Probiotics-loaded nanoparticles attenuated colon inflammation, oxidative stress, and apoptosis in colitis

Abstract

Promising therapy is needed for treating inflammatory bowel diseases (IBD) to overcome current treatment that inefficient and associated with unnecessary health risks. Recently, the concept of incorporating natural products into nanocarriers has been intended as a promising therapy for treating IBD via modulating their stability and bioavailability. Thus, we aimed to explore the potential alleviating effects of dietary nano-supplement combined with bacillus strains (Bacillus amyloliquefaciens; BANPs) in colitis model. Rats were orally gavaged by 5% DSS and the efficacy and mechanistic actions of BANPs were evaluated by assessing the severity of clinical signs and inflammatory and apoptosis response, histopathological and immunohistochemistry examination in colonic tissues. The severity of clinical signs was successfully alleviated and fecal Lcn-2 levels, an important colitic marker, were decreased in BANPs then free BA treated groups. In contrast, inflammatory markers overexpression IL-6, IL-1β, TNFα, COX-2, and iNOS in the colitic group were reduced more prominently in BANPs treated group, unlike free BA. The amelioration of BANPs to colon injury was also correlated with oxidative stress suppression along with restoring total antioxidant capacity. Interestingly, BANPs treatment modulated apoptotic markers as proved by downregulation of cytochrome c, and caspase-3 and upregulation of Bcl-2 and Bax than free BA. The severity of the histopathological alterations in the colon was greatly reduced in BANPs than free BA groups. Remarkably, over-expression of ki67 and IL-6 in colonic tissues were suppressed in BANPs group. These findings together highlighted the beneficial efficacy of BANPs in IBD treatment which are evidenced by colonic inflammation alleviation. Taken together, these results recommend that BANPs is a promising agent that encourages its possible therapeutic role in colitis treatment.

Figure 1

Effects of orally administered B. amyloliquefaciens (BA) or BA-nanoparticles (BANPs) in non-colitic and colitic Signs. (a) Body weight gain. (b) Disease activity index score. (c) Colon length. (d) Spleen weight. Non-colitic groups including Control, BA and BANPs groups where rats were orally gavaged PBS, B. amyloliquefaciens and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day), respectively, for 7 days. Colitic groups: DSS, BA and BANPs groups where rats were orally gavaged dextrane sodium sulphate (DSS), B. amyloliquefaciens + DSS and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day) + DSS, respectively, for 7 days. All groups orally gavaged by 5% DSS. Values are expressed as mean ± SE, ^a,b,cMeans of the bars with different letters were significantly different among groups (P < 0.05).

Figure 2

Effects of orally administered B. amyloliquefaciens (BA) or BA-nanoparticles (BANPs) in non-colitic and colitic rats on fecal lipocalin-2 (Lcn-2) levels 7 days post DSS induction. Non-colitic groups including Control, BA and BANPs groups where rats were orally gavaged PBS, B. amyloliquefaciens and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day), respectively, for 7 days. Colitic groups: DSS, BA and BANPs groups where rats were orally gavaged dextrane sodium sulphate (DSS), B. amyloliquefaciens + DSS and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day) + DSS, respectively, for 7 days. All groups orally gavaged by 5% DSS. Values are expressed as mean ± SE, ^a,b,cMeans of the bars with different letters were significantly different among groups (P < 0.05).

Figure 3

Effects of orally administered B. amyloliquefaciens (BA) or BA-nanoparticles (BANPs) in non-colitic and colitic rats on colonic myeloperoxidase (MPO) activity activity (a) and serum C-reactive protein (CRP) levels 7 days post DSS induction. Non-colitic groups including Control, BA and BANPs groups where rats were orally gavaged PBS, B. amyloliquefaciens and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day), respectively, for 7 days. Colitic groups: DSS, BA and BANPs groups where rats were orally gavaged dextrane sodium sulphate (DSS), B. amyloliquefaciens + DSS and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day) + DSS, respectively, for 7 days. All groups orally gavaged by 5% DSS. Values are expressed as mean ± SE, ^a,b,cMeans of the bars with different letters were significantly different among groups (P < 0.05).

Figure 4

Effects of orally administered B. amyloliquefaciens (BA) or BA-nanoparticles (BANPs) in non-colitic and colitic rats on lipid peroxides expressed as malondialdehyde; MDA. (a) Nitric oxide; NO. (b) Total antioxidant capacity; TAC (c) 7 days post DSS induction. Non-colitic groups including Control, BA and BANPs groups where rats were orally gavaged PBS, B. amyloliquefaciens and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day), respectively, for 7 days. Colitic groups: DSS, BA and BANPs groups where rats were orally gavaged dextrane sodium sulphate (DSS), B. amyloliquefaciens + DSS and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day) + DSS, respectively, for 7 days. All groups orally gavaged by 5% DSS. Values are expressed as mean ± SE, ^a,b,cMeans of the bars with different letters were significantly different among groups (P < 0.05).

