Cornin (purity >99.0%; CAS no. 548-37-8; molecular formula, C₁₇H₂₄O₁₀; molecular weight, 388.37) was provided by Shandong Engineering Research Center for Nature Drug (Shangdong, China) was dissolved in sterile physiological (0.9%) saline to make a stock solution. Dilutions were prepared according to the different administration doses.

The Akt (cat. no. 8805; 1:1,000 dilution), phosphorylated (p)-Akt (cat. no. 38449; 1:500 dilution), mTOR (cat. no. 2732; 1:2,000 dilution), p-mTOR (cat. no. 109268; 1:1,000 dilution), apoptosis regulator Bcl-2 (cat. no. 32124; 1:1,000 dilution), apoptosis regulator BAX (Bax) (cat. no. 32503; 1:1,000 dilution), β-actin (cat. no. 8226; 1:500 dilution), microtubule-associated proteins 1A/1B light chain 3B (LC3) (cat. no. 128025; 1:1,000 dilution) and Beclin-1 (cat. no. 62557; 1:2,000 dilution) antibodies were purchased from Abcam (Cambridge, UK). The following pharmacological agents were used: The PI3K/Akt inhibitor LY294002 and the mTOR inhibitor rapamycin (both Merck KGaA, Darmstadt, Germany).

The human neuroblastoma SH-SY5Y cell line was obtained from the Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences (Beijing, China). Short-tandem repeat analysis was performed by Shanghai Saily Biotechnologies Co., Ltd. (Shanghai, China) to ascertain that the cell line used in the present study was of human origin (sample no. 20160921-01). SH-SY5Y cells were maintained in Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences) and antibiotics (100 U/ml penicillin G and 100 µg/ml streptomycin; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) in a humidified atmosphere of 5% CO₂ at 37°C. The control group was treated with dimethyl sulfoxide (DMSO). In order to study the mechanism of the effect of cornin on autophagy, SH-SY5Y cells were incubated with cornin (9 µM) for 24 h prior to OGD, followed by 10 µM LY294002 or 10 µM rapamycin for 6 h.

In order to simulate OGD in vitro, SH-SY5Y cells were incubated in a hypoxia solution for 6 h. The hypoxia solution contained 0.9 mM NaH₂PO₄, 6.0 mM NaHCO₃, 1.0 mM CaCl₂, 1.2 mM MgSO₄, 40 mM sodium lactate, 20 mM HEPES, 98.5 mM NaCl and 10.0 mM KCl (pH adjusted to 6.8), and was bubbled with N₂ for 30 min prior to application. The O₂ pressure of the hypoxia solution was adjusted to 64.0 kPa. Hypoxic conditions were produced by placing the plates of cultured SH-SY5Y cells in a hypoxic incubator (Kendro; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with the oxygen adjusted to 1.0% and the CO₂ to 5.0%. Prior to hypoxia, SH-SY5Y cells were pretreated with various concentrations (3, 9 and 27 µM) of cornin for 24 h. Normal culturing (DMEM containing 2% FBS under 20% oxygen and 5% CO₂) served as the negative control, and the hypoxia solution culture served as the control.

SH-SY5Y cells were incubated with or without cornin in the hypoxia solution for 6 h and cell viability was assessed using an MTT assay. MTT solution (5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added into the cells and the cells were incubated for 4 h in 37°C. Following the removal of the medium, DMSO was added to dissolve the blue-colored formazan product. The absorbance was measured at a wavelength of 490 nm to determine the optical density value of each well. LDH, an indicator of cellular injury, was detected according to manufacturer's protocol of the LDH assay kit (Beijing Zhongsheng Bioreagent, Beijing, China). The formula LDH leakage rate (%)=Ae/At ×100 was used, in which Ae indicated extracellular LDH (cell culture fluid) and At indicated intracellular and extracellular LDH (cell lysate).

