Anat Cell Biol.  2020 Jun;53(2):228-239. 10.5115/acb.20.014.

The beneficial influence of rhubarb on 5-fluorouracil-induced ileal mucositis and the combined role of aquaporin-4, tumour necrosis factor-α, nuclear factor-kappa B & matrix metalloproteinase-9 in rat model: histological study

• Kamar SS
• Baky MH
• Omar AI

• KMID: 2503452
• DOI: http://doi.org/10.5115/acb.20.014

Abstract

A 5-fluorouracil (5-FU) is used for cancer treatment despite its cytotoxic sequelae on healthy cells, especially the rapid proliferating ones. Intestinal mucositis is one of the most frequent chemotherapeutic debilitating sequelae. Rhubarb (Rh), an ancient herb, is known for its curing effect on gastrointestinal complications. This study aims to detect the role of aquaporin-4 (AQP-4), tumour necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), and matrix metalloproteinase-9 (MMP-9) in 5-FU-induced ileal histological and biochemical changes and the potential therapeutic effect of Rh water extract on these changes in rats. A 45 rats were divided into 3 groups; control, 5-FU (single intraperitoneal injection of 150 mg/kg/rat) and Rh-treated (oral 20 mg/kg/day/rat for 8 days). The change in animals’ weight, incidence of diarrhoea and AQP-4 and TNF-α values in ileal homogenates were measured. Ileal sections were subjected to hematoxylin and eosin stain, periodic acid Schiff (PAS)-reaction and MMP-9, NF-κB and AQP-4 immunohistochemical staining. A 5-FU group revealed marked ileal mucosal damage associated with a significant decrease in the mean body weight, AQP-4 level and area percent of PAS and AQP-4 positive reaction. Significant increase in the mean incidence of diarrhoea, TNF-α value and area percent of MMP-9 and NF-κB was detected. These changes were significantly corrected with Rh administration. A 5-FU resulted in severe ileal mucositis through TNF-α, NF-κB, MMP-9, and AQP-4 disturbances. Rh treatment was highly effective in preventing such mucositis.

Keyword

Aqueous rhubarb extract; Intestinal mucositis; Aquaporin-4; Matrix metalloproteinase-9; Nuclear factor-kappa B

Figure

• 

  Fig. 1 Photomicrograph of H&E stained ileal sections showing: (A–D) control group: normal tissue architecture demonstrating M (Vi, c), muscularis M (curved arrow), submucosa (*) containing PP and ME (ic, ol). The villi are displaying a core of LP and EP formed of simple columnar cells, E, with brush border and G. The intestinal c are separated by LP and lined with E, G, and basally located P. Intra-epithelial L are noted in the epithelial lining of the Vi and the c. (E, F) A 5-FU group: obvious mucosal damage with most of the epithelial cells showing multiple V, some of them seemed with deeply eosinophilic cytoplasm and shrunken darkly stained nuclei (arrow heads), and some appearing swollen and dissolute while others are nearly absent (wavy arrow). Some of the Vi appeared F while others showed areas of mucosal U with complete loss of the epithelial lining. Disorganization of the c and marked mononuclear inflammatory cell In within the mucosal connective tissue are noted. Some of the BV are dilated and/or disrupted. (G, H) Rh-treated group: nearly normal tissue architecture, yet few epithelial cells with cytoplasmic V and few pyknotic nuclei, some inflammatory cellular In and slightly disorganized c are noted. Scale bars=200 μm (A), 100 μm (B, E, G), and 20 μm (C, D, F, H). BV, blood vessels; c, crypts; E, enterocytes; EP, epithelium; F, fused; G, goblet cells; ic, inner circular; In, infiltration; L, lymphocytes; LP, lamina propria; M, mucosa; ME, muscularis externa; ol, outer longitudinal; P, Paneth cells ; PP, Peyer’s patches; Rh, rhubarb; U, ulceration; V, vacuolations; Vi, villi; 5-FU, 5-fluorouracil.

• 

  Fig. 2 Photomicrograph picture of PAS reaction in the ileal sections of the experimental groups: (A) control group: abundant+ve PAS reaction filling the cytoplasm of the G (wavy arrow), in addition to+ve reaction in the brush border (arrowhead) of the enterocytes. (B) A 5-FU group: minimal+ve PAS reaction in a few G (wavy arrow) and the brush border (arrowhead). (C) Rh-treated group: abundant+ve PAS reaction in the cytoplasm of many G (wavy arrow) and the brush border (arrowhead). Scale bars=20 μm. G, goblet cells; PAS, periodic acid Schiff; 5-FU, 5-fluorouracil.

• 

  Fig. 3 Photomicrograph of immunohistochemistry in the ileal sections for MMP-9, NF-κB and AQP-4. Anti-MMP-9 immunohistochemistry showing: (A) control group: minimal+ve immunoreaction (arrows) in some cells of the mucosal LP (B) 5-FU group: abundant+ve cytoplasmic immunoreaction (arrows) in the inflammatory cells within the mucosal C.T. and the epithelial cells covering the Vi. (C) Rh-treated group: minimal+ve immunoreaction (arrows) in few inflammatory and epithelial cells. Anti-NF-κB immunohistochemistry showing: (D) control group: few+ve cytoplasmic immunoreactions (arrows) in some cells of the mucosal C.T. (E) A 5-FU group: obvious increased+ve immunoreaction (arrows) involving both the cytoplasm and the nuclei of most of the epithelial and inflammatory cells (F) Rh-treated group: some epithelial and inflammatory cells showing+ve immunoreaction (arrows) which is mainly cytoplasmic with few nuclear immunoreactions. Anti-AQP-4 immunohistochemistry showing: (G) control group: obvious+ve AQP-4 immunoreaction (arrows) mostly in the basolateral regions in addition to the cytoplasm of the enterocytes. (H) A 5-FU group: minimal+ve AQP-4 immunoreaction (arrows) in the epithelial cells (I) Rh-treated group: increased+ve AQP-4 immunoreaction (arrows) mainly in the basolateral regions in addition to the cytoplasm of the enterocytes. Scale bars= 20 μm. AQP-4, aquaporin-4; C.T., connective tissue; LP, lamina propria; MMP-9, matrix metalloproteinase-9; NF-κB, nuclear factor-kappa B; Rh, rhubarb; Vi, villi; 5-FU, 5-fluorouracil.

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