Adult female C57BL/6 mice (aged 8-10 weeks, weighing 20-25 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All animal care and experimental procedures were performed at the Animal experimental Center of the First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology and all procedures were approved by the Animal Ethics Committee of the First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology. Mice were maintained under controlled conditions with a 12-h light-dark cycle, at 23°C, ~40% humidity with access to food and water. GP-17 (cat. no. PHL83506) used in the present study was purchased from Sigma-Aldrich, Merck KGaA. The chemical structure of GP-17 is illustrated in Fig. 1A.

Mice were randomly divided into four groups (n=10 per group) as follows: i) The sham-operated (sham) group; ii) SCI group; iii) SCI + GP-17 group (10 mg/kg); and iv) the SCI + GP-17 group (50 mg/kg). All mice in the SCI model group were anesthetized by an intraperitoneal injection of 50 mg/kg pentobarbital sodium, after which a 3-cm skin incision along the median line on the back of the animals was made. Subsequently, a laminectomy was performed using Mouse Laminectomy Forceps (Fine Science Tools GmbH) at the T9 level, followed by a mechanically controlled compression injury using a mouse spinal cord compression device based on a previous study (23). The mice in the sham group were subjected to the same surgical procedure, but sustained no impact injury. Following surgery, the mice were immediately treated with GP-17 (10 and 50 mg/kg i.g. Winherb Medical S&T Development) administered daily for 28 days. Schematic diagrams of the in vivo experimental designs are presented Fig. 1B.

In another experiment, the mice were randomly divided into four groups as follows: The SCI group, SCI + GP-17 group, SCI + GP-17 + antagomir-21 group, and SCI + GP-17 + antagomir-negative control (NC) group. In the SCI + GP-17 + antagomir-21 group/antagomir-NC group (n=10/group), the mice were subjected to SCI and then treated with antagomir-21/antagomir-NC (5 nmol/g/day, Guangzhou RiboBio Co., Ltd.) via intrathecal injection beginning 15 min after SCI for 3 consecutive days. On the following day after SCI, GP-17 (50 mg/kg, i.g.) was administered to the mice daily until they were sacrificed.

Pentobarbital sodium (50 mg/kg, intraperitoneal injection) was used for anesthesia prior to each surgery, and all efforts were made to minimize animal suffering. The health and behavior of the mice were monitored twice a day. No mice were found dead during the anesthesia process. Following recovery from anesthesia, the mice exhibited paralysis of both hind limbs and urinary disorder. Their bladders were manually expressed twice a day until the recovery of reflex voiding. The animals were euthanized when the defined humane endpoint requirements were met: A loss of >15% of body weight within 1-2 days or of >20% loss in body weight in overall; the observation of apparent signs of suffering, such as lethargy, squinted eyes, dehydration or a hunched back. Euthanasia was performed by an intraperitoneal injection of pentobarbital sodium (50 mg/kg) followed by cervical dislocation, and animal death was confirmed by the cessation of respiration and heartbeat (24). At 28 days post-injury, the mice were euthanized, and the spinal cord tissues (1 cm with injury epicenter located centrally) were then harvested, and fixed in 4% paraformaldehyde (PFA) in 4°C in PBS (pH 7.4) for 20 min, embedded in paraffin, and sectioned at a thickness of 4 µm for immunohistochemistry.

Subsequently, the fixed sections were blocked with 10% bovine serum for 30 min at room temperature following incubation in 3% H₂O₂ for 15 min at room temperature. The sections were then incubated with cleaved caspase-3 antibody (1:200; cat. no. 9664, Cell Signaling Technology, Inc.) overnight at 4°C. Subsequently, the sections were incubated with anti-rabbit IgG, HRP-linked antibody (1:200; cat. no. 7074, Cell Signaling Technology, Inc.) at room temperature for 60 min. Finally, images were acquired using a microscope with a digital camera (VHX-5000, Keyence Corporation) at ×200 magnification.

The locomotor activity was evaluated using the BBB scoring method at 1, 7, 14, 21 and 28 days following SCI as previously described (25). The scores were recorded by two independent and well-trained investigators according to the BBB scale.

