Tetrahydroxystilbene Glucoside Protects Against 6-OHDA-induced Apoptosis In PC12 Cells Through Regulation Of ROS-NO Pathway

Parkinson's disease (PD) is a common degenerative disease of the nervous system, its main pathological characteristic is pigment Dopaminergic (DAergic) neurons selective degeneration missing in substantia nigra pars compacta (SNpc), its main clinical characteristics as static tremors, muscle rigidity, attitude instability and slow motion. At presently, the etiology of PD is still unclear, but a lot of studies have revealed that oxidative stress and mitochondrial dysfunction are closely related to the pathogenesis of PD.6-hydroxydopamine (6-OHDA), a hydroxylated analogue of dopamine, is used to in mode systems to mimic PD. It possesses neurotoxicity, which can induce damage dopamineergic neurons in vivo and in vitro. PC12 cells, a clonal rat adrenal gland pheochromocytoma cell line, possesses dopamine synthesis, metabolism, and transporting systems and retain the features of DAergic neurons in the midbrain. Tetrahydroxystilbene glucoside (TSG), is one of effectively active constituents extracted from Chinese herbal medicine, Polygonum multiflorum thunb (PM). It possesses anti-oxidation, anti-inflammatory and improving learning and memory function. It is confirmed TSG can improve the learning and memory effects in mice Alzheimer's disease (AD), indicating it has a neuroprotective effect. Rencently, our research has shown that TSG has neuroprotceive effect on 1-methyl-4-phenylpyridinium (MPP+) induced apopto- sis of PC12cells.To further study neuroprotection effect of TSG, in this study, we selecte PC12 cells that has similar characteristics with dopaminergic neurons as model in vitro in order to observe the influence of TSG on 6-OHDA-induced cell damage in PC12 cells, and preliminarily explore the mechanism of its action.【Objective】1. Establish the mode of Parkinson's disease in vitro, screen the effective concentration of 6-OHDA-induced apoptosis in PC12 cells and TSG protect 6-OHDA-induced apoptosis in PC12 cells.2. Observe the effect of TSG on the apoptosis of PC12 cells induced by 6-OHDA.3. Study the mechanisms of TSG on 6-OHDA-induced apoptosis in PC12 cells.【Methods】1. Seted on control group, 25,75,100,125,150,200 and 300μmol/L6-OHDA respectetively exposured to PC12 cells for 24h to screen experimentally effective concentrations of 6-OHDA, which is 75μmol/L; Seted on control group, 1,5,10,20,50,100 and 200μmol/LTSG respectetively exposured to PC12 cells for 24h to observe the effect of TSG alone on PC12 cells; Seted on control group, 1,5,10,20,50,100 and 200μmol/LTSG respectetively exposured to PC12 cells for 24h, and then 6-OHDA(75μmol/L) treatment cells for an additional 24h to screene experimentally effective concentrations of TSG, which are 10,20 and 50μmol/L.2. Set on normal control group, TSG alone treatment cells group, 6-OHDA group and the different concentrations of TSG pretreatment(10,20,50μmol/L) groups, cell viability was measured by MTT assay, the changes in nuclear morphology of cells are observed by labeling the cells with the nuclear stain Hoechst 33258,apoptosis rate was measured by flow cytometry,determination of the DNA fragmentation was measured by terminal deoxynucleotidyl transferaseTdT)-mediated dUTP biotin nick end-labeling(TUNEL)staining cells.3. Intracellular ROS and NO were detected by the Automatic fluorescence microplate.4. The level of 3-NT was detected by Elisa Assay Kit.5. The expression of iNOS and nNOS protein in PC12 cells was detected by the Western Blot method.【Results】1. The MTT detection observated that the cell vitality is (52.6±1.3) % (P< 0.01) after 75μmol/L 6-OHDA treatment PC12 cells for 24h, compared with the control. While the cell vitality in 10, 20, 50μmol/LTSG pretreatment group were up by (66.6±3.2) % (P<0.01), (73.0±1.6)% (P<0.01), (83.9±1.5)% (P<0.01) respectively after TSG pretreatment cells for 24h followed by 6-OHDA treatment cells for 24h, compared with 6-OHDA group (52.6±1.3) %.TSG (1, 5, 10, 20 and 50μmol/L) alone has no obviously effect on PC12 cells. But high concentration of TSG (100, 200μmol/L) show a inhibition trend on PC12 cells.2. It is observed by Hoechst33258 staining that normal nuclei showed blue, uniform light coloring, smooth edges and tidy, while treatment with 6-OHDA nucleis were dense hyperchromatic, chromatin, cell shrinkage, displayed bright blue fluorescent, and even rupturethe nucleus can be seen, showed the dense granules Fluorescence. But the characteristics of apoptosis was significantly improved by the TSG (10, 20, 50μmol/L) pretreated cells. But TSG alone has no obviously effect on PC12 cells3. Results measured by FCM showed that the percentage of apoptotic cells were respectively 1.8%, 1.6%, 35.2%, 30.7% ,18.7% and 13.6% in the normal control group, TSG alone treatment group, 6-OHDA treatment group and TSG (10,20,50μmol/L) pretreatment group.4. TUNEL staining showed the number of TUNEL-positive cells increased (30.6±2.0) % (P<0.01) in 6-OHDA group, compared with the normal control group (4.8±1.4)%, and TSG (10,20,50μmol/L) group TUNEL positive cells decreased to 22.8±1.0% (P<0.01), 19.4±0.9% (P<0.01) and 11.6±1.1% (P <0.01). But TSG alone has no obviously effect on PC12 cells5. It is observed by the Automatic fluorescence microplate for detecting intracellular ROS and NO that levels of intracellular ROS increased to (178.4±9.4)%(P<0.01)in 6-OHDA group, compared with levels of intracellular ROS (99.8±3.1)% in the normal control group, and ROS levels drop respectively for,(169.6±8.8)% ,(156.0±9.1)%(P<0.01),(119.2±7.4)%(P<0.01)in 10, 20, 50μmol/L TSG pretreatment group. But TSG alone has no obviously effect on PC12 cells. The levels of intracellular NO increased to (308.6±23.6)% (P<0.01) in 6-OHDA group, compared with the NO level (98.8±4.1)% in the normal control group, and the level of intracellular NO respectively reduced to (287.6±23.2)%, (236.0±34.8)%, (158.0±14.6)% (P<0.01) after pretreatment cells with 10, 20, 50μmol/LTSG. But TSG alone has no obviously effect on PC12 cells.6. Results of Elisa for 3-NT detection shows the level of 3-NT increased to (1.82±0.8)% (P<0.01) in the 6-OHDA group, compared with normal control group (4.4±0.3)%, and 3-NT levels drop respectively for (16.7±0.9)%, (14.6±1.3)%(P<0.05), (10.1±0.7)%(P<0.01) in 10,20,50μmol/LTSG pretreatm- ent group. But TSG alone has no obviously effect on PC12 cells7. Results of Western blot indicate that expressive level of nNOS and iNOS obviously abated in a dose-dependent manner after 10, 20, 50μmol/L TSG pretreatment cells, compared with 6-OHDA treatment groups.【Conclusion】1. TSG can attenuated 6-OHDA-induced apoptosis in PC12 cell in a dose-dependent feature in some concentration range.2. The mechanisms of TSG protective effects on 6-OHDA-induced apoptosis in PC12 cell may be related to regulation of ROS-NO pathway.