ICA (purity > 98%) was purchased from Nanjing Zelang Biological Technology Co., Ltd. (Nanjing, China). 6-OHDA was obtained from Sigma-Aldrich (St. Louis, MO). Sytox green nucleic acid fluorescence stain was bought from Bio-Rad (Hercules, CA, USA). Anti-tyrosine hydroxylase (TH), HO-1, NQO1, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-Kelch-like ECH-associated protein 1 (Keap1), Nrf2, proliferating cell nuclear antigen (PCNA), ionized calcium-binding adapter molecule-1 (Iba-1), glia fibrillary acidic protein (GFAP), inducible nitric oxide synthase (iNOS), TNF-α, and β-actin antibodies were bought from Proteintech Group (Chicago, IL, USA). Nrf2-siRNA and control-siRNA were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Animals and treatment
Male wild type (WT) mice and male homozygous Nrf2 knockout (Nrf2 KO) mice (25–30 g, 8–10 weeks) were purchased from the Model Animal Research Centre of Nanjing University (Nanjing, China). All experimental procedures were carried out in accordance with Chinese Guidelines of Animal Care and Welfare and this study received an approval from the Animal Care and Use Committee of Zunyi Medical University (Zunyi, China). WT and Nrf2 KO mice received intragastric administration with ICA (60 mg/kg) once daily for 10 consecutive days. On the third day, mice were injected stereotactically with 6-OHDA (4 μg, in 0.2% ascorbic acid) in the SN on the left side of the brain with coordinates from Bregma: AP − 2.2 mm, ML 1.4 mm, DV − 4.7 mm . Normal control animals accepted equal volume saline.
The rotarod test was widely used to detect the muscular coordination and balance . Prior the test start, all mice were trained to stay at 0 rpm for a while, then steadily increased to 10 rpm in 30 s and 5 rpm per 30 s until the mice slid off the steps. Animal behavioral activity was detected for 3 repeated trials on 1 day, and the average duration of stay on the rod was recorded.
Open field test
Open field test was performed at the last ICA application for evaluating the levels of anxiety emotionality of animals . Mice were placed on the open field and each mouse was placed in a separate area and its behavioral parameters were recorded during the 5 min. The device was washed with 75% alcohol solution before each next behavioral test in order to eliminate the odors from the previous mouse. The distance of mice in central and peripheral activities was recorded by computer. After the experiment, the total distance of mouse movement was counted.
Ultra-high-performance-liquid chromatography (UHPLC) analysis
Mouse striatum levels of DA, DOPAC, and HVA were detected by Agilent 1290 Infinity II series UHPLC System (Agilent Technologies). Striatum tissues were sonicated in extract solvent (acetonitrile-methanol-water, 2:2:1, 2% formic acid). The homogenate was centrifuged, and a 100 μl aliquot of the clear supernatant was transferred to an auto-sampler vial for UHPLC-MS/MS analysis. The mobile phase A contained 10 mmol/L ammonium acetate/0.1% formic acid, and the mobile phase B was acetonitrile.
Thirty-five-micrometer-thick brain sections stained with the corresponding antibodies. Briefly, brain slices were incubated with 0.3% Triton X-100 and closed with goat serum. Subsequently, brain slices concentrated with anti-TH (1:500), Iba-1 (1:200), and GFAP (1:300) antibodies at 4 °C overnight, respectively. Then, the slices were incubated with goat anti-rabbit antibody (green) secondary antibodies (1:1500) for 1 h. Digital images of TH-positive neurons, Iba-1-positive microglia, and GFAP-positive astroglia were obtained via Olympus microscope (Olympus, Tokyo, Japan). DA damage was assessed through the quantification of TH-positive neurons.
