Involvement Of Apoptosis-inhibition In Protective Effect Of Puerarin To The Models Of Parkinson's Disease

IntroductionParkinson's disease is one kind of neurodegenerative disease, mainly after Alzheimer's disease, with high incidence and serious social effects. The hallmarks of PD are degeneration of dopaminergic neurons in the substantia nigra and depletion of striatal dopamine. It is of particular interest to find therapeutic drugs targeting the dopaminergic neurons.Puerarin isolated from Pueraria lobata (Willd.) and Ohwi Pueraria thomsonii Benth, possesses an anti-oxidative property and has a variety of biological actions in cardiovascular diseases, gynecology disease, osteoporosis, cognitive capability. Clinical and experimental model of Parkinson's disease indicated prominent oxidative species in the SN and striatum, implying potential therapeutic effect of Puerarin. Accumulating evidence suggested that Puerarin possesses potential anti-oxidative effect in various diseases. While whether apoptosis-inhibiting was involved in the protective effect need further elucidate. Also, other scientists reported that Puerarin could obviously attenuate the injury to midbrain dopaminergic neurons elicited by MPP+, since the anti-oxidative effect. Recently, apoptosis was documented as an important pathogenesis in PD, while apoptosis could elicited by oxidative injury. Whether apoptosis-inhibiting was involved in the protective effect of Puerarin to PD is the basic objective of this study.ObjectiveTo study neuroprotective effect of puerarin to models of Parkinson's disease and underlying mechanisms in both in vivo and in vitro.Methods1 Cell culture and drug treatment and cell viability assaySH-SY5Y human neuroblastoma cells cultured in DMEM were classified into five groups exposured to MPP+ with or without different doses of Puerarin for 48 h .Then trypan blue dye exclusion test was applied to detect the cell viability.2 Mitochondrial membrane potentialUsing JC-1 dye kit, the mitochondrial membrane potential of different cell groups were examined by FCM.3 Release of Cyt c assayed by ELISAThe release of cyt c from mitochondrial to cytosol was assayed by ELISA.4 Western BlotCells were lysised and ultracentrifuged to obtain supernatants. Equal amounts of protein were resolved on 8% SDS-PAGE and transferred onto PVDF membrane. A standard Western blot procedure was followed for immunoblot with polyclonal Cyt c antibody, An ECL kit was used for provided in the assay kit. The blot was reprobed with monoclonalβ-actin antibody to confirm equal protein loading.5 Flow cytometer methodPI-only stained the cells and the dyeing cells were detected by Flow cytometry.6 Caspase-3, Caspase-8 and Caspase-9 activities Caspase-3, Caspase-8 and Caspase-9 activities were determined according to the instructions of the user's manual for assay kits. The supernatant obtained by a centrifugation of lysed cells was added to the reaction mixture containing dithiothreitol and caspase-3, Caspase-8 and Caspase-9 substrates Ac-DEVD-pNA (2mM), Ac-IETD–pNA and Ac-LEHD–pNA, respectively. The absorbance was measured at a test wavelength of 405 nm. The protein concentration was determined by the Bradford method.7 DNA ladder testThe test was done to verify the apoptotic cells.8 Animal experimentsThe nigrostriatal neurons of the Sprague-Dawley rats were selective lateral induced by 6-hydroxydopamine, and then Puerarin administered the rats those passed APO-rotation tests. The 6-OHDA injected rats were randomly separated into (1) the only 6-OHDA treated group; (2) Low-dose puerarin group. After 6-OHDA injected 3 weeks, Puerarin was injected into rat's intraperitoneal, the dose was 40mg?Kg-1. (3) High-dose puerarin group. After 6-OHDA injected 3 weeks, Puerarin was injected into rat's intraperitoneal, the dose was 120mg?Kg-1. The term of puerarin-administrated was 10d and only one time a day. The control group with no drug treatment and the pseudo-control treated wih Vitc to replace 6-OHDA toxin.All animal behavior by apomorphine-induced were detected before killed. Take 2 rats of each group prepared for the pathological ultrastructure analysis by EM. The methods adopted by the routine.Rats were killed 24h after the last injection, the brains were removed quickly and fixed with 4% formaldehydum polymerisatum and then dehydrated. Brain were prepared for paraffin sections according the universal methodsThe substantia nigra was sliced with 8μm thick for HE staining and for TH, TUNEL Bcl-2, Bax TH mRNA tests. And the GDNF were tested in the striatum slices. The IHC, Tunel and ISHH results were analyzed by a pathological imagine system.9 Statistic analysisResults were presented as x±s; SPSS11 was used for data