SH-SY5Y cells were cultured for 24 h, washed twice with ice-cold PBS and lysed in NP40 lysis buffer (BioSource International, Inc., Camarillo, CA, USA) (50 mM Tris, pH 7.4; 250 mM NaCl; 5 mM EDTA; 50 mM NaF; 1 mM Na₃VO₄; 1% NP-40; and 0.02% NaN₃) supplemented with 1 mM phenylmethylsulfonyl fluoride and 1X protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Equal amounts of cellular protein (40 µg) were separated by 12 or 10% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% (w/v) skimmed milk for 2 h at room temperature. The membranes were then incubated with the following specific antibodies: Anti-Akt (cat. no. 8805; 1:1,000 dilution), anti-p-Akt (cat. no. 38449; 1:500 dilution), anti-mTOR (cat. no. 2732; 1:2,000 dilution), anti-p-mTOR (cat. no. 109268; 1:1,000 dilution), anti-LC3 (cat. no. 128025; 1:1,000 dilution), anti-beclin-1 (cat. no. 62557; 1:2000 dilution), anti-Bcl-2 (cat. no. 32124; 1:1,000 dilution), anti-Bax (cat. no. 32503; 1:1,000 dilution), anti-caspase-3 (cat. no. 13847; 1:1,000 dilution) and anti-β-actin (cat. no. 8226; 1:500 dilution) as a loading control at 4°C overnight. Subsequently, they were incubated for 2 h at room temperature with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat no. A0208; 1:5,000 dilution; Beyotime Institute of Biotechnology). Protein bands were visualized using enhanced chemiluminescence regent (EMD Millipore, Billerica, MA, USA). The optical densities of the bands were scanned and quantified using a Gel Doc2000 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Data were normalized against those of the corresponding β-actin bands. Results are expressed as a fold increase compared with the control.

All of the experiments were performed in triplicate. Quantitative data from experiments are expressed as the mean ± standard deviation. Significance was determined by one-way analysis of variance followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

The cell viability of OGD-treated SH-SY5Y cells was markedly decreased compared with the normal cultured cells. However, the cell viability of OGD-treated cells was markedly increased following treatment with cornin (3–27 µM), as presented in Table I. In order to further investigate the protective effect of cornin, the LDH leakage rate was estimated. A significant increase in the LDH leakage rate in SH-SY5Y cells was observed following OGD. Incubation with various concentrations of cornin significantly inhibited the OGD-induced LDH release in a concentration-dependent manner.

The Bcl-2 and caspase families are the important mediators of apoptosis. Bcl-2, caspase-3, and Bax protein levels were determined to elucidate whether cornin was able to protect SH-SY5Y cells from OGD-induced apoptosis. The protein levels of Caspase-3 and Bax were significantly increased, and Bcl-2 was decreased, by treatment with OGD, compared with normal cells. However, cornin increased Bcl-2, and decreased Bax and caspase-3 levels significantly in OGD-treated cells (Fig. 1).

In order to determine the protective mechanisms of cornin in OGD-treated cells, the present study analyzed the effects of cornin on autophagy in the protection of SH-SY5Y cells. Previous studies have demonstrated that autophagy activation is involved in ischemic stroke (19,20); however, the role of cornin in regulating autophagy in ischemic stroke has not been clearly defined. In the model used in the present study, the regulatory effect of cornin on LC3 and beclin-1, which are frequently used as indicators of autophagy, was investigated. LC3 and beclin-1 expression in the OGD model were detected by western blot analysis. As presented in (Fig. 2), beclin-1 and the ratio of LC3-II/LC3-I increased in the OGD model compared with the control group. This effect was significantly inhibited by treatment with cornin for 24 h prior to OGD, in a concentration-dependent manner.

Previous studies demonstrated that cornin inhibited OGD-induced cell damages by activating PI3K/Akt signaling (16,18). Therefore, the present study aimed to further examine whether cornin may affect OGD-induced autophagy through the Akt/mTOR pathway. SH-SY5Y cells were treated with 3, 9, or 27 µM cornin, and control groups were treated with DMSO. A total of 24 h subsequently, the phosphorylation levels of Akt and mTOR were examined using western blotting. The results demonstrated that cornin was able to upregulate the protein levels of p-Akt, and p-mTOR under OGD, as presented in Fig. 3.

To investigate whether cornin affected OGD-induced autophagy via the PI3K/Akt/mTOR signaling pathway, the PI3K/Akt inhibitor LY294002 and the mTOR inhibitor rapamycin were used in western blotting. The protein expression levels of p-Akt, p-mTOR, LC3-II/LC3-I and beclin-1 were decreased by treatment with LY294002. Rapamycin increased mTOR phosphorylation, and increased LC3-II/LC3-I and beclin-1 protein expression, as presented in (Fig. 4). These results of the present study indicated that cornin reduced OGD-induced autophagy by affecting Akt and mTOR phosphorylation levels.