PC12 cells were obtained from the American Type Cell Culture Collection (ATCC; Manassas, VA, USA) and were maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and streptomycin) in a humidified incubator at 37°C with an atmosphere of 5% CO₂ and 95% air (15). Cells were supplied with fresh medium three times per week and were passaged at a 1:3 ratio twice per week.

Adenoviral vectors expressing SH2B1 (Ad-SH2B1) or green fluorescent protein (Ad-GFP) were synthesized and provided by Shanghai GeneChem, Inc. (Shanghai, China). Sequences were obtained from https://blast.ncbi.nlm.nih.gov/Blast.cgi (Accession no. 89817). In brief, the entire coding region of target gene SH2B1 was cloned into shuttle vector GV135 (Shanghai GeneChem, Inc.). The co-transfection of 293T cells (ATCC) with a SH2B1 recombinant shuttle vector and an auxiliary packaging plasmid (Microbix Biosystems Inc., Toronto, ON, Canada) was performed using an AdMax Adenovirus Packaging system (AdMax Local, Los Angeles, CA, USA) according to the manufacturer's protocol (16). By applying Cre/loxP enzymes, the recombinant adenovirus Ad-SH2B1 and the Ad-GFP negative control were obtained. Subsequently, adenoviruses were amplified, purified by an Adeno-X™ Virus Purification kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocols, and routinely titrated to 1×10¹⁰ PFU/ml.

For viral transfection, PC12 cells (1×10⁵ cells per 24-well plate) were incubated with Ad-SH2B1 or Ad-GFP for 4 h at 37°C, prior to glucose deprivation as described below, at different multiplicities of infections as calculated via: Viral titer × viral volume/cell number (0, 10, 20, 40 and 80 MOI), and finally, 20 MOI was selected for the following experiments due to ~95% transfection efficiency with no significant effects on cell viability as determined by flow cytometry described below. After 4 h of incubation at 37°C, medium was removed, and PC12 cells were further incubated at 37°C with complete medium (DMEM+FBS) for 48 h prior to OGD/R treatment (17). Each experiment was repeated in triplicate.

To determine the association between SH2B1 and OGD/R-induced neuronal impairment, PC12 cells were randomly allocated to four groups: i) Control group (untransfected, no OGD/R); ii) OGD/R group; iii) Ad-SH2B1 transfection plus OGD/R (Ad-SH2B1 + OGD/R); and iv) Ad-GFP transfection plus OGD/R (Ad-GFP + OGD/R). To further detect the underlying mechanisms of the anti-OGD/R effects conferred by SH2B1 in PC12 cells, an additional experiment was conducted in which Ad-SH2B1 or Ad-GFP was transduced into PC12 cells as aforementioned in the absence or presence of a JAK2 inhibitor (AG490, 10 µM, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). These cells were then subjected to OGD/R treatment as described (17). The inhibitor was added into the medium 30 min at 37°C prior to the OGD procedure (17).

To mimic the I/R procedure in vitro, oxygen deprivation was induced in an anaerobic chamber containing 95% N₂ and 5% CO₂ at 37°C, and glucose deprivation was concurrently induced by maintaining cells in a glucose-free Hanks' Balanced Salt Solution (Invitrogen; Thermo Fisher Scientific, Inc.) for 6 h at 37°C. Subsequently, PC12 cells from the OGD/R-treated groups were removed from the anoxia incubator and cultured under normal conditions as aforementioned to induce reoxygenation for an additional 24 h (17,18).

Cell injury was assessed by measuring the content of lactate dehydrogenase (LDH) in the culture medium using an LDH diagnostic kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol (17). LDH activity was then calculated using a microplate spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at a wavelength of 440 nm. Alternations in absorbance were expressed as concentration units per liter (17).

Cell viability was measured using a Cell Counting kit-8 (CCK-8) assay according to the manufacturer's protocol (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) (15). Briefly, PC12 cells from all four groups were seeded into 96-well plates (1×10⁴ cells/well) and were washed with PBS.CCK-8 solution (10 µl) was then added to each well for an additional 4 h at 37°C. The absorbance at 450 nm was determined with a microplate reader (Biotek Instruments, Inc., Winoski, VT, USA). Cell viability was expressed as a percentage of the control group cells.

