Rhubarb Monomers Protect Intestinal Mucosal Barrier in Sepsis via Junction Proteins

Abstract

Background: Leakage of the intestinal mucosal barrier may cause translocation of bacteria, then leading to multiorgan failure. This study hypothesized that rhubarb monomers might protect the gut mucosal barrier in sepsis through junction proteins.

Methods: Healthy male Sprague-Dawley rats (weighing 230-250 g) under anesthesia and sedation were subjected to cecal ligation and perforation (CLP). After surgical preparation, rats were randomly assigned to eight groups (n = 6 or 8 each group): sham group (Group A: normal saline gavage); sepsis group (Group B: normal saline gavage); Group C (intraperitoneally, dexamethasone 0.5 mg/kg) immediately after CLP surgery; and rhubarb monomer (100 mg/kg in normal saline)-treated groups (Group D: rhein; Group E: emodin; Group F: 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid; Group G: 1-O-caffeoyl-2-(4-hydroxy-O-cinnamoyl)-D-glucose; and Group H: daucosterol linoleate). Animals were sacrificed after 24 h. Intestinal histology, lactulose, mannitol concentrations were measured, and zonula occludens (ZO)-1, occludin and claudin-5 transcription (polymerase chain reaction), translation (by Western blot analysis), and expression (by immunohistochemistry) were also measured.

Results: Intestinal histology revealed injury to intestinal mucosal villi induced by sepsis in Group B, compared with Group A. Compared with Group A (0.17 ± 0.41), the pathological scores in Groups B (2.83 ± 0.41, P < 0.001), C (1.83 ± 0.41, P < 0.001), D (2.00 ± 0.63, P < 0.001), E (1.83 ± 0.41, P < 0.001), F (1.83 ± 0.75, P < 0.001), G (2.17 ± 0.41, P < 0.001),and H (1.83 ± 0.41, P < 0.001) were significantly increased. Lactulose/mannitol (L/M) ratio in Group B (0.046 ± 0.003) was significantly higher than in Group A (0.013 ± 0.001, P< 0.001) while L/M ratios in Groups C (0.028 ± 0.002, P< 0.001), D (0.029 ± 0.003, P< 0.001), E (0.026 ± 0.003, P< 0.001), F (0.027 ± 0.003, P< 0.001), G (0.030 ± 0.005, P< 0.001), and H (0.026 ± 0.002, P< 0.001) were significantly lower than that in Group B. ZO-1, occludin and claudin-5 transcription, translation, and expression in Group B were significantly lower than that in Group A (P < 0.001), but they were significantly higher in Groups C, D, E, F, G, and H than those in Group B (P < 0.05).

Conclusion: Rhubarb monomer treatment ameliorated mucosal damage in sepsis via enhanced transcription, translation, and expression of junction proteins.

Figure 1

The survival rates in sham, CLP, and rhubarb-treated CLP groups. CLP: Cecal ligation and perforation.

Figure 2

Hematoxylin and eosin staining in rat intestinal mucosa (slice thickness 5 μm; original magnification ×100): (a) Group A; (b) Group B; (c) Group C; (d) Group D; (e) Group E; (f) Group F; (g) Group G; and (h) Group H. (i) Mucosal pathological scores among groups. Data are shown as mean ± SD. *P < 0.05, versus Group B; ^†P < 0.05, versus Group A; ^‡P < 0.05, versus Group C. SD: Standard deviation.

Figure 3

Lactulose/mannitol ratios among groups. Data are shown as mean ± SD. *P < 0.05, versus Group B; ^†P < 0.05, versus Group A. SD: Standard deviation.

Figure 4

Relative messenger RNA expression of junction protein ZO-1 (a), occludin (b), and claudin-5 (c). Data are shown as mean ± SD. *P < 0.05, versus Group B; ^†P < 0.05, versus Group A. SD: Standard deviation. ZO-1: Zonula occludens-1.

Figure 5

Western blot analysis and band grayscale of ZO-1 (a and b), occludin (c and d); and claudin-5 (e and f) expressions. Data are shown as mean ± SD. *P < 0.05, versus Group B; ^†P < 0.05, versus Group A. SD: Standard deviation; ZO-1: Zonula occludens-1.

Figure 6

Immunohistochemical determination of the expression of junction protein ZO-1 (slice thickness 5 μm; original magnification ×250). ZO-1: Zonula occludens-1.

Figure 7

Immunohistochemical determination of the expression of junction protein occludin (slice thickness 5 μm; original magnification ×250).

Figure 8

Immunohistochemical determination of the expression of junction protein claudin-5 (slice thickness 5 μm; original magnification ×250).