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Abstract

Objective:

C1q/TNF-related protein 3 (CTRP3), a member of CTRP family, has been found to have neuroprotective effect. In the current study, we investigated the protective role of CTRP3 in hippocampal neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R).

Materials and methods:

The mRNA and protein levels of CTRP3 in OGD/R-stimulated hippocampal neurons were measured using qRT-PCR and western blot analysis, respectively. CCK-8 assay was performed to assess cell viability. ROS production was measured using the fluorescence probe 2′,7′-dichlorofluorescein diacetate (H₂DCFDA). The activities of SOD and GPx were determined using ELISA. Cell apoptosis was assessed. Luciferase reporter assay was carried out to assess the activation of ARE). The levels of p-AMPK and Nrf2 were measured using western blot.

Results:

Our results showed that the expression of CTRP3 was significantly downregulated in hippocampal neuronal cells exposed to OGD/R. Overexpression of CTRP3 improved cell viability of OGD/R-induced hippocampal neurons. In addition, overexpression of CTRP3 attenuated the OGD/R-caused oxidative stress with decreased ROS production and increased activities of SOD and GPx. Moreover, CTRP3 caused a significant increase in bcl-2 expression and decreases in bax expression and caspase-3 activity. Furthermore, CTRP3 overexpression significantly upregulated the levels of p-AMPK and Nrf2, as well induced the activation of ARE in OGD-R-induced hippocampal neurons. CTRP3 upregulated the mRNA expression levels of HO-1, NQO-1 and GPx-3. Additionally, treatment with the inhibitor of AMPK partially reversed the neuroprotective effect of CTRP3 in OGD/R-exposed neurons.

Conclusion:

CTRP3 exerted protective effect on OGD/R-induced cerebral injury, which was regulated by AMPK/Nrf2/ARE pathway.

Keywords

Cerebral ischemia/reperfusion (I/R) injury CTRP3 oxidative stress apoptosis AMPK/Nrf2/ARE signaling pathway

Introduction

Cerebral ischemia/reperfusion (I/R) injury is a severe pathologic condition that may lead to stroke, which is a major public health problem with rapid progression and serious consequences.^1,2 Although a series of studies have been performed to explore new approaches, effective therapeutics for cerebral I/R injury are still lacking.³ It has been documented that I/R injury is characterized by overproduction of reactive oxygen species (ROS), excessive inflammatory response and apoptosis.^4,5 However, the precise molecular mechanisms remain largely unknown.

C1q/TNF-related protein (CTRP) family is a group of proteins that are pleiotropic and secreted mediators involved in various processes such as energy metabolism, inter-organ signaling, inflammation, immune-regulation, hormones-regulation functions.^6,7 In recent years, CTRP family has attracted much interest because of these effects. CTRP3, also known as CORS26/cartducin, is a well-studied member of CTRP family that has been found to be expressed in adipocytes, adipose stromal cells and other tissues.^8,9 Importantly, circulating CTRP3 level is confirmed to be positively correlated with adiponectin, as well as negatively correlated with waist circumference, blood pressure, fasting glucose, triglycerides, and cholesterol.⁸ Feng et al. reported that CTRP3 promotes mitochondrial energy production through targets of reactive oxygen species (ROS) and peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α in vascular smooth muscle cells.¹⁰ Zhang et al. showed that CTRP3 attenuates pressure overload-induced cardiac hypertrophy by suppressing the p38/CREB pathway.¹¹ Additionally, CTRP3 was found to exhibit neuroprotective effect in intracerebral hemorrhage (ICH) model.^12,13 However, the role of CTRP3 in cerebral I/R injury has not been investigated.

AMP-activated protein kinase (AMPK) is a cellular energy sensor that has a key role in maintaining the cellular homeostasis in response to variety of conditions, such as nutrient starvation (especially glucose), hypoxia and toxins exposure.^14,15 It has been documented that AMPK acts as a principal regulator of oxidative stress and inflammatory responses via regulating downstream signaling pathways including Nrf2/ARE.^16
–18 Activation of AMPK/Nrf2/ARE has been found to be associated with I/R injury.