The apoptotic rate was detected by flow cytometry with an apoptosis detection kit (BD Pharmingen; BD Biosciences) (15). Briefly, following the corresponding interferences, cells were digested by 0.25% trypsin, collected from 24-well culture plates, washed twice with PBS and centrifuged at 20,000 × g at 4°C for 5 min. Then, PC12 cells from all four groups were incubated with 5 µl of 7-aminoactinomycin D (7-AAD) dye at room temperature for 15 min. Subsequently, 1 µl Annexin V-allophycocyanin (APC) was added into the cell suspension, and maintained for 15 min in the dark. Finally, cells were analyzed by fluorescence-activated cell sorting via a flow cytometric analysis (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and CytExpert 1.0 software (Beckman Coulter, Inc., Brea, CA, USA). Cells that stained positively for Annexin V-APC or 7-AAD were considered as apoptotic cells.

RT-qPCR was performed to detect mRNA expression as previously described (15,17,18). Briefly, total RNA from all four PC12 cells group was extracted and purified with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and reverse-transcribed into cDNA with a commercial PrimeScript™ II 1st strand cDNA synthesis kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. RT-qPCR was performed with an ABI Prism 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in the presence of primers and SYBR^® Green Supermix (Bio-Rad Laboratories, Inc.). The following thermocycling conditions were used for the PCR: Initial denaturation at 50°C for 2 min; 40 cycles of denaturation at 95°C for 30 sec; annealing at 56°C for 30 sec; and extension at 72°C for 30 sec. The mRNA expression levels of targeted genes were normalized to the reference gene β-actin, and calculated using the comparative quantification method (2^−ΔΔCq) (19). The primer sequences used in the present study are listed as follows: SH2B1, forward 5′-CCCGTCTTAGCATTCCCTGT-3′ and reverse, 5′-GGCTTAGGCACTCTTGGATGT-3′; β-actin, forward 5′-CACGATGGAGGGGCCGGACTCATC-3′ and reverse, 5′-TAAAGACCTCTATGCCAACACAGT-3′.

Western blotting was performed to measure protein expression as previously described (15,17,18). In brief, PC12 cells of all four groups were lysed with ice-cold radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Jiangsu, China), supplemented with 1 mmol/l PMFS, 1 µg/ml leupeptin and 1 µg/ml pepstatin as the protease inhibitors (Beyotime Institute of Biotechnology). Following centrifugation at 20,000 × g at 4°C for 5 min, total extracted protein was collected from the supernatant, and the protein concentration was measured with a commercial Bicinchoninic Acid protein assay kit (Beyotime Institute of Biotechnology). A total of 50 µg protein lysate was loaded in each lane and separated by 8–12% SDS-PAGE. Targeted proteins were then transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA) via an electroblotting technique. Non-specific bindings were blocked with 5% non-fat milk dissolved in tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature. Following three washes with TBST, membranes were incubated with primary antibodies overnight at 4°C against SH2B1 (1:800; sc-136065; Santa Cruz Biotechnolgy, Inc., CA, USA), Bax (1:500; ab32503; Abcam, Cambridge, UK), Bcl-2 (1:600; cat. no. 2876S; Cell Signaling Technology, Inc., Danvers, MA, USA), cleaved caspase-3 (1:400; cat. no. 9661L; Cell Signaling Technology, Inc.), cleaved caspase-9 (1:500; cat. no. 9507S; Cell Signaling Technology, Inc.), total (t)-JAK2 (1:500; cat. no. 3230S; Cell Signaling Technology, Inc.), phosphorylated (p)-JAK2 (1:800; cat. no. 3776S; Cell Signaling Technology, Inc.), t-STAT3 (1:800 dilution; 4904P; Cell Signaling Technology, Inc.) and p-STAT3 (1:600; cat. no. 9134S; Cell Signaling Technology, Inc.). GAPDH (1:1,000; ab181602; Abcam) was utilized as an internal reference control. Subsequently, the membranes washed three times with TBST and were incubated with a horseradish peroxidase-conjugated rabbit anti-rat IgG secondary antibody (1:1,000; BA1058; Wuhan Boster Biological Technology Ltd., Wuhan, China). The protein bands were visualized by an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.), and the signal intensities were calculated by BandScan 5.0 software (Glyko, Inc., Novato, CA, USA).

