Icariin protects against sodium azide—induced neurotoxicity by activating the PI3K/Akt/GSK-3β signaling pathway

Reagents

Reagent grade ICA (purity ≥ 98% by HPLC analysis) was obtained from Nanjing Zelang Medical Technology Corporation Ltd. (Nanjing, China) and dissolved in dimethyl sulfoxide (DMSO); the final concentration of DMSO in the media was less than 0.1% (v/v). NaN₃ (A0639) was purchased from Amresco (Solon, OH, USA). RIPA buffer (high) (R0010) and protein phosphatase inhibitor (P1260) were purchased from Solarbio Life Science (Beijing, China). A glucose oxidase assay kit (E10160) and antibodies against GSK-3β (9315), p-Ser9-GSK-3β (9323) and p-PI3K (p85) (4228) were obtained from Cell Signaling Technology (Beverly, MA, USA). Goat anti-mouse IgG-HRP (SA00001-1), goat anti-rabbit IgG-HRP (SA00001-2), and antibodies against GAPDH (60004-1-Ig), and PI3K p110α (21890-1-AP) were obtained from Proteintech Group (Wuhan, China). Antibodies against PHF1 (ab184951) and PI3K (ab86714) were obtained from Abcam (Cambridge, MA, USA). PageRuler prestained protein ladder (26616) and antibodies against p-T217 (44-744) and TAU-5 (MA5-12808) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). RPMI 1640 HyClone™ cell culture medium (SH30809.01) was purchased from GE Healthcare (Chicago, IL, USA).

Cell culture and treatment

Rat adrenal pheochromocytoma PC12 cells were purchased from the American Type Culture Collection (Rockville, MD, USA).

The cells were cultured in RPMI 1640 medium supplemented with 10% horse serum (16050-122; Gibco™, Carlsbad, CA, USA), 5% FBS (16000-044; Gibco™, Carlsbad, CA, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml) (P1400; Solarbio™, Beijing, China) and maintained at 37 °C and 5% CO₂. The PC12 cells (1.5 × 10⁵ cells/mL) were plated overnight at 37 °C for 24 h. The cells were pretreated with ICA for 2 h and thereafter exposed to 50 mM NaN₃ (dissolved in saline) for an additional 24 h. Then, the cells were subjected to subsequent experiments and assays.

Cell viability determination

Cell viability was detected by CCK-8 assay (CA1210; Solarbio™, Beijing, China); which uses (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), to produces a water-soluble formazan dye upon bioreduction in the presence of an electron carrier. Briefly, PC12 cells (1.5 × 10⁵ cells/mL) were seeded in each well of a 96-well plate for 24 h. After the end of the treatments, CCK-8 solution (10 μL) was added to each well of the 96-well plate and incubated for 2 h at 37 °C. The absorbance was measured at 450 nm with an automatic microplate reader (Multiskan™ GO, Waltham, MA, USA).

Measurement of lactate dehydrogenase release

The effects of ICA on the LDH leakage rate in NaN₃-induced PC12 cells were detected by an LDH (C0016; Beyotime™, Beijing, China) assay kit. Briefly, according to the manufacturer’s instructions, after treatment, the supernatant of each well of a 96-well plate was collected. The positive control showing total release (100% LDH release) was treatment of cells with an LDH release agent (C0016-1). The optical density was measured at 490 nm with an automatic microplate reader (Multiskan™ GO, Waltham, MA, USA).

Detection of mitochondrial membrane potential

MMP was measured by using a JC-1 (a fluorescent cationic probe) kit (C2006; Beyotime™, Beijing, China). Briefly, PC12 cells (1.5 × 10⁵ cells/mL) were seeded in 24-well plates, and after treatment, the cells were washed with PBS three times. A total of 0.5 ml culture medium and 0.5 ml JC-1 staining solution were added and incubated at 37 °C for 20 min. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as the control of MMP decrease (Dong et al., 2016). The cells were rinsed with binding buffer twice and observed under an inverted fluorescence microscope (IX73; Olympus™, Shinjuku City, Tokyo, Japan).

Glucose consumption rate

The concentration of glucose was detected using the glucose oxidase method (Li et al., 2015). The assay kit (E1010; Applygen Technologies, Beijing, China) was purchased from Applygen Technologies (Beijing, China). After treatment, the PC12 cells were collected, and the concentration of total protein was extracted using a total protein extraction kit (P1250; Applygen, Beijing, China) and detected using a BCA protein assay kit (P0012; Beyotime, Beijing, China). Then, according to the instructions, the absorbance was measured at 570 nm with an automatic microplate reader and calculated against a glucose standard curve.

