Neuroprotective Effects Of EGCG On Paraquat-induced Apoptosis In PC12 Cells And Its Mechanism

Parkinson's disease (PD) is a neurological disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta. Although the etiology of PD remains unclear, several risk factors have been associated with the disease, including age, genetic and environmental factors. Exposure to agricultural chemicals via living in a rural environment, drinking well water, or occupational exposure has been postulated to be a potential environmental risk factor.Recently, a widely used herbicide PQ has triggered people's interests because its chemical structure is very similar to MPP+, the active form of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is a neurotoxin that is not naturally occurring but which leads to parkinsonism in animals and humans. So, the structure similarity between PQ and MPP+ indicates that PQ may be one of the potential environmental factors in PD. Epidemiological studies have found that there is an association between the use of PQ in agriculture and incidence of PD. Furthermore, previous studies indicate that PQ exposure causes selective degeneration of dopaminergic neurons and produces a neurobehavioral syndrome in animal models.Green tea is becoming more and more popular for its health benefits, particularly with respect to its potential for preventing and treating cancer, cardiovascular diseases, inflammatory diseases, and neurodegenerative diseases. Recently, the neuroprotective properties of green tea are rapidly becoming focused. Research indicated that reduced risk of PD associated with consumption of 2 or more cups of tea daily. Research has also suggested the neuroprotection of green tea extracts in the neuronal cell model of PD.EGCG, the main ingredient found in green tea polyphenols, is thought to be responsible for the majority of the biological activity of green tea extracts. Studies show that EGCG can protect neuronal cells damage mediated by neuropoison, ischemia, hypoxia, serum withdrawl and so on. Some researchers reported that EGCG prevents MPTP-induced dopaminergic neurodegeneration in mice. Then studies showed that the preventive effects of EGCG against MPTP may be explained by the inhibition of nNOS in the substantia nigra. However, whether EGCG could provide protection against injury induced by PQ in dopaminergic neurons or not is still unknown. The central hypothesis guiding this study is that EGCG may play a protective role in PQ induced injury of dopaminergic neurons. PC12 cells retaining dopaminergic characteristic, was used as an in vitro model. The possible effect of EGCG was investigated and the related mechanism was probed. Experimental contents as follows:Experiment 1Objective: To sieve the effective concentrations of PQ inducing injury and the protective concentrations of EGCG against the injury in PC12 cells. Methods: Setting control group and 200, 400, 600, 800, 1000μmol/L PQ groups, PC12 cells were respectively exposed to various concentrations of PQ for 12, 24, 36, 48h; To choose protective concentrations of EGCG, different concentrations of EGCG (1, 5, 10, 50, 100, 200μmol/L) protective groups, PQ group (800μmol/L) and control group were set. The cell viability was measured by MTT assay. Results: After exposed to a range of concentrations of PQ for various periods of time, there was a dose- and time-dependent decrease in cell viability as measured by MTT assay. In the experimental group treated with 800μmol/L PQ for 24h, cell viability was reaching 53±3.2% compared with control group (P<0.001). PC12 cells were incubated with different concentrations of EGCG for 2h, then exposed to 800μmol/L PQ for another 24h. It is observed that pretreatment with EGCG (1, 5, 10μmol/L) caused a significant decrease in the level of cell death compared with PQ-treated cells (54.6±3.5%). After exposure to EGCG, cell viability was reaching 66.5±2.7% (P>0.05), 74.4±2.6% (P<0.05), 83.7±3.2% (P<0.01), respectively. But high concentrations of EGCG (50, 100, 200μmol/L) didn't show protective effect. Conclusion: PC12 cells can be injured by PQ and the