Huaiyu pill (槐榆片) alleviates inflammatory bowel disease in mice via blocking toll like receptor 4/ myeloid differentiation primary response gene 88/ nuclear factor kappa B subunit 1 pathway

doi:10.19852/j.cnki.jtcm.20240719.001

Abstract

Abstract:

OBJECTIVE: To investigate the therapeutic effects of Huaiyu pill (槐榆片, HYP) on inflammatory bowel disease (IBD) and the underlying mechanisms have not been elucidated.

METHODS: To establish the IBD model, mice were administered with dextran sulfate sodium (DSS). Mice were intragastrically pre-treated with sulfasalazine (SASP) and HYP. Disease activity index (DAI) and colon length were monitored, and the colonic tissues were subjected to hematoxylin-eosin staining. Pro-inflammatory factors and vascular inflammation-related proteins were determined using enzyme-linked immunosorbent assay (ELISA). The potential mechanisms of HYP were examined using network pharmacology analysis.The expressions of zona occludens 1 (ZO-1), occludin, toll like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MYD88), and nuclear factor kappa B p65 subunit (NF-κB p65) in colon tissues were examined using Western blotting or immunohistochemical analyses.

RESULTS: Pre-treatment with HYP enhanced the colon length, decreased DAI scores, and mitigated histopathological alterations in DSS-treated mice. HYP alleviated intestinal inflammation by downregulating the levels of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α) and interleukin 17 (IL-17). Additionally, HYP suppressed the disruption of the gut barrier by upregulating the ZO-1, occludin, and mucin 2 (MUC2) levels and downregulating the endothelin 1 (ET-1) and erythropoietin (EPO) levels. Network pharmacological analysis and experimental results revealed that HYP downregulated the colonic tissue levels of TLR4, MYD88, and NF-κB p65 in DSS-treated mice.

CONCLUSION: This study investigated the in vivotherapeutic effects of HYP on IBD and the underlying molecular mechanisms. These findings provide an experimental foundation for the clinical application of HYP.

Key words: inflammatory bowel diseases, network pharmacology, intestinal inflammation, intestinal damage, Huaiyu pill

YANG Chunyan, LUO Jia, PENG Weijie, DAI Weibo. Huaiyu pill (槐榆片) alleviates inflammatory bowel disease in mice via blocking toll like receptor 4/ myeloid differentiation primary response gene 88/ nuclear factor kappa B subunit 1 pathway[J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 916-925.

Figures/Tables 5

Figure 1 HYP relieved DSS-induced IBD symptoms in mice A: experimental design of the animal study and the time-course of changes in body weight. B: representative colon images in each group. C: the colon length in each group. D: the DAI in different group. E: colon tissue with hematoxylin-eosin staining (× 200, scale bars = 100 μm, the red box indicates the intestinal crypt structure, the red arrow indicates the inflammatory cells infiltration, and the green arrow indicates the glandular structure); E1: colon tissues of the control group; E2: colon tissues of the model group; E3: colon tissues of the SASP group; E4: colon tissues of the HYP low-dose group; E5: colon tissues of the HYP high-dose group. CTL: normal control group (fed on standard chow); Model: model group (DSS + standard chow); SASP: positive control group (DSS + 40 mg/20 mL/kg SASP); HYP-L: low-dose group (DSS + 25 g/20 mL/kg HYP); HYP-H: high-dose HYP group (DSS + 50 g/20 mL/kg HYP). HYP: Huaiyu pill; DSS: dextran sulfate sodium; IBD: inflammatory bowel disease; SASP: sulfasalazine. Student t-test and one-way analysis of variance were used for comparison analysis. Statistical significance between the groups was determined by Dunnett's T3 analysis. The data were illustrate d in mean ± standard deviation (n = 8). aP<0.01 compared with the control group; bP<0.01 compared with the model group; cP<0.05 compared with the model group.

