rectally,10 min, twice daily for 14 d; AAFL group: Hemorrhoid-induced rats; treated with 37 ℃ AAFL granule solution (200 g/1000 mL),10 min, twice daily for 14 d; Positive group: Hemorrhoid-induced rats; treated with 37 ℃ potassium permanganate (1:5000) 10 min, twice daily for 14 d. AAFL: An’s anorectal fumigation lotion; HE: hematoxylin-eosin; IL-1β: interleukin-1 beta; IL-6: interleukin-6; TNF-α: tumor necrosis factor-alpha.

Figure 3 Expression of NF-κB p65, IκB, and IκK in hemorrhoid tissues A: expression level of NF-κB p65, IκB, and IκK in hemorrhoid tissues after 7-days treatment; A1: expression level of NF-κB p65, IκB, and IκK determined by Western blotting, using GADPH as a loading control; A2: Western blot analysis of NF-κB p65; A3: Western blot analysis of IκB; A4: Western blot analysis of IκK; A5: expression level of NF-κB p65 mRNA; A6: expression level of IκB mRNA; A7: expression level of IκK mRNA. Control group: normal rats; saline-soaked cotton balls applied rectally, 10 min, twice daily for 7-d;Model group: hemorrhoid-induced rats; saline-treated cotton balls applied rectally,10 min, twice daily for 7-d; AAFL group: hemorrhoid-induced rats; treated with 37 ℃ AAFL granule solution (200 g/1000 mL), 10 min, twice daily for 7-d; Positive group: hemorrhoid-induced rats; treated with 37 ℃ potassium permanganate (1:5000) 10 min, twice daily for 7-days. GADPH: glyceraldehyde-3-phosphate dehydrogenase; AAFL: An’s anorectal fumigation lotion; NF-κB p65: nuclear factor kappa-B; IκB: inhibitor of kappa-B; IκK-β: inhibitor of NF-κB kinase; ANOVA: analysis of variance. ANOVA was performed for normal distribution data. The data were presented as the mean ± standard deviation from 3 independent experiments. aP < 0.01, eP < 0.05 vs control group; bP < 0.01, vs model group; cP < 0.05, dP < 0.01, vs positive group.

Figure 4 Expression of NF-κB p65, IκB, and IκK in hemorrhoid tissues A: expression level of NF-κB p65, IκB, and IκK in hemorrhoid tissues after 14-d treatment; A1: expression level of NF-κB p65, IκB, and IκK determined by Western blotting, using GADPH as a loading control; A2: Western blot analysis of NF-κB p65; A3: Western blot analysis of IκB; A4: Western blot analysis of IκK; A5: expression level of NF-κB p65 mRNA; A6: expression level of IκB mRNA; A7: expression level of IκK mRNA. Control group: normal rats; saline-soaked cotton balls applied rectally,10 min, twice daily for 14-d; Model group: hemorrhoid-induced rats; saline-treated cotton balls applied rectally,10 min, twice daily for 14-d; AAFL group: hemorrhoid-induced rats; treated with 37 ℃ AAFL granule solution (200 g/1000 mL), 10 min, twice daily for 14-d; Positive group: hemorrhoid-induced rats; treated with 37 ℃ potassium permanganate (1:5000) 10 min, twice daily for 14-d. GADPH: glyceraldehyde-3-phosphate dehydrogenase; AAFL: An’s anorectal fumigation lotion; NF-κB p65: nuclear factor kappa-B; IκB: inhibitor of kappa-B; IκK-β: inhibitor of NF-κB kinase; ANOVA: analysis of variance. ANOVA was performed for normal distribution data. The data were presented as the mean ± standard deviation from 3 independent experiments. aP < 0.01, fP < 0.05, vs control group; bP < 0.01, cP < 0.05, vs model group; dP < 0.05, eP < 0.01, vs positive group.

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