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Introduction

Stroke, also known as cerebral vascular accident, is characterized by sudden onset and the rapid occurrence of localized or diffuse brain dysfunction (1). Stroke includes ischemic stroke and hemorrhagic stroke, of which ischemic stroke accounts for 67.3-80.5% of cases, which seriously threatens human health and creates a large social burden (1). The main treatments for ischemic stroke are intravascular thrombolysis and mechanical thrombectomy, aiming to restore the blood supply of ischemic brain tissue through reperfusion (2). However, there is often an aggravation of tissue damage called cerebral ischemia-reperfusion injury (CIRI) when the blood flow is restored (3,4). The mechanisms of CIRI include abnormal signal transduction, mitochondrial damage, oxidative stress, autophagy, inflammation and apoptosis (5,6). Thus, avoiding the injury of CIRI is required in the early treatment of ischemic stroke.

Tissue inhibitor of metalloproteinases (TIMP)-3 is a member of the tissue inhibitors of metalloproteinases family, which prevent extracellular matrix degradation by inhibiting the activity of matrix metalloproteinases (MMPs) (7). Moreover, TIMP3 has been demonstrated to exert a role in regulating apoptosis in tumor cell lines and myocardial infarction, and also in inhibiting angiogenesis in addition to targeting MMPs (8-11). The overexpression of TIMP3 could attenuate oxidative stress in metabolic disorders (12). Similarly, TIMP3 also serves a notable role in ischemia-reperfusion (I/R) injury. A previous study has demonstrated that knockout of TIMP3 can aggravate the I/R injury of hepatocytes (13). By contrast, TIMP3-overexpression protects myocardial I/R injury by inhibiting cardiomyocyte apoptosis (11). However, the role of TIMP3 in CIRI remains unknown.

Due to the nerve growth factor (NGF) receptor on the cell membrane, PC12 cells can grow neurites and differentiate into neurons after being induced by NGF at the physiological level. Therefore, PC12 cells are widely used as in vitro model of cerebral ischemia injury (14,15). In the current study, PC12 cells were used as a cell model of cerebral ischemia to study the effect of TIMP3 on oxygen glucose deprivation/reoxygenation (OGD/R)-induced PC12 cell injury. Additionally, numerous studies have indicated that the Akt pathway mediates the occurrence and development of cerebral ischemia injury, and AKT activity is significantly inhibited after cerebral ischemia damage (16,17). Meanwhile, TIMP3-mediated neuroprotection mainly depends on the activation of AKT signaling pathway (18). The present study aimed to investigate whether TIMP3 prevented cerebral ischemia/reperfusion and to explore the role of TIMP3.

Materials and methods

Animals

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.

Models

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).

TTC staining

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).

Immunohistochemistry

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.).

Induction of OGD/R model

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.

Cell transfection

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.

Semi-quantitative PCR and reverse transcription-quantitative (RT-q)PCR analysis

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.

Western blotting

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.).

Detection of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA)

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.

TUNEL assay

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.

Statistical analysis

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.

Results

TIMP3 expression is downregulated in OGD/R-induced PC12 cells and cerebral tissues after I/R injury

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.

Figure 1 TIMP3 expression is downregulated in OGD/R-induced PC12 cells and cerebral tissues after I/R injury. (A) Model mice of cerebral I/R injury and the control group received TTC staining. (B and C) Semi quantitative RT-PCR was used to detect the expression of TIMP3 after I/R injury in mouse cerebral tissue. (D) Immunohistochemical staining was used to evaluate the TIMP3-positive cells between sham and I/R groups. (E) Expression levels of TIMP3 in cerebral tissues after I/R injury was detected by RT-quantitative PCR analysis. (F) Western blotting of TIMP3 expression in OGD/R-induced PC12 cells. **P<0.01 and ***P<0.001 vs. sham or NC. TIMP3, tissue inhibitor of metalloproteinases-3; OGD/R, oxygen glucose deprivation and reoxygenation; I/R, ischemia-reperfusion; RT, reverse transcription; ns, no significance; NC, negative control.

TIMP3 inhibits OGD/R-induced PC12 apoptosis and oxidative stress

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.

Figure 2 TIMP3 inhibits OGD/R-induced PC12 apoptosis and oxidative stress. (A) Western blotting was used to confirm the expression of TIMP3 by Ad-TIMP3. (B) TUNEL immunofluorescence staining was used to examine apoptosis-positive cells in different groups (magnification, x400). (C) Western blotting was performed to detect the expression of apoptosis proteins, Bax and Bcl-2. (D) ROS generation, MDA level and SOD activity were detected after different treatments using corresponding commercial kits. *P<0.05, **P<0.01 and ***P<0.001 as indicated. TIMP3, tissue inhibitor of metalloproteinases-3; OGD/R, oxygen glucose deprivation and reoxygenation; Ad, adenovirus; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase; NC, negative control; con, control.

TIMP3 regulates the expression of phosphorylated (p)-AKT

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.

Figure 3 TIMP3 regulates the expression of phosphorylated AKT. Western blotting was performed and quantified to examine AKT and p-AKT (Thr308 and Ser473) protein expression levels after (A) transfection with Ad-TIMP3 or (B) treatment with p-AKT inhibitor (AZD5363). *P<0.05, **P<0.01 and ***P<0.001 as indicated. TIMP3, tissue inhibitor of metalloproteinases-3; p, phosphorylated; Ad, adenovirus; OGD/R, oxygen glucose deprivation and reoxygenation.

