Study of the Possible Alleviated Role of Atorvastatin on Irinotecan-Induced Lingual Mucosal Damage: Histological and Molecular Study

Abstract

Background: Oral mucositis is the most debilitating and troublesome adverse effect of irinotecan (CPT-11) treatment. It adversely affects the patient quality of life. The aim of this work was to study the histological, immunohistochemical, and molecular changes in the oral mucosa by CPT-11 and the possible alleviated role of atorvastatin.

Methods: Rats were randomly divided into control, CPT-11-treated group, and CPT-11+ atorvastatin-treated group. At the end of the experiment, the anterior two-thirds of the tongue was dissected out and divided into two parts: one part for light microscopic examination and the second for molecular study.

Results: CPT-11-treated group revealed loss of normal mucosal organization, areas of ulceration and inflammation, and loss of architecture of lingual papillae. A significant decrease in immunohistochemical and molecular gene expression of Ki-67 and antiapoptotic Bcl-2 levels was observed. A significant increase in NF-κB immunohistochemical and mRNA gene expression level and a nonsignificant increase in Nrf2 gene expression were detected. Coadministration of atorvastatin showed remarkable improvement in the histopathological picture with a significant increase in Ki-67 and Bcl-2, a significant decrease in NF-κB protein and gene expression, and a significant increase in Nrf2 gene expression.

Conclusion: Atorvastatin substantially attenuates CPT-11-induced oral mucositis through the initiation of the antiapoptotic gene, modulation of the inflammatory, and antioxidant gene expression.

Figure 1

H&E-stained sections from the tongue of the control group (a), the dorsal surface showing regularly arranged conical shape filiform papillae (arrow) covered by stratified squamous keratinized epithelium with pointed tips (curved arrow) and underlying connective tissue cores continue with the lamina propria (LP). A characteristic fungiform papilla has broad top and narrow base with vascular connective tissue core (BV) and is covered by stratified squamous keratinized epithelium (arrowhead) contains barrel-shaped pale stained taste buds (TB). Bundles of lingual muscle fibers (M) run in various directions and surround by endomysium and perimysium that are continued with the connective tissue corium of both surfaces. The ventral surface is covered by smooth stratified squamous keratinized epithelium (crossed arrow) without papillae. CPT-11-treated group (b), the dorsal surface reveals the area of mucosal ulceration (arrow), other areas with short or absent filiform papillae (thick-tailed arrow), and blunt-ended tops (B) with inflammatory cell infiltration (double arrow) and congested blood vessels (BV) in the lamina propria (LP). The cells of stratified epithelium reveal vacuolation (zigzag arrow) and nuclear changes; chromatin margination with crescent formation (black arrow), pyknosis (thick white arrow), and chromatin condensation (double arrowheads). The fungiform papillae (arrowhead) show loss of taste buds and congested vascular core (LP) with inflammatory cell infiltration (double arrow). The papillae are covered by a thick layer of keratin (curved arrow) with areas of keratin separation (white asterisks). Disturbed organization of muscle fibers (M) with wide separation (black asterisks). The ventral surface shows thinning of the stratified epithelium with thin keratin layer (crossed arrow), and the lamina propria (LP) showed congested blood vessels (BV) with inflammatory cell infiltration (double arrow). CPT − 11 + atorvastatin treated group (c), the dorsal surface reveals normal orientation of the lingual papillae (arrow). Most of the filiform papillae are long with pointed tips and covered by stratified squamous keratinized epithelium (curved arrow), few papillae with loss of their tips (double arrowheads). Most of the cells are normal with few ones with marginated chromatin (black arrow) and few pyknotic nuclei (thick white arrow). Normal fungiform papillae (arrowhead) with taste bud (TB), normal thickness of keratin layer (curved arrow), and connective tissue core (LP) are observed. The organization of muscle layer (M) and the ventral surface (crossed arrow) closely resemble those of the control. (H&E a1, b1, c1 X100, a2 − 5, b2 − 5, c2 − 5 X400).

Figure 2

Ki-67 immunohistochemical staining. (a) (The control group) the dorsal surface shows frequent cells with positive immunoexpression in their nuclei in the basal (white arrow) and suprabasal layers (black arrow). (b) CPT-11-treated group reveals few positive cells mainly in the basal epithelial layer (white arrow). (c) CPT − 11 + atorvastatin treated group shows numerous immunostaining cells in the basal (white arrow) and suprabasal layers (black arrow) epithelial layer. (Ki-67 a-c X400).

Figure 3

Bcl2 immunohistochemical staining. (a) The control group shows strong positive cytoplasmic immunoexpression to Bcl2 in most of the dorsal epithelial cells (arrow). (b) CPT-11-treated group reveals negative cytoplasmic reaction in most epithelial cells (arrow). (c) CPT − 11 + atorvastatin − treated group shows positive cytoplasmic immunoexpression with moderate density (arrow) in most of the epithelial cells. (Bcl2 a-c X400).

Figure 4

NF-κB immunohistochemical staining. (a) The control group shows negative immunoexpression to NF-κB in the cytoplasm of dorsal epithelial cells (arrow). (b) CPT-11-treated group reveals a strong positive cytoplasmic reaction in most epithelial cells (arrow). (c) CPT − 11 + atorvastatin treated group shows weak positive to negative cytoplasmic immunoexpression (arrow) in most of the epithelial cells. (NF-Κb a-c X400).

Figure 5

PCR level of KI-67 within the control and the experimental groups. (a) Comparison in relation to (-ve control DW) group. (b) Comparison in relation to (-ve control buffer) group. (c) comparison in relation to (+ve control atorvastatin) group. (d) Comparison in relation to (CPT-11) group.

Figure 6

PCR level of BCL2 within the control and the experimental groups. (a) Comparison in relation to (-ve control DW) group. (b) Comparison in relation to (-ve control buffer) group. (c) Comparison in relation to (+ve control atorvastatin) group. (d) Comparison in relation to (CPT-11) group.

Figure 7

PCR level of NF-Kb level within the control and the experimental groups. (a) Comparison in relation to (-ve control DW) group. (b) Comparison in relation to (-ve control buffer) group. (c) Comparison in relation to (+ve control atorvastatin) group. (d) Comparison in relation to (CPT-11) group.

Figure 8

Nr-F2 level within the control and the experimental groups. (a) Comparison in relation to (-ve control DW) group. (b) Comparison in relation to (-ve control buffer) group. (c) Comparison in relation to (+ve control atorvastatin) group. (d) Comparison in relation to (CPT-11) group.