Icarrin-mediated Regulation Of Mitochondrial Function And Its Role In Neuroprotection Of Rotenone-induced Rat Parkinson’s Disease Model

Objective: To investigate the protective effects of Icariin(ICA)on Rotenone(ROT)-induced mitochondrial dysfunction and neuronal cell loss,and explore the molecular mechanism behind ICA-mediated cellular protection.Methods:(1)100 male SD rats were randomly divided into five groups(20 in each group): control,ICA(30 mg/kg/day),ROT(1 mg/kg/day),ROT+ ICA(15 mg/kg/day),and ROT+ ICA(30 mg/kg/day).ICA was administrated by oral gavage,while ROT was given by subcutaneous injection once per day at a dose of 1 mg/kg.The control group was subject to an equivalent volume of vehicle.The animals were treated for 5 weeks.At the end of the treatment period,all animals were behaviorally assessed,and subsequently,other molecular and biochemical parameters were examined.(1)Animal behavior was evaluated by rotarod test.(2)DA neurons were examined through TH staining.(3)Mitochondrial function was assessed by measuring oxygen consumption of isolated rat brain mitochondria.(4)RT-PCR was used to detect the expression level of cortex PGC-1α and TFAM.(5)Western blot was used to detect cortex and striatum α-synuclein protein accumulation,determine protein level of cortex and striatum autophagy relational proteins: beclin-1,LC3-II,and SQSTTM1/P62,as well as autophagy regulation proteins in cortex and striatum: m TOR,p-m TOR,P70 S6 Kinase,and p-P70 S6 Kinase expression.Further,the molecular mechanism of ROT cytotoxicity was examined in PC12 cells:(i)cytotoxicity of ROT was evaluated through LDH assay.(ii)PC12 cell through Oxygraph-2k detecting cell mitochondrial function was assessed by measuring oxygen consumption.(iii)Western blot was employed to detect the protein expression of LC3-II,SQSTTM1/P62,m TOR,p-m TOR,P70 S6 Kinase,and p-P70 S6 Kinase.Results:(1)Animal experiment: the riding time was significantly shorter in ROT group compared to the control group,while administration of ICA markedly increased riding time of ROT-treated animals.ROT caused notable loss of DA neurons,accompanied by a reduction in mitochondrial oxygen consumption,both of which were significantly reversed by administration of ICA.ICA also increased significantly the gene expression level of PGC-1α and TFAM compared to ROT treatment alone.ROT treatment resulted in significant increase in the accumulation of α-synuclein protein in cortex and striatum,which was notably diminished in the animals co-administrated with ICA.Analysis of autophagy flux found that ROT treatment significantly inhibited the protein expression of LC3-II and beclin-1,while enhanced the protein levels of P62,p-m TOR,and p-P70.All these protein expressions were reversed by co-administration with ICA,indicating the improvement of autophagy flux by ICA.(2)PC12 cell experiments: when treated with ROT,LDH release was significant elevated,and mitochondria respiration was suppressed,accompanied by dysregulation of autophagy flux,while co-treatment with ICA markedly prevented LDH release,increased mitochondrial respiration and improved autophagy flux compared to ROT treatment alone.Conclusion: ICA protects against ROT-induced neuronal cell death through promoting autophagy flux and maintaining mitochondrial function.