The Neuroprotective Effects Of Candisartan Cilexextil And Salubrinal On Rotenone-induced Dopaminergic Cells

Parkinson’s diseae (PD) is a common adult-onset neurodegenerative characterized by the relatively selective death of dopaminergic neurons in the substantia nigra pars compacta and the present of intracellular a-synuclein inclusions. It’s entity typically diagnosed by its cardinal motor symptoms, including resting tremor, bradykinesia, rigidity, and postural instability. The health,social and economic impact resulting from Parkinson’s diease will continue to increase alongsid the longevity of the population. Because the menchanisms underlying the selective neuronal death in PD are not fully understood, there is currently no treatment that slows the degenerative process. Growing evidence from studies in human PD brains, in addition to genetic and toxicological models, indicates that endoplasmic reticulum (ER) stress is acommon feature of the diseae and contributes to neurodegeneration. Recent studies place ER dysfunction as an early compont of PD pathogenesis. So the strategies to mitigate ER stress could provide new insight into clinical therapeutics for PD.There are a local renin-angiotensin system (RAS), and angiotensin Ⅱ (Ang Ⅱ) type1(AT1) receptors and AT2receptors have been located in DA neurons of the brain. It is indicated that DA cell loss induced by DA neurotoxins is enhanced by Ang Ⅱ via AT1receptors. Most studies have shown that AT1receptors blockers reduce oxidative stress and protect DA cell in6-OHDA and MPTP in vivo models of parkinsonism. But the effect of AT1receptors blockers on the ER stress is still poorly understood. Most studies have shown that ATI receptors blockers reduce oxidative stress and protect DA cell in6-OHDA and MPTP models of parkinsonism. But the effect of AT1receptors blockers on the ER stress is still poorly understood.The effects of ATF4, a member of the ATF/CREB family of basic leucine zipper transcription factors, on neuronal survival or death are complex. Previous studies found that rotenone induces PERK phosphorylation, which results in ATF4and CHOP (CCAAT-enhancer-binding protein (C/EBP) homologous protein) protein induction in neuronal PC12cells. However, many target genes are regulated by ATF4, including parkin, which is transcriptionally upregulated by ER stress and can protect cells from ER stress-induced cell death. Moreover, a dominant-negative ATF4mutant prevents the ER stress-induced upregulation of parkin. And a recent report showed that ATF4-parkin pathway protects against neuronal death induced by6-hydroxydopamine (6-OHDA) and1-methyl-4-phenyl-pyridinium (MPP+). However, it is unknown whether ATF4-parkin pathway is beneficial or harmful in the rotenone model.Therefore, in the first part of our study we explored that the effect of candesartan cilexetil on the rotenone-induced PD rat and determined whether candesartan cilexetil reduces DA neuronal apoptosis via ATF4-CHOP-Puma signaling pathway.And in the second part, we used rotenone-induced apoptosis in SH-SY5Y cells as an in vitro model of dopaminergic neuronal degeneration. We investigate the effects and mechanisms of action of salubrinal on this model of neuronal cell death. Part Ⅰ Candesartan cilexetil guards rotenone-induced dopaminergic neuronal apoptosis in PD rat modelAIM:To investigate whether candesartan cilexetil protects against rotenone-induced dopaminergic (DA) neuronal apoptosis in PD rat model and explore the underlying mechanisms.METHODS:Experimental rats (10weeks old) were randomly divided into four groups:vehicle, rotenone (Rot), rotenone+candesatan cilexetil (Rot+CC), and CC group. Rats received2.5-3.0mg/kg/day rotenone in DMSO:PEG via the osmotic minipumps implanted under the skin of the back to established rat PD model. Rats was orally administered candesartan cilexetil suspended in0.5%metyl cellulose beginning24hours after the osmotic minipumps were implanted. Four weeks after candesartan cilexetil administration, the behavioral profile of animals was accessed by catalepsy tests. Immunohistochemistry was performed to detect the staining of tyrosine hydroxylase (TH) and the expression of α-synuclein in substantia nigra (SN). Western blotting confirmed the expression of TH and cleaved caspase-3in SN. The mRNA and protein of GRP78, ATF4, CHOP, and Puma was measured respectively by RT-PCR and western blotting.RESULTS:(1) Chronic rotenone treatment resulted in a significantly prolonged descent latency compared with the vehicle group (P<0.01), and catalepsy induced by rotenone was significantly antagonized by treatment with candesartan cilexetil (P<0.01). Moreover, the rat’s weight reduced by rotenone was also reversed by candesartan cilexetil, but the blood pressure of four groups was not different significantly.