Protective Effects And Mechanisms Of Tranilast In Dopaminergic Neurons

Objective:Parkinson’s disease(PD)is a neurodegenerative disease characterized by degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain.With the incidence rate increasing year by year,the limitation that simply supplementing dopamine can only improve symptoms in a short time makes the treatment strategy turn to protect the progressive loss of "primary" dopaminergic neurons.Oxidative stress and neuroinflammation are important drivers of dopaminergic neuron death.Tranilast is an anti-allergic drug used in clinic,it has multiple effects such as anti-inflammatory,anti-fibrosis and anti-oxidation,but it has not been studied in PD.Based on the close relationship between the pharmacological effect of Tranilast and the mechanism of dopaminergic neuron injury,this experiment aims to explore whether Tranilast can protect dopaminergic neurons and its possible mechanism.Methods:(1)To investigate whether Tranilast can reduce the damage of MPP+induced SH-SY5Y cells:intervention of MPP+induced SH-SY5Y cells with Tranilast,cell viability and toxicity were detected by MTT and lactate dehydrogenase(LDH)detection kit;Hoechst staining,flow cytometry and Western blot were used to detect the changes of cell morphology,the number of apoptotic cells and the level of apoptosis related proteins.Tetramethylrhodamine ethyl ester(TMRE)and DCFH-DA dye were used to detect the changes of mitochondrial membrane potential and intracellular superoxide anion level.Malondialdehyde(MDA)and Western blot were used to determine the changes of intracellular MDA,SOD,thioredoxin reductase1(TrxR1)and extracellular signal regulated kinases(ERK1/2)protein levels.At the same time,explore whether the inhibitor of ERK1/2 protein sch772984 can block the protective effect of tranilast.(2)To investigate whether Tranilast can reduce the inflammatory level of BV2 cells induced by LPS:intervention of LPS induced BV2 cells with Tranilast,cell viability was detected by CCK-8,IL-1β level in cell supernatant was detected by Elisa.The level of NLRP3 proteins was detected by Western blot.(3)To verify the protective effect and mechanism of Tranilast on MPTP induced PD mice:MPTP induced PD mice were treated with Tranilast,pole,rotarod and balance beam tests were used to evaluate motor function.The damage of dopaminergic neurons,the activation of microglia and the changes of related protein levels in lesion area were detected by immunofluorescence and Western blot.Results:(1)In the MPP+ induced SH-SY5Y cell injury model in vitro,Tranilast can significantly enhance cell viability,reduce the level of cytotoxicity,improve cell morphology,reduce the rate of apoptosis and the level of apoptosis related proteins.It can also increase the level of mitochondrial membrane potential,reduce the level of intracellular reactive oxygen species and Increase the level of endogenous superoxide dismutase.However,administration of ERK1/2 inhibitor SCH772984 weakened the protective effect of Tranilast.(2)In the in vitro inflammation model of BV2 induced by LPS,Tranilast can reduce the binding of LPS to TLR4 and NF-kB nuclear transcription,reduce the expression level of NLRP3,iNOS and COX-2.Moreover,the level of IL-1β in cell supernatant was also decreased.(3)In the MPTP induced PD mouse model in vivo,Tranilast can shorten the climbing time,increase the running time on the roller and reduce the number of hind foot slips when mice cross the balance beam.In addition,Tranilast reversed the reduction of TH,TrxR1,phosphorylated ERK1/2 in the lesion area of mice and reduce the expression level of NLRP3 inflammatory complex related protein.Conclusion:Tranilast plays a neuroprotective role in experimental model of PD induced by MPP+/MPTP in vivo and in vitro.The mechanism may be related to reducing oxidative damage and neuroinflammation related to NLRP3 activation.