It was reported that CTRP3 significantly enhanced AMPK phosphorylation in the hematoma border zone 7 days after intracerebral hemorrhage (ICH).¹² A study by Yan et al. reported that CTRP3 administration attenuated high glucose high lipid (HGHL)-induced VCAM-1 production in an AMPK-dependent manner in cultured human retinal microvascular endothelial cells (HRMECs).¹⁹ In addition, Zhang and He reported that CTRP3 enhanced the activation of Nrf2 pathway in high glucose-stimulated ARPE-19 cells.²⁰ Thus, we hypothesized that CTRP3 might play an important role in I/R injury through AMPK/Nrf2/ARE signaling pathway. Herein, we investigated the role of CTRP3 in hippocampal neuronal cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R).

Materials and methods

Cell culture

All animal experiments were approved by the ethics committee of Shaanxi Provincial People’s Hospital (Xi’an, China) and performed in accordance with the guidelines developed by the care and use of laboratory animals. Hippocampi were dissected from newborn SD rats (<24 h old, 5 ± 2 g; Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China). The hippocampi tissues were digested with 0.25% trypsin solution (Solarbio, Beijing, China) for 10 min at 37°C. The dissociated cells were maintained in neurobasal medium (Invitrogen, Carlsbad, CA, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1 mM glutamine (Invitrogen), 2% B27 (Invitrogen), 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL streptomycin (Sigma-Aldrich). The primary hippocampal neurons were maintained in a humidified incubator with 5% CO₂ at 37°C.

OGD/R model

To initiate OGD, the primary hippocampal neurons were kept in glucose-free medium under a gas mixture consisting of 95% N₂ and 5% CO₂ for 4 h. After the hypoxia treatment, the culture medium was returned to a standard medium under a normoxic atmosphere for up to 24 h.²¹

Cell transfection

The CTRP3 cDNA was cloned into the eukaryotic expression vector pcDNA3.0 to generate the CTRP3-overexpressing vector pcDNA3.0-CTRP3. Cells at 70% confluence were transfected with pcDNA3.0-CTRP3 or pcDNA3.0 using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instruction. The experiment was repeated at least three times.

qRT-PCR

Total RNA from hippocampal neurons was isolated by TRIzol reagent (Invitrogen). The first-strand cDNA was synthesized by reverse transcription using PrimeScript RT Master Mix (Takara, Dalian, China). Then the real-time PCR was performed using SYBR green Master Mix (Takara) on a 7500 Real Time PCR System. The PCR primers used in the study were listed as following: β-actin forward, 5′-AAC CCTAAGGCCAACCGTGAAAAG-3′, reverse, 5′-TGGCGTGAGGGAGAG CATAGC-3′; CTRP3 forward, 5′-CCACGTCCTACAGCCACCGACTGA-3′, reverse, 5′-AAGGAACCAGATCACGGGTCAGAT-3′; heme oxygenase-1 (HO-1) forward, 5′-ATCCTATCCACCAACGAACC-3′ and reverse, 5′-GTGGTCTGTTATTTGCGCTA-3′; NAD(P)H: quinone oxidoreductase 1 (NQO-1) forward, 5′-TGGCCGATTCAGAGTGGCAT-3′ and reverse, 5′-AAACAGGCTGCTTGGAGCAAAA-3′; glutathione peroxidase 3 (GPx-3) forward, 5′-TTAGCCCAAAGCTTCAAGAGG-3′ and reverse, 5′-TGCCTAGCAGAAGAAGCTACA-3′. The experiment was repeated at least three times.

Western blot analysis

Total protein was extracted from hippocampal neurons using protein extraction reagent (Pierce Biotechnology, Rockford, IL, USA). After determination of protein concentration using a bicinchoninic acid (BCA) protein assay kit (Pierce), equal amount of protein (30 μg/lane) was loaded onto 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were probed with monoclonal antibodies against CTRP3, HIF-1α, bax, bcl-2, p-AMPK, AMPK, nuclear Nrf2 and lamin B1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by the incubation of secondary horseradish peroxidase (HRP)-conjugated antibody (1: 2000; Santa Cruz). The bands were detected by electrochemiluminescence (ECL) reagent (Pierce Biotechnology). The experiment was repeated at least three times.

