Protective effect of rosuvastatin on cyclophosphamide-induced oral toxicity in rats: Histological and immunohistochemical Study

 

Jawnaa K. Mammdoh*, Rana KA Attarbashee, Alhan DH Al mola

College of Dentistry, University of Mosul, Mosul, Iraq.

 

ABSTRACT:

Background and Objective: Cyclophosphamide is a well-known alkylating anticancer drug that is used to treat a variety of cancers, both malignant and non-malignant. Cyclophosphamide can have a number of side effects, including oral toxicity. Rosuvastatin, a statin, has anti-inflammatory and antioxidant properties in addition to its anti-hyperlipidemic properties. The goal of this trial was to see if rosuvastatin could help prevent cyclophosphamide-induced tongue lesions. Methods: Twenty-four Wister-albino rats, weighing 300-400grams and aged 12-16 weeks, were used. The animals were divided into three groups: group I considered as control. Group 2 was given cyclophosphamide 150mg\kg and in group 3 was given cyclophosphamide 150mg/kg, and rosuvastatin (20mg/kg). The microscopic parameter was estimated, and the oxidative stress marker malondialdehyde (MDA) in the tongue was measured. On day 15, the animals (eight per group) were slaughtered, and the tongue was removed from the oral cavity for histological and immunohistochemical investigation. Results: At day 15, rosuvastatin significantly reduced the severity of the cyclophosphamide-induced tongue lesion in terms of histological score and immunohistochemistry expression of MDA (P 0.05). Conclusion: Rosuvastatin, at a dose of 20mg/kg/day, provided antioxidant and histological grade-reducing protection against cyclophosphamide-induced tongue lesion, and hence could be utilized as a preventive drug against cyclophosphamide-induced tongue lesion.

 

 

 

INTRODUCTION: 

Cyclophosphamide is alkylating anticancer drug that is used to treat a variety of cancers, both malignant and non-malignant¹. It's also used to treat autoimmune illnesses and as an immunosuppressive medication to avoid organ rejection². Cyclophosphamide, although its widespread usage in clinical practice, can have a number of side effects, including diarrhea, myelosuppression, foot syndrome, alopecia, nausea, vomiting, and dermatitis³. Oral toxicity is a common and terrible side effect of chemotherapy that can occur even at standard doses, and cyclophosphamide is linked to oral mucositis and tongue hyperpigmentation^4,5.

 

 

Rosuvastatin belongs to the statin class of antihyperlipidemic drugs. It operates by inhibiting the enzyme 3-hydroxy-3-methylbutaryl reductase⁶. Rosuvastatin has recently been discovered to have antioxidant and anti-inflammatory qualities in addition to its anti-hyperlipidemic capabilities⁷. Several studies have shown that rosuvastatin reduces oxidative stress in patients suffering from indomethacin-induced gastropathy⁸, amikacin-induced kidney injury⁹, and trinitrobenzene sulfonic acid-induced colitis¹⁰. In the liver and kidney tissues of fipronil-intoxicated animals, we previously demonstrated antioxidant and anti-apoptotic effects¹¹. The antioxidant and anti-apoptotic properties of cyclophosphamide against cyclophosphamide-induced mouth ulcers are yet to be explored. As a result, the goal of this study was to see if rosuvastatin could protect against cyclophosphamide-induced mouth ulcers.

 

 

MATERIALS AND METHODS:

Four male adult albino rats (300-400g) were placed in a cage for seven days before the experiment.  Allowed to eat laboratory pellets and drink tap water as they pleased. All research was done after receiving previous approval from the Institutional Animal Ethics Committee of the College of Dentistry/University of Mosul (UoM.Dent/A, L12/22).

 

Baxter Corporation provided cyclophosphamide for treatment.

Another alternative is AstraZeneca's rosuvastatin. Mode of experimentation.

A total of 24 adult male rats were used in this study. Three groups of rat (each with eight rat) were formed. The first group got no therapy and functioned as a control group, while the other two were given cyclophosphamide (150mg/kg) intraperitoneally. Group III received rosuvastatin 20 mg/kg orally seven days before and after cyclophosphamide induction.

 

Prior to injection, cyclophosphamide and rosuvastatin were freshly primed on the day of the trial. The determined medicines were produced as distilled water suspensions. A previous study was used to estimate the rosuvastatin dose (20mg/kg). Oral chemotherapy-induced toxicity has been treated with cyclophosphamide (150mg/kg).

 

All of the experimental rat were maintained in the animal house for seven days without any treatment to allow them to adjust to the new surroundings. The chemotherapeutic drug cyclophosphamide (150mg/kg) was administered intraperitoneally to cause tongue lesion.

 

Group III was given rosuvastatin at a dose of 20 mg/kg seven days before starting cyclophosphamide treatment and continued for another seven days following induction (for a total of 15 days of treatment).

 

Animals were anesthetized with ketamine (50mg/kg) plus xylazine (5mg/kg) in the same syringe after the experiment ended, and their tongues were removed for histological and immunohistochemical analyses.

 

The tongue samples were fixed with 10% formalin, then dried, embedded in paraffin, decaffeinated with xylene, cut into thin slices, and stained with hematoxylin and eosin (H&E). To assess histological changes, slides were examined and scored. The slides were coded during the review to avoid observer bias. A skilled histopathologist analyzed all tissue slices in a blinded manner. According to prior research, it was categorized as follows: 1 = vasodilation of epithelial and connective tissue, absence or low cellular infiltration, no edema, ulceration, and abscess. 2 = sporadic vasodilation, areas of reperfusion, diffuse cell infiltration with mononuclear leukocytes, absence of hemorrhages, edema, ulcers, and abscesses. 3 = moderate vasodilation, hydro-epithelial degeneration (vacuole), moderate infiltration of cells dominated by polymorphonuclear leukocytes, presence of hemorrhagic areas, edema. 5= Severe vasodilation and inflammatory infiltration with abscesses, ulcers, and disseminated neutrophils.