Overexpression of TIMP3 inhibits OGD/R-induced PC12 apoptosis and oxidative stress via the AKT pathway

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.

Figure 4 TIMP3 inhibits OGD/R-induced PC12 apoptosis and oxidative stress via the AKT pathway. (A) TUNEL immunofluorescence staining was used and quantified to detect the apoptosis-positive cells in different groups (magnification, x400). (B) Western blotting was conducted to determine the expression levels of apoptosis proteins Bax and Bcl-2. (C) ROS generation, MDA level and SOD activity after different treatments were detected by corresponding commercial kits. *P<0.05, **P<0.01 and ***P<0.001 as indicated. TIMP3, tissue inhibitor of metalloproteinases-3; OGD/R, oxygen glucose deprivation and reoxygenation; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase; con, control; Ad, adenovirus; NC, negative control.

Discussion

CIRI is one of the important factors that aggravates brain injury after cerebral ischemia. Reducing reperfusion injury helps protect neurons in the ischemic area and reduces neuron necrosis and apoptosis (2). Various mechanisms have been demonstrated to be involved in the development of CIRI, including apoptosis and oxidative stress (3). Thus, treatments that improve apoptosis and oxidative stress may have potential therapeutic effects for CIRI.

TIMP3 is a member of the TIMP family of proteins, which are characterized as inhibitors of MMPs (7). However, with increasing research, the role of TIMP3 in regulating apoptosis and alleviating oxidative stress has attracted much attention (11,12). For example, Liu et al (11) revealed that TIMP3 upregulation could protect against cardiac I/R injury by inhibiting myocardial apoptosis. In the present study, the expression of TIMP3 was significantly decreased in the cerebral tissues after I/R injury and in OGD/R-induced PC12 cells. The results indicated that TIMP3 may have a pathological role in CIRI.

Mitochondrial dysfunction and oxidative stress are responsible for neuronal damage, which eventually leads to a cascade of apoptosis (23). Brain injury is associated with an increase of apoptosis, which is characterized by the increased expression of Bax and the decreased expression of Bcl-2; meanwhile, inhibition of apoptosis has been demonstrated to attenuate neuronal damage in CIRI (24). The overproduction of ROS and the weakening of the antioxidant mechanism are notable pathological events in the process of CIRI (25). The increase of ROS leads to the oxidation of lipids, proteins and nucleic acids, which changes the function of cells (26). MDA is one of the essential products of membrane lipid peroxidation, thus, its expression reflects the degree of oxidative stress (26). Therefore, the clearance of ROS is particularly important in CIRI. SOD has a significant scavenging effect on ROS produced in the process of oxidation (26). In the present study, overexpression of TIMP3 reduced the number of TUNEL-positive cells and attenuated the Bax/Bcl-2 ratio in OGD/R-induced PC12 cells. In addition, overexpression of TIMP3 could suppress the levels of ROS and MDA, and also elevate SOD activity in the OGD/R-induced neurocytes. These data indicated that overexpression of TIMP3 could protect neurocytes from OGD/R-induced apoptosis and oxidative stress in vitro. The effect of TIMP3 in cerebral tissues after I/R injury in vivo would need to be further investigated to confirm these results.

The AKT pathway serves a notable role in regulating CIRI (27); TIMP3 exerts its function by regulating the AKT pathway (18). The results of the current study indicated that TIMP3-overexpression could reverse the downregulation of p-AKT (Thr308 and Ser473) in OGD/R-treated PC12 cells. Further rescue studies indicated that the anti-apoptosis and anti-oxidative stress role of TIMP3 in OGD/R-induced PC12 cells could be partially abolished after AZD5363 treatment. This demonstrated that TIMP3 attenuated CIRI apoptosis and oxidative stress in neurocytes by regulating the AKT pathway. However, the specific binding molecules and their sites of binding to TIMP3 to modify the phosphorylation of AKT in I/R injury were unknown, which will be investigated further in the future. It has been previously demonstrated that the AKT pathway is activated in hypoxia-induced neuronal injury, which is contrary to the findings of the present study (28). These inconsistent results may be caused by the different models applied. Continuous lack of glucose oxygen was possibly a factor in AKT phosphorylation. Thus, the opposite effect on AKT activity would be observed under glucose and oxygen-glucose deprivation condition.

In conclusion, the present study demonstrated that TIMP3 exerted anti-apoptotic and anti-oxidative stress roles in cerebral I/R injury and provided a potential novel therapeutic target for the treatment of CIRI.

Acknowledgements

Not applicable.

Funding

Funding: The work was supported by The Xingtai Science and Technology Support Plan Project (grant no. 2018ZC191).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

LM, YZ and FG proposed the project, aims and objectives. LM, YZ, DL and XS conducted the experiments and collected the data. LM, YZ, XH, XC and FG analyzed the results and wrote the manuscript. FG submitted the manuscript. LM and FG confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

All animal experiments were conducted according to the ethical guidelines of The Xingtai People's Hospital and the ‘3R’ principle (Replacement, Reduction, and Refinement). All efforts were made to minimize animal suffering. The present study was approved by The Ethics Committee of Xingtai People's Hospital (Xingtai, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References