| Objective: To study the protective effect of icariin(ICA)on the degeneration of dopaminergic neurons induced by rotenone(ROT)and explore the possible molecular mechanism.Methods: 1.In vivo animal study design.75 male SD rats were divided into five groups: control,ICA(30 mg/kg),ROT(1 mg/kg),ROT+ICA(15 mg/kg),and ROT+ICA(30 mg/kg)groups.ICA was administrated by oral gavage(15 mg/kg or 30mg/kg),while ROT was given by subcutaneous injection once per day at a dose of 1mg/kg.The behavioral function of rats was measured by rotarod test;the number of dopamine neurons in the substantia nigra of rats was detected by immunohistochemical analysis;and the protein levels of SIRT3 and PGC-1? protein in rat substantia nigra were detected by Western blot.2.In vitro PC12 cell experiments.(1)Cells were divided into control,ICA(4 ?M),ROT(0.5 ?M),ROT+ICA(2 ?M),and ROT+ICA(4 ?M)groups.(1)To observe the effect of ICA on ROT-induced cytotoxicity,cells were pretreated with ICA for 2 h,followed by exposure to ROT for 24 h.MTT assay was used to detect cytotoxicity,and high-resolution oxygraph was used to detect the mitochondrial respiratory function.Cellular ROS content was measured by flow cytometry,and measurement of cellular SOD activity was determined by a commercial SOD activity kit.The protein expression levels of SIRT3 and PGC-1? proteins in PC12 cell were determined by Western blot.(2)To determine the effect of modulation of PGC-1? expression on the protective effect of ICA on ROT-induced cytotoxicity,PC12 cells were treated with PGC-1? siRNA for 24 h,followed by exposure to ROT for 24 h.Upon the completion of the treatment,MTT assay was used to detect cytotoxicity.The protein expression levels of PGC-1? and SIRT3 proteins were measured by Western blot.(3)To explore the involvement of SIRT3 in the ICA-mediated protective effect on ROT-inducedcytotoxicity,PC12 cells were treated with ROT in combination with SIRT3 inhibitor3-TYP for 24 h.Following the treatment,MTT assay was used to detect cytotoxicity,and high-resolution oxygraph was used to detect mitochondrial respiratory capacity.The cellular ROS content was measured by flow cytometry,and measurement of cellular SOD activity was conducted by a commercial SOD activity kit.The protein expression levels of SIRT3 and PGC-1? were determined by Western blot.Results: 1.In vivo animal study design.Rats treated with ROT exhibited a marked loss of DA neurons and a decline in motor function,along with a decrease in protein expression levels of SIRT3 and PGC-1? in substantia nigra.Administration of ICA significantly alleviated the loss of DA neurons and improved behavioral function,and concomitantly enhanced the expressions of SIRT3 and PGC-1?.2.(1)In vitro studies in PC12 cells showed that ROT treatment resulted in loss of cell viability,a decrease in mitochondrial respiratory function,a lower SOD activity,and a higher cellular ROS production,accompanied by a lower expression levels of SIRT3 and PGC-1? proteins,in the relative to the control cells.However,after the intervention of ICA,the cell viability,mitochondrial respiratory function,activity of SOD,the expressions of SIRT3 and PGC-1? proteins were significantly increased,and the content of ROS was lower in the cells treated with ROT + ICA than the cells treated with ROT alone.(2)Knockdown of PGC-1? by siRNA suppressed ICA-mediated protective effect on ROT-induced cytotoxicity but did not affect the expression of SIRT3.(3)The cytoprotective effect of ICA was markedly abolished in ROT-treated cells by SIRT3 inhibitor 3-TYP,along with a resultant decrease in PGC-1? protein expression.Inhibition of SIRT3 protein resulted in a decrease in intracellular SOD activity,inhibition of cellular mitochondrial respiratory function,and an increased in cellular ROS production.Conclusion: ICA exerts neuroprotective effects on ROT-induced dopaminergic neurodegeneration.The underlying mechanism of ICA-mediated neuroprotection involves the upregulation of SIRT3 and improvement in mitochondrial function and oxidative stress. |
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