Figure 5

Effects of orally administered B. amyloliquefaciens (BA) or BA-nanoparticles (BANPs) in non-colitic and colitic rats on measurement of cytokines protein levels by ELISA kits (a,b) and heat map illustrating mRNA of various inflammatory markers including cytokines by real time PCR (c,d) 7 days post DSS induction. Interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor α (TNFα), transforming growth factor-beta (TGF-β), cyclooxygenase-2 (COX-2) and Inducible nitric oxide synthase (iNOS). Non-colitic groups including Control, BA and BANPs groups where rats were orally gavaged PBS, B. amyloliquefaciens and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day), respectively, for 7 days. Colitic groups: DSS, BA and BANPs groups where rats were orally gavaged dextrane sodium sulphate (DSS), B. amyloliquefaciens + DSS and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day) + DSS, respectively, for 7 days. All groups orally gavaged by 5% DSS. Values are expressed as mean ± SE, ^a,b,cMeans of the bars with different letters were significantly different among groups (P < 0.05). The scale bar on the right side described the unit for each color as blue and light green squares corresponded to significant upregulation and downregulation, respectively of genes relative to the control groups (P < 0.05).

Figure 6

Heat map illustrating the effects of orally administered B. amyloliquefaciens (BA) or BA-nanoparticles (BANPs) in non-colitic and colitic rats on mRNA expression of caspase-3, heat shock protein-70 (HSP-70), Cytochrome c, Bax and Bcl-2, 7 days post DSS induction. Non-colitic groups including Control, BA and BANPs groups where rats were orally gavaged PBS, B. amyloliquefaciens and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day), respectively, for 7 days. Colitic groups: DSS, BA and BANPs groups where rats were orally gavaged dextrane sodium sulphate (DSS), B. amyloliquefaciens + DSS and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day) + DSS, respectively, for 7 days. All groups orally gavaged by 5% DSS. Values are expressed as mean ± SE, ^a,b,cMeans of the bars with different letters were significantly different among groups (P < 0.05). The scale bar on the right side described the unit for each color as blue and light green squares corresponded to significant upregulation and downregulation of genes relative to the control groups (P < 0.05).

Figure 7

Effects of orally administered B. amyloliquefaciens (BA) or BA-nanoparticles (BANPs) in non-colitic and colitic rats on abundance of Bacillus, Bacteroides, Firmicutes, Enterobacteriaceae and Bifidobacterium populations (log₁₀ CFU), 7 days post DSS induction. Non-colitic groups including Control, BA and BANPs groups where rats were orally gavaged PBS, B. amyloliquefaciens and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day), respectively, for 7 days. Colitic groups: DSS, BA and BANPs groups where rats were orally gavaged dextrane sodium sulphate (DSS), B. amyloliquefaciens + DSS and B. amyloliquefaciens loaded nanoparticles (BA at level of 1.0 × 10¹⁰ CFU/kg in 1 mL of PBS/rat/day) + DSS, respectively, for 7 days. All groups orally gavaged by 5% DSS.

Figure 8

Histological changes following oral administration of B. amyloliquefaciens (BA) or BA-nanoparticles (BANPs) in non-colitic and colitic rats. Non-colitic groups including Control (a,b), BA (c,d) and BANPs (e–g) groups. While, colitic groups include DSS (h,j), DSS + BA (k,l) and DSS + BANPs (m,n) groups. Red rectangle = magnification of selected area, Arrow = normal colon crypt structure, black triangle = normal muscularis mucosa layer, yellow circle = diffuse leukocytic infiltration, zigzag arrow = desquamated colon epithelium, thick yellow arrow = mucosal abscess within the colonic crypts, arrow head = apparently normal colon crypts with increased number of goblet cells, star = prominent lymphoid aggregation within the submucosa.

Figure 9

Immunohistochemical staining for IL-6 positive cells within the colonic mucosa and submucosa following oral administration of B. amyloliquefaciens (BA) or BA-nanoparticles (BANPs) in non-colitic and colitic rats. Non-colitic groups including Control (a), BA (b) and BANPs (c) groups. While, colitic groups include DSS (d), DSS + BA (e) and DSS + BANPs (f) groups.

Figure 10

Immunohistochemical staining for Ki-67 positive cells within the colonic mucosa and submucosa following oral administration of B. amyloliquefaciens (BA) or BA-nanoparticles (BANPs) in non-colitic and colitic rats. Non-colitic groups including Control (a), BA (b) and BANPs (c) groups. While, colitic groups include DSS (d) , DSS + BA (e) and DSS + BANPs (f) groups.

Figure 11

Transmission electron microscopy (A) and zeta potential distribution (B) of B. amyloliquefaciens loaded nanoparticles.