Glutamine protects against oxidative stress injury through inhibiting the activation of PI3K/Akt signaling pathway in parkinsonian cell model

1-Methyl-4-phenylpyridinium (MPP⁺) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and the purity of MPP⁺ was ≥ 97%. LY294002 (PI3K/Akt inhibitor) and glutamine (Gln) also were obtained from Sigma-Aldrich, and the LY294002 was used at a concentration of 20 μM. IGF-1 was purchased from Apexbio Technology LLC (Houston, TX, USA) and used at a concentration of 100 ng/ml. Additional reagents employed in the present study were commercially available and of analytical purity.

Cells of the rat pheochromocytoma tumor cell line PC12 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). PC12 cells were cultured in RPMI-1640 medium (without glutamine; Procell Life Science & Technology Co., Ltd., Wuhan, China) supplemented with 10% fetal bovine serum (Clark Bioscience, MD, USA), 5% horse serum (Hyclone, UT, USA), and 1% penicillin–streptomycin (Hyclone, UT, USA) at 37 °C in a humidified atmosphere containing 5% CO₂.

PC12 cells were suspended and cultured in 96-well plates at a density of 6 × 10³/well. Then, cells were cultured in 10% Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) diluted in fresh medium for 1 h. The absorbance value was calculated by using a microplate reader (Thermo Fisher Scientific, MA, USA).

PC12 cells were suspended and seeded onto chamber slides in six-well plates at a density of 2 × 10⁵/well and incubated for 48 h. Following treatment, the cells were washed with phosphate buffered saline three times and fixed in 4% paraformaldehyde for 20 min. Cell nuclei were stained with acridineorange (AO) and ethidium bromide (EB) for 5 min. The images of the cells were captured under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

The harvested cells were washed with phosphate buffered saline and lysed with lysis buffer (Boster, Wuhan, China) to obtain total cellular protein. Protein concentration was determined by using BCA Protein Assay kit (Boster, Wuhan, China). The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF; Millipore Corporation, MA, USA) membrane through a Bio-Rad II System (Bio-Rad Laboratories, Inc.). Then, the membranes were sealed with 5% skimmed milk powder at room temperature for 1 h and incubated with rabbit monoclonal antibody against PI3K, P-Akt, Akt, P-mTOR, mTOR, and β-actin (1:1000; Cell Signaling Technology, MA, USA) at 4 °C overnight and goat anti-rabbit IgG at room temperature for 1 h. β-actin was used as inner loading control. The epitope was visualized by an ECL detection reagent (Millipore Corporation, MA, USA) according to the manufacturer’s instructions. The gray value was analyzed by Image-ProPlus software (Media Cybernetics, Inc., MD, USA).

The harvested cells were washed with phosphate buffered saline twice and digested with trypsin. Following the cells was disrupted by an ultrasonic cell disruptor at 4 °C and the lysate was centrifuged at 1000 r/min at 4 °C for 10 min. A total of 100 μl supernatant was obtained to detect the OD values using a microplate reader according to the instructions of SOD, GSH-Px, and MDA kit, and the activity and content were calculated, respectively.

In order to study the cytotoxicity of MPP⁺ on PC12 cells, the cells were treated with various concentrations of MPP⁺ for 24, 48, and 72 h and the inhibition of cell proliferation was detected via a CCK-8 assay. As shown in Fig. 1a, MPP⁺ decreased the viability of PC12 cells in a dose- and time-dependent manner, with IC₅₀ values of 1552.21 ± 125.38, 227.91 ± 23.08, and 109.82 ± 13.02 μM at 24, 48, and 72 h, respectively. Thus, 227 μM (M0) and 48 h were selected as the intervention concentration and time of MPP⁺ in subsequent experiments. To determine whether the cytotoxicity of MPP⁺ against PC12 cells induces apoptosis, the present study analyzed nuclear morphological changes by AO/EB staining following 48 h of treatment with MPP⁺. As shown in Fig. 1b, the PC12 cells exhibited karyopyknosis and were notably stained with red following treatment with MPP⁺, the viable cells exhibited uniformly green. These results indicated that MPP⁺ could increase the apoptosis of PC12 cells in a dose-dependent manner.

To evaluate whether glutamine has protective effect on MPP⁺-induced PC12 cell injury, cells were pre-treated with different concentrations of glutamine for 1 h, followed by incubation with MPP⁺ (227 μM) for 48 h. As shown in Fig. 2a, the viability of PC12 cells was significantly increased following pre-treatment with glutamine, and glutamine at 64 μM had the most significant protective effect on PC12 cells. Additionally, AO/EB staining demonstrated that glutamine combined with MPP⁺ could decrease cell apoptosis, indicating that glutamine plays a protective role against MPP⁺-induced PC12 cell apoptosis (Fig. 2b).

As shown in Fig. 3, compared with the control group, the activity of SOD and GSH-Px was significantly decreased, while the content of MDA was markedly increased in the M0 group. Compared with the M0 group, the G0+M0 group displayed increased SOD and GSH-Px activity and reduced MDA content. These results indicated that glutamine could improve the antioxidant capacity of a PD cell model.

The western blot results indicated that the MPP⁺ could upregulate the expression levels of PI3K, P-Akt, Akt, P-mTOR, and mTOR, compared with the control group (Fig. 4). The expression levels of PI3K, P-Akt, Akt, P-mTOR, and mTOR in the M0+G0 group were significantly decreased compared with those in the M0 group, while markedly increased compared with the control group. These results suggested that glutamine could inhibit the activation of PI3K/Akt signaling pathway, but cannot reverse it.

To elucidate whether PI3K/Akt/mTOR signaling pathway involved in glutamine protects PC12 cell against oxidative stress, LY294002 and IGF-1, PI3K/Akt signaling pathway inhibitor and agonist were used, respectively. As shown in Fig. 5a, b, the protein expression levels of PI3K, P-Akt, and Akt were significantly decreased in the LY294002 group and significantly increased in the IGF-1 group, compared with those in the control group, indicating that the PI3K/Akt/mTOR signaling pathway was successfully inhibited by LY294002. Compared with the control group, SOD and GSH-Px activity was significantly decreased, while MDA content was markedly increased in the M0 group. Compared with the M0 group, the activity of SOD and GSH-Px was significantly increased, while MDA content was markedly decreased in the M0+G0 and M0+LY294002 groups, indicating that both glutamine and LY294002 could decrease the oxidative stress level of PC12 cells. Compared with the M0 group, the SOD and GSH-Px activity was significantly decreased, while MDA content was markedly increased in the M0+IGF-1 group, indicating that the activation of PI3K/Akt/mTOR signaling pathway could increase the oxidative stress level of PC12 cells. Compared with the M0+G0 group, the SOD and GSH-Px activity was significantly increased in the M0+G0+LY294002 group and decreased in the M0+G0+IGF-1 group; MDA content was the opposite, indicating that the inhibition of the PI3K/Akt/mTOR pathway can enhance the antioxidant capacity of glutamine in the PD cell model (Fig. 5c, d).