Cell viability assay

Cell ability of hippocampal neurons was assessed by Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) following the instructions of manufacture. Cells were added with 10 µl CCK-8 and incubated for 4 h under the condition of 95% and 5% CO₂ at 37°C. Cell viability was assessed by detecting the optical density (OD) value (450 nm) using a Microplate reader (BioTek Instruments, Winooski, VT, USA). The experiment was repeated at least four times.

Assessment of reactive oxygen species (ROS) level

ROS production was measured using the ROS-sensitive dye 2′,7′-dichlorofluorescein diacetate (H₂DCFDA).²² Hippocampal neurons were incubated with 10 µM H₂DCFDA (Invitrogen) for 30 min at 37°C. The H₂DCFDA is rapidly oxidized to form highly fluorescent DCF in the presence of ROS. The fluorescence intensity was quantified using a Becton Dickinson FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA). The experiment was repeated at least five times.

Enzyme-linked immunosorbent assay (ELISA)

The superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities of hippocampal neurons were assessed using corresponding commercially available ELISA kits (Invitrogen) following the provided instructions. The caspase-3 activity was measured using a Caspase-3 ELISA Kit (Invitrogen) according to the instructions. The measurement of absorbance at 450 nm was carried out with a microplate reader (Bio-Rad). The experiment was repeated at least five times.

Luciferase reporter assay

To assess the activation of Nrf2/ARE, the pGL4.37 [luc2P/ARE/Hygro] vector (Promega, Madison, WI, USA) containing four copies of ARE were transfected into hippocampal neurons with pcDNA3.0-CTRP3 or pcDNA3.0. Finally, the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s protocol. The experiment was repeated at least three times.

Statistical analysis

All data were expressed as means ± standard deviation (SD). Statistical analysis was conducted using Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey post hoc test using SPSS 13.0 software (SPSS, Chicago, IL, USA). A p value less than 0.05 was considered statistically significant.

Results

CTRP3 was lowly expressed in OGD/R-induced hippocampal neurons

To investigate the role of CTRP3, the mRNA and protein levels of CTRP3 in hippocampal neurons were measured after OGD/R stimulation. The results from qRT-PCR showed that the mRNA level of CTRP3 was significantly decreased in OGD/R-induced hippocampal neurons (p = 0.001; Figure 1(a)). Western blot analysis proved that the protein level of CTRP3 in OGD/R-induced hippocampal neurons was markedly lower than that in control hippocampal neurons (p = 0.000; Figure 1(b)).

Figure 1.

The mRNA and protein levels of CTRP3 in hippocampal neurons. The mRNA and protein levels of CTRP3 in hippocampal neurons were measured after OGD/R stimulation using qRT-PCR (a) and western blot analysis (b), respectively. n = 3. Differences were determined by Student’s t-test. **p < 0.01 versus control hippocampal neurons.

Overexpression of CTRP3 suppressed OGD/R-induced oxidative stress in hippocampal neurons

To further explore the role of CTRP3, the hippocampal neurons were transfected with pcDNA3.0-CTRP3 or pcDNA3.0. The protein expression of CTRP3 was markedly increased in hippocampal neurons transfected with pcDNA3.0-CTRP3 under OGD/R condition (p = 0.000; Figure 2(a)). Figure 2(b) showed that the cell viability of OGD/R-induced hippocampal neurons was significantly decreased when compared to control cells (p = 0.000). However, the decreased cell viability was improved by CTRP3 overexpression (p = 0.001). In addition, OGD/R-induced significant increase in ROS production was prevented by CTRP3 (p = 0.000; Figure 2(c)). The decreased activities of SOD and GPx in OGD/R-induced hippocampal neurons were elevated after transfection with pcDNA3.0-CTRP3 (p = 0.000; Figure 2(d); p = 0.000, Figure 2(e)). Furthermore, we observed that the protein expression level of HIF-1α was significantly increased after OGD/R injury (p = 0.002); however, this effect was reversed by CTRP3 overexpression (p = 0.001; Online Supplemental Figure 1).

