All experimental animal procedures were approved by the Animal Ethics Committee of the Tianjin Medical University. C57BL/6 mice (male; age, 10–12 weeks; weight, 21–23 g; n=8 in each group; Charles River Laboratories, Inc.) were housed at 22±2°C and 50±15% relative humidity and a 12-h light/dark cycle with adequate food and water.

Animals were randomly divided into the following two groups (n=10 per group): i) MCAO; and ii) sham-operated. To establish the MCAO mouse model, mice were anesthetized by the intraperitoneal injection of 45 mg/kg sodium pentobarbital (2%). An uncoated 6-0 monofilament nylon suture (diameter, 0.20 mm) was inserted to occlude the MCA for 1 h. Subsequently, the suture was removed for 24 h of reperfusion. Mice in the sham-operated group underwent the same procedure, but the suture was not inserted. Mice were euthanized 24 h following ischemia by intraperitoneal injection of 150 mg/kg sodium pentobarbital (2%). Death was verified by dilated pupils and cessation of the heartbeat. The infarct ipsilateral hemisphere brain was isolated and used for subsequent experiments.

The HT22 mouse hippocampal neuronal cell line was purchased from Procell Life Science & Technology Co., Ltd. Cells were incubated in a humidified incubator in a normal culture medium containing DMEM solution (Gibco; Thermo Fisher Scientific, Inc.) mixed with 10% fetal serum (Gibco; Thermo Fisher Scientific, Inc.) and 7.5% horse serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C and 5% CO₂. HT22 cells were seeded at a density of 4×10⁵ cells/well were transfected with 100 mmol/l H19 small interfering (si)RNA or scrambled siRNA negative control (si-NC) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 24 h at 37°C with 5% CO₂. The siRNA sequences were as follows: i) H19 siRNA, 5′-CCCUCAAGAUGAAAGAAAUTTAUUUCUUUCAUCUUGAGGGTT-3′; ii) si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′. Subsequently, HT22 cells were exposed to OGD to mimic ischemic-like conditions. Briefly, cells in the OGD group were cultured in a hypoxic incubator with 95% N₂ and 5% CO₂ for 3, 6 or 9 h. For reperfusion, cells were transferred to normal culture medium for 24 h and kept at 37°C in an incubator with 5% CO₂.

HT22 cell viability was assessed by performing an MTT assay. Briefly, HT22 cells were seeded (5×10⁴ cells/ml) into 96-well plates. Following OGD and H19 siRNA transfection, 20 µl MTT (5 mg/ml) was added to each well and incubated for 4 h at 37°C. Subsequently, 150 µl DMSO was used to dissolve the purple formazan. The absorbance was measured at a wavelength of 490 nm using a microplate reader. Cell viability in the control group was set at 100% and cell viability in OGD and H19 siRNA transfection groups were normalized to the control group.

Flow cytometry was performed to detect the rate of apoptosis. Cell apoptosis was assessed using the Annexin V-FITC/propidium iodide double staining kit (Beijing Solarbio Science & Technology, Co., Ltd.) according to the manufacturer's protocol.

The concentrations of inflammatory cytokines, including interleukin (IL)-6, IL-1β, TNF-α, IL-10 and TGF-β1, were measured using ELISA kits (R&D Systems, Inc.) according to the manufacturer's protocols.

Total RNA was extracted from mouse brain tissue and HT22 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix kit (Takara Biotechnology Co., Ltd.). Subsequently, qPCR was performed using the SYBR Premix Ex Taq II kit (Takara Biotechnology Co., Ltd.). The following primers were used for qPCR: H19 forward, 5′-GTCAAACAGGGCAAGATGGG-3′ and reverse, 5′-ATTACGGTGGGTGGGATGTT-3′; miR-29b forward, 5′-CAGACCTGTAGCACCATTTGAA-3′ and reverse, 5′-TATCCTTGTTCACGACTCCTTCAC-3′; SIRT1 forward, 5′-TCCTTGGAGACTGCGATGTT-3′ and reverse, 5′-ATATGAAGAGGTGTTGGTGGC-3′; PGC-1a forward, 5′-CAATACCTCATGGGACAGCG-3′ and reverse, 5′-GCCTCCAGGGAAAGCAAA-3′; U6 forward, 5′-CGCTTCGGCAGCACATATAC-3′ and reverse, 5′-AAATATGGAACGCTTCACGA-3′ and GAPDH forward, 5′-AGGTCGGTGTGAACGGATTTG-3′ and reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′. All samples were run in triplicate. miRNA and mRNA expression levels were quantified using the 2^−ΔΔCq method and normalized to the internal reference genes U6 and GAPDH, respectively.

