PROTECTIVE ROLE OF PROTOCATECHUIC ACID IN NEVIRAPINE-INDUCED HEPATOTOXICITY AND APOPTOSIS IN MALE WISTER RATS

Abstract

Nevirapine is a Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) used in HIV-I treatment. Although efficacious, it produces toxic responses such as hepatotoxicity which is characterised by generation of free radicals. Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA), a phenolic antioxidant compound from edible plants is known to be hepatoprotective by mechanisms of action associated with inhibition of free radical generation, regulation of inflammation, and apoptosis. This study was designed to determine the protective role of PCA against nevirapine-induced hepatotoxicity. Seventy-two male Wistar rats (150-170g) were randomly assigned into six groups of twelve animals each. The animals were treated orally with distilled water alone (control), PCA (50.0 mg/kg), PCA (100.0 mg/kg), nevirapine alone (5.7 mg/kg), nevirapine (5.7 mg/kg) with PCA (50.0 mg/kg) and nevirapine (5.7 mg/kg) with PCA (100.0mg/kg) daily for three weeks, respectively. Enzyme activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic levels of reduced glutathione (GSH) and malondialdehyde (MDA) were determined spectrophotometrically. The serum concentrations of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), prostaglandin E₂ (PGE₂), caspase 3, caspase 9 and cytochrome C were assessed using ELISA. The expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were measured by immunohistochemistry. Histology of liver was determined by microscopy and apoptosis by TUNEL assay. Data were analysed using Student t test and ANOVA at p-0.05. Treatment with nevirapine alone caused significant elevations of serum activities of ALT (8.1±0.5 U/L) and AST (10.3±0.2 U/L) compared with control (3.2±0.7 and 8.1±0.8 U/L respectively). However, treatment with PCA (50.0 mg/ kg and 100.0 mg/kg respectively) significantly lowered serum activities of ALT (7.5±0.7 and 6.4±1.3 U/L) and AST (9.0±2.9 and 9.5±1.7 U/L). Nevirapine lowered GSH level (0.4±0.1 mg/mL) compared with control (1.6±0.4 mg/mL) and significantly increased MDA level (366.0±39.0 nmol/g) compared with control (188.0±8.7 nmol/g). Treatment with PCA (50.0 mg/kg and 100.0 mg/kg respectively) however, caused significant increase in GSH (1.4±0.7 and 0.7±0.12 mg/mL) and decrease in MDA (245.0±20.2 and 262.9±9.0 nmol/g). Nevirapine elevated serum PGE₂ (56.0±5.2 mg/mL), TNF-α (20.3±1.4 mg/mL) and 1L,-1β (68.2±1.2 mg/mL) compared to control (37.9±4.6, 15.9±0.7 and 53.8±1.4 mg/mL respectively). Protocatechuic acid (50.0 mg/kg and 100.0 mg/kg respectively) significantly reduced serum PGE₂ (54.4±2.4 and 42.8±4.1 ng/mL), TNF-α (15.6±0.4 and 16.0±0.3 ng/mL) and IL.1β (59.5±2.0 and 55.3±1.0 ng/mL). Nevirapine induced COX-2 and iNOS expressions, increased serum caspase 3 (2.0±0.8 ng/mL) relative to control (1.5±0.3 ng/mL). and significantly elevated serum caspase 9 (159.0±3.0 ng/mL) and cytochrome C (215.0±51.2 ng/mL) compared to control (127.3±19.8 and 116.6±27.8 ng/L respectively). Protocatechuic acid (50.0 mg/kg and 100.0 mg/kg respectively) reversed the increased COX-2 and iNOS expressions, significantly lowered serum caspase 9 (108.0±37.0 and 112.3±18.5 ng/mL), cytochrome C (107.4±6.9 and 106.0±7.2 ng/mL) and caspase 3 (1.3±0.1 and 1.4±0.6 ng/mL). Nevirapine also induced hepatic apoptosis while protocatechuic acid at both concentrations reduced the apoptosis. Histology showed severe hepatic necrosis in nevirapine-treated group which was reduced with PCA treatment. Protocatechuic acid ameliorated nevirapine-induced hepatotoxicity by its antioxidant, anti-inflammatory and anti-apoptotic properties.