Neuroprotective Effects Of Geniposide In The Model Of Pakinson's Disease

Background and Purpose:Parkinson's disease(PD)is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra.It involves multiple neuronal systems and results from changes developing in a few susceptible types of nerve cells.PD is associated with resting tremor,postural rigidity,and progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta(SNpc).Characteristic pathological features of PD include Lewy bodies(LBs),which are juxtanuclear ubiquitinated proteinaceous inclusions in neuronal perikarya,and Lewy neurites(LNs),which are similar protein aggregates found in neuronal processes.Genomic triplication of the a-synuclein gene(SNCA)has been reported to cause hereditary early-onset parkinsonism with dementia.The pathogenesis of PD is not very clear,and many researches show cell apoptosis is a main mechanism causing the degeneration of dopaminergic neurons.At present,the main principle of treatment for PD is to improve the clinical symptoms.Levodopa drugs,dopamine agonists and cholinergic inhibitors can improve clinical symptoms,but they can't delay the apoptosis of dopaminergic neuron or improve the function of dopaminergic neurons that has been damaged.Moreover,long-term application had obvious adverse reactions.Therefore,developing a new drug on treating PD to prolong,prevent and even reverse the degeneration of dopamine neurons is urgent.Geniposide(GP)is a major iridoid glycoside extracted from the fruits of Gardenia jasminoides which is widely used as a traditional Chinese medicine for the treatment of hepatic and inflammatory diseases,contusions and brain disorders.It's been reported that GP has favorable nerve protective effect on mouse model with Parkinson's disease induced by MPTP.The neuroprotective effects of geniposide are also proved in other neurodegenerative diseases like Alzheimer's disease(AD),and the effects depend on the RAGE-mediated signaling.MicroRNAs(MiRNAs)are gaining recognition as important regulators of gene expression with the capability to modulate cellular events.The complex network with their selective targets genes paves way for the regulation of many physiological processes.Anti-apoptotic microRNA-21(miR-21)can protect neuron from death by targeting cell apoptosis and inducing ligand FASLG,and it's been reported that miR-21 level could be regulated by S100P/RAGE mediated pathways.Together with the aforesaid fact that RAGE-mediated signaling pathway was involved in the neuroprotective effect,we decided to focus on miR-21 in PD,another neurodegenerative disease like AD.Researches also shows that miR-21 is also able to target lysosomal membrane receptor LAMP2A(lysosome-associated membrane protein 2)and the high expression of miR-21 would significantly decrease the expression of LAMP2A in PD model mice.LAMP2A can result in the increase of chaperone-mediated autophagy(CMA)activities mediated by molecular chaperone,and CMA is a main pathway that degrades ?-synuclein.In PD,whether geniposide play its neuroprotection effect by regulating the expression of miR-21 is still unclear.In the present study,in vitro and in vivo experiments were designed to confirm the neuroprotective effect of GP in PD models and to explore the underlying mechanism from a certain point of view,which is expected to clarify the roles of miR-21,LAMP2A and a-synuclein in the neuroprotection of GP in PD.Part one Establishing a stable cell model of Parkinson,s diseaseSH-SY5Y cells were treated with different concentration of MPP+(0?1?2?4)for 48hours,the appropriate MPP+ concentration was selected to establish an acute PD cell model;The cellular viablity of SH-SY5Y cells treated with MPP+was determined using CCK-8 assay;and Intracellular ROS production was assess by ROS detection assay.Result1.SH-SY5Y cells were induced with different concentrations of MPP +,with increasing concentration the cell damage decreased significantly;2.Parkinson's disease cell model were established with ImM MPP+;3.1 mM MPP+ acted on SH-SY5Y cells,and the expression of ROS in the cells increased significantly.4.11mM MPP+ acted on SH-SY5Y cells,and the intracellular LDH release increased significantly.Conclusion:1mM MPP+ has great effect on the viability of SH-SY5Y cells,and increased intracellular ROS levels,upregulated LDH activity,which was selected for establishing an acute cellular PD model.Part Two Explore the effect of geniposide on miR-21,LAMP2A and a-synuclein expression in Parkinson's cell modelIn vitro,human neuroblastoma cell line SH-SY5Y was induced to differentiate and subsequently treated with DMSO,MPP+(N-Methyl-4-phenylpyridinium Iodide),and MPP+ + GP(MPP+ together with GP).To identify the role of miR-21 in the regulation of LAMP2A(lysosome-associated membrane protein 2)and a-synuclein,SH-SY5Y cells pretreated with MPP+ were respectively transfected with miR-21 mimic and miR-21 inhibitor.In order to identify whether GP could reduce the level of a-synuclein via miR-21/LAMP2A,the SHSY5Y cells,which were pretreated with GP,were treated with miR-21 mimic or miR-21 inhibitor;meanwhile,a luciferase reporter assay was performed to confirm the direct target of miR-21.LAMP2A was over-expressed using a pCMV6-XL5-LAMP2A vector to confirm the role of LAMP2A in the regulation of a-synuclein by miR-21.In these in vitro experiments,the expressions level of miR-21mRNAand/oprotein,LAMP2A and a-synuclein in SH-SY5Y cells were determined by qRT-PCR and western blotting,respectively.Result:1.The level of miR-21 in the cells was significantly higher than that in normal cells,and it could be significantly downregulated by GP.2.GP raise the protein and mRNA expressions of LAMP2A and reduce the protein level of ?-synuclein in PD models.LAMP2A overexpression abolished the upregulating effect of miR-21 mimic on a-synuclein.3.Mir-21 mimics/agomir reversed the GP induced downregulation of a-synuclein;miR-21 inhibitor strengthened the downregulation of a-synuclein caused by GP.miR-21 directly targets LAMP2A and reduces its mRNA level.Conclusion:In Parkinson's disease,the decrease of a-synuclein expression level by geniposide may be related to the decrease of miR-21 expression and increase of LAMP2A protein and gene expression levels.Part Three Establishment of Parkinson's mouse model,explore the effect of geniposide on miR-21,LAMP2A and ?-synuclein expression in Parkinson's mice modelmethodsIn vivo,PD model mice were established through the intraperitoneal injection with MPTP(N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).The mice were treated with saline,MPTP,MPTP + GP and MPTP + GP + miR-21 agomir,respectively.The numbers of TH+ cells in substantia nigra in different groups of mice were counted and compared.The RNA and/or protein expressions of miR-21?LAMP2A anda-synuclein mRNA in different groups were also determined.Result1.MPTP significanttly decreases the number of TH+ cells.GP significanttly ihibits the decrease of ipsilateral TH+ cells caused by MPTP.2.miR-21 agomir decreases the number of TH+ cells caused by GP.3.The highly expression of miR-21 reverses the reducing effect of GP on the protein levels of ?-synuclein,and that the effect can be effectively increased by inhibiting miR-21.Conclusion:GP exhibits neuroprotective properties by inhiting a-synuclein in PD modles through the expression of miR-21/LAMP2A?...