A total of 16 male C57BL/6 mice (8-10-weeks-old; weight, 20±2 g) were purchased from the Model Animal Research Center of Nanjing University. The Ethics Committee of Xingtai People's Hospital (Xingtai, China) approved all animal experiments. All mice were kept in a climate-controlled room (temperature, 25±1˚C; relative humidity, 50-60%; free access to food and water; 12 h light/dark cycle). Mice were randomly allocated to the sham group (n=6) or the I/R group (n=10), including two mice that died after the transient middle cerebral artery occlusion (MCAO) with unknown exact cause. Animal health and behavior were monitored once a day before model construction, and once every 30 min after MCAO. Proper anesthetic procedures were used to ensure that the mice did not suffer unnecessarily during or after the experimental procedure.

MCAO was conducted according to a previous study (19). In brief, the mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg) until loss of limb reflexes. Subsequently, unilateral MCAO was carried out by introducing a 4-0 nylon thread into the internal carotid artery after cutting the common carotid artery. The origin of the middle cerebral artery was occluded when a mild resistance was felt for 2 h followed by reperfusion. The mice in the sham group underwent the same surgery without occlusion. At 24 h after reperfusion, following sacrifice by anesthetic overload with intraperitoneal 500 mg/kg ketamine and 50 mg/kg xylazine (no breathing and heartbeat were used to verify mortality), the brain tissues were obtained for assays. The methods of euthanasia conformed to the American Veterinary Medical Association Guidelines for the Euthanasia of Animals, 2020 Edition (20).

The brain tissues were kept overnight at -80˚C and sliced into 2-mm thick sections, which were cultured with 1% TTC for 15 min at 37˚C to visualize the infarct area. The white area manifested infarcted brain tissues. Infarct area was measured with computer-assisted planimetry (ImageJ version 1.57; National Institutes of Health).

The brain tissues were fixed with 10% formalin at 4˚C overnight and embedded in paraffin before being cut into 5-µm sections. After that, the sections were deparaffinized with xylene, rehydrated in a graded alcohol series and heated until boiling in a microwave oven at 1200w for 5 min for antigen retrieval. Endogenous peroxidase activity was blocked with 3% H₂O₂ at 37˚C for 20 min. Subsequently, these sections were treated with TIMP3 primary antibody (1:300; cat. no. 5673S; Cell Signaling Technology, Inc.). After incubation at 4˚C for one night and washing with PBS, the sections were cultured with a HRP polymer-bound goat anti-rabbit secondary antibody (1:5,000; cat. no. ab7090; Abcam) at room temperature for 1 h. These samples were then stained with 3, 3-diaminobenzidine solution at room temperature for 10 min and hematoxylin at room temperature for 5 min. Finally, the slides were observed using a light microscope and the positively stained cells were counted using GraphPad Prism 6 software (GraphPad Software, Inc.).

NGF-induced PC12 cells were obtained from the American Type Culture Collection and cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO₂ for 24 h. PC12 cells were then washed with glucose-free-Hanks' balanced salt solution (Gibco; Thermo Fisher Scientific, Inc.) twice and placed in glucose-free DMEM (Gibco; Thermo Fisher Scientific, Inc.) in a hypoxic incubator chamber (1% O₂; 5% CO₂; 94% N₂) at 37˚C for 2, 4 and 8 h. The cells were transferred to normal growth conditions (DMEM with 10% fetal bovine serum at 37˚C with 5% CO₂) for 24 h for oxygen-glucose deprivation and reoxygenation (OGD/R) (21). PC12 cells without OGD/R treatment were used as the control.

Recombinant adenovirus [human adenovirus (Ad) 5] of TIMP3 (Ad: TIMP3) and empty adenovirus (human adenovirus 5, Ad5) vector as control (Ad: Con) were purchased commercially from Hanbio Biotechnology Co., Ltd. PC12 cells were maintained at 37˚C in a humidified atmosphere with 5% CO₂. The cells were transfected with Ad: TIMP3 or Ad: Con (100x10⁸ PFU/ml) using polybrene (Sigma-Aldrich; Merck KGaA) for 48 h and treated with or without AKT inhibitor AZD5363 (Selleck Chemicals) for another 48 h. The cells were then subjected to the associated experiments.

