Protective Effects And Mechanistic Study Of Pinostrobin On 6-OHDA-Induced Parkinson’s Disease

Background:Parkinson’s Disease(PD)is a common neurodegenerative disease and is mainly characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta,which leads to loss of dopamine in striatum and clinical motor symptoms including static tremor and muscle rigidity.Accumulating evidence suggests that oxidative stress is one of the essential factors that induce the onset of PD.In addition,abnormal levels of reactive oxygen species and unbalanced activities of antioxidant enzymes have been shown to induce cellular dysfunction and neuronal apoptosis,including in PD.Therefore,interventions that attenuate oxidative stress may inhibit degeneration of dopaminergic neurons and slow down the progression of PD.At present,modern medical strategies for treatment of PD have shown some disadvantages,whereas traditional Chinese medicine have indicated some specific effects in PD treatment,contributing to developing more effective drugs for the treatment of PD.The leaves of Cajanus cajan have been widely used as traditional herbal medicine which includes antioxidant and anti-inflammatory activity.In addition,the total extracts of Cajanus cajan leaves show neuroprotective effects in PD models.At present,there is little researches on flavonoids from leaves Cajanus cajan.Based on the idea of"kidney-brain treatment",the potential compounds of flavonoids from Cajanus cajan leaves will be further explored for the treatment of PD.In our previous study,we demonstrated that PSB is a neuroprotective bioflavonoid that mainly exists in Cajanus cajan,whereas its pharmacological effects and specific mechanisms of neuroprotection in 6-OHDA-induced PD models remain unclear.Objective:In this paper,the classical nerve agent 6-OHDA was used as an inducer in Parkinson’s disease models.The neuroprotective effects of flavonoid PSB from leaves of Cajanus cajan were identified via zebrafish.The potential mechanism of PSB in protecting neurons from oxidative stress was further explored.Methods:PD in vivo model was established via inducing neurotoxicity in zebrafish with 250μM 6-OHDA.The neuroprotection of PSB was determined by tyrosine hydroxylase immunofluorescence staining and locomotor assays.Nomifensine(30μM)was used in positive control group.PD model in vitro was established in human neuroblastoma cells SH-SY5Y via 6-OHDA(50μM)treatment.With PSB treatment in different concentrations(0.1,1,5,25,100,200μM),the expressions levels of Bcl-2,Bax,Cleaved Caspase-3,Cleaved PARP,Nrf2,HO-1,and NQO1 were detected by RT-q PCR and Western Blot.The cell viability and toxicity were assessed via MTT assay and lactate dehydrogenase release assay.ROS levels,mitochondrial membrane potential,early apoptosis rate and Nrf2 nuclear translocation were detected by fluorescent staining.The activity of ARE promoter was detected by dual luciferase reporter assay.ELISA was used to detect DNA fragmentation.Intracellular levels of MDA,GSH,GSSG,NAD~+,NADH,GSH-Px and SOD was assessed by corresponding kits.Nrf2 was silenced by si RNA in SH-SY5Y cells,which was determined by Western Blot.AKT and ERK protein kinase were blocked by inhibitors in SH-SY5Y cells.ROS levels,cell viability,LDH release levels,early apoptosis rate,and mitochondrial membrane potential were detected in Nrf2-KO SH-SY5Y cells.The effects of AKT/ERK protein kinase inhibitors on intranuclear expression levels of Nrf2,as well as phosphorylation and total AKT/ERK protein expression levels were assessed via Western Blot.The effects of AKT/ERK protein kinase was clarified by detecting ARE promoter activity,cellular ROS levels,cell viability and early apoptosis rates.Results:6-OHDA induced significant reductions in TH-positive cell densities and moving distances in zebrafish,whereas PSB treatment or NOM treatment significantly mitigated 6-OHDA-induced neurotoxicity.SH-SY5Y cells treated by 6-OHDA showed significant decreases in cell viability,mitochondrial membrane potential,Bcl-2 protein expressions levels,GSH/GSSG ratio,NAD~+/NADH ratio,GSH-Px and SOD enzyme activities,and increase in LDH release,early apoptosis rates,DNA fragmentation,the expression levels of Bax,Cleaved Caspase-3 and Cleaved PARP proteins,ROS and MDA levels,which was reversed by PSB treatment.Furthermore,PSB also significantly induced nuclear translocation of Nrf2 and the expressions of HO-1 and NQO1.Nrf2-KO SH-SY5Y cells were established via specific si RNA,in which cell viability andΔψm were significantly decreased and the early apoptosis rates,LDH release and ROS levels were significantly increased with 6-OHDA treatment,however,PSB treatment did not reverse the condition.The neuroprotection of PSB was abolished.Compared with PSB groups,the effects of PSB-induced increase in ARE promoter activity and the promotion of Nrf2 nuclear translocation were abolished by AKT/ERK protein kinase inhibitors,and the effect of PSB in improving cell survival,reducing ROS levels and inhibiting early apoptosis were reversed.Conclusion:Pinostrobin ameliorates 6-OHDA-induced dopaminergic neuronal loss and locomotor deficits in zebrafish;pinostrobin attenuates 6-OHDA-induced oxidative stress and apoptosis in SH-SY5Y cells by activating the Nrf2/ARE signaling pathway;AKT/ERK protein kinase is involved in the activation of Nrf2/ARE signaling pathways regulated by pinostrobin.