neurotoxicity is in a dose- and time-dependent manner. The biphasic mode of biological activity of EGCG relies on its concentration-dependent window of pharmacological action: EGCG exhibits protective activity at low doses whereas at high concentrations it shows no protective effect.Experiment 2Objective: To explore the neurotoxic effect of PQ on PC12 cells and observe the protective effect of EGCG against the apoptosis induced by PQ. Methods: MTT assay was used to detect the cell viability, 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: MTT assay showed that 1, 5, 10μmol/L EGCG inhibited the decrease of cell viability induced by 800μmol/L PQ for 24 h. Compared with PQ-treated cells (54.6±3.5%), after pretreatment with EGCG, cell viability was reaching 66.5±2.7% (P>0.05), 74.4±2.6% (P<0.05), 83.7±3.2% (P<0.01), respectively; Hoechst 33258 staining demonstrated nuclear condensation, one of the typical hallmarks of apoptosis. Nuclei of normal cells appeared with regular contours and were round and large in size, which showed a homogeneous and diffused staining. While exposed to PQ for 24 h, most cells exhibited an asymmetric and bright blue fluorescence. Compared with PQ group (43.5±8.4%), in the cells pretreated with EGCG, condensed nuclei was decreased to 30.7±7.9% (P<0.05), 17.9±6.8% (P<0.001), 11.2±4.4% (P<0.001), respectively; Compared with control cells (5.2±1.9%), TUNEL-positive cells in PQ-treated group increased to 32.4±6.7% (P<0.001). While, prior to incubation with EGCG, the number of TUNEL-positive cells was markedly reduced to 25.9±7.7% (P>0.05), 16.6±3.2% (P<0.01) and 9.6±3.2% (P<0.001) compared with that of PQ group; FCM results indicated that, after PQ treatment, the percentage of apoptotic cells (39.9%) was increased compared with control (4%), but was dropped respectively to 32.6%, 20.1% and 10.4% with 1, 5 or 10μmol/L EGCG pretreatment. Conclusion: EGCG can relieve the apoptosis induced by PQ in PC12 cells.Experiment 3 Objective: To explore the related mechanisms of the protective effect of EGCG on PQ-induced apoptosis in PC12 cells. Methods: Mitochondrial membrane potential (MMP) was detected by Rhodamine 123 staining, Caspase-3 activity was measured with a colorimetric Caspase-3 assay kit, the expression of pro-apoptotic protein Cyto C and Smac in cytosol was observed by immunocytochemical staining and Western blotting. Results: The collapse of MMP in PC12 cells was observed with the probe rhodamine 123. Incubating cells with 800μmol/L PQ for 24h reduced the fluorescent intensity significantly compared with control (P<0.001). EGCG dose-dependently attenuated the MMP changes in PC12 cells. Compared with PQ group (26.2±2.8%), cells fluorescent intensity increased to 41.3±3.6% (P<0.01), 56.7±5.4% (P<0.001) and 82.9±4.2% (P<0.001) after 1, 5, 10μmol/L EGCG pretreatment; A significant increase in Caspase-3 activity was induced after PQ exposure compared with controls (P<0.001), while this activation was reduced by 1, 5, 10μmol/L EGCG incubation. After pretreatment with EGCG, the Caspase-3 activity reached to 179.9±7.6 (P<0.001), 123.2±8.1 (P<0.001), 68.5±6.1 (P<0.001), respectively, compared with group treated with PQ alone (216.5±9.6); In control group, the staining cells were light-coloured and their morphous was more integrity.After incubating with PQ for 24h, the expression of Cyto C and Smac significantly increased in PC12 cells. Most cells were stained and their morphous changed. The staining cells were dark yellowish-brown and the positive stain distributed in cytoplasm. Immunocytochemical staining indicated that EGCG downregulated the over-expression of Cyto C and Smac in the cytosol induced by PQ and improved the morphous of PC12 cells.Western blotting revealed an increased expression of Smac in cytosol after the exposure to PQ and EGCG pre-treatment reduced the expression.Conclusion: Apoptosis of PC12 cells was induced by PQ, and this effect could be attenuated by EGCG. The possible mechanism may be through maintaining MMP, inhibiting Caspase-3 activity and down-regulating the expression of pro-apoptotic protein Cyto C and Smac in cytosol.