Total RNA from brain tissues and PC12 cells was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.). The purity of RNA was used to detect the absorbance ratio of 260/280 nm by a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.). Reverse transcription of RNA was performed using a PrimeScript RT kit (Takara Bio, Inc.). The temperature protocol used for RT was 37˚C for 15 min and 85˚C for 15 sec, then the sample was kept at 4˚C for immediate use or -20˚C for long-term storage. RT-PCR was used to measure the expression of TIMP3 mRNA. The primers used in the study were as follows: TIMP3, forward 5'-GGCAACTGTGCTGAACAGGAT-3', and reverse, 5'-GATGGCCAGCGTGACACTT-3'; 18s ribosomal RNA forward 5'-GTTGTGCTGACGGCGCA-3' and reverse, 5'-CCGGCTTCGTGGCAGCA-3'; β-actin, forward 5'-GTAAAGACCTCTATGCCAACA-3', and reverse, 5'-GGACTCATCGTACTCCTGCT-3'.

For semi-quantitative PCR, reactions used a PCR Master mix (cat. no. K0171; Thermo Fisher Scientific, Inc.) and were performed on a StepOnePlus system (Applied Biosystems; Thermo Fisher Scientific, Inc.), starting with denaturation at 94˚C for 3 min, followed by 35 cycles of 94˚C for 30 sec, 55˚C for 30 sec and 72˚C for 30 sec, with a final extension at 72˚C for 7 min. Amplified DNA fragments were analyzed by electrophoresis on 1% agarose gels with 5% ethidium bromide at 100 V for 30 min at room temperature, and these ethidium bromide stained DNA fragments were observed under UV illumination.

For RT-qPCR, the experiment was carried out using the Power SYBR^® Green PCR Master mix (cat. no. 4309155; Thermo Fisher Scientific, Inc.) with ABI7300 detector (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction parameters were as follows: 95˚C For 10 min, and 40 cycles at 95˚C for 15 sec and 60˚C for 30 sec. The mRNA expression levels were quantified using the 2^-ΔΔCq method (22) and normalized to the internal reference genes β-actin.

The brain tissues and PC12 cells were lysed with lysis buffer (Cell Signaling Technology, Inc.) at 4˚C for 30 min before centrifuging (4˚C; 14462 x g; 5 min). The total proteins were in the supernatant. Concentrations of proteins extracted from cerebral tissues or PC12 cells were then determined using the BCA protein assay kit (Bio-Rad Laboratories, Inc.). After that proteins (10 µg per lane) were separated using a 10% SDS-PAGE gel, then they were transferred to PVDF membranes (MilliporeSigma) followed by blocking with 5% non-fat milk (Bio-Rad Laboratories, Inc.) at room temperature for 1 h. Next, the membrane was probed with the following primary antibodies: TIMP3 (1:1,000; cat. no. 5673S), Bax (1:1,000; cat. no. 5023S), Bcl-2 (1:1,000; cat. no. 15071S), caspase-3 (1:1,000; cat. no. 9662S), AKT Thr308 (1:500; cat. no. 13038S), AKT Ser473 (1:500; cat. no. 4060S), AKT (1:1,000; cat. no. 2920S), GAPDH (1:1,000; cat. no. 5174S) and β-actin (1:2,000; cat. no. 3700T) (all Cell Signaling Technology, Inc.). Afterwards, the membrane was cultured with HRP-conjugated IgG anti-mouse (1:2,000; cat. no. 7076) or anti-rabbit secondary antibodies (1:2,000; cat. no. 7074) (both Cell Signaling Technology, Inc.) at room temperature for 2 h and washed with TBST (0.05% Tween). Finally, the blots were analyzed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.) and visualized via a Chemiluminescence Imaging system (ChemiScope 3600 Mini; Clinx Science Instruments Co., Ltd). The protein bands were quantified using GraphPad Prism 6 software (GraphPad Software, Inc.).

The levels of ROS were detected using a ROS Assay kit (cat. no. S0033S; Beyotime Institute of Biotechnology), the level of total SOD was evaluated by Superoxide Dismutase (SOD) assay kit (cat. no. A001-3-2; Nanjing Jiancheng Bioengineering Institute), and the level of MDA was measured by Malondialdehyde (MDA) assay kit (cat. no. A003-1-2; Nanjing Jiancheng Bioengineering Institute). These kits were all used according to the manufacturers' instructions.

