Huang Lian Jie Du decoction attenuated colitis via suppressing the macrophage Csf1r/Src pathway and modulating gut microbiota

Abstract

Introduction: Ulcerative colitis, a subtype of chronic inflammatory bowel disease (IBD), is characterized by relapsing colonic inflammation and ulcers. The traditional Chinese herbal formulation Huang Lian Jie Du (HLJD) decoction is used clinically to treat diarrhea and colitis. However, the mechanisms associated with the effects of treatment remain unclear. This study aims to elucidate the molecular mechanistic effects of HLJD formulation on colitis.

Methods: Chronic colitis in mice was induced by adding 1% dextran sulfate sodium (DSS) to their drinking water continuously for 8 weeks, and HLJD decoction at the doses of 2 and 4 g/kg was administered orally to mice daily from the second week until experimental endpoint. Stool consistency scores, blood stool scores, and body weights were recorded weekly. Disease activity index (DAI) was determined before necropsy, where colon tissues were collected for biochemical analyses. In addition, the fecal microbiome of treated mice was characterized using 16S rRNA amplicon sequencing.

Results: HLJD decoction at doses of 2 and 4 g/kg relieved DSS-induced chronic colitis in mice by suppressing inflammation through compromised macrophage activity in colonic tissues associated with the colony-stimulating factor 1 receptor (Csf1r)/Src pathway. Furthermore, the HLJD formula could modify the gut microbiota profile by decreasing the abundance of Bacteroides, Odoribacter, Clostridium_sensu_stricto_1, and Parasutterella. In addition, close correlations between DAI, colon length, spleen weight, and gut microbiota were identified.

Discussion: Our findings revealed that the HLJD formula attenuated DSS-induced chronic colitis by reducing inflammation via Csf1r/Src-mediated macrophage infiltration, as well as modulating the gut microbiota profile.

Keywords: Csf1r; Src; colitis; gut microbiota; macrophage.

Figure 1

HLJD decoction improved chronic colitis conditions in the DSS-induced mouse model. (A) Timeline of the animal experiment. (B) Body weight changes among different groups (n = 12). (C, D) Representative colon samples and colon lengths measured from each group (n = 12). Scale bar, 1 cm. (E, F) Stool consistency (n = 12) and blood scores (n = 12) recorded during treatment measured from each group, respectively. (G) DAI scores (n = 12). NC, normal control group; H, HLJD at the dose of 2 g/kg daily by oral gavage; DSS, mice given 1% DSS 5 days a week for 8 weeks; DHL and DHH, mice given 1% DSS were treated with HLJD at 2 and 4 g/kg daily by oral gavage, respectively. Values were presented as means ± SEM. ***p < 0.001 vs. NC; ##p < 0.01, ###p < 0.001 vs. DSS.

Figure 2

HLJD decoction attenuated colonic damage in the DSS-induced mouse model. Representative H&E staining of both distal end (A) and proximal end (C) of colon sections from different treatment groups (scale bar, 100 µm). (B, D) Calculated histological scores of distal ends (n = 6) and proximal ends (n = 10) of colon sections based on collected H&E images. Colon D, distal end of colon tissue; Colon P, proximal end of colon tissue. NC, normal control group; H, HLJD at the dose of 2 g/kg daily by oral gavage; DSS, mice given 1% DSS 5 days a week for 8 weeks; DHL and DHH, mice given 1% DSS were treated with HLJD at 2 and 4 g/kg daily by oral gavage, respectively. Data were expressed as mean ± SEM. **p < 0.01, ***p < 0.001 vs. NC; #p < 0.05, ##p < 0.01 vs. DSS.

Figure 3

HLJD decoction ameliorated colonic inflammation in the DSS-induced chronic colitis mouse model. (A) Representative immunofluorescent staining images of F4/80 in the distal end of colon tissues from each group. Scale bar, 50 µm. (B) Quantification of F4/80-positive macrophages in the colon (n = 6). (C) Representative IHC staining images of F4/80 in the distal end of colon tissues from each group. Scale bar, 50 µm. (D–G) Relative mRNA expression of Tnfa, Cxcl1, Cxcl10, and Ccl3 normalized to Actb (n = 7). NC, normal control group; H, HLJD at the dose of 2 g/kg daily by oral gavage; DSS, mice given 1% DSS 5 days a week for 8 weeks; DHL and DHH, mice given 1% DSS were treated with HLJD at 2 and 4 g/kg daily by oral gavage, respectively. Data were expressed as mean ± SEM. *p < 0.05 vs. NC; #p < 0.05 vs. DSS.

Figure 4

HLJD decoction inhibited Csf1r/Src levels in the DSS-induced chronic colitis mouse model. (A to C) Relative mRNA expression of Csf1 (n = 6), Il34 (n = 6), and Csf1r (n = 9) normalized to Actb. (D) The representative Western blot image of Csf1R, p-Src, Src, and Gapdh. (E) Quantitative analyses of blots (n = 3). (F) Representative IHC staining image of Csf1r in colon tissues taken from each group. Scale bar, 50 µm. (G) Quantification of Csf1r expression in the colon (n = 3). NC, normal control group; H, HLJD at the dose of 2 g/kg daily by oral gavage; DSS, mice given 1% DSS 5 days a week for 8 weeks; DHL and DHH, mice given 1% DSS were treated with HLJD at 2 and 4 g/kg daily by oral gavage, respectively. Data were expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs. NC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. DSS.

Figure 5

HLJD decoction inhibited Csf1r levels in F4/80-positive macrophages of the DSS-induced chronic colitis mouse model. Scale bar, 50 µm. NC, normal control group; H, HLJD at the dose of 2 g/kg daily by oral gavage; DSS, mice given 1% DSS 5 days a week for 8 weeks; DHL and DHH, mice given 1% DSS were treated with HLJD at 2 and 4 g/kg daily by oral gavage, respectively (n = 5).

Figure 6

Alterations in the gut microbiota composition from different groups. (A) The Venn diagram representing the operational taxonomic units (OTUs) in samples obtained from different groups. (B) PCoA of the bacterial community composition based on Bray–Curtis distances. Dots represent individual samples. (C) Sobs index of the OTU level. (D, E) Abundant phyla and genera found among groups, respectively (n = 6). NC, normal control group; H, HLJD at the dose of 2 g/kg daily by oral gavage; DSS, mice given 1% DSS 5 days a week for 8 weeks; DH, mice given 1% DSS were treated with HLJD at 2 g/kg by oral gavage.

Figure 7

Cladogram of the LEfSe analysis of the gut microbiota composition in different groups. (A) The microbial compositions were compared at different evolutionary levels. (B–E) Comparison of relative abundant microbes at the genus level in different groups. NC, normal control group; H, HLJD at the dose of 2 g/kg daily by oral gavage; DSS, mice given 1% DSS 5 days a week for 8 weeks; DH, mice given 1% DSS were treated with HLJD at 2 g/kg by oral gavage. Data were expressed as mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs. the indicated group, Kruskal–Wallis test.

Figure 8

Spearman correlation analysis between disease-associated parameters and intestinal bacterial flora at the genus level after HLJD treatment. NC, normal control group; H, HLJD at the dose of 2 g/kg daily by oral gavage; DSS, mice given 1% DSS 5 days a week for 8 weeks; DH, mice given 1% DSS were treated with HLJD at 2 g/kg by oral gavage. Data were expressed as mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 vs. the indicated group.