analysis. P-values were determined using Student's t-test for single comparisons of two samples. One-way ANOVA was completed.Results1 Puerarin pretented against MPP+-elicited SH-SY5Y cells death To determine the protective effect of puerarin on MPP-induced SH-SY5Y cell death, typan blue assay was applied to detect cell viability. The results suggested that no apparent difference was observed in single Puerarin treated SH-SY5Y cells. While single treatment of MPP indicated that MPP+ significantly inhibited the cell viability with a dose-dependent manner and the inhibitory rate could attain 44% in the dose of 1 mM. Pretreatment with 25μM and 50μM Puerarin could apparently increase the viability of the SH-SY5Y cells (p<0.05). Furthermore, the results of the phase-contrast microscope were in line with the results measured by trypan blue exclusion test.2 Involvement of apoptosis inhibiting in protective effect of puerarin on MPP+-elicited cell apoptosisTo verify the potential role of apoptosis in the protective effect, FCM, Western blot and ELISA were applied in the experiment. Based on the result of JC-1 assay, MPP+ significantly attenuated MMP of the SH-SY5Y cells; pretreatment with puerarin inhibited the deterioration of the MMP. Both ELISA and Western blotting tests indicated that Puerarin prevented against the release of cyt c from the mitochondrial interior to the cystol elicited by MPP+. Flow cytometer results apparently showed that Puerarin negatively regulated the apoptosis ratio, compared with the MPP+ group. DNA ladder showed that typical DNA ladder was present in the MPP+-induced SH-SY5Y cells. In addition, activity of caspase-9 and caspase-3, initiator and effector of the apoptosis respectively, were not at the same level among different groups. Puerarin decreased the up-regulated activity of caspase-9 and caspase-3 activated by MPP. However, no obvious difference was observed in the activity of Caspase-8.3 Pathological morphology and the results of TH-ICH, TH-ISHH, Tunel, Bcl-2, Bax in SNc and the GDNF expression in striatumTo examine the protective effect of Puerarin on 6-OHDA-induced lesion, we applied electron microscope and HE staining method to observe morphological changes of neuron and the expression of TH of SN. After 34 days of intrastriatal 6-OHDA injection, dopaminergic neurons of SN were obviously damaged with the characteristics of condensed chromatin and cellular damage. However, co-application of Puerarin could significantly prevent 6-OHDA-induced lesion. The data of HE staining further supported the protective effect of Puerarin.To investigate whether apoptosis was potential pathway involved in 6-OHDA lesioned rat, we applied TUNEL assay to assess DNA fragmentation in the substantia nigra. The results significantly indicated that 6-OHDA induced apoptosis of neurons of SN. Pretreatment with Puerarin negatively mediated the apoptotic neurons. Simultaneously, the results of immunohistochemical method indicated that TH positive neurons of SN were up-regulated by Puerarin in comparison with 6-OHDA lesion, which further verify the quantitative change of TH positive neurons. To further examine the potential apoptotic mechanisms, we detected the level of Bcl-2 and Bax. In our study, no obvious change in the expression of Bcl-2 was observed in different groups. However,the expression of Bax took on different trends. Expression of Bax increased after 6-OHDA-induced (compared with the blank control, p <0.01), and co-application with Puerarin had a significant down-regulated function on Bax expression.After 34 days intrastriatal 6-OHDA injection, the levels of GDNF in striatum were significantly decreased in rats compared with the control group based on immunohistochemistry,while the level of GDNF in Puerarin pretreatment group increased more significant than single 6-OHDA injection.Conclusion1 Puerarin apparently improved the viability of the SH-SY5Y cells harmed by MPP+. The mechanism is involved in the cell apoptosis which mediated by decreasing of mitochondrial membrane potential, induction of cytochrome c releasing, systematic cascade reactions and some other apoptosis protease-activating factors, etc. Puerarin, can stabilize the mitochondrial membrane potential, inhibit the releasing of cytochrome c, negatively regulate the activity of caspase-3 and caspase-9, consequently, and maintain normal penetrability of mitochondria membrane. The above ways and other paths co-operate to intervene the apoptotic proceeding.2 Puerarin could protect the rat's neurons from the toxicity of 6-OHDA, down-regulate the expression of Bax and up-regulate the ratio of Bcl-2/Bax. Also, it could actively promote the secretion of GDNF in the rat's corpus striatum.