PC12 cells were fixed in PBS with 4% polyformaldehyde at room temperature for 15 min. The cells were washed once with PBS and then, apoptosis of PC12 cells in the different groups was detected by TUNEL via a in situ Cell Death Detection kit (Roche Applied Science). The kit was used in accordance with the manufacturer's protocol, and the ratio of apoptotic to total cells was calculated under fluorescence microscopy by observing three fields of view (magnification, x400). After that, ImageJ software (version 1.53, National Institutes of Health) was used to quantify the apoptosis rate.

SPSS version 20.0 statistic software (IBM Corp.) was used to analyze the data, which was expressed as mean ± SD (unless otherwise presented). Statistical analysis was carried out using unpaired Student's t-test between two groups. Comparisons among two or more groups were analyzed using one-way ANOVA followed by Turkey's multiple comparisons post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the expression difference of TIMP3 after cerebral I/R injury in vivo, a model of cerebral I/R injury was constructed in mice. The morphology of the cerebral I/R injury model is presented in Fig. 1A. As demonstrated in Fig. 1B and C, the expression levels of TIMP3 in I/R-induced injury in mice cerebral tissues were significantly downregulated compared with the sham group. Moreover, immunohistochemical results indicated that the expression of TIMP3-positive cells decreased significantly in the I/R group (Fig. 1D). RT-qPCR and western blotting results indicated that the TIMP3 expression in OGD/R-induced PC12 cells was significantly downregulated in a time-dependent manner (Fig. 1E and F). These results demonstrated that TIMP3 expression was downregulated in I/R injury in vivo and in vitro.

PC12 cells were transfected with Ad-TIMP3 before OGD 8h/R treatment to confirm whether overexpression of TIMP3 could protect neurocytes from OGD/R-induced apoptosis and oxidative stress. The expression of TIMP3 was markedly overexpressed by Ad-TIMP3 therapy for 48 h (Fig. 2A). The number of TUNEL-positive cells in OGD/R-induced cells significantly increased after OGD/R, but was significantly reduced by TIMP3-overexpression (Fig. 2B). Western blotting indicated that OGD/R increased the Bax/Bcl-2 ratio, and caspase-3 expression compared with the NC; by contrast, this was significantly attenuated by TIMP3-overexpression (Fig. 2C). In addition, the levels of ROS and MDA were significantly elevated in OGD/R-induced PC12 cells compared with control cells, while this was significantly suppressed by TIMP3-overexpression. Conversely, SOD activity decreased significantly in PC12 cells after OGD/R treatment, and overexpression of TIMP3 significantly elevated SOD activity in the OGD/R-induced neurocytes (Fig. 2D). The data revealed that TIMP3 inhibited apoptosis and oxidative stress of OGD/R-induced PC12.

As TIMP3 was demonstrated to exert its function by regulating the AKT pathway, the phosphorylation levels of AKT (Thr308 and Ser473) were detected. Western blotting demonstrated that p-AKT (Thr308 and Ser473) was significantly downregulated in OGD/R-treated PC12 cells, which could be significantly reversed by TIMP3-overexpression. However, TIMP3-overexpression did not significantly affect p-AKT expression in cells without OGD/R treatment (Fig. 3A). In addition, in order to further conduct the rescue experiment in the following mechanism study, the p-AKT inhibitor (AZD5363) was used to inhibit p-AKT (Thr308 and Ser473) activity (Fig. 3B). The experiments demonstrated that the expression of p-AKT (Thr308 and Ser473) was regulated by TIMP3.

After confirming that TIMP3 could elevate the expression levels of p-AKT (Thr308 and Ser473) that had been decreased by OGD/R treatment, whether the function of TIMP3 could be mediated via p-AKT overexpression was investigated. TIMP3-overexpression significantly reduced the number of TUNEL-positive cells that were elevated in OGD/R-induced neurocytes compared with the OGD/R + Ad-control group. This effect of TIMP3 in OGD/R-induced PC12 cells was partially but significantly reversed after AZD5363 treatment (Fig. 4A). Western blotting indicated that TIMP3 downregulated the expression of Bax and upregulated the expression of Bcl-2 in OGD/R, which were partially reversed after AZD5363 treatment (Fig. 4B). Consistent with the aforementioned results, the anti-oxidative stress role of TIMP3 in OGD/R-induced PC12 cells was also partially blocked by AZD5363 treatment (Fig. 4C). Thus, it is confirmed that TIMP3 inhibits apoptosis and oxidative stress via AKT pathway in vitro.