Tetrahydroxystilbene Glucoside Reduces The MPP + -induced Apoptosis In PC12 Cells By Regulating Ros-related JNK And Mitochondrial Pathway

Parkinson's disease (PD) is the second most common neurodegenerative disorder next to Alzheimer's disease (AD). PD is clearly an age-related disease: it rarely occurs before the age of 50 and its prevalence increases with age up to 4% in the people over 85 years. Currently about 200 million people in China suffer from this disease. The pathology of PD can be attributed to the selective loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNPc). The etiology of PD has not been completely understood as yet, may be related to aging, environmental factors, genetic. Large number of studies had confirmed that the apoptosis of DAergic neuronal may be related to oxidative stress, mitochondrial dysfunction and the transcription factor, a variety of related kinases and phosphatases.The neurotoxin 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis in PC12 cells is a classic cellular model of PD. It is believed that MPP+, which is the active metabolite of 1-methyl-4-phenyl-2, 3, 6-tetrahydropyridine (MPTP), causes mitochondrial dysfunction, oxidative stress, energetic failure and activation of genetic programs leading to cell death, finally resulting in a phenomenon that closely resembles PD. PC12 cells—a clonal rat adrenal gland pheochromocytoma cell line—possess dopamine synthesis, metabolism, and transporting systems and retain the features of DAergic neurons; therefore, they have been used as an in vitro model for conducting studies on PD.Currently, there is no proven drug or strategy to slow or stop the neurodegenerative process. Only symptomatic treatment, mainly in the form of dopamine replacement and adjuvant surgical therapy, are considered efficacious in PD. However, anti-PD with levodopa-based drugs can only control the symptoms, not prevent disease progression, and there are many adverse reactions and side effects after long-term use. Therefore, there are a large number of neuroprotective drugs on the exploratory literature in recent years. Among them, more and more people concern the neuroprotective effect of the herbal extract in vivo and in vitro PD model.The root of Polygonum multiflorum Thunb.(PM), which is also called heshouwu,is a famous traditional Chinese medicinal herb and has been used since a long time as a monarch drug in traditional Chinese medicines to treat the age-related diseases. The results from recent medical studies have indicated that PM can treat the age-related diseases. It is reported that the PM extract had neuroprotective effect on nigrostriatal DA degeneration induced by paraquat and maneb in mice. The PM extrac can improve learning and memory in aging AD mice through affecting superoxide dismutase activity and decrease lipofuscin and malondialdehyde content, decreased cellular oxidative damage, improve the pathological changes of the hippocampus.The mechanism of its action may be attributed to its antioxidant activity.A monomer of stilbene, 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), is one of the main active ingredients of PM. In the Chinese pharmacopoeia (2005), TSG has been defined as the ingredient used to assess the quality of PM. It has been reported that TSG exhibits antioxidative and anti-in?ammatory effects. Previous studies have suggested that TSG improves learning and memory ability in both APP transgenic mice and aged rats, and protects in vitro and in vivo model against cerebral ischemia. In addition, It's reporte that TSG pretreatment can protect against cerebral ischemia/reperfusion injury in in vitro and in vivo ischemic models, and the mechanisms of neuroprotection of TSG are inhibition of JNK and NF-κB pathways and inhibition of intracellular ROS generation. However, whether TSG affords the neuroprotetion the in PD model is unclear. Viewing of the neuroprotetion of TSG on the AD model, and the important role of ROS, JNK and NF-ΚB signaling pathway in PD pathogenesis, we speculated that TSG can protect PC12 cells against MPP+-induced apoptosis.Experiment 1Objective: To sieve the effective concentrations of MPP+-induced injury and the protective concentrations of TSG against the injury in PC12 cells. Methods: To sieve the effective concentrations of MPP+ -induced injury: the cells were set control group and different concentrations of MPP+ (50,100,250,500 and 1000 M) treated group,PC12 cells were respectively exposed to various concentrations of MPP+ for 12,24,36 and 48h; To sieve the protective concentrations of TSG against the injury in PC12 cells: the cells were set control group, MPP+ (500 M) treated group and different concentrations of TSG((1,5 and 10 M) pretreated groups. Cell viability was measured by quantitative colorimetric assay with MTT. Results: There