Neuroprotective effects of the standardized extract of Centella asiatica ECa233 in rotenone-induced parkinson’s disease model

Abstract

The standardized extract of Centella asiatica ECa233 consists of asiaticoside and madecassoside. Previous studies reported various pharmacological effects of, ECa233, especially in the central nervous system disorders. The present study aimed to investigate the neuroprotective effects and protective mechanisms of ECa233 against rotenone-induced neurotoxicity in rats and in neuroblastoma SH-SY5Y cells. In animal study, male Wistar rats were divided into five experimental groups (n=10); control, PD, PD-ECa10, PD-ECa30, and PD-EC100. On days 1-20, control and PD rats received 0.5% carboxymethylcellulose (CMC) (1 mL/kg, p.o.) while ECa233 10, 30 and 100 mg/kg (p.o.) were given to PD-ECa10, PD-ECa30, and PD-EC100, respectively. Rotenone (2.5 mg/kg, i.p.) was given to PD and PD-ECa groups on days 15-20 while controls received 2% Dimethylsulfoxide (DMSO) (i.p.) Locomotor activity was performed on days 1, 14, 17, and 20, and apomorphine challenge was tested on day 21. Brains were collected for further analysis of tyrosine hydroxylase (TH)-positive neurons in the nigrostriatal pathway, mitochondrial complex I activity, malondialdehyde (MDA) levels and superoxide dismutase (SOD) and catalase protein expression. The results showed that locomotor activity of rats was significantly reduced, while ECa233 (30 mg/kg) pretreatment significantly alleviated motor deficits in PD-ECa233 compared to PD rats on day 20 (p<0.01). On day 21, the locomotor deficit was responded to apomorphine challenge, suggesting dopamine depletion in the nigrostriatal pathway. ECa233 (30 mg/kg) protected against rotenone-induced dopaminergic neuronal loss in the substantia nigra (p<0.001) and preserved dopaminergic axonal fibers in the striatum (p<0.0001). ECa233 (30 mg/kg) also decreased brain MDA levels (p<0.05) while increased mitochondrial complex I activity (p<0.05) and SOD and catalase protein expression (p<0.05). To investigate the molecular mechanisms of ECa233, SH-SY5Y cells were treated with ECa233 (10, 25, 50, and 100 µg/mL) for 12 h, followed by rotenone for 48 h. Cell viability, mitochondrial membrane potential, and mitophagy were measured using resazurin assay, tetramethylrhodamine, ethyl ester (TMRE) assay, and mitophagy detection, respectively. To determine intracellular reactive oxygen species (ROS) levels by dichloro-dihydro-fluorescein diacetate (DCFH-DA) technique, cells were treated with ECa233 for 12 h followed by rotenone for either 6, 24 or 48 h. Autophagy and apoptosis-associated proteins were measured after 12 h ECa233 pretreatment followed by 24 h rotenone incubation. The results showed that ECa233 (10-100 µg/mL) significantly increased cell viability compared to rotenone-treated alone (p<0.01). ECa233 (50-100 µg/mL) decreased intracellular ROS levels in rotenone-treated cells at 24 (p<0.05), but not 6 and 48 h. In addition, ECa233 (25-100 µg/mL) prevented against rotenone-induced the reduction of mitochondrial membrane potential (p<0.01) and mitophagy (p<0.05). The expressions of autophagic-associated proteins, Beclin-1 and LC3II/I, were significantly increased in ECa233 (50-100 µg/mL) pretreatment groups (p<0.05). In addition, ECa233 (25-100 µg/mL) significantly increased Bcl-2 expression (p<0.05) while decreased cleaved-caspase-3/caspase-3 ratio (p<0.05). To summarize, ECa233 protects against rotenone-induced neurotoxicity in vivo and in vitro. The underlying neuroprotective mechanisms are mitochondrial complex I protection, lipid peroxidation reduction, induction of antioxidant enzymes, prevention of mitochondria membrane potential reduction, enhancing mitophagy, and prevention of neuronal apoptosis. Taken together, the current study suggests that ECa233 is a potential compound in drug development for the treatment of Parkinson’s disease.