Data were presented as the mean ± standard deviation. All statistical analyses were performed using standard SPSS software (version 19.0, IBC Corp., Armonk, NY, USA). A Student's t-test was utilized for between-group comparisons. Multiple comparisons were conducted with one-way analysis of variance, followed by a Student-Newman-Keuls-q-test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the association between SH2B1 and cerebral I/R injury, mRNA and protein expression levels of SH2B1 were detected in PC12 cells following OGD/R. Compared with the control group, SH2B1 expression at both the mRNA and protein levels was significantly suppressed in OGD/R-treated untransfected PC12 cells (P<0.05; Fig. 1). Ad-SH2B1 transfection significantly increased SH2B1 expression (Ad-SH2B1 + OGD/R group vs. OGD/R group; P<0.05), indicating efficient transduction of the adenovirus. In addition, SH2B1 expression was not significant altered between the Ad-GFP + OGD/R and OGD/R groups (P>0.05).

To determine whether neuronal SH2B1 upregulation alleviated OGD/R-induced injury, cell viability and LDH (necrotic biomarker) release in PC12 cells were measured by CCK-8 and ELISA, respectively. Compared with the control group, OGD/R treatment induced neuronal cell death as observed by the decrease in cell viability (Fig. 2A); however, LDH activity was increased (P<0.05; Fig. 2B). Conversely, these effects were significantly reversed by SH2B1 upregulation under the OGD/R condition (Ad-SH2B1 + OGD/R group vs. OGD/R group; P<0.05). The aforementioned parameters demonstrated no significant differences between the OGD/R and Ad-GFP + OGD/R groups (P>0.05). These data indicated that SH2B1 may serve neuroprotective roles in cerebral I/R injury.

To investigate the potential mechanisms of the neuroprotective effects of SH2B1 in OGD/R, the apoptotic rate and apoptotic mediators, including Bax, Bcl-2 and cleaved caspase-9/3 were detected by flow cytometry and western blotting, respectively. In line with the flow cytometry results, the expression levels of Bax and cleaved caspase-9/3 revealed similar pattern of alternations. As presented in Fig. 3, OGD/R-induced untransfected PC12 cells demonstrated an aggravated apoptotic rate, and upregulated Bax and cleaved caspase-9/3 expression levels; however, Bcl-2 anti-apoptotic effector expression levels were lower compared with in the control group (P<0.05). When OGD/R-inducted cells were pre-treated with Ad-SH2B1, the aforementioned parameters were significantly inversed (Ad-SH2B1 + OGD/R group vs. OGD/R group, P<0.05). Additionally, no significant differences were observed between the Ad-GFP + OGD/R and OGD/R groups (P>0.05). These results suggested that the effects of SH2B1 on OGD/R-induced PC12 cells may be associated with the deactivation of the apoptotic cascade.

The JAK2/STAT3 signaling pathway was extensively investigated as a major anti-apoptotic mediator in OGD/R injury (12,13). To investigate whether the JAK2-STAT3 axis was involved in the favorable effects of SH2B1 on OGD/R, the expression levels of JAK2 and STAT3 in PC12 cells were analyzed. As presented in Fig. 4, following OGD/R induction, protein expression levels of p-JAK2 and p-STAT3 were significantly reduced (OGD/R group vs. control group, P<0.05); however, Ad-SH2B1 delivery into OGD/R-treated PC12 cells significantly increased p-JAK2 and p-STAT3 expression levels compared with in the OGD/R group (P<0.05). Ad-GFP transfection resulted in no significant alterations in the expression levels of p-JAK2 and p-STAT3 (Ad-GFP + OGD/R group vs. OGD/R group, P>0.05). In addition, t-JAK2 and t-STAT3 revealed no significant differences in expression levels among the four groups (P>0.05).

To further examine whether the inhibitor effects of SH2B1 on OGD/R injury were dependent upon the activation of the JAK2/STAT3 signaling pathway, virus-transfected and OGD/R-treated PC12 cells were exposed to the JAK2 inhibitor AG490. As presented in Fig. 5, the expression levels of p-JAK2/STAT3 (Fig. 5A), cell viability and LDH activity (Fig. 5B), as well as the apoptotic rate (Fig. 5C) were significantly different between the Ad-GFP and Ad-SH2B1 groups under the OGD/R + PBS condition; however, significantly differences were also observed between the Ad-SH2B1 + OGD/R + PBS group compared with the presence of AG490. Treatment with AG490, however, abrogated the effects of SH2B1 overexpression on p-JAK2/STAT3 expression levels, viability and cell death relative to OGD/R + PBS + Ad-SH2B1-treated PC12 cells (P<0.05). Overall, these data demonstrated that the protective effects of SH2B1 against OGD/R may be largely dependent upon the activation of the JAK2/STAT3 signaling pathway and the suppression of apoptosis.