At 28 days post-injury, the spinal tissues were immediately weighed, and then dried at 80°C for 48 h. After the dry weight was measured, the spinal cord water content was evaluated as follows: Water content=[(wet weight-dry weight)/wet weight] ×100%.

Spinal cord samples were harvested and homogenized in phosphate-buffered saline (PBS), and then centrifuged at 5,000 × g at 4°C for 10 min. The protein expression levels of tumor necrosis factor (TNF)-α (cat. no. PT516), interleukin (IL)-6 (cat. no. P1328), IL-1β (cat. no. P1303) and IL-10 (cat. no. PI525) in the supernatant were detected by ELISA, using protocols supplied by the manufacturer (Beyotime Institute of Biotechnology).

A microarray dataset was obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67515). GEO2R (www.ncbi.nlm.nih.gov/geo/geo2r/), an interactive web tool, was applied to compare the samples in two different groups under the same experimental condition. Differentially expressed miRNAs (DE-miRNAs) were then identified based on the fold change. The heatmap of the DE-miRNAs was created using a method of hierarchical clustering using GeneSpring GX, version 7.3 (Agilent Technologies, Inc.).

Total RNA was isolated from spinal cord tissues using a mirVana™ miRNA Isolation kit (Thermo Fisher Scientific, Inc.). The reverse transcription of miRNA and mRNA was generated using the PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd.) and PrimeScript RT kit (Takara Biotechnology Co., Ltd.), respectively. qPCR was performed using the SYBR Premix Ex Taq (Takara Bio, Inc.) on an ABI Prism7500 Sequence Detection System (Thermo Fisher Scientific, Inc.). U6 was used as an internal control for miRNAs, and GAPDH for phosphatase and tensin homologue (PTEN), respectively. The thermocycling conditions were as follows: Pre-denaturation at 95°C for 30 sec, 40 cycles of denaturation at 95°C for 5 sec, annealing at 58°C for 30 sec, followed by extension at 72°C for 15 sec. The primer sequences were listed in Table I. Analyses of gene expression were performed using the 2^−ΔΔCq method (26).

Immortal BV-2 murine microglial cells were obtained from Procell Life Science & Technology Co., Ltd. (cat. no. CL-0493) and cultured in DMEM/F12 supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, Inc.), and 1% penicillin and streptomycin (Sigma-Aldrich, Merck KGaA) in 5% CO₂ at 37°C.

Agomir-21, agomir-NC, antagomir-21 and antagomir-NC were purchased from the Shanghai GenePharma Co., Ltd. BV-2 cells were transfected with 100 nM agomir-21 or antagomir-21 or controls using Lipofectamine 2000^® (Invitrogen, Thermo Scientific, Inc.) according to manufacturer's instructions. The cells were harvested at 24 h after transfection for testing.

The miRNA target prediction tools PicTar version 2007 (https://pictar.mdc-berlin.de/) and TargetScan Release 7.0 (http://targetscan.org/) were used to search for putative targets of miR-21.

The fragment of the 3′-UTR of PTEN [wild-type (wt) or mutant (mut)] was amplified and cloned into the pGL3 control vector (Promega Corporation). Site-directed mutagenesis of the PTEN 3′-UTR at the putative miR-21 binding site was performed using a QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies, Inc.). BV-2 cells were treated with antagomir-21/antagomir-NC and these luciferase reporter plasmids using Lipofectamine 2000^® (Invitrogen, Thermo Scientific, Inc.). At 48 h post-transfection, luciferase activity was assessed using the dual luciferase reporter kit (Promega Corporation). Renilla activity was used to normalize Firefly luciferase activity.

