Ling Zhi-8, a fungal immunomodulatory protein in Ganoderma lucidum, alleviates CPT-11-induced intestinal injury via restoring claudin-1 expression

Abstract

CPT-11 (Irinotecan) remains an important chemotherapeutic agent against various solid tumors nowadays. Potential adverse effects, especially gastrointestinal toxicities, are the main limiting factor for its clinical utility. Ling Zhi-8 (LZ-8), a fungal immunomodulatory protein in Ganoderma lucidum mycelia, has potential for drug development due to its multiple bioactivities and functions. This study aimed to explore the influence of LZ-8 on CPT-11-treated IEC-6 cells in vitro and on mice with CPT-11-induced intestinal injury in vivo. The mechanism through which LZ-8 exerted its protective effects was also investigated. In the in vitro study, the viability and claudin-1 expression of IEC-6 cells decreased gradually with increasing concentrations of CPT-11, but LZ-8 treatment had no obvious influence on their viability, morphology, and claudin-1 expression. Pretreatment of LZ-8 significantly improved CPT-11-decreased cell viability and claudin-1 expression in IEC-6 cells. In mice with CPT-11-induced intestinal injury, LZ-8 treatment could ameliorate symptoms and mitigate intestinal damage. Meanwhile, LZ-8 restored claudin-1 expression in the intestinal membranes in CPT-11-treated mice. Collectively, our results demonstrated the protective effects of LZ-8 against CPT-11 damage in both IEC-6 cells and mice. LZ-8 can restore claudin-1 expression in intestinal cells following CPT-11 treatment, suggesting the role of claudin-1 in the scenario.

Keywords: CPT-11; Ganoderma lucidum; Ling Zhi-8; claudin-1; irinotecan.

Figure 1

Protective effects of LZ-8 against CPT-11-induced cytotoxicity in IEC-6 cells. CCK-8 assay was used to determine the viability of IEC-6 cells. For control, the absorbance of untreated cells was considered 100%. LZ-8 treatment had no obvious influence on the viability and morphology of IEC-6 cells, but cell death in a dose-dependent manner was noted in IEC-6 cell treated with CPT-11. LZ-8 pretreatment improved the cell viability and morphology of IEC-6 cells following CPT-11 treatment. Data were presented as mean ± SD, and one-way ANOVA was used for statistical analysis. * indicates P < 0.05, compared with the untreated cells; # indicates P < 0.05, compared with the CPT-11-treated cells. n = 5. (A) The viability of IEC-6 cells after 24-hour incubation of LZ-8 (left panel), 24-hour incubation of CPT-11 (middle panel), or 24-hour pretreatment of LZ-8 following 24-hour incubation of CPT-11 (right panel). (B) The viability of IEC-6 cells after 48-hour incubation of LZ-8 (left panel), 48-hour incubation of CPT-11 (middle panel), or 24-hour pretreatment of LZ-8 following 48-hour incubation of CPT-11 (right panel). (C) Effects of LZ-8 on CPT-11-induced cell morphological changes.

Figure 2

Restoration of claudin-1 mRNA expression by pretreatment of LZ-8 in IEC-6 cells following CPT-11 treatment, assessed by qPCR analysis. After normalization with β-actin, relative expression levels were shown with the mean value of untreated cells in the control group set at 1. LZ-8 treatment had no obvious influence on the expression levels of claudin-1 mRNA in IEC-6 cells, but CPT-11-treated IEC-6 cells expressed lower levels in a dose-dependent manner. LZ-8 pretreatment increased claudin-1 mRNA expression in IEC-6 cells following CPT-11 treatment. Data were presented as mean ± SD, and one-way ANOVA was used for statistical analysis. * indicates P < 0.05, compared with the untreated cells; # indicates P < 0.05, compared with the CPT-11-treated cells. n = 5.

Figure 3

Improvement of claudin-1 protein expression by pretreatment of LZ-8 in IEC-6 cells following CPT-11 treatment, assessed by Western blotting. After normalization with β-actin, relative expression levels were shown with the mean value of untreated cells in the control group set at 1. LZ-8 treatment had no obvious influence on the expression levels of claudin-1 protein in IEC-6 cells, but CPT-11-treated IEC-6 cells expressed lower levels. LZ-8 pretreatment increased claudin-1 protein expression in IEC-6 cells following CPT-11 treatment. Data were presented as mean ± SD, and one-way ANOVA was used for statistical analysis. * indicates P < 0.05, compared with the untreated cells; # indicates P < 0.05, compared with the CPT-11-treated cells. n = 4.

Figure 4

Beneficial effects of LZ-8 on body weight loss in mice with CPT-11-induced intestinal injury. Relative weight was calculated for each animal relative to its weight on day 4. There were no apparent changes of body weight profiles in the control and LZ groups, but body weights decreased significantly in mice of the CPT and LZ+CPT groups. The decline in body weights was milder in mice of the LZ+CPT group than the CPT group. One-way ANOVA was used for statistical analysis. * indicates P < 0.05, compared with the control group; # indicates P < 0.05, compared with the CPT group. n = 5 mice/group.

Figure 5

Protective effects of LZ-8 on intestinal damage by CPT-11 in mice. (A) Lengths of the total intestine, small intestine, and colon in mice following different treatments. No obvious changes of the intestinal lengths were observed in mice of the LZ group, but significant shortening of the intestinal lengths was noted in mice of the CPT group. The reduction in intestinal lengths was ameliorated in mice of the LZ+CPT group, compared to the CPT group. Data were presented as mean ± SD, and one-way ANOVA was used for statistical analysis. * indicates P < 0.05, compared with the control group; # indicates P < 0.05, compared with the CPT group. n = 5 mice/group. (B) Microscopic histopathology of the small intestine and colon tissue sections from mice with different treatments (H&E staining, 200×). Normal intestinal architecture was noted in the control and LZ groups. The small intestine section of the CPT and LZ+CPT groups showed an edematous change of the intestinal villi with hyperemia (arrowhead) and prominent infiltration of inflammatory cells into the lamina propria. The major abnormal finding in the colon section was the increase of inflammatory cells in the lamina propria (arrowhead). Compared to the CPT group, the histopathologic alterations were attenuate in the LZ+CPT group, with nearly normal architecture of the colon.

Figure 6

Restoration of claudin-1 expression in the intestinal membranes by LZ-8 treatment in mice with CPT-11-induced intestinal injury, assessed by immunohistochemical staining. (A) The small intestine sections (40× and 200×). (B) The colon sections (40× and 200×). Homogeneous strong membrane staining (3+) was noted in the control and LZ groups. The intensity diminished in the CPT group, only 1+ in the small intestine section (arrowhead) and 2+ in the colon section (arrowhead). Compared to the CPT group, the intensity increased in the LZ+CPT group, showing 2+ in the small intestine section (arrowhead) and 3+ in the colon section (arrowhead).

Figure 7

The dosing schedule of CPT-11 and LZ-8 in the animal study of CPT-11-induced intestinal injury. n = 5 mice/group.