Icariin Protects Against Rotenone-induced Dopamine Neurotoxicity Via Induction Of SIRT3 And Increase Of Mitochondrial Complex ? Activity

Objective: To investigate the neuroprotective effect of icariin(ICA)on rotenone(ROT)-induced neurotoxicity and its potential mechanism.Methods: In the in vivo experiments,50 male SD rats weighing 200-250 g were randomly divided into the following five groups: Control,ICA alone(30 mg/kg/day),ROT(1 mg/kg/day),ROT+ low-ICA(15 mg/kg/day),and ROT+ High-ICA(30 mg/kg/day).ROT was subcutaneously injected once per day,while ICA was administered by oral gavage.The rats in the control group were given ddH2 O by gavage according to their body weight,and the solvent was injected subcutaneously.The weight of rats was measured throughout the course of the treatment.After 5 weeks,the behavioral performance of rats was assessed by the rotarod test.Immunohistochemical staining was used to quantify the number of dopaminergic neurons in the substantia nigra.TH expression in the rats' brain tissue was detected by Western blot.The activities of complex ? and SIRT3 were determined by corresponding commercial assay kits.In the in vitro experiments,PC12 cells were randomly divided into four groups as follows: Control,ROT(0.6 ?M),ROT+ICA(4 ?M),and ROT+ICS ?(0.75 ?M).Cells were pretreated with ICA or ICS ? for 30 min.ROT was then added into the medium and incubated for 24 h.The cytotoxicity was detected by MTT assay and measurement of lactate dehydrogenase(Lactic dehydrogenase,LDH)activity.The level of cellular reactive oxygen species(ROS)was detected by flow cytometry.The contents of GSH and GSSG in cells were detected and the ratio of GSH/GSSG was calculated.The expression of autophagy's substrate,SQSTTM1/P62,and autophagy protein,beclin-1,was detected by Western blot.Autophagosomes were detected by Ad-GFP-LC3 B.To further clarify the protection effect and mechanism of ICA,PC12 cells were randomly divided into Control,ROT(0.6 ?M),ROT+ Low-ICA(2 ?M)group,and ROT+ High-ICA(4 ?M)groups.Following 24 h treatment,high-resolution oxygraphy was used to detect mitochondrial respiratory capacity and a commercial assay kit was used to detect mitochondrial complex ? activity.The protective effect of ICA was further explored using 3-TYP,a SIRT3 inhibitor.PC12 cells were incubated with 3-TYP and ICA for 30 min,then ROT was added.After 24-hour incubation,cytotoxicity was evaluated by MTT.The activity of SIRT3 and mitochondrial complex ? in cells were detected by commercial assays kits.The contents of GSH and GSSG were determined and the ratio of GSH/GSSG was calculated.Results: In vivo experimental: Compared with the control group,the rats in the model group lost weight significantly,while the rats receiving a high dose of ICA(30 mg/kg/day)in combination with ROT gained weight significantly compared to the rats treated with ROT alone.The results of rotarod test showed that a marked decrease in the motor activity of rats administered with ROT,while the motor activity was significantly improved in the rats given both ICA and ROT.The results of immunohistochemistry and Western blot suggested that the expression of TH was decreased and DA neurons were significantly lost in the model group.After the administration of ICA,TH protein expression was increased and the number of DA neurons were increased.Analysis of the activities of complex ? and SIRT3 found that both of them decreased after ROT treatment.On the contrary,both activities of C ? and SIRT3 were significantly increased when ICA was co-administrated.In vitro study: ROT treatment resulted in the loss of cell viability,an increase of LDH release,and the accumulation of ROS along with a decrease in the ratio of GSH/GSSG.ICA and ICS ? were able to improve the cell viability,reduce LDH release and cellular ROS level,and increase the GSH/GSSG ratio.Analysis of Western blot and examination of LC3 B fluorescence found that while ROT treatment significantly enhanced the protein expression of SQSTTM1/P62,it inhibited the expression of Beclin-1 and decreased the formation of autophagosomes in the model group.These results suggested that autophagy flow was blocked by ROT treatment.By co-administration of ICA or ICS ?,all these protein expressions were reversed.Respiratory function analysis found that ROT treatment resulted in significant inhibition of cellular oxygen consumption and a decrease in the activity of C ?,while the treatment of ICA and ICS ? resulted in a significant improvement in the oxygen consumption and the CII activity.The analysis using isolated mitochondrial further revealed that ROT treatment significantly reduced mitochondrial complex II activity in the model group,while the activity of C ? was significantly increased in the presence of ICA.However,3-TYP the protective effect of ICA on ROT-induced neurotoxicity was noticeably reduced,as evidenced by MTT assay and the measurement of LDH activity.Detection of SIRT3 activity found that the effect of ICA on the activation of SIRT3 was significantly inhibited in the presence of 3-TYP.Furthermore,the ICA-mediated increase in the activity of C II was attenuated,and the ratio of GSH /GSSG decreased when cells were co-treated with 3-TYP.Conclusion: The present study demonstrates that ICA is protective against dopaminergic neuron damage induced by rotenone,which may be mediated through the activation of SIRT3,increasing complex ? activity,reducing the accumulation of ROS,and promoting autophagy flow.