Gastrodin injections were purchased from Nanchong Central Hospital (Nanchong, China). Rotenone and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rotenone was dissolved in DMSO and stored at −20°C.

Lewis rats (weight, 200–250 g) were purchased from the Shanghai Laboratory Animal Centre (Chinese Academy of Sciences, Shanghai, China) and maintained in specific pathogen-free conditions. The rats were acclimated and maintained at 23°C under a 12-h light/dark cycle (lights on, 08:00–20:00). Rats were housed in standard laboratory cages with free access to food and water. The rats were randomly divided into experimental (n=6) and control (n=12) groups. The experimental group subcutaneously received rotenone and gastrodin (2.5 and 5.0 mg/kg, respectively, in Panacet) and the control group received Panacet only. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (27) and were approved by the animal care committee of China Medical University (Shenyang, China).

Primary astrocytes were prepared from the brains of neonatal Wistar rats (age, 1–2 days) (28), which were purchased from Shanghai SLAC Laboratory Animal Co. (Shanghai, China). Briefly, the brains were digested with 0.05% trypsin-EDTA at 37°C for 10 min, dissociated by gentle pipetting and passed through a 100-μm-pore nylon mesh. Cells were plated onto 75-cm² plastic flasks and grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% v/v fetal bovine serum and 1% penicillin/streptomycin, at 37°C in a humidified 5% CO₂ atmosphere. The medium was changed every three days. Cells were harvested when they reached 80% confluence and seeded into a secondary culture. The purity of the primary astrocyte cultures was determined by immunocytochemical staining using an antibody against an astrocyte-specific marker, glial fibrillary acidic protein (GFAP; dilution, 1:1000; product number G 3893, Sigma) or a microglia-specific marker (anti-CD11b; dilution, 1:200; Serotec, Oxford, UK). At 30 days in vitro, 99% of the primary cultured cells were GFAP-positive and no detectable CD11b-positive cells (microglia) were identified (29). Cultured astrocytes were treated with rotenone (8 nM) and gastrodin (10 or 20 nM; molecular formula, C₁₃H₁₈O₇; molecular weight, 286.25), or with 8 nM rotenone only for 2 days.

The quantitative FRAP assay for GJIC was performed as previously described (23), using a laser-scanning confocal microscope (LSCM, Olympus Fluoview FV300; Olympus, Ltd., Beijing, China). Following the bleaching of randomly selected cells with a micro-laser beam, the rate of transfer of 5,6-carboxyfluoresceindiacetate (Sigma-Aldrich) from adjacent labeled cells back into the bleached cells was calculated. The recovery of fluorescence was examined after 0.5 min and the recovery rate (RR) was calculated as the percentage of photobleached fluorescence/min. The RR was adjusted for the loss of fluorescence measured in unbleached cells and the results are expressed as the fold increase in the RR compared with that of the untreated control cells (23).

Cells were grown in 6-cm cell culture dishes for ≥48 h. The cells were trypsinized and suspended in DMEM containing 10% fetal calf serum. Total RNA was isolated from the cells using the QIAshredder and RNeasy mini kits (Qiagen, Inc., Almeda, CA, USA). The initial strand of cDNA was synthesized from 500 ng RNA extracts in a volume of 20 μl using AMV reverse transcriptase XL (Takara Biotechnology Co., Ltd., Dalian, China) priming with random nonamer primers (9-mers) at 42°C for 10 min. The cDNA strand was stored at 20°C until use. The expression of Cx43 mRNA was determined by qPCR. PCR was performed in an ABI Prism 7900 sequence detector (Applied Biosystems, Foster City, CA, USA) in a final volume of 20 μl. The PCR mixture contained 10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM dNTP mixture, 0.5 units Ampli Taq gold enzyme (Applied Biosystems) and 0.2 M primers. The primer and probe sequences for gene amplification were as follows: Cx43 forward, 5′-ATCAGCATCCTCTTCAAGTCTGTCT-3′ and reverse, 5′-CAGGGATCTCTCTTGCAGGTGTA-3′ (22); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-CCCTTCATTGACCTCAACTAC-3′ and reverse, 5′-CCACCTTCTTGATGTCATCAT-3′. GAPDH was used as an internal control. The Ampli Taq gold enzyme was activated by heating for 10 min at 95°C, and all genes were amplified by 50 cycles of heating for 15 sec at 95°C, followed by 1 min at 60°C.

For the construction of standard curves for the positive controls, the total RNA of the primary astrocytes was reverse transcribed into cDNA and serially diluted in water in five or six log steps to achieve four-fold serial dilutions of cDNA from ~100 ng to 100 pg. These cDNA serial dilutions were stored at −20°C. The coefficient of linear regression for each standard curve was calculated, and the cycle threshold value of a sample was substituted into the formula for each standard curve to calculate the relative concentration of Cx43 or GAPDH. To normalize the differences in the quantity of total RNA added to each reaction mixture, GAPDH was used as an endogenous control. The data represent the average expression of target genes relative to GAPDH, from three independent cultures.