Total protein was extracted from the spinal cord specimens using RIPA lysis buffer (Beyotime Institute of Biotechnology) and quantified with a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). The proteins (30 µg/lane) were then separated by 10% SDS-PAGE gels and transferred to PVDF membranes (EMD Millipore). Thereafter, the membranes were blocked with 5% skim milk solution for 1 h at room temperature and then incubated with specific primary antibodies to PTEN (#9188, 1:1,000), cleaved caspase-3 (#9661, 1:1,000), caspase-3 (#14220, 1:1,000), AKT (#4685, 1:1,000), p-AKT (#9611, 1:1,000), mammalian target of rapamycin (mTOR; #2983, 1:1,000) p-mTOR (#5536, 1:1,000) and β-actin (#3700, 1:1,000 dilution) at 4°C overnight. Subsequently, the corresponding anti-rabbit secondary antibodies (cat. no. 3678, 1:2,000) were added to the membranes for 2 h at room temperature. All antibodies were obtained from Cell Signaling Technology, Inc. The bands were visualized using an ECL kits (EMD Millipore). Semi-quantification was performed using ImageJ version 1.46 (NIH).

Statistical analysis was conducted using SPSS software (version 18.0; SPSS, Inc.). Data are expressed as the mean ± SD. Statistical significance analysis was performed using an unpaired Student's t-test between two groups and one-way ANOVA among multiple groups followed by Tukey's post hoc test. Differences were considered statistically significant when P<0.05.

First, the mouse model of SCI was established as described above. As shown in Fig. 1C and D, the BBB score was decreased and the water content in the spinal cord was increased in the tissues from mice with SCI, compared with the sham group, indicating that the mouse model of SCI was successfully established. To investigate the role of GP-17 in mice with SCI, GP-17 (10 and 50 mg/kg) was administered to the mice with SCI by gavage daily for 28 days. Following treatment with GP-17, the BBB score was significantly increased, and the water content was markedly reduced in the GP-17 group compared with the SCI group (P<0.01; Fig. 1C and D). To determine whether GP-17 prevents neuronal apoptosis, the expression levels of cleaved caspase-3, the key intracellular cysteine protease of the cascade of events associated with apoptosis, were examined by western blot analysis in mice with SCI. As shown in Fig. 1E and F, the expression of cleaved caspase-3 was significantly increased in the SCI group compared with the sham group, whereas the expression of cleaved caspase-3 in the GP-17 group was markedly decreased compared with that in the SCI group. Similar results were obtained by IHC (Fig. 1G). Notably, the potency of 50 mg/kg GP-17 was greater than that of 10 mg/kg GP-17. Collectively, these results suggest that GP-17 may improve motor functional recovery by reducing neuronal apoptosis in mice.

Subsequently, the effects of GP-17 on the inflammatory response in mice with SCI were examined. The results revealed that the expression levels of the anti-inflammatory cytokine, IL-10, were significantly decreased, while the levels of pro-inflammation cytokines, including IL-1β, TNF-α and IL-6 were markedly increased in mice with SCI compared with those in sham group; however, these effects were attenuated following GP-17 treatment (P<0.01; Fig. 2). Therefore, the data indicated that GP-17 improved the inflammatory response in mice with SCI.

In order to investigate the potential mechanisms of GP-17 in regulating the apoptosis and inflammatory response in SCI, differentially expressed miRNAs were analyzed through the retrieval of the gene expression dataset, GSE67515. As shown in Fig. 3A, a great number of differentially expressed miRNAs (24 miRNAs were significantly downregulated and 21 miRNAs were upregulated) were found in spinal cord tissues following SCI. A total of seven miRNAs (miR-21, miR-34a-5p, miR-494, miR-543-3p, let-7, miR-142 and miR-155-5p) were selected and further confirmed through RT-qPCR. The results revealed that miR-21, miR-34a-5p, miR-494 and miR-543-3p were markedly decreased, while let-7, miR-142 and miR-155-5p were significantly increased; these findings are in line with those of previous reports, further attesting to the reliability of the current microarray results (27-33). Of note, among these miRNAs, miR-21 was the only significantly upregulated miRNA when GP-17 was administered to the mice with SCI, whereas the other miRNAs exhibited no significant difference in expression in the mice with SCI following GP-17 treatment (P<0.01; Fig. 3B). Previous studies have reported the anti-apoptotic and anti-inflammatory effects of miR-21 in SCI animal and cell models (27,34,35). Therefore, it was hypothesized that the alteration of miR-21 expression may have contributed to the protective effects of GP-17 against SCI.