Western blot assay

After 24 h of treatment with different concentrations of ICA or 50 µM NaN₃ at 37 °C, the cells were harvested and lysed with RIPA lysis buffer, followed by centrifugation at 14,000 rpm at 4 °C for 10 min. The total protein concentration was measured by BCA protein assay kit (P0012; Beyotime, Beijing, China). Then, the proteins were heat-denatured at 100 °C for 5 min. Equal amounts of total protein (15 µg per lane) were loaded. And we used 8–12% SDS-PAGE to separate the proteins. The gel was run at 60 V for the stacking gel and 100 V to separate the proteins until the dye ran off the bottom of the gel. Then, the sandwich was transferred to polyvinylidene difluoride (PVDF) membranes for western blot procedure at 4 °C in 1× buffer at 220 mA for 90 min. Following three washes with TBST. Then, 5% nonfat milk was used for blocking. Then, the cells were incubated with the following primary antibodies at 4 °C for 12 h: anti-PI3K-110α (1:1,000), anti-p-PI3K (p85) (1:1,000), anti-PI3K (1:1,000), anti-Akt (1:1,000), anti-p-Ser473-Akt (1:1,000) anti-p-GSK-3β-Ser9 (1:1,000), anti-GSK-3β (1:1,000), anti-PHF-1 (1:5,000), anti-p-T217 (1:1,000) and anti-TAU-5 (1:200). Following three washes with TBST, the membranes were incubated with secondary antibody goat anti-rabbit IgG-HRP (1:2,000) at room temperature for 1 h. Membranes were developed using hydrogen peroxide and Supersignal West Pico Luminol (Pierce; Thermo Fisher Scientific, Waltham, MA, USA). Finally, the membranes were visualized using chemiluminescence reagent ECL Plus (E003-100; 7Sea Biotech, Shanghai, China).

Statistical analysis

All data are expressed as the mean ± SEM. The data were analyzed statistically by SPSS 22.0 statistics software via one-way ANOVA. When ANOVA test results for all data were significant, post hoc least significant difference (LSD) tests were used to determine individual differences. Statistical significance was set as P < 0.05.

ICA attenuated NaN₃-induced damage in PC12 cells

Cell viability was detected by CCK-8 assay. First, based on our previous study (Huang et al., 2019) and OD/A450 of approximately 0.8, the appropriate cell seeding concentration was 1.5 × 10⁵ cells/mL (Fig. 1A). Then, we determined that ICA (0.01–5 μM) had no significant toxic effects on PC12 cells, except for 10 μM ICA for 24 h (Fig. 1B). Combined with previous studies by our laboratory, ICA at concentrations of 0.01–1 μM was used in the subsequent experiments. As shown in Fig. 1C, NaN₃ decreased cell viability, and the viability of PC12 cells decreased by approximately 50% compared with that of the control at a final concentration of 50 mM. Furthermore, we investigated whether ICA protected PC12 cells from NaN₃-induced reductions in cell viability. The WST-8 contained in the CCK-8 reagent is reduced to a highly water-soluble yellow formazan product by dehydrogenase found in the mitochondria of living but not dead cells. Therefore, cell viability can be indirectly determined by detecting the absorbance of the lysed formazan. The results indicated that pretreatment with ICA reduced NaN₃-induced cell damage (Fig. 1D).

Effect of ICA on NaN₃-induced LDH release in PC12 cells

We found that ICA pretreatment prevented NaN₃-induced morphological damage (Figs. 2A–2E). LDH leakage is an indicator of cell membrane integrity. When the cell membrane is damaged, intracellular LDH is released into the cell culture medium. Therefore, the leakage rate of LDH reflects the level of cell damage. The results showed that LDH release was higher in NaN₃-treated cells than in control cells, while ICA significantly reduced the LDH leakage rate in PC12 cells (Fig. 2F).

Effect of ICA on the NaN₃-induced change in MMP in PC12 cells

The change in MMP was assessed by using JC-1. The change in MMP is detected by the change from red fluorescence to green fluorescence. As shown in Fig. 3, NaN₃-treated cells showed a decrease in MMP in comparison with that of the control, which was consistent with the CCCP group. ICA pretreatment increased red fluorescence and decreased green fluorescence. These results suggest that ICA pretreatment increases the MMP of PC12 cells.

ICA ameliorated the NaN₃-induced reduction in the glucose consumption rate in PC12 cells

Mitochondria are cellular power plants and the energy required for the activity of neurons is mainly provided by the glucose metabolism of brain mitochondria. To investigate the glucose metabolism level, we examined the glucose content in PC12 cells. The NaN₃-administered group exhibited a reduction in glucose consumption compared with that of the control group, leading to an increase in glucose concentration. However, ICA pretreatment ameliorated this effect (Fig. 4).

ICA activated the PI3K/Akt/GSK-3β signaling pathway in PC12 cells

To determine whether the PI3K/Akt/GSK-3β signaling pathway is involved in the protective effects of ICA against NaN₃-induced neuronal damage, we examined the expression of related proteins. As shown in Fig. 5, the protein levels of p-PI3K (p85), PI3K-110α, p-Ser473-Akt and p-Ser9-GSK-3β were markedly decreased in PC12 cells after NaN₃ treatment for 24 h, whereas these effects were reverted after pretreatment with ICA. These results suggest that the protective effect of ICA on NaN₃-induced mitochondrial dysfunction is related to the PI3K/Akt/GSK-3β signaling pathway (Fig. 5).

ICA reduced the levels of tau phosphorylation

Excessive activation of GSK-3β promotes abnormal hyperphosphorylation of Tau. Therefore, we detected the phosphorylation level of Tau in PC12 cells at PHF-1 (identified Ser396/404 sites) and p-T217. As shown in Fig. 6, Tau phosphorylation at the Ser396/404 and Thr217 sites was significantly higher in NaN₃-treated cells than in control cells. In contrast, the phosphorylation of Tau at Ser396/404 and Thr217 was decreased by pretreatment with ICA.