Atorvastatin mitigates cyclophosphamide-induced hepatotoxicity via suppression of oxidative stress and apoptosis in rat model

Abstract

Cyclophosphamide (CP), as a chemotherapy drug, induces hepatotoxicity through causing oxidative stress. Atorvastatin (ATV) at a low dose has antioxidant and anti-inflammatory properties. The present study was designed to investigate the protective effects of ATV against CP-induced hepatotoxicity in rat. In this experimental study, 32 rats were treated with ATV orally at a dose of 10 mg/kg for 10 consecutive days, 5 days before and 5 days after the administration of a single intraperitoneal injection of CP (150 mg/kg). The hepatoprotective effect of ATV was evaluated by measuring liver function markers, oxidative markers, histological and immunohistochemical assays. The biochemical results showed that administration of CP increased hepatic biomarkers enzymes as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels. CP increased malondialdehyde (MDA), protein carbonyl (PC) and decreased glutathione (GSH) content in rats. Moreover, administration of CP was associated with periportal leucocyte infiltration, dilation sinusoids, hepatocyte vacuolation, congestion and hemorrhage in livers of rats. CP significantly increased immunoreactivity of caspase-3 as a marker of apoptosis in liver tissue. ATV markedly mitigated liver injury through reduction in oxidative stress biomarkers, histopathological findings and apoptosis. The antioxidant and anti-apoptotic activities of ATV are main proposed mechanisms involved in its hepatoprotective effects against CP-induced hepatic injury.

Keywords: Atorvastatin; Caspase-3; Cyclophosphamide; Hepatotoxicity; Oxidative stress.

Fig. 1

A, Malondialdehyde (MDA); B, glutathione (GSH); C, protein carbonyl (PC) levels; and D, represent cell viability in all groups. Rats treated with cyclophosphamide (CP) showed an increase in the MDA, PC, and a decrease in GSH content and MTT compared with control group. Pretreatment with atorvastatin (ATV) in CP- treated rats significantly decreased concentration of MDA, PC and increased GSH content and MTT in the liver tissue compared with CP group. All values are expressed as mean ± SD. **, ***significantly different from control group (P < 0.01, P < 0.001, respectively); ^$$$ significantly different from ATV (P < 0.001); and ^### significantly different from CP groups (P < 0.001).

Fig. 2

Photomicrographs showed the effect of atorvastatin (ATV) pre-treatment and cyclophosphamide (CP) on the histological architecture of liver in various groups. A, Control; B and C, CP; and D, ATV + CP groups. Normal structure in control group, disorganization, hepatic tissue periportal inflammation (B, white arrow), dilation sinusoids (C, black arrow), hemorrhage, congestion (B, black arrow) and vacuolization (C, white arrow), in CP group. Treatment with ATV improved these changes. Hematoxylene and eosin (H & E), liver sections with magnification; A, B, and D ×40, C ×100. Scale bar = 100 μm.

Fig. 3

Liver injury scores in liver tissue. Data are presented as mean ± SD. The highest score belongs to cyclophosphamide (CP) group. Atorvastatin (ATV) was able to reduce liver injury score in the ATV + CP group, but was not significant. * and *** significantly different from the control and ^$ and ^$$$ significantly different from ATV groups. * and ^$P < 0.05, *** and ^$$$P < 0.001.

Fig. 4

A, Immunohistochemical staining demonstrated no caspase-3 immunoreactivity in the control group. B, Cyclophosphamide (CP) increased caspase-3 immunoreactivity that were remarkable in hepatocyts. C, Atorvastatin (ATV) treatment diminished caspase-3 immunoreactivity in CP treated mice. D, Densitometry analysis of immunohistochemical staining for caspase-3. Data are presented as a percentage of total tissue area. Immunoreactivity level of caspase-3 in the control and ATV alone groups was similar. Data are presented as mean ± SD. *** significantly different from the control, ^$$$ significantly different from ATV, and ^## significantly different from CP groups. ^##P < 0.01 and *** and ^$$$P < 0.001.