To clarify the role of miR-21 in the GP-17-induced protective effects, the mice with SCI were treated with antagomir-21/antagomir-NC via intrathecal injection, followed by GP-17 treatment. First, the efficacy of antagomir-21 in vivo was evaluated by RT-qPCR. As shown in Fig. 4A, compared with Sham group, SCI resulted in a significant decrease in the expression of miR-21, while GP-17 treatment reversed the inhibitory effects induced by SCI on the expression levels of miR-21. Moreover, antagomir-21 injection significantly decreased the levels of miR-21 compared with the SCI + GP-17 group (P<0.01; Fig. 4A). As mentioned above, GP-17 treatment improved motor function and spinal cord edema, but failed to do so in the mice with SCI injected with antagomir-21 (P<0.01; Fig. 4B and C). Functional experiments revealed that GP-17 significantly downregulated the expression levels of cleaved caspase-3 in mice with SCI, while the inhibitory effect of GP-17 was partially reversed by antagomir-21 (P<0.01; Fig. 4D and E). Of note, the results of ELISA revealed that the suppressive effects of GP-17 on the inflammatory response were also reversed by antagomir-21 in mice with SCI, as evidenced by the promotion of the levels of IL-1β, TNF-α and IL-6, and the decreased IL-10 levels in the group injected with antagomir-21 (P<0.01; Fig. 4F-I). In total, the data proved that miR-21 was at least partially responsible for the suppression of GP-17 in SCI-induced apoptosis and the inflammatory response.

To elucidate the mechanisms through which miR-21 mediates the protective effects of GP-17 on SCI, potential targets of miR-21 were predicted using the bioinformatics tools, PicTar version 2007 (https://pictar.mdc-berlin.de/) and TargetScan Release 7.0 (http://targetscan.org/). Bioinformatics analysis indicated that PTEN was a potential target of miR-21 (Fig. 5A). Previous research has reported that PTEN plays an important role in tissue pathology and apoptosis, leading to secondary damage after the initial SCI (36). In the present study, a dual luciferase reporter assay was performed in order to validate whether PTEN was a direct target of miR-21. First, it was confirmed that miR-21 expression in BV-2 cells was significantly increased/decreased following transfection with agomir-21 or antagomir-21 compared with that of cells transfected with agomir-NC or antagomir-NC (Fig. 5B). Subsequently, as shown in Fig. 5C, miR-21 inhibition significantly increased, while miR-21 upregulation markedly decreased the luciferase activity of wt PTEN 3′-UTR in BV-2 cells (P<0.01). However, these effects were not observed in cells the transfected with the vector bearing the mutant PTEN 3′-UTR. Furthermore, the results of western blot analysis revealed that the overexpression of miR-21 significantly decreased the expression of PTEN, while the PTEN expression levels were markedly increased by miR-21 knockdown (P<0.01; Fig. 5D). To further investigate whether GP-17 affects the expression levels of PTEN in vivo, the mice with SCI were treated with antagomir-21/antagomir-NC via intrathecal injection, followed by GP-17 treatment. As shown in Fig. 5E, PTEN expression was significantly increased in the SCI group compared with the sham group, whereas GP-17 treatment led to a marked reduction in PTEN expression compared with the SCI group. However, the suppressive effect of GP-17 on PTEN expression was significantly attenuated by antagomir-21 (P<0.01). All these data suggest that GP-17 suppresses PTEN expression by upregulating miR-21.

It is well-known that PTEN is a negative regulator of the AKT/mTOR pathway that has been implicated in the apoptosis and inflammatory response during SCI (37,38). Thus, further experiments were designed to examine the influence of GP-17 on the activation of PTEN/AKT/mTOR pathway in vivo. Therefore, the expression level changes of AKT/mTOR pathway-related proteins were analyzed in the spinal cords of mice with SCI. It was found that SCI led to a significant reduction in the expression of p-AKT and p-mTOR, whereas this inhibitory effect was attenuated by GP-17 treatment, suggesting that GP-17 can re-activate the AKT/mTOR pathway in SCI. However, the activation of the AKT/mTOR pathway induced by GP-17 was blocked by antagomir-21 (P<0.01; Fig. 6A and B). All these data indicated that GP-17 exerts its protective effects by regulating the activation of the miR-21/PTEN/AKT/mTOR pathway.