Bacillus amyloliquefaciens Enriched Camel Milk Attenuated Colitis Symptoms in Mice Model

Abstract

Fermented camel's milk has various health beneficial prebiotics and probiotics. This study aimed to evaluate the preventive efficacy of Bacillus amyloliquefaciens enriched camel milk (BEY) in 2-, 4- and 6-Trinitrobenzenesulfonic acid (TNBS)-induced colitis mice models. To this end, the immune modulatory effects of Bacillus amyloliquefaciens (BA) on TNF-α challenged HT29 colon cells were estimated using the cell proliferation and cytokines ELISA method. BEY was prepared using the incubation method and nutritional value was quantified by comparing it to commercial yogurt. Furthermore, TNBS-induced colitis was established and the level of disease index, pathological scores, and inflammatory markers of BEY-treated mice using macroscopic and microscopic examinations, qPCR and immunoblot were investigated. The results demonstrate that BA is non-toxic to HT29 colon cells and balanced the inflammatory cytokines. BEY reduced the colitis disease index, and improved the body weight and colon length of the TNBS-induced mice. Additionally, Myeloperoxidase (MPO) and pro-inflammatory cytokines (IL1β, IL6, IL8 and TNF-α) were attenuated by BEY treatment. Moreover, the inflammatory progress mRNA and protein markers nuclear factor kappa B (NFκB), phosphatase and tensin homolog (PTEN), proliferating cell nuclear antigen (PCNA), cyclooxygenase-2 (COX-2) and occludin were significantly down-regulated by BEY treatment. Interestingly, significant suppression of PCNA was observed in colonic tissues using the immunohistochemical examination. Treatment with BEY increased the epigenetic (microRNA217) interactions with PCNA. In conclusion, the BEY clearly alleviated the colitis symptoms and in the future could be used to formulate a probiotic-based diet for the host gut health and control the inflammatory bowel syndrome in mammals.

Keywords: Bacillus amyloliquefaciens; PCNA; TNF-α; inflammatory bowel disease; probiotics.

Figure 1

Effect of probiotics on colon cells: (A) BA 10⁸ microbial loads treated with ht29 colon cells and incubated for 2 h; after incubation, the 6-well plates were aspirated with ampicillin 20 U/mL, and the treated cells were quantified for cell viability using 4% trypan blue. (B) TNF-α-induced HT29 cells were treated with BA for 12 h and the cell-free supernatant was collected and used for cytokine IL1, IL4, IL6 and IL8 estimation and the values were expressed as pg/mL. All of the data were collected from three individual experiments and pooled and expressed as mean ± SD (p < 0.05). (C) Phase-contrast images of TNF-α-induced HT29 cells that were treated with BA for 2 h; the magnification in the microscopic image was 200×. * p < 0.05 represents significance compared to the TNFα vs. TNFα + BA group.

Figure 2

BEY alleviated TNBS-induced colitis in C57Bl6j mice. TNBS induction was made at 0 and 4 days of induced mice; BA 10⁸/mL was administered as oral gavage at alternative days from 0 to 14. (A) the body weight of BA-treated TNBS-induced colitis mice and the values were expressed in g. The BA alone group was analyzed in parallel with the TNBS-induced group. (B) Colon lengths of TNBS-induced colitis mice and BEY-treated mice were evaluated and values were expressed in cm. (C) The fecal microbial load was quantified in TNBS-induced colitis mice and BA-treated mice, and values were expressed as log CFU/mL. (D) Microscopic examination of distal colon of TNBS-induced colitis was carried out, and the pathological changes were observed using the H&E staining method. The pathological score was evaluated in TNBS-induced colitis mice and BA-treated mice. The cellular infiltration and inflammation were observed, and the magnification in the microscopic image was 200×. (E) The pathological score distal colon was evaluated in TNBS-induced colitis mice and Bacillus-treated mice. Cellular infiltration and inflammation were observed. All of the data were collected from three individual experiments and pooled and expressed as mean ± SD (p < 0.05). * p < 0.05 represents significance compared to the TNBS vs. TNBS + BEY group.

Figure 3

Effect of BEY on inflammatory markers in TNBS-induced colitis mice. The myeloperoxidase-infiltration marker was quantified in TNBS-induced colitis mice and BEY-treated mice after 14 days of the experiment. The values are expressed in pg/mg protein. (A) the neutrophil infiltration marker MPO was quantified; (B,C) TNF-α and IL-6 (pro-inflammatory markers) were quantified in TNBS-induced colitis mice and BEY-treated mice after 14 days of the experiment. The values are expressed in pg/mL. (D) The lymphocytes degradation marker (C-reactive protein) was quantified in TNBS-induced colitis mice and BEY-treated mice after 14 days of the experiment. The values are expressed in mg/dL. (E,F) IL4 and IL8 (anti-inflammatory and macrophage markers, respectively) were quantified in TNBS-induced colitis mice and BEY-treated mice after 14 days of the experiment. The values are expressed in pg/mL. All data were collected from three individual experiments and pooled and expressed as mean ± SD (p < 0.05). * p < 0.05 represents significance compared to the TNBS vs. TNBS + BEY group.

Figure 4

Effect of Bacillus on TNBS-induced colitis. Bacillus enhanced the intestinal barrier function of colitis mice by regulating inflammatory markers. (A) Detection of FITC-dextran in serum of mice by fluorometric ELISA. (B) qPCR of NFκB, PTEN, PCNA, COX-2 and occludin mRNA in colonic tissues on the 14th day of TNBS-induced mice (all values were normalized to the TNBS-induced group). (C,D) Immunoblot and densitometric analysis of NFκB, PTEN, PCNA, COX-2 and occludin protein expression normalized with actin internal control. Data are representative of at least three independent experiments. Data are shown as mean ± SD of the mean (n = 3). * p < 0.05 represents significance compared to the TNBS vs. TNBS + BEY group.

Figure 5

Immunohistochemical expression of PCNA in colon tissues of control and experimental groups of mice. PCNA protein expression is illustrated as brown staining. (A) Microscopic imaging of colon tissue with scale bar: 100 µm. (B) The quantitative data expressing the high-grade dysplasia marker PCNA protein level illustrate that there was significant downregulation in the TNBS with Bacillus and Bacillus alone groups compared with the TNBS alone disease group. (C,D) The epigenetic marker miR-217 was quantified using real-time PCR. Data are shown as mean ± standard error of the mean (n = 3). * p < 0.05 represents significance compared to the TNBS vs. TNBS + BEY group.