Effect of salvianolate on intestinal epithelium tight junction protein zonula occludens protein 1 in cirrhotic rats

Abstract

Aim: To study the effect of salvianolate on tight junctions (TJs) and zonula occludens protein 1 (ZO-1) in small intestinal mucosa of cirrhotic rats.

Methods: Cirrhosis was induced using carbon tetrachloride. Rats were randomly divided into the untreated group, low-dose salvianolate (12 mg/kg) treatment group, medium-dose salvianolate (24 mg/kg) treatment group, and high-dose salvianolate (48 mg/kg) treatment group, and were treated for 2 wk. Another 10 healthy rats served as the normal control group. Histological changes in liver tissue samples were observed under a light microscope. We evaluated morphologic indices of ileal mucosa including intestinal villi width and thickness of mucosa and intestinal wall using a pathological image analysis system. Ultrastructural changes in small intestinal mucosa were investigated in the five groups using transmission electron microscopy. The changes in ZO-1 expression, a tight junction protein, were analyzed by immunocytochemistry. The staining index was calculated as the product of the staining intensity score and the proportion of positive cells.

Results: In the untreated group, hepatocytes showed a disordered arrangement, fatty degeneration was extensive, swelling was obvious, and disorganized lobules were divided by collagen fibers in hepatic tissue, which were partly improved in the salvianolate treated groups. In the untreated group, abundant lymphocytes infiltrated the fibrous tissue with proliferation of bile ducts, and collagen fibers gradually decreased and damaged hepatic lobules were partly repaired following salvianolate treatment. Compared with the untreated group, no differences in intestinal villi width between the five groups were observed. The villi height as well as mucosa and intestinal wall thickness gradually thickened with salvianolate treatment and were significantly shorter in the untreated group compared with those in the salvianolate treatment groups and normal group (P < 0.01). The number of microvilli decreased and showed irregular lengths and arrangements in the untreated group. The intercellular space between epithelial cells was wider. The TJs were discontinuous, which indicated disruption in TJ morphology in the untreated group. In the treated groups, the microvilli in the intestinal epithelium were regular and the TJs were gradually integrated and distinct. The expression of ZO-1 decreased in the small intestine of the untreated cirrhotic rats. The high expression rate of ZO-1 in ileal mucosa in the untreated group was significantly lower than that in the medium-dose salvianolate group (21.43% vs 64.29%, χ(2) = 5.25, P < 0.05), high-dose salvianolate group (21.43% vs 76.92%, χ(2) = 8.315, P < 0.01) and normal group (21.43% vs 90%, χ(2) = 10.98, P < 0.01).

Conclusion: Salvianolate improves liver histopathological changes, repairs intestinal mucosa and TJ structure, and enhances ZO-1 expression in the small intestinal mucosa in cirrhotic rats.

Keywords: Cirrhosis; Gut barrier; Salvianolate; Tight junction; Zonula occludens protein 1.

Figure 1

Liver histopathology in cirrhotic rats. A: Untreated group; B: Low-dose salvianolate-treated group; C: Medium-dose salvianolate-treated group; D: High-dose salvianolate-treated group (hematoxylin and eosin staining, × 200).

Figure 2

Tight junction structural morphology in ileal mucosa. A: Normal control group; B: Untreated group; C: Low-dose salvianolate-treated group; D: Medium-dose salvianolate-treated group; E: High-dose salvianolate-treated group (transmission electron microscopy, × 26  500).

Figure 3

Immunohistochemical staining for zonula occludens protein 1 in ileal mucosa. A: Normal control group; B: Untreated group; C: Low-dose salvianolate-treated group; D: Medium-dose salvianolate-treated group; E: High-dose salvianolate-treated group (× 40).

Figure 4

Expression of zonula occludens protein 1 in ileal mucosa. A: Normal control group; B: Untreated group; C: Low-dose salvianolate-treated group; D: Medium-dose salvianolate-treated group; E: High-dose salvianolate-treated group. The staining index was calculated as the product of the staining intensity score and the proportion of positive cells. We evaluated zonula occludens protein 1 (ZO-1) expression in the five groups by determining the staining index with scores of 0, 1, 2, 3, 4, 6, or 9. An optimal cutoff value was identified as follows: a staining index score of ≥ 6 was used to define high ZO-1 expression and a staining index score of < 3 was used to indicate low ZO-1 expression.