Nuclear and cytosol fractions were extracted from the mice midbrain tissues with Nuclear-Cytosol Extraction Kit (Solarbio, Beijing, China) following the manufacture’s protocols. Total proteins were isolated by RIPA lysis solution (Solarbio, Beijing, China). Protein levels were quantified using BCA assay. Equal amounts of protein (10–30 μg/lane) were separated on 10% Bis-Tris Nu-PAGE gel. Then, the membranes were incubated with primary antibodies: TH (1:2000), Iba-1 (1:800), GFAP (1:1000), Nrf2 (1:1500), Keap1 (1:2000), HO-1 (1:10000), NQO1 (1:2000), PCNA (1:2000), TNF-α (1:800), iNOS (1:1500), GDNF (1:800), BDNF (1:1000), and β-actin (1:4000). Next day, the membranes were incubated with anti-rabbit IgG secondary antibodies at 1:5000 for 1 h. Finally, the blots were detected using ECL substrate. β-actin was used as an internal standard to monitor loading errors. Densitometric analysis of immunoblots was performed using the Quantity One (Bio-Rad, Hercules, CA, USA) software system. The ratio of densitometry values of purpose protein with β-actin was analyzed and normalized to each respective control groups.
Real-time RT-PCR assay
The total RNA of mouse midbrain tissues was prepared using RNeasy kit and the detailed steps of Real-time RT-PCR were described previously . Nrf2, Keap1, HO-1, NQO1, and β-actin genes were tested. Accordingly, the genes’ expression was normalized with β-actin. The primer sequences were as follows.
Forward primer (5′-3′)
Reverse primer (5′-3′)
Primary rat midbrain neuron-glia and neuron-enriched cultures
Female pregnant SD rats (250-300 g) were purchased from the Experimental Animal Center of the Third Military Medical University (Chongqing, China). Primary midbrain neuron-glia cultures were prepared from the ventral mesencephalic tissues of embryonic rats 14–15 days age . Then, the rat whole brains were dissected and the midbrain were isolated. After excision of blood vessels and meninges, the midbrain tissues were mechanically triturated and the dissociated cells were planted in poly-d-lysine-coated 24-well plates or 96-well plates. Seven-day-old cultures were employed for drug treatment. During treatment, immunocytochemical staining demonstrated that primary neuron-glia co-cultures composed of 50% astrocytes, 10% microglia, 40% neurons, and 1% DA neurons. In addition, midbrain neuron-enriched cultures were prepared by adding cytosine β-D-arabinofuranoside (8 μM) in primary neuron-glia cultures to inhibit glial cells proliferation . ICA treatment was performed with 7 days of cultures followed by 6-OHDA (40 μM) intervention .
Primary rat mixed-glia cell cultures
Primary rat mixed-glia cultures were prepared from the whole brains of 1-day-old rat pups . The meninges and blood vessels were isolated, the brain tissues were homogenized and the cells (2 × 106/well) were planted in a poly-d-lysine-coated 6-well plate. Seven-day-old mixed-glial cells were cultured for drug treatment.
Primary mixed-glia cells were transfected with Nrf2-siRNA (40 nmol/L) or control-siRNA (40 nmol/L) for 24 h. Western blotting and real-time RT-PCR were used to test the transfection rate.
Neuron-glia reconstituted cultures using transwell
First, primary neuron-enriched cultures were planted in 24-well culture plates. Mixed-glia cultures were planted in the transwell. Seven days later, the mixed glial cells in transwell were processed by Nrf2-siRNA for 1 day. Then, the mixed glial cells were transferred to 7-day neuron-enriched cultures within fresh medium. Accordingly, the reconstituted neuron-glia cultures were treated with ICA and 6-OHDA for 7 days. DA neurons damage was assessed by DA neuronal quantification and TH protein expression detection.
Cells were fixed with 4% paraformaldehyde followed by permeabilization using Triton X-100 and closed with goat serum. DA neurons were labeled with anti-TH (1:500) antibody at 4 °C overnight and then incubated with anti-rabbit-IgG (1:1500) antibody for 1 h. TH-positive neurons numbers were calculated from three wells, and three randomly selected areas were analyzed for each group.
All data were presented as mean ± SEM. Statistical comparisons were analyzed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA) by one-way ANOVA. After ANOVA expressed significant differences, the Bonferroni’s post hoc test was used for all pairwise comparisons among means. Statistical significance was considered as p < 0.05.