was a dose- and time-dependent decrease in the cell viability following MPP+ exposure as assayed by MTT. A final concentration of 500 M MPP+ and an incubation time of 24 h were selected as optimal concentration and time for the induction of deleterious effects on the viability of PC12 cells (the cell viability of treated cells decreased to 51.4±3.0% compared to that of the control group, P < 0.01). Cells were pretreated with TSG for 24 h, washed, and then treated with 500 M MPP+ for an additional 24 h. The viability of the cells pretreated with 1 M, 5 M, and 10 M TSG recovered to 62.9±2.5% (P >0.05), 77.6±2.3% (P < 0.05) and 85.1±1.9% (P < 0.01), respectively. Conclusion: The results showed that TSG attenuated the MPP+-induced decrease in cell viability in a dose-dependent manner.Experiment 2Objective: To explore the neurotoxic effect of MPP+ on PC12 cells and observe the protective effect of TSG against the apoptosis induced by MPP+. Methods: Hoechst33258 staining was employed to observe morphological changes of the cell nuclear, FCM was used to detect the apoptosis ratio,and TUNEL staining was adopted to observe the DNA fragment of PC12 cells. Results: Hoechst33258 staining showed that homogeneous and diffused staining with regular contours and was round in shape, while the apoptotic cells exhibited reduced nuclear size, chromatin condensation, intense fluorescence, and nuclear fragmentation. Exposure to 500 M of MPP+ alone for 24 h induced apoptosis in PC12 cells. The number of apoptotic nuclei was significantly lesser in the cells pretreated with 1 M, 5 M, and 10 M TSG. Treatment with TSG alone did not induce apoptosis. FCM results indicated that, after 24h of incubation with MPP+, results demonstrated that the percentage of early apoptosis increased significantly and TSG showed a dose-dependent protective effect against MPP+-induced apoptosis. Treatment with TSG alone did not induce apoptosis. TUNEL staining demonstrated that, compared with the TUNEL-positive cells in untreated control cells (5.7±1.2%), those in the MPP+-treated group increased to 52.6±4.7% (P < 0.01). On the other hand, when the cells were pretreated with 1, 5, and 10 M TSG, the number of TUNEL-positive cells markedly reduced to 36.9±4.5% (P <0.05), 28.8±5.5% (P < 0.01), and 17.0±2.8% (P < 0.01), respectively. TSG alone did not induce any TUNEL-positive cells. Conclusion: TSG can relieve the apoptosis induced by MPP+in PC12 cells.Experiment 3Objective: To explore the related mechanisms of the protective effect of TSG on MPP+-induced apoptosis in PC12 cells. Methods: The intracellular ROS level was examined by using DCF-DA. MMP was measured by the lipophilic cationic probe rhodamine 123. The expression of pro-apoptotic protein (Cyto C, JNK, p-JNK, Bcl-2 and caspase-3) in cytosol was observed by immunocytochemical staining and Western blotting. Results: The intracellular ROS level was examined by using DCF-DA. Exposure of PC12 cells to 500 M MPP+ for 24 h led to a significant increase in DCF signal compared with the control group (P<0.01). Such increase in DCF fluorescence was eliminated in a concentration-dependent manner by pretreatment with TSG in the 1-10 M rang. MMP was measured by the lipophilic cationic probe rhodamine 123.After 24h exposure to MPP+, the fluorescence intensity of Rh 123 in PC12 cells was rapidly reduced, representing a fall in the mitochondrial membrane potential. However, the MMP decrease recovered after incubation with TSG in a dose-dependent manner. Western blot showed that, Cyt C was released following treatment with 500μM MPP+ at 24h. This release was dose-dependently inhibited by TSG pretreatment. We examined the role of Bcl-2 protein, a major member of the Bcl-2 family of proteins. We showed that MPP+ (500μM) treatment caused a dramatic decrease in the levels of the anti-apoptotic protein, Bcl-2. Pretreatment with TSG prevented the decrease in Bcl-2 caused MPP+ by in a dose-dependent manner. We observed an increase in the expression of caspase-3 induced by MPP+ (500μM) treatment. TSG pretreatment significantly reduced elevated caspase-3 expression in a dose dependent manner. Western blot analysis revealed an increased expression of phosphorylated JNK after exposure to MPP+ alone, and TSG pretreatment attenuated the expression of the phosphorylated protein. Interestingly, inhibition of the activation of JNK by SP600125 led to a dose-dependent decrease in the MPP+-induced cell viability loss and apoptosis. Conclusion: Apoptosis of PC12 cells were induced by MPP+, and this effect could be attenuated by TSG. The possible mechanism may be through inhibiting ROS generation, maintaining MMP, modulating JNK activation, preventing the decrease in Bcl-2 caused MPP+, inhibiting caspase-3 activity and down-regulating the expression of pro-apoptotic protein Cyto C in cytosol.