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. 2015 Jul 21;21(27):8314-25.
doi: 10.3748/wjg.v21.i27.8314.

TLR4-HMGB1-, MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury

Jie Wang  1 , Gui-Zhen He  1 , Yu-Kang Wang  1 , Qian-Kun Zhu  1 , Wei Chen  1 , Tai Guo  1
Affiliations

TLR4-HMGB1-, MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury

Jie Wang et al. World J Gastroenterol. .

Abstract

Aim: To characterize high-mobility group protein 1-toll-like receptor 4 (HMGB1-TLR4) and downstream signaling pathways in intestinal ischemia/reperfusion (I/R) injury.

Methods: Forty specific-pathogen-free male C57BL/6 mice were randomly divided into five groups (n = 8 per group): sham, control, anti-HMGB1, anti-myeloid differentiation gene 88 (MyD88), and anti-translocating-chain-associating membrane protein (TRIF) antibody groups. Vehicle with the control IgG antibody, anti-HMGB1, anti-MyD88, or anti-TRIF antibodies (all 1 mg/kg, 0.025%) were injected via the caudal vein 30 min prior to ischemia. After anesthetization, the abdominal wall was opened and the superior mesenteric artery was exposed, followed by 60 min mesenteric ischemia and then 60 min reperfusion. For the sham group, the abdominal wall was opened for 120 min without I/R. Levels of serum nuclear factor (NF)-κB p65, interleukin (IL)-6, and tumor necrosis factor (TNF)-α were measured, along with myeloperoxidase activity in the lung and liver. In addition,morphologic changes that occurred in the lung and intestinal tissues were evaluated. Levels of mRNA transcripts encoding HMGB1 and NF-κB were measured by real-time quantitative PCR, and levels of HMGB1 and NF-κB protein were measured by Western blot. Results were analyzed using one-way analysis of variance.

Results: Blocking HMGB1, MyD88, and TRIF expression by injecting anti-HMGB1, anti-MyD88, or anti-TRIF antibodies prior to ischemia reduced the levels of inflammatory cytokines in serum; NF-κB p65: 104.64 ± 11.89, 228.53 ± 24.85, 145.00 ± 33.63, 191.12 ± 13.22, and 183.73 ± 10.81 (P < 0.05); IL-6: 50.02 ± 6.33, 104.91 ± 31.18, 62.28 ± 6.73, 85.90 ± 17.37, and 78.14 ± 7.38 (P < 0.05); TNF-α, 43.79 ± 4.18, 70.81 ± 6.97, 52.76 ± 5.71, 63.19 ± 5.47, and 59.70 ± 4.63 (P < 0.05) for the sham, control, anti-HMGB1, anti-MyD88, and anti-TRIF groups, respectively (all in pg/mL).Antibodies also alleviated tissue injury in the lung and small intestine compared with the control group in the mouse intestinal I/R model. The administration of anti-HMGB1, anti-MyD88, and anti-TRIF antibodies markedly reduced damage caused by I/R, for which anti-HMGB1 antibody had the most obvious effect.

Conclusion: HMGB1 and its downstream signaling pathway play important roles in the mouse intestinal I/R injury, and the effect of the TRIF-dependent pathway is slightly greater.

Keywords: C57BL/6 mouse; High-mobility group protein 1; Intestinal ischemia-reperfusion injury; Myeloid differentiation gene 88; Nuclear factor-κB translocating-chain-associating membrane protein.

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Figures

Figure 1
Figure 1
High-mobility group protein 1-toll-like receptor 4 and downstream signaling pathways. IFN: Interferon; IL: Interluekin; MyD88: Myeloid differentiation gene 88; NF-κB: Nuclear factor-κB; TNF: Tumor necrosis factor; TRIF: Translocating-chain-associating membrane protein; HMGB1: High-mobility group protein 1; TLR4: Toll-like receptor 4.
Figure 2
Figure 2
Levels of myeloperoxidase activity in the lung and liver. S: Sham group; C: Control group; H: Anti-high-mobility group protein 1 group; M: Anti-myeloid differentiation gene 88 group; T: Anti-translocating-chain-associating membrane protein group; n = 4; aP < 0.05 vs sham group; bP < 0.05 vs control group; cP < 0.05 vs anti-HMGB1 group. MPO: Myeloperoxidase.
Figure 3
Figure 3
Hematoxylin and eosin staining of lungs in all groups (magnification × 400). A: Sham group; B: Control group; C: Anti-high-mobility group protein 1 group; D: Anti-myeloid differentiation gene 88 group; E: Anti-translocating-chain-associating membrane protein group (magnification × 400; scale bars = 50 μm).
Figure 4
Figure 4
Hematoxylin and eosin staining of the jejunum and ileum in all groups (magnification × 100). Representative images from jejunum (A1-E1) and ileum (A2-E2). A: Sham group; B: Control group; C: Anti-high-mobility group protein 1 group; D: Anti-myeloid differentiation gene 88 group; E: Anti-translocating-chain-associating membrane protein group (magnification × 100; scale bars = 200 μm).
Figure 5
Figure 5
Expression of HMGB1 and NF-κB in lung, jejunum, and ileum in all groups. HMGB1 expression in: Lung (A1); Jejunum (A2); and Ileum (A3); respectively, in all groups; NF-κB expression in: Lung (B1); Jejunum (B2); and Ileum (B3). S: Sham group; C: Control group; H: Anti-HMGB1 group; M: Anti-myeloid differentiation gene 88 group; T: Anti-translocating-chain-associating membrane protein group. n = 4,aP < 0.05 vs sham group; bP < 0.05 vs control group; cP < 0.05 vs anti-HMGB1 group. HMGB1: High-mobility group protein 1; NF-κB: Nuclear factor-κB.
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