| Parkinson's disease(PD)is an age-related progressive neurodegenerativeisease which defining pathological features are selective loss of dopaminergiceurons in substantia nigra pars compacta and subsequent decrease of dopamineevels in the striatum,the main target innervated by these neurons.Although thetiology of PD remains unclear,both exogenous and endogenous neurotoxicubstances are known to provide partial explanation of these processes.Severalactors are suspected to participate in the onset of PD that includesnvironmental exposure to pesticides,metals and hydrocarbons.Paraquat(1,1,-dimethyl-4,4,-bypiridinium,Parquat PQ),a nonselectiveerbicide widely used in agriculture,has extremely similar chemical structureith MPP+(1-methyl-4-pheny1-pyridium,MPP+),the active metabolite ofPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)which is a knowneurotoxin.The structural similarity between MPP+and PQ confers to theseindings a potential interest in the study of the etiological causes of PD.pidemiological studies have found that there is an association between the use of PQ in agriculture and incidence of PD. Furthermore, research using animal models has also indicated the neurotoxicity induced by PQ in nigrostriatal dopaminergic cells.Ginsenosides, the pharmacologically active components found in ginseng, and may modulate neurotransmission. Both Rg1 and Rb1 are capable of partially reversing scopolamine induced amnesia by improving cholinergic activity and having partial neurotrophic and neuroprotective effects. Ginsenosides Rg1 has a lot of beneficial effects, such as improving learning and memory, enhancing immunity, and delaying apolexis. It seems to be an interesting drug for the neuroprotective effect of Ginsenosides Rg1.PC12 cells, the rat adrenal pheochromocytoma cell line, which possesses dopamine synthesis, metabolism and transporting systems. The membrane receptors and synthesizing transmitters of PC12 cell close to dopaminergic neurons in midbrain. Therefore, it has been used as a cellular model of PD. In this study, we investigated whether Ginsenosides Rg1 can protect against PQ-induced apoptosis in cultured PC12 cells as model of PD in vitro, and related mechanisms of the neuroprotective effect.Experiment 1Objective: To sieve the effective concentration of PQ inducing injury and the protective concentration of Ginsenosides Rg1 to the injury in PC12 cells. Methods: Setting control group and 100, 200, 400, 600, 800, 1000μmol/L PQ groups, PC12 cells were respectively exposed to various concentrations of PQ for 12, 24, 36, 48 h; Ginsenosides Rg1 protective groups were set control,PQ group(800μmol/L),preventive groups: pretreated with Ginsenoside Rg1(final concentration: 5, 10, 20μmol/L) for 24 h, then PQ(final concentration: 800μmol/L) was added for an additional 24 h. The cell viability was measured by MTT chromometry and LDH-release 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 24 h, cell viability was reaching 44.8±6.9% compared with control group (P<0.05). PC12 cells were incubated with different concentrations of Ginsenoside Rg1 for 24 h then exposed to 800μmol/L PQ for another 24 h. Compared with control group, it is observed that pretreatment with Ginsenoside Rg1(5, 10, 20μmol/L) caused a significant decrease in the level。of cell death compared with PQ-treated cells(49.5±4.2%). After incubated with Ginsenoside Rg1, cell viability was reaching 55.7±2.8%,71.2±3.8%,84.4±3.2%, respectively(P<0.05). Different concentrations of Ginsenoside Rg1 had protective effects on the PQ damage, in certain scope the protective effects potentized along with the increase of Ginsenoside Rg1 concentration. Conclusion: PQ can injure PC12 cells, and the injury is in a dose and time dependent manner. In certain scope Ginsenoside Rg1 has protective effects on the injury induced by PQ in PC12 cells, which potentize along with the increase of Ginsenoside Rg1 concentration.Experiment 2Objective: To explore the neurotoxic effect of PQ on PC12 cells and observe the protective effect of Ginsenoside Rg1 against the apoptosis induced by PQ. Methods: MTT assay was used to detect the cell viability; FCM was used to detect the apoptosis ratio; Hoechst 33258 staining was employed