Ginnalin A Protects SH-SY5Y Cells Against 6-OHDA-induced Parkinson’s Disease Model Via Activation Of The Keap1-Nrf2-ARE Pathway

Parkinson’s disease(PD)is the second most prevalent neurodegenerative disease,and it’s main pathological hallmarks are the degeneration of dopaminergic neurons in substantia nigra pars compacta(SNpc)and dopamine deficiency in striatum.PD is characterized by both motor symptoms including tremor,rigidity and bradykinesia,and non-motor symptoms such as memory deterioration and cognitive decline.The pathology of PD is complicated,including misfolding of alpha-synuclein,mitochondria disfunction,oxidative stress and neuroinflammation,etc..Oxidative stress has been firmly established as a major contributor to the progression of PD.Excessive accumulation of reactive oxygen species(ROS)and deficiency in antioxidant defense system could cause the degeneration of nucleic acids,proteins,lipids,etc.,and ultimately induce oxidative stress damage in neurons.Natural compounds from plants can protect cells by scavenging intracellular ROS or by activating the antioxidant cell defense system.Therefore,discovery of agents from plants,that can recover the oxidative stress damage of neuronal cells,has become an important strategy in the development of new PD drugs.Ginnalin A(GA),a natural polyphenol abundant in red maple(Acer tataricum subsp.ginnala),is reported have many functions,including antioxidation,anti-inflammatory,anti-cancer.However,whether GA could protect neural cells from6-hydrogen dopamine(6-OHDA)induced PD model remains unclear.Herein,we investigated the protective effect of GA on the PD cell model induced by 6-OHDA.Firstly,the protective effect of GA on SH-SY5 Y cells was evaluated by cytotoxicity assay and LDH release,and DCFH-DA probes were utilized to detect intracellular ROS level.We found that he SH-SY5 Y cell viability was decreased to 58.9% following 6-OHDA treatment.Pretreatment of GA could protect about 25.3% cells from apoptosis or death and reduce LDH release by 47.5% compared with the 6-OHDA-only group Meanwhile,the pretreatments with10 and 20μM GA drastically reduced the intracellular ROS levels,by 61.2% and 88.5%,respectively.Then,real-time quantitative PCR,western blot,immunofluorescence assay(IFA)and glutathione(GSH)assay were employed to determine the m RNA and proteins levels of the targets downstream Nrf2-ARE pathway.The results indicated that the expression of Nrf2 and its downstream antioxidative targets(NQO1、HO-1 and GCLC)were upregulated by GA.In addition,GA pretreatment significant promote the production of GSH,an endogenous antioxidative peptide,indicating that GA promotes the antioxidant capacity of SH-SY5 Y cells.Moreover,GA pretreatment promoted the Nrf2 translocation into nucleus.RNA interfering(RNAi)was used to knock down Nrf2 to confirm the protective mechanism of GA.Knocking down of Nrf2 using si RNA significantly abrogates GA protection against the 6-OHDA-induced oxidative damage of SH-SY5 Y.Finally,molecular docking calculation was performed to gain further insight into the protective effect role of GA.The results revealed that GA can competitively bind with the P1,P2,and P3 sub-pockets of Kelch domain in Keap1 via hydrogen bonds and hydrophobic interaction,promote Nrf2 release from the preexisting Nrf2-Keap1 complex,accumulate in cell and translocate into nucleus.For the first time,we found that the natural polyphenol GA could competitively interact with Keap1,induce the dissociation of Nrf2 from Keap1,promote Nrf2 accumulation and translocation into nucleus,and activate the antioxidative Nrf2-ARE pathway.GA could induce the expression of f Nrf2 and its downstream antioxidative genes to eliminate the excessive intracellular ROS,in turn protect cells from oxidative stress damage.Therefore,as a food additive and traditional medicine,GA is a promising agent to treat PD,and deserves more researches.