Total protein was extracted from HT22 cells. Protein concentrations were determined using a Protein Assay kit (Bio-Rad Laboratories, Inc.). Proteins were separated via SDS-PAGE and transferred onto nitrocellulose membranes, which were blocked with 5% skimmed milk in TBS-Tween-20 (TBST) at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies targeted against: SIRT1 (1:1,000; GeneTex, Inc.), PGC-1α (1:500; GeneTex, Inc.) and GAPDH (1:5,000; OriGene Technologies, Inc.). Following washing three times for 10 min each time, the membranes were incubated with a goat anti-rabbit secondary antibody (1:2,000; GeneTex, Inc.) at room temperature for 1 h. The membranes were washed three times with TBST for 10 min each time. Protein expression was semi-quantified via densitometry (Bio-Rad Laboratories, Inc.) with GAPDH as the loading control.

Data are presented as the mean ± SD. Comparisons among multiple groups were analyzed using one-way ANOVA analysis followed by the Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether lncRNA H19 mRNA expression level were altered in ischemic brain tissue isolated from the MCAO mouse model, RT-qPCR was performed. The results indicated that lncRNA H19 expression levels were significantly elevated in ischemic brain tissue isolated from the MCAO mouse model compared with the sham group (P<0.05; Fig. 1A). Furthermore, miR-29b, SIRT1 and PGC-1α expression levels were significantly decreased in the MCAO mouse model compared with the sham group (P<0.05; Fig. 1B).

To investigate the effect of OGD treatment on HT22 cells, cell viability and lncRNA H19 expression levels were measured following OGD treatment at different times. Following treatment for 3, 6 or 9 h, OGD significantly decreased HT22 cell viability in a time-dependent manner compared with the control group (P<0.05; Fig. 2A). The RT-qPCR results indicated that lncRNA H19 expression levels were significantly elevated in OGD-treated cells compared with control cells (P<0.05; Fig. 2B). The aforementioned results indicated that elevated lncRNA H19 mRNA expression levels may aggravate cerebral ischemia injury.

To investigate the role of lncRNA H19 in OGD-induced cytotoxicity, H19 siRNA was used to knock down lncRNA H19 expression. H19 siRNA significantly decreased lncRNA H19 expression levels compared with the si-NC group (P<0.05; Fig. S1). The MTT assay results demonstrated that OGD treatment for 6 h significantly reduced cell viability compared with the control group (P<0.05), which was significantly ameliorated by H19 knockdown (P<0.05; Fig. 3A). Flow cytometry was performed to evaluate the effects of lncRNA H19 on cell apoptosis. The results indicated that OGD treatment for 6 h significantly increased cell apoptosis compared with the control group (P<0.05), which was also significantly ameliorated by H19 knockdown (P<0.05; Fig. 3B). The results suggested that OGD may induce cell injury by increasing lncRNA H19 expression levels.

Increasing evidence has demonstrated that inflammatory cytokines participate in the pathogenesis of cerebral ischemia (28,29). In the present study, the concentrations of several inflammatory cytokines were detected to investigate whether H19 knockdown altered the inflammatory response in HT22 cells (Fig. 4). IL-6, IL-1β, TNF-α, IL-10 and TGF-β1 concentrations were significantly increased following OGD treatment for 6 h compared with the control group (P<0.05). However, H19 knockdown significantly inhibited OGD-induced increases in IL-6, IL-1β, TNF-α and IL-10 concentrations following treatment for 6 h (P<0.05). IL-10 is an anti-inflammatory cytokine (30). IL-10 concentrations were significantly increased by OGD treatment for 6 h compared with the control group (P<0.05), which suggested a potential self-protection or compensation mechanism. The results indicated that lncRNA H19 may prompt the inflammatory response in ischemia stroke.

The effects of lncRNA H19 on miR-29b, SIRT1 and PGC-1α expression levels in OGD-treated HT22 cells were investigated by performing RT-qPCR and western blotting. Following OGD treatment for 6 h, miR-29b, SIRT1 and PGC-1α expression levels were significantly decreased compared with the control group (P<0.05); however, OGD-mediated effects on expression were significantly inhibited by H19 knockdown (P<0.05; Fig. 5). Consistent with the RT-qPCR results, the western blotting results demonstrated that OGD treatment for 6 h significantly decreased SIRT1 and PGC-1α protein expression levels compared with the control group (P<0.05; Fig. 6). However, H19 knockdown significantly increased SIRT1 and PGC-1α expression levels compared with the OGD 6 h group (P<0.05).