to observe morphological changes of the cell nuclear. Results: MTT assay showed that 5, 10, 20μmol/L Ginsenoside Rg1 inhibited the decrease of cell viability induced by 800μmol/L PQ for 24 h. Compared with PQ-treated cells (46.4±3.6%), after pretreatment with Ginsenoside Rg1, cell viability was reaching 53.6±3.3%, 73.2±3.1%, 82.2±2.6%(P<0.05), respectively; FCM results indicated that, after PQ treatment, the percentage of apoptotic cells (48.9%) was increased compared with control (12.8%), but was dropped respectively to 39.8%, 20.1%,and 12.3% with 5, 10, 20μmol/L Ginsenoside Rg1 pretreatment; 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 fluorescent fragment could be seen in some cells in PQ group. 5, 10, 20μmol/L Ginsenoside Rg1 significantly inhibited PQ-induced cell damage, and condensed nuclei decreased markedly compared with PQ-induced group. Conclusion: Ginsenoside Rg1 can relieve the apoptosis induced by PQ in PC12 cells.Experiment 3Objective: To explore the related mechanisms of the protective effect of Ginsenoside Rg1 on PQ-induced apoptosis in PC12 cells. Methods: The active of superoxide dismutase(SOD),γ-glutamylcysteinylglycine(GSH) were (unit:U/mg prot) in PQ-induced cells decreased markedly from 130.51±5.99 to 53.12±2.67 and 107.87±4.84 to 49.50±2.54, respectively, compared with controls. However, the activities of SOD, GSH increaseed by 80.58±3.36, 105.08±4.92, 118.35±3.69,and 61.53±3.90, 72.90±4.32, 87.39±4.54 with 5, 10, 20μmol/L Ginsenoside Rg1 treatment, severally(P<0.05). Conclusion: The mechanisms of the protective effect of Ginsenoside Rg1 was partially dependent on antioxidative stress effects, inhibiting decrease the activation of SOD, GSH in PC12 cells induced by PQ.Experiment 4Objective: To explore the related mechanisms of the protective effect of Ginsenoside Rg1 on PQ-induced apoptosis in PC12 cells. Methods: Mitochondrial membrane potential(MMP) was detected by Rh123 staining; Caspase-3 activity was measured with a colorimetric Caspase-3 assay kit; the expression of pro-apoptotic protein Cyt C and Bcl-2 in cytosol was observed by immunocytochemical staining, and the expression of Bcl-2, Bax was observed by Western blotting. Results: Control cells exhibited numerous brightly staining mitochondria that emitted green fluorescence,which was indicative of normal high membrane potential, PQ treatment induced a transition in mitochondria permeability and a significant loss of membrane potential. Ginsenoside Rg1 treatment inhibited the collapse of mitochondrial membrane potential with increasing dosage, as indicated via the reappearance of brightly green mitochondrial staining; A significant increase in Caspase-3 (unit: pmol.min-1.mg-1) activity was induced after PQ exposure compared with controls (P<0.001), while this activation was reduced by 5, 10, 20μmol/L Ginsenoside Rg1 incubation. After pretreatment with Ginsenoside Rg1, the Caspase-3 activity reached to 1219.7±28.7, 1083.8±82.7, 925.0±29.1(P<0.001), respectively, compared with group treated with PQ alone (1600.6±55.7); In control group, the staining cells were light-coloured and their morphous was more integrity. After incubating with PQ for 24 h, the expression of Cyt C and Bcl-2 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 5, 10, 20μmol/L Ginsenoside Rg1 downregulated the over-expression of Cyt C and significantly further enhanced anti-apoptotic Bcl-2 protein levels in the cytosol induced by PQ and improved the morphous of PC12 cells. Western blotting revealed an increased expression of Bcl-2, Bax in PQ treated group. Treatment of PC12 cells with Ginsenoside Rg1(5, 10, 20μmol/L) for 24 h, significantly further enhanced anti-apoptotic Bcl-2 protein levels in PC12 cells as compared with controls. Protein levels of pro-apoptotic Bax were significantly reduced, and the Bax/Bcl-2 ratio was significantly decreased in PC12 cells. Conclusion: Apoptosis of PC12 cells was induced by PQ, and this effect could be attenuated by Ginsenoside Rg1. The possible mechanism may be through maintaining MMP, inhibiting Caspase-3 activity and regulating the expression of pro-apoptotic protein protein Cyt C, Bcl-2 and Bax in cytosol.
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