Icariin attenuates neuroinflammation and exerts dopamine neuroprotection via an Nrf2-dependent manner

Abstract

Background: Oxidative stress and neuroinflammation are considered the major central events in the process of Parkinson's disease (PD). Nrf2 is a key regulator of endogenous defense systems. New finds have contacted activation of Nrf2 signaling with anti-inflammatory activities. Therefore, the outstanding inhibition of neuroinflammation or potent Nrf2 signaling activation holds a promising strategy for PD treatment. Icariin (ICA), a natural compound derived from Herba Epimedii, presents a number of pharmacological properties, including anti-oxidation, anti-aging and anti-inflammatory actions. Recent studies have confirmed ICA exerted neuroprotection against neurodegenerative disorders. However, the underlying mechanisms were not fully elucidated.

Methods: In the present study, mouse nigral stereotaxic injection of 6-hydroxydopamine (6-OHDA)-induced PD model was performed to investigate ICA-conferred dopamine (DA) neuroprotection. In addition, adult Nrf2 knockout mice and primary rat midbrain neuron-glia co-culture was applied to elucidate whether ICA-exerted neuroprotection was through an Nrf2-dependent mechanism.

Results: Results indicated that ICA attenuated 6-OHDA-induced DA neurotoxicity and glial cells-mediated neuroinflammatory response. Furtherly, activation of Nrf2 signaling pathway in glial cells participated in ICA-produced neuroprotection, as revealed by the following observations. First, ICA enhanced Nrf2 signaling activation in 6-OHDA-induced mouse PD model. Second, ICA failed to generate DA neuroprotection and suppress glial cells-mediated pro-inflammatory factors production in Nrf2 knockout mice. Third, ICA exhibited neuroprotection in primary neuron-glia co-cultures but not in neuron-enriched cultures (without glial cells presence). Either, ICA-mediated neuroprotection was not discerned after Nrf2 siRNA treatment in neuron-glia co-cultures.

Conclusions: Our findings identify that ICA attenuated glial cells-mediated neuroinflammation and evoked DA neuroprotection via an Nrf2-dependent manner.

Keywords: Icariin; Neuroinflammation; Neuroprotection; Nrf2; Parkinson’s disease.

Fig. 1

ICA ameliorated 6-OHDA-caused DA neuronal damage. Mice were intragastrically given ICA (60 mg/kg) for 10 consecutive days and a single intranigral injection of 6-OHDA (4 μg) in the left side of SN beginning 3 days after ICA treatment. After the last treatment of ICA, rotarod test (a) and open-field test (b) were performed. TH-positive neurons were measured by immunofluorescence staining (c). TH protein expression in midbrain was tested by western blot assay (d). The levels of DA, DOPAC and HVA in striatal tissues were detected by UHPLC. Meanwhile, the DA metabolite ratio [(DOPAC + HVA)/DA × 100] was computed (e). Results were mean ± SEM from 5 to 6 mice. *p < 0.05 compared with the control group, ^#p < 0.05 compared with 6-OHDA-treated group. Scale bar = 200 μm

Fig. 2

ICA activated Nrf2 signaling pathway. After ICA treatment for 10 days, the mRNA levels of Nrf2, Keap1, HO-1, and NQO1 in midbrain SN were detected by real-time RT-PCR (a and b). The protein expressions of nuclear Nrf2, cytosol Nrf2, HSP90, total Nrf2, Keap1, HO-1, and NQO1 in midbrain were detected via western blotting (c and d). Results were mean ± SEM from 5 to 6 mice. *p < 0.05 compared with the control group, ^#p < 0.05 compared with 6-OHDA-treated group

Fig. 3

Nrf2 signaling participated in ICA-mediated neuroprotection. Nrf2 KO mice were treated with ICA (60 mg/kg) daily for 10 consecutive days and a single intranigral injection of 6-OHDA (4 μg) in the left side of SN 3 days after ICA treatment. Then, mouse brains were harvested and the knockout efficiency was tested by western blotting (a). The mRNA and protein expressions of Keap1, HO-1, and NQO1 in midbrain were detected by real-time RT-PCR (b) and western blotting (c), respectively. The rotarod test and open-field test were performed (d, e). TH-positive neurons were measured by immunofluorescence staining (f). TH protein expression in mesencephalon was detected by western blot assay (g). The levels of DA, DOPAC, and HVA in striatal tissue were tested by UHPLC. The DA metabolite ratio [(DOPAC + HVA)/DA × 100] was computed (h). Results were mean ± SEM from 5 to 6 mice. *p < 0.05 compared with the control group. Scale bar = 200 μm

Fig. 4

ICA depressed glial cells activation through activating Nrf2 signing. WT and Nrf2 KO mice were treated with ICA (60 mg/kg/d) with a single intranigral injection of 6-OHDA (4 μg) in the SN on the left side of the brain beginning 3 days after ICA treatment. Later, brain sections from WT and Nrf2 KO mice were immunostained with anti-Iba-1 (a) and GFAP (c) antibodies, respectively. The protein expressions of Iba-1 (b) and GFAP (d) in WT and Nrf2 KO mice were measured by western blotting. Results were mean ± SEM from 5 to 6 mice. *p < 0.05 compared with the control group, ^#p < 0.05 compared with 6-OHDA-treated group. Scale bar = 100 μm

Fig. 5

The role of Nrf2 signaling on ICA-affected functions of glial cells. After ICA treatment for 10 days, the protein levels of pro-inflammatory mediators, such as TNF-α and iNOS (a), and the production of neurotrophic factors, such as GDNF and BDNF (b), in the midbrain of WT and Nrf2 KO mice were measured by western blotting. Results were mean ± SEM from 5 to 6 mice. *p < 0.05 compared with the control group, ^#p < 0.05 compared with 6-OHDA-treated group

Fig. 6

ICA targeted Nrf2 in glial cells to produce DA neuroprotection. Primary rat midbrain neuron-enriched and neuron-glia cultures were pretreated with ICA (0.1 μM) for 30 min followed by 6-OHDA (40 μM) stimulation for 7 days. TH protein expression was measured by western blot analysis (a). Primary mixed-glia in transwells were treated with Nrf2-siRNA (40 nM) for 24 h and the silence rate was verified by real-time RT-PCR and western blotting (b). Then, mixed-glial cells after silence were transferred to neuron-enriched cultures. After that the reconstituted neuron-glia co-cultures were treated with ICA and 6-OHDA for 7 days. DA neurons quantification was determined by immunocytochemical staining and western blotting with anti-TH antibody (c and d). Results were mean ± SEM from 3 independent experiments performed in triplicate. *p < 0.05 compared with the control cultures, ^#p < 0.05 compared with 6-OHDA-treated cultures, ^△p < 0.05 compared with 6-OHDA+ICA-treated cultures. Scale bar = 100 μm