Icariin-mediated activation of autophagy confers protective effect on rotenone induced neurotoxicity in vivo and in vitro

Abstract

Rotenone (ROT) is an environmental neurotoxin which has been demonstrated to cause characteristic loss of dopamine (DA) neurons in Parkinson's disease (PD). Icariin (ICA) is a flavonoid glucoside isolated from Herba Epimedii that has been shown to display neuroprotective functions. The present study evaluated protective effects of ICA on ROT-induced neurotoxicity and determined the modulation of ICA on the regulation of autophagy in vivo and in vitro. Rats were treated with ROT (1.0 mg/kg/day) with a co-administration of ICA (15 or 30 mg/kg/day) for 5 weeks. Immunohistochemical analysis showed a significant loss in DA neurons in the substantia nigra (SN) of rats treated with ROT, accompanied by an increase in the accumulation of α-synuclein and a compromised mitochondrial respiration. However, co-administration of ICA potently ameliorated the ROT-induced neuronal cell injury and improved mitochondrial function and decreased the accumulation of α-synuclein. ROT treatment resulted in a decrease in the protein expression of LC3-II and Beclin-1, and an increase in the protein level of P62, and upregulated the activation of mammalian target of rapamycin (mTOR), whereas ICA significantly reversed these aberrant changes caused by ROT. Furthermore, the neuroprotective effect of ICA was further verified in PC12 cells. Cells treated with ROT displayed an increased cytotoxicity and a decreased oxygen consumption which were rescued by the presence of ICA. Furthermore, ROT decreased the protein expression level of LC3-II, enhanced Beclin-1 expression, and activated phosphorylation of mTOR, whereas ICA markedly reversed this dysregulation of autophagy caused by ROT in the PC12 cells. Collectively, these results suggest that ICA mediated activation of autophagic flux confers a neuroprotective action on ROT-induced neurotoxicity.

Keywords: Autophagy; BCA, bicinchoninic acid; DA, dopamine; DMEM, Dulbecco's modified Eagle's medium; HRP, horseradish peroxidase; ICA, icariin; Icariin; LDH, lactate dehydrogenase; Mitochondrial function; Neurotoxicity; OCR, oxygen consumption rate; PD, Parkinson`s disease; PE, phosphatidylethano-lamine; ROT, rotenone; Rotenone; SN, substantia nigra; mTOR, mammalian target of rapamycin.

Fig. 1

Protective effect of ICA on ROT-induced loss of DA cells. The animals were administrated with ROT and ICA for 5 weeks. ICA (15 and 30 mg/kg) was given by oral gavage, and ROT was administered by subcutaneous injection once per day at the dose of 1 mg/kg. Twenty-four hours following the last treatment, rats were sacrificed, and the brains were collected. Brain tissues were processed as described in the Material and methods, and brain sections were immunostained with an anti-TH antibody. (A) and (B), Immunostaining of DA cells and quantification of TH-positive cells in SN, respectively. The results were the mean ± SEM from three rats (n = 3). *, p <  0.05 as compared with control; #, p < 0.05 as compared with ROT group.

Fig. 2

Effect of ICA on ROT-induced of protein expression levels of α-synuclein in SN. After treatment, the SN of rats were collected, then the protein expression level of α-synuclein was determined by Western blot. The results were the mean ± SEM from three rats (n = 3). *, p <  0.05 as compared with control; #, p <  0.05 as compared with ROT group.

Fig. 3

Protective effect of ICA on brain mitochondrial respiration. Mitochondria were isolated from the whole brain tissues as described in the Materials and methods. Mitochondrial respiration flux was detected by high-resolution respirometry. The results were the mean ± SEM from three rats (n = 3). *, p <  0.05 as compared with control; #, p <  0.05 as compared with ROT group.

Fig. 4

Effects of ROT and ICA on the expression levels of autophagic protein LC3-II, Beclin-1, P62, mTOR and p-mTOR in SN. After treatment, the SN of rats were collected, then the protein expression levels of LC3-II, Beclin-1, P62, mTOR and p-mTOR were determined by Western blot. The results were the mean ± SEM from three rats (n = 3). *, p <  0.05 as compared with control; #, p <  0.05 as compared with ROT group.

Fig. 5

Protective effects of ICA on ROT-induced neurotoxicity in PC12 cells. PC12 cells were treated with ICA for 2 h, then exposed to ROT for 24 h. LDH release was measured by the LDH assay kit and cellular respiration was detected by high-resolution respirometry. The results were the mean ± SEM from three independent experiments (n = 3). *, p <  0.05 as compared with control; #, p <  0.05 as compared with ROT group.

Fig. 6

Effects of ROT and ICA on LC3-II, P62, mTOR and p-mTOR protein expression in PC12 cells. PC12 cells were treated with ICA for 2 h, then exposed to ROT for 24 h. The protein expression levels of LC3-II, P62, mTOR and p-mTOR were determined by Western blot. The results were the mean ± SEM from three independent experiments (n = 3). *, p <  0.05 as compared with control; #, p <  0.05 as compared with ROT group.