Cells and rat brains were lysed in ice-cold buffer (50 mmol/l Tris-HCl, pH 7.4; 150 mmol/l NaCl; 1% [v/v] NP40; 5 mmol/l EDTA; 5% [v/v] glycerol; 10 μg/ml leupeptin; 10 μg/ml aprotonin; 1 mmol/l phenylmethylsulfonyl fluoride and 1 mmol/l Na₃VO₄) using a polytron. The lysates were then sonicated, the samples were diluted 1:4 in water and their protein concentrations were determined using the Bradford method (30), with affinity-purified bovine serum albumin as a standard. Samples (10 g) were dissolved in Laemmli sample buffer (60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue), separated on 12% acrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes. Blots were incubated with anti-Cx43 antibody (Shengshizhongfang BioSci and Tech. Co., Ltd., Beijing, China) overnight at 4°C, followed by three 15 min washes with phosphate-buffered saline and 0.1% Triton X-100 (PBST). As an internal control, to determine whether equal amounts of protein had been loaded onto the gel, the PVDF membranes were stripped and reprobed with anti-tubulin (T5168; Sigma-Aldrich). Blots were incubated with goat anti-rabbit antibody conjugated horseradish peroxidase (AP307P, Merck Millipore, Billerica, MA USA). The immunoreactive bands were visualized by enhanced chemiluminescence (ECL; GE Healthcare, Shanghai, China) and quantified by densitometry with ImageJ 1.45 software (National Institutes of Health, Bethesda, USA) according to the manufacturer’s instructions.

The correlation between Cx43 levels and gastrodin and rotenone treatment in the different groups was compared by a one-way analysis of variance followed by post hoc analysis with a protected Fisher’s least significant difference test. P<0.05 indicates a statistically significant difference.

Western blot analysis demonstrated that three forms of the Cx43 immunoreactive protein (Mr 40,000–43,000) were observed in all samples; a fast-migrating band (non-phosphorylated form, P0; Fig. 1) and two slower migrating bands (phosphorylated forms, P1 and P2; Fig. 1). Phosphorylated Cx43 was observed to localize at the plasma membrane and gap junctions (23). Densitometric analysis demonstrated that rotenone induced a significant dose- and time-dependent increase in phosphorylated Cx43 levels compared with that of the control cells (23). The levels of the non-phosphorylated form, P0, appeared to marginally change (Fig. 1). The effect of rotenone on Cx43 protein levels was also determined, and the phosphorylated Cx43 level was demonstrated to be modulated by rotenone treatment. The expression of phosphorylated Cx43 reached high levels when the astrocytes were treated with 8 nm rotenone for 48 h (Fig. 1). However, the enhanced expression level of phosphorylated Cx43 was inhibited by gastrodin. The increased inhibitory rate was correlated with an increasing concentration of gastrodin, and was greatest when 20 nM gastrodin was added (Fig. 1).

Quantification analysis also demonstrated that gastrodin inhibited the rotenone-induced levels of Cx43 expression in astrocytes. The effect of rotenone on Cx43 mRNA levels was also investigated by qPCR. Rotenone treatment was observed to modulate the Cx43 mRNA levels. Following the treatment of astrocytes with 8 nm rotenone for 48 h, the Cx43 mRNA levels increased (Fig. 2). The enhanced mRNA levels of Cx43 were shown to be inhibited by gastrodin. In addition, the increased inhibitory rate was correlated with increasing concentrations of gastrodin; levels of inhibition were greatest following the addition of 20 nM gastrodin (Fig. 2).

The effect of rotenone and gastrodin on GJIC in cultured astrocytes was observed. The GJIC was quantitatively assessed in living cells by a FRAP assay, as previously described (23), in terms of the RR. Following photobleaching, sequential scans detected the recovery of fluorescence in the bleached cells as the dye was transferred from the surrounding non-bleached cells to the photobleached cells through GJIC. The RR at 48 h of treatment showed a dose-dependent increase up to 8 nM rotenone (23). In addition, time course analysis showed a time-dependent increase in GJIC following rotenone treatment (23). The quantity of GJIC was consistent with the expression levels of phosphorylated Cx43 induced by rotenone (Figs. 1 and 3). The results suggested that rotenone treatment of cultured astrocytes generated increased levels of phosphorylated proteins and a broadened membrane distribution of Cx43, which in turn led to the enhancement of GJIC.

By contrast, the concentration of gastrodin was inversely correlated with the levels of GJIC. The data from Fig. 1 suggested that gastrodin treatment of cultured astrocytes generated reduced levels of phosphorylated Cx43, and this in turn led to the reduction in GJIC (Fig. 3). Thus, gastrodin may prevent PD by inhibiting the phosphorylation of Cx43 and reducing the expression of Cx43.

To investigate whether Cx43 levels may be altered in Parkinsonism, the Cx43 protein level in the rotenone-induced model of PD in rats was observed. In this model, Cx43 was identified in all regions (although at different levels) and the Cx43 protein level was significantly lower in the striatum and hippocampus than in the other brain regions (data not shown) (23). The levels of phosphorylated Cx43 were markedly enhanced in the striatum of the treated group. Significant differences in the total Cx43 levels were observed in the striatum of rotenone-treated rats at 1, 2 and 4 weeks, as well as in the hippocampus of rotenone-treated rats at those weeks (P<0.01; Fig. 4). However, no significant changes were observed in other regions (data not shown) (23). The results from Fig. 4 suggested that treatment of the rat model with gastrodin reduced the protein levels of phosphorylated Cx43, which was lower than that of the control group (P<0.01).

By contrast, the concentration of gastrodin was inversely correlated with the expression of phosphorylated Cx43 in the PD model induced by rotenone. The results from Fig. 4 suggested that gastrodin treatment in the rat model generated reduced protein levels of phosphorylated Cx43 (P<0.01), which may be less than those of the control group (Fig. 4). Thus, gastrodin may be used for the prevention of PD by inhibiting the phosphorylation of Cx43 and reducing the expression of Cx43 in the PD model.