Figure 2 Examination of the molecular mechanisms underlying the therapeutic efficacy of HYP A: venn diagram illustrates the shared targets between HYP and IBD. B: KEGG enrichment analysis of shared targets; C: functional module statistics for KEGG enrichment results; D: the pathways enriched for the top 10 number of gene counts within the immune system; E: compounds-targets-pathways network in IBD. HYP: Huaiyu pill; IBD: inflammatory bowel disease; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Table 1 Effect of HYP on mice colon tissues inflammatory cytokines levels (pg/mg, $\bar{x}±s$)

Group	n	IL-1β	IL-6	IL-17	TNF-α
CTL	8	6±5	15±4	31±9	25±5
Model	8	24±13^a	28±7^a	68±12^a	50±11^a
SASP	8	11±4^b	18±4^b	31±5^b	27±5^b
HYP-L	8	16±7^c	20±7^b	31±4^b	33±8^b
HYP-H	8	13±7^b	17±5^b	31±6^b	28±9^b

Figure 3 HYP attenuates intestinal damage caused by increased intestinal permeability in the DSS-induced IBD mouse model A: representative western blotting brands of ZO-1 and occludin in the colon tissues; B: normalized against β-actin; B1: quantitative analysis of ZO-1; B2: quantitative analysis of occluding; C: the contents of ET-1 and EPO in colon tissues were measured by ELISA; C1: the contents of ET-1 in colon tissues; C2: the contents of EPO in colon tissues; D: Mean density of MUC2 protein in colon tissues; E: Representative images of MUC2 immunostaining of colon tissues in each group (× 200, bar = 100 μm); E1: Colon tissues of the control group; E2: Colon tissues of the model group; E3: Colon tissues of the SASP group; E4: Colon tissues of the HYP low-dose group; E5: Colon tissues of the HYP high-dose group. CTL: normal control group (fed on standard chow); Model: model group (DSS + standard chow); SASP: positive control group (DSS + 40 mg/20 mL/kg SASP); HYP-L: low-dose group (DSS + 25 g/20 mL/kg HYP); HYP-H: high-dose HYP group (DSS + 50 g/20 mL/kg HYP). HYP: Huaiyu pill; DSS: dextran sulfate sodium; SASP: sulfasalazine; ZO-1: occludens 1; ET-1: endothelin 1; EPO: erythropoietin; MUC2: mucin 2. Student t-test and one-way analysis of variance were used for comparison analysis. Statistical significance between the groups was determined by the least significant difference method. The data was illustrated in mean ± standard deviation (n = 3). aP<0.01 and dP<0.05 compared with the control group; bP<0.01 and cP<0.05 compared with the model group.

Figure 4 HYP suppresses intestinal inflammation and damage by inhibiting TLR4/MyD88/NF-κB signaling pathway A: the expressions of TLR4, MYD88 and NF-κB p65 in colon tissue of mice by western blotting; B: normalized against β-actin; B1: quantitative analysis of TLR4; B2: quantitative analysis of MYD88; B3: quantitative analysis of NF-κB p65; C: mean density of TLR4, NF-κB p65 and MYD88 protein in colon tissues; C1: mean density of TLR4 protein in colon tissues. C2: mean density of MYD88 protein in colon tissues. C3: mean density of NF-κB p65 protein in colon tissues; D: representative images of TLR4 immunostaining of colon tissues in each group (× 200, bar = 100 μm); E: representative images of MYD88 immunostaining of colon tissues in each group (× 200, bar = 100 μm); F: representative images of NF-κB p65 immunostaining of colon tissues in each group (× 200, bar = 100 μm); D1, E1, F1: colon tissues of the control group; D2, E2, F2: colon tissues of the model group; D3, E3, F3: colon tissues of the SASP group; D4, E4, F4: colon tissue of the HYP low-dose group; D5, E5, F5: colon tissues of the HYP high-dose group. CTL: normal control group (fed on standard chow); Model: model group (DSS + standard chow); SASP: positive control group (DSS + 40 mg/20 mL/kg SASP); HYP-L: low-dose group (DSS + 25 g/20 mL/kg HYP); HYP-H: high-dose HYP group (DSS + 50 g/20 mL/kg HYP). HYP: Huaiyu pill; DSS: dextran sulfate sodium; SASP: sulfasalazine; TLR4: toll like receptor 4; MYD88: myeloid differentiation primary response gene 88; NF-κB p65: nuclear factor kappa B p65 subunit. Student t-test and one-way analysis of variance were used for comparison analysis. Statistical significance between the groups was determined by the least significant difference method. The data was illustrated in mean ± standard deviation (n = 3). aP<0.01 compared with the control group; bP<0.01 and cP<0.05 compared with the model group.

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