Side-stream smoking reduces intestinal inflammation and increases expression of tight junction proteins

Abstract

Aim: To investigate the effect of side-stream smoking on gut microflora composition, intestinal inflammation and expression of tight junction proteins.

Methods: C57BL/6 mice were exposed to side-stream cigarette smoking for one hour daily over eight weeks. Cecal contents were collected for microbial composition analysis. Large intestine was collected for immunoblotting and quantitative reverse transcriptase polymerase chain reaction analyses of the inflammatory pathway and tight junction proteins.

Results: Side-stream smoking induced significant changes in the gut microbiota with increased mouse intestinal bacteria, Clostridium but decreased Fermicutes (Lactoccoci and Ruminococcus), Enterobacteriaceae family and Segmented filamentous baceteria compared to the control mice. Meanwhile, side-stream smoking inhibited the nuclear factor-κB pathway with reduced phosphorylation of p65 and IκBα, accompanied with unchanged mRNA expression of tumor necrosis factor-α or interleukin-6. The contents of tight junction proteins, claudin3 and ZO2 were up-regulated in the large intestine of mice exposed side-stream smoking. In addition, side-stream smoking increased c-Jun N-terminal kinase and p38 MAPK kinase signaling, while inhibiting AMP-activated protein kinase in the large intestine.

Conclusion: Side-stream smoking altered gut microflora composition and reduced the inflammatory response, which was associated with increased expression of tight junction proteins.

Keywords: Inflammation; Intestine; Microbiota; Side-stream smoking; Tight junction protein.

Figure 1

Cecal microflora composition of Con and side-stream smoking mice. ^aP < 0.05, ^bP < 0.01 vs control group (mean ± SEM; n = 6 per group).

Figure 2

NF-κB signaling pathway in large intestine of Con and side-stream smoking mice. A: Phos-p65 and p65; B: Phos-IκBa and IκBa; C: Phos-IKKα/β and IKKβ. ^aP < 0.05, ^bP < 0.01 vs control group (mean ± SEM; n = 6 per group).

Figure 3

Xanthine oxidase, superoxide dismutase 1 and heat shock protein 60 content in large intestine of Con and side-stream smoking mice. ^bP < 0.01 vs control group (mean ± SEM; n = 6 per group). XO: Xanthine oxidase; SOD1: Superoxide dismutase 1; HSP60: Heat shock protein 60.

Figure 4

MAP kinase signaling pathways in large intestine of Con and side-stream smoking mice. A: JNK; B: MAP kinase p38. ^aP < 0.05, ^bP < 0.01 vs control group (mean ± SEM; n = 6 per group).

Figure 5

Total AMP-activated protein kinase α subunit content and its phosphorylation at Thr 172 in large intestine of Con and side-stream smoking mice. ^bP < 0.01 vs control group (mean ± SEM; n = 6 per group). AMPK: Total AMP-activated protein kinase.

Figure 6

Tight junction protein content in large intestine of Con and side-stream smoking mice. A: mRNA expression; B: Protein content. ^aP < 0.05, ^bP < 0.01 vs control group (mean ± SEM; n = 6 per group).