Ulva pertusa Modulated Colonic Oxidative Stress Markers and Clinical Parameters: A Potential Adjuvant Therapy to Manage Side Effects During 5-FU Regimen

Abstract

One of the most used chemotherapy agents in clinical practice is 5-Fluorouracil (5-FU), a fluorinated pyrimidine in the category of antimetabolite agents. 5-FU is used to treat a variety of cancers, including colon, breast, pancreatic, and stomach cancers, and its efficacy lies in its direct impact on the patient's DNA and RNA. Specifically, its mechanism blocks the enzymes thymidylate synthetase and uracil phosphatase, inhibiting the synthesis of uracil, which cannot be incorporated into nuclear and cytoplasmic RNA. Despite being one of the most used drugs in oncology, it is associated with several significant side effects, including inflammation of the mouth, loss of appetite, and reduction in blood cells. In our study, we examined the reduction of side effects in a 5-FU regimen administered at doses of 15 mg/kg and 6 mg/kg for 14 days in 6-week-old male Sprague-Dawley rats. On the 14th day, the rats were treated orally for 2 weeks with 100 mg/kg of Ulva pertusa, a well-known seaweed from the Ulvaceae family, which has demonstrated powerful biological properties. The administration of this green alga alleviated the side effects of 5-FU, improving several parameters including body weight, food intake, and diarrhea index. It also helped reduce side effects in the blood, kidneys, and liver. Histological and molecular analyses were conducted on serum and colon tissues from the rats, examining changes in colon structure and the release of oxidative stress markers such as iNOS, COX-2, and nitrotyrosine. Several biochemical indicators, including SOD, CAT, GSH, MDA, and ascorbic acid, were also evaluated. Overall, our data indicated Ulva pertusa to be a promising therapeutic against 5-FU's adverse effects, therefore, it could be worthwhile to investigate the possibility of using this alga in safer cancer treatment formulations. Certainly, future preclinical and clinical studies could assess the alga's efficacy in diverse cancer treatment regimens, exploring its role as an adjuvant therapy that may reduce chemotherapy-related toxicity without compromising therapeutic outcomes.

Keywords: 5-Fluorouracil (5-FU); Ulva pertusa; anti-inflammatory and antioxidant markers; clinical biochemistry parameters; fluoropyrimidines; side effects.

Figure 1

Effect of the green alga Ulva pertusa on body weight loss, food intake, and diarrhea index. Graphical representation of body weight (A), food intake (B), and diarrhea index (C). The number of rats in every experimental group was eight. Data are expressed as means ± SD. Two-way ANOVA test. ND: Not Detectable; *** p < 0.001 vs. Sham; ## p < 0.01 vs. 5-FU + vehicle; ### p < 0.001 vs. 5-FU + vehicle.

Figure 2

Positive modulation of photophobia and pain sensitivity by Ulva pertusa. Behavioral analysis of the rats such as light/dark test (A) and von Frey test (B) at time 0, after 15 days and 30 days. The number of rats in every experimental group was eight. Data are expressed as means ± SD. One-way ANOVA test. *** p < 0.001 vs. Sham; ### p < 0.001 vs. 5-FU + vehicle.

Figure 3

Evaluation of colon damage and neutrophil infiltration. H&E representatives’ images of colon from Sham (A,D), 5-FU + vehicle (B,D), and 5-FU + Ulva pertusa 100 mg/kg (C,D). Neutrophilic activity was assessed by MPO assay (E). The number of rats in every experimental group was eight. The histological evaluations are represented at 20× magnification. Data are expressed as means ± SD. One-way ANOVA test. ND: Not detectable; *** p < 0.001 vs. Sham; ### p < 0.001 vs. 5-FU + vehicle.

Figure 4

Evaluation of COX-2 expression and MDA levels in colon tissues. Immunohistochemical staining of COX-2 in colon tissue from Sham (A,A1), 5-FU + vehicle (B,B1), and 5-FU + Ulva pertusa 100 mg/kg (C,C1). Immunohistochemical score (D). ELISA kit for COX-2 (E). MDA assay (F). The number of rats in every experimental group was eight. Black arrows indicate immunopositivity for COX-2 in the colonic cells. The pictures are displayed at 20× and 40× magnification. Data are expressed as means ± SD. One-way ANOVA test. *** p < 0.001 vs. Sham; ### p < 0.001 vs. 5-FU + vehicle.

Figure 5

Evaluation of iNOS expression in colon tissues. IHC staining showed the trend of iNOS expression in rat colon tissues from Sham (A,A1), 5-FU + vehicle (B,B1), and 5-FU + Ulva pertusa 100 mg/kg (C,C1). Immunohistochemical score (D). ELISA kit for iNOS (E). The number of rats in every experimental group was eight. Black arrows indicate immunopositivity for COX-2 in the colonic cells. The pictures are presented at 20× and 40× magnification. Data are expressed as means ± SD. One-way ANOVA test. *** p < 0.001 vs. Sham; ### p < 0.001 vs. 5-FU + vehicle.

Figure 6

Immunolocalization of nitrotyrosine in colon tissues. Immunohistochemical staining for nitrotyrosine in the colon tissues from Sham group (A,A1), 5-FU + vehicle group (B,B1), and 5-FU + Ulva pertusa 100 mg/kg group (C,C1). Immunohistochemical score (D). ELISA kit for ROS quantification (E). The number of rats in every experimental group was eight. Black arrows indicate immunopositivity for COX-2 in the colonic cells. The pictures are presented at 20× and 40× magnification. Data are expressed as means ± SD. One-way ANOVA test. *** p < 0.001 vs. Sham; ### p < 0.001 vs. 5-FU + vehicle.

Figure 7

Blood cells count. The red (A) and white (B) blood cells and platelets (C) were evaluated on day 15 and 30 by using an automated cell counter. The number of rats in every experimental group was eight. Data are expressed as means ± SD. One-way ANOVA test. *** p < 0.001 vs. Sham; # p < 0.05 vs. 5-FU + vehicle.

Figure 8

Renal and hepatic evaluations. Using ELISA kits, AST (A), ALT (B), uric Acid (C), creatinine (D) and urea (E) were evaluated. The number of rats in every experimental group was eight. The results are expressed as means ± SD. One-way ANOVA test. *** p < 0.001 vs. Sham; # p < 0.05 vs. 5-FU + vehicle.

Figure 9

Biochemical analysis of antioxidant defense markers. Serum analysis of GSH (A), CAT (B), SOD (C) and Vitamin C (D). The number of rats in every experimental group was eight. Data are expressed as means ± SD. One-way ANOVA test. *** p < 0.001 vs. Sham; ### p < 0.001 vs. 5-FU + vehicle.