Neuroprotection Of Resveratrol Against Rotenone Induced Oxidative Injury And Its Molecular Mechanism

Parkinson’s disease (PD) is the second largest neurodegenerative disease. The main characteristics of PD are progressive degeneration and loss of dopamine neurons, as well as the formation of Lewy body in the substantia nigra, which is mainly constituted of alpha-synuclein, ubiquitin, neurofilament protein. In the wake of accelerating aging of population in China, the incidence of PD increases annually. PD seriously endangers the health of the elderly. Thus, elucidating the pathogenesis of PD is an urgent event. Many studies have confirmed that the familial PD is associated with genetic factors, but the causes of 90% of sporadic PD is not clear, and especially the specific degeneration of the substantia nigra has been unable to be fully explained. In 1983, because of smoking heroin containing N-methyl-4-phenyl-5-four hydrogen pyridine (MPTP), heroin abusers showed typical symptoms of PD. As a result, environmental factor has graduallybecome the focus of PD study.Epidemiological investigations show that long-term exposure to pesticides and herbicides is apt to inducing PD. Rotenone, one of common pesticides, may promote the incidence and development of Parkinson’s disease via inhibiting the activity of mitochondrial respiratory chain complex I and decreasing ATP levels. In addition, rotenone can induce Reactive Oxygen Species (ROS) in the mitochondria. The accumulation of excess ROS in mitochondria leads to oxidative damage, and eventually trigger apoptosis pathway, causes apoptosis and induce neural degenerative diseases. However, we are still not clear how rotenone induces the occurrence of Parkinson’s disease, and the specific mechanism of the process.Resveratrol, a natural polyphenolic compound, enriches in peanuts and grape, could defendplants against external attack, and is generally considered to be the chemical preventer of cardiovascular disease. Recently, a wide range of literatures consider that resveratrol is concerned for its extensive beneficial biological effects including anti-inflammatory, antioxidant, anti-cancer and anti-aging characteristics. Studies have found that resveratrol can activate silent signal transduction factors (Silence signal regulation fatorl, SIRT1), and has neuroprotective effects. The over expression of SIRT1 enhances cell metabolism, prolongs life and reduces the incidence of neurodegenerative disease. Serine threonine kinase Akt1 is a downstream molecule of SIRT1, and plays key roles in the regulation of multiple cellular functions ranging from cell proliferation and survivalto protein synthesis. Previous studies have reported that SIRT1 may reduce oxidative stress, protect the blood vessel function, and raise the catalase activity, as well as overexpression of SIRT1 significantly increases the tolerance of neurons on toxicity of oxygen free radical damage. As downstream molecular targets of SIRT1, Aktl can be acetylatedby SIRT1 to p-Aktl, Aktl is closely related to the synthesis of cell apoptosis protein, and oxidative stress, furthermore, studys find that p-Aktl activity in patients with PD reduced. Whether SIRT1/Aktl signaling pathways mediates the neuroprotective effects of resveratrol on rotenone-induced neuronal damage is undetermined till now.Based on above studies, we designed the following experiments to investigate the neurotoxicity effects of rotenone, elucidate the protective effects of resveratrol on rotenone-induced cell toxicity, and further explore the underlying molecular mechanism of resveratrol against rotenone induced neurotoxicity.Firstly, we established rotenone-injured cell model. PC12 cells were exposed to different concentrations of rotenone. According to morphology changes, the results of CCK8 assay and lactate ehydrogenase (LDH) activity assay, we selected the suitable concentration of rotenone. The results showed that exposed to different concentrations of rotenone, PC 12 cells showed different degree of damage. To determine the protective concentration of resveratrol, PC 12 cells were pretreated by different concentrations of resveratrol for two hours, before exposure to rotenone. As mentioned above, CCK8 assay and LDH activity assay were used to select the best protective concentration of resveratrol. Pretreated with different concentrations of resveratrol for 2 hours, PC 12 cells showed obvious improving of cellular morphology and 25μM turn out to be the optimal concentration. Compared to rotenone-injury group, the neurites length of cells were increased, cell body turn to be round and the cell viability improved.Then, we detected reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and cell apoptosis. Cellular ROS levels are detected by DCFH-DA. JC-1 staining is used to determine the change of mitochondrial membrane potential. and cell apoptosis is detected by Hoechst 33342/PI double dye. Compared to the control, the production of ROS significantly increased and ΔΨm declined in rotenone exposure group, while the fluorescence intensity of ROS can be reduced and ΔΨm rised by 25μM resveratrol. Cellular apoptosis rate and mortality rate in resveratrol group were obviously lower than that in rotenone injury group.Next, mRNA and protein expression of SIRT1 and Aktl in PC12 cells were respectively detected by the Real-time PCR and Western Blotting. OneμM rotenone treatment decreased SIRT1 and Aktl expressions at mRNA and protein levels. However, these effects are reversed by 25μM resveratrol.Finally, To further reveal the protective mechanism of resveratrol against rotenone cytotoxicity whether via SIRT1/Akt1 pathway or not, we used nicotinamide and LY294002 to specifically block SIRT1 and Akt1 activities. the results indicated that cell activity of PC12 was decreased, the neuroprotective effects of resveratrol significantly attenuated, and SIRT1 and Akt1 expressions decreased. Furthermore, another new type of Akt1 inhibitor MK-2206 was consistent with LY294402. In the study, we found that both LY294402 and MK-2206not only inhibited Akt1 activation but also blocked SIRT1 expression, which suggested the mutual relationship between SIRT1 and Akt1.In conclusion:Our study shows that rotenone can induce apoptosis of neurons, ROS production, and reduce cell viability. Resveratrol protects PC12 cells against rotenone induced neurotoxicity, improves mitochondrial function, and inhibits neuronal apoptosis through activation of the SIRT1/Aktl signal pathway.