 

Immunohistochemistry (IHC) revealed the presence of cells in tissues that were directly affected. Development of specific antibodies for immunohistochemical responses, as well as evaluation of the production of a variety of biochemical markers in paraffin-embedded gut samples for the measurement of the oxidative stress marker (MDA). IHC was scored using the following semi-quantitative scores based on the proportion of positively stained cells: 0, no staining; 1, 25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%.

 

To summarize, evaluate, and present the data, two software applications were used: SPSS (Statistical Package for the Social Sciences) version 23 and Microsoft Office Excel 2013. The mean and standard error were used to express the quantitative (numerical) variables.

 

Figure 1. Rosuvastatin blocked cyclophosphamide induced damage indicated by histology and immunohistochemical examination. The slides prepared from tissue slices of tongue. CPA=cyclophosphamide, Rosu=Rosuvastatin.

 

Table 1. Histopathological and immunohistochemical scores of oxidative stress (MDA) in animals

Letters are used to express the comparison. Different letters imply a significant difference. Calculate the values' mean and standard error (SE).

 

The difference in mean dependent variables between two groups was determined using the paired t-test. The difference in the mean of quantitative variables between more than two groups was investigated using the ANOVA approach, which was followed by a post hoc least significant difference (LSD) test to determine the mean difference between any two groups. P>0.05 was used as the level of significance.

 

RESULTS:

The histological abnormalities in tongue tissues in the cyclophosphamide-treated group are depicted in Figure 1, with infiltration of inflammatory cells, particularly large cells, necrosis of skeletal muscle cells, and atrophy and edema between them.

 

Furthermore, as shown in Table (1), the rosuvastatin-treated group demonstrated a significant decrease (p0.01) in microscopic degree as determined by healthy skeletal muscle cells, reduced inflammatory infiltration, and edema (3). The cyclophosphamide group had a highly significant link for MDA level (p0.05) when compared to the rosuvastatin group, as shown in Table 1 and figure 1.

 

DISCUSSION:

Cyclophosphamide, an alkylating drug used in cancer chemotherapy, has been linked to a number of negative side effects¹². Such as taste abnormalities, which can lead to a lack of appetite, malnutrition, poor recuperation, and a lower quality of life. Previous mouse studies have found evidence that CYP causes a two-stage change in taste behavior: one that occurs right after the drug is taken and another that appears several days later¹³. As a result, chemotherapeutic changes in taste may pose nutritional challenges at a time when a cancer patient desperately needs a well-balanced, high-energy diet.

 

Cyclophosphamide toxicity is linked to the synthesis of acrolein, one of its metabolites¹⁴ and antioxidants have been shown to protect normal cells against cyclophosphamide toxicity. According to studies, oxidative stress is induced by a shift in the equilibrium between the prooxidant and antioxidant systems, which occurs as a result of excessive production of free oxygen radicals and neutrophil infiltration^15,16. It plays a significant part in many diseases' chronic consequences¹⁷.

 

The administration of cyclophosphamide considerably increased the amount of MDA in this study, indicating that cyclophosphamide causes oxidative stress. Free radicals produced from oxygen have been proven in studies to play a significant impact in tissue injury¹⁸.

 

The major reactive oxygen species formed during oxidative stress are super anions (O₂ percent), hydroxyl radicals (OH percent), and hydrogen peroxide            (H₂O₂)^19-21. Cyclophosphamide produced oxidative damage in this study, suggesting a significant increase in MDA. Rosuvastatin, on the other hand, reduced the oxidative damage produced by cyclophosphamide and improved microscopy in tongue tissues.

 

In the cyclophosphamide-treated group, histopathology revealed infiltration of inflammatory cells, particularly large macrophage cells, as well as necrosis of skeletal muscle cells, atrophy, and edema between them. These findings are similar to those of Yahya-Zadeh²² who employed cyclophosphamide as a model for inducing hypoglossal adenitis in male Wistar rats in order to investigate the preventive impact of curcumin.

 

Rosuvastatin, a member of the statin family, is an inhibitor of HMG-CoA reductase that is primarily used to decrease cholesterol²³. It has recently been discovered to have anti-inflammatory and antioxidant properties in addition to its hypolipidemic properties^24-26.

 

When HMG-CoA is suppressed by lowering cholesterol biosynthesis, a compensatory process is triggered by exogenous receptor exposure, which increases cholesterol absorption and lowers cholesterol levels in the blood²⁷. It has been shown to reduce inflammation by inhibiting the production of cytokines and the recruitment of inflammatory cells²⁸. The antioxidant potential is thought to be mediated by the NADPH-dependent suppression of O2 percent, a precursor of H₂O₂ and percent OH, as well as the up-regulation of cellular antioxidants such as GSH and CAT^24,29. Based on the therapeutic effects, we observe that the treatment can help to reduce cyclophosphamide-induced oral toxicity, as indicated by significant reductions in histological lesions and improved oxidative state³⁰.

 

CONCLUSION:

The histological effect of cyclophosphamide on the tongue of rats and the antioxidant effect of rosuvastatin on the tongue lesion was discovered in a few research. Rosuvastatin has an antioxidant effect and a reduction in histological grade, which protects the tissues of the tongue. Rosuvastatin protects the body by acting as an antioxidant and lowering the histological grade.

ACKNOWLEDGMENT:

The authors thank the College of Dentistry/University of Mosul for the facilities provided to accomplish this work.

 

CONFLICT OF INTEREST:

The authors declare that no conflict of interest exists for this research.

 

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Accepted on 14.05.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):759-762.