(2) In rotenone-treated rats, the number of TH-positive neurons and Nissl stained cells in the SNc was reduced significantly (P<0.05). However, the death rate of TH-positive cells and Nissl stained cells in SN of the Rot+CC group was reduced compared with the rotenone group (P<0.05). Dietary treatment with candesartan cilexetil abrogated the effect of rotenone on TH levels in SN determined by western blotting. And our data showed that expression levels of cleaved caspase-3in rotenone-treated rats were increased significantly compared with vehicle rats, but decreased significantly after subsequent candesartan cilexetil treatment (P<0.05).(3) The mRNA and protein of GRP78, ATF4, CHOP, and Puma was significantly increased in rotenone-treated rats, when PD rats treated with candesartan cilexetil displayed a considerable decrease in expression of GRP78, ATF4, CHOP, and Puma (P<0.05).CONCLUSION:In this study, we clarified that rotenone triggered ER stress and induced DA neuron death in the PD rat model. And candesartan cilexetil protected dopminergic neuron by reducing ER stress. Additionally, the effect of candesartan cilexetil may be via inhibiting activation of the ATF4-CHOP-Puma signaling pathway.Part Ⅱ Salubrinal protects against rotenone-induced SH-SY5Y cell death via ATF4-parkin pathwayAIM:To investigate whether salubrinal, an ER stress inhibitor, protects against rotenone-induced SH-SY5Y cell death, and explore its underlying mechanisms.METHODS:SH-SY5Y cells were treated with rotenone in the presence or absence of salubrinal at different time. Cell viability was assessed using MTT, and quantitative evaluation of apoptosis was performed using flow cytometry after double labeling with an FITC Annexin V Apoptosis Detection kit. To investigate the effect of ATF4and parkin on salubrinal-mediated cytoprotection of SH-SY5Y cells, we knocked down the expression of ATF4and parkin using ATF4siRNA or parkin siRNA, respectively. Western blotting was used to analysis the expression of GRP78, eIF2a, p-eIF2a, ATF4, CHOP, parkin, cleaved caspase-3and C/EBPβ.RESULTS:(1) Rotenone induced a significant ER stress response and caused apoptosis, which can be inhibited by salubrinal.(2) The expression of ATF4was upregulated by salubrinal. ATF4knockdown, using siRNA, significantly increased rotenone cytotoxicity and decreased salubrinal’s protective effect.(3) The protein level of parkin was significantly declined after cells treatment with rotenone, which was rescued by salubrinal, and parkin siRNA significantly increased the apoptosis percent and inhibited the cell viability, and salubrinal did not reduce the apoptosis percent and increase the cell viability in parkin siRNA cells.(4) Rotenone did not change the expression of bZIP protein C/EBP isoform LIP in SH-SY5Y cells, but the levels of LIP decreased significantly upon treatment with salubrinal.CONCLUSION:Our study indicates that salburinal protects against rotenone-induced SH-SY5Y cell death and provides an insight into the potential involvement of the ATF4-parkin pathway in the salubrinal-mediated neuroprotection.In summary, our present study has the following new concerns:1. Candesartan cilexetil protects against rotenone-induced DA neurons in the rat model of Parkinsonism involving ameliorating ER stress, possibly via inhibiting activation of the ATF4-CHOP-Puma signaling pathway, which could provide new insight into clinical therapeutics for PD.2. Salubrinal protects against rotenone-induced dopaminergic neuron death. that the role of the cytoprotection may depend on the activation of ATF4. Although the effect of ATF4activation on neuronal survival is complex, our study provides an insight into the potential involvement of ATF4-parkin pathway in the salubrinal-mediated neuroprotection of rotenone-induced dopaminergic cell death.