Figure 2.

Effect of CTRP3 overexpression on OGD/R-induced oxidative stress in hippocampal neurons. Hippocampal neurons were transfected with pcDNA3.0-CTRP3 or pcDNA3.0 for 48 h, and then exposed to OGD/R injury. (a) The protein level of CTRP3 was measured using western blot analysis. n = 3. (b) CTRP3 overexpression improved the cell viability of OGD/R-induced hippocampal neurons. n = 4. (c) CTRP3 overexpression prevented ROS production in OGD/R-induced hippocampal neurons. n = 5. (d and e) The decreased activities of SOD and GPx were elevated by CTRP3 overexpression. n = 5. Statistical analysis was carried out with one-way ANOVA followed by Tukey post hoc test. **p < 0.01 versus control hippocampal neurons. ^## p < 0.01 versus OGD/R-induced hippocampal neurons.

Overexpression of CTRP3 inhibited OGD/R-induced cell apoptosis in hippocampal neurons

Next, we assessed the effect of CTRP3 on OGD/R-induced cell apoptosis in hippocampal neurons. The results of western blot analysis indicated that OGD/R caused a significant decrease in bcl-2 expression (p = 0.000) and an increase in bax expression (p = 0.000), which could be reversed by CTRP3 overexpression (p = 0.000; Figure 3(a)). Besides, the caspase-3 activity was significantly increased in OGD/R-induced hippocampal neurons (p = 0.000). Overexpression of CTRP3 obviously inhibited the caspase-3 activity (p = 0.001; Figure 3(b)).

Figure 3.

Effect of CTRP3 overexpression on OGD/R-induced cell apoptosis in hippocampal neurons. Hippocampal neurons were transfected with pcDNA3.0-CTRP3 or pcDNA3.0 for 48 h, and then exposed to OGD/R injury. (a) CTRP3 overexpression caused significant decrease in bax expression and increase in bcl-2 expression. n = 3. (b) CTRP3 overexpression inhibited the caspase-3 activity in OGD/R-induced hippocampal neurons. n = 5. Statistical analysis was carried out with one-way ANOVA followed by Tukey post hoc test. **p < 0.01 versus control hippocampal neurons. ^## p < 0.01 versus OGD/R-induced hippocampal neurons.

Overexpression of CTRP3 increased the activation of AMPK/Nrf2/ARE pathway in hippocampal neurons under OGD/R condition

To uncover the molecular mechanism underlying the role of CTRP3, western blot was performed to detect the expression levels of p-AMPK and Nrf2. The results revealed that overexpression of CTRP3 significantly enhanced OGD/R-caused increase in the expression levels of p-AMPK (p = 0.001) and Nrf2 (p = 0.000; Figure 4(a)). Luciferase reporter assay showed that CTRP3 overexpression significantly upregulated the luciferase activity of hippocampal neurons (p = 0.000; Figure 4(b)).

Figure 4.

Effect of CTRP3 overexpression on AMPK/Nrf2/ARE pathway in hippocampal neurons under OGD/R condition. (a) Western blot demonstrated that overexpression of CTRP3 induced the expression levels of p-AMPK and Nrf2. n = 3. (b) Luciferase reporter assay proved that CTRP3 overexpression promoted the activation of ARE. n = 3. Statistical analysis was carried out with one-way ANOVA followed by Tukey post hoc test. *p < 0.05 versus control hippocampal neurons. **p < 0.01 versus control hippocampal neurons. ^## p < 0.01 versus OGD/R-induced hippocampal neurons.

Overexpression of CTRP3 upregulated the expression levels of HO-1, NQO-1, and GPx-3 in hippocampal neurons under OGD/R condition

Next, we evaluated the mRNA expression levels of Nrf2/ARE downstream genes including HO-1, NQO-1 and GPx-3. We found that OGD/R-induced the mRNA expression levels of HO-1 (p = 0.006), NQO-1 (p = 0.001) and GPx-3 (p = 0.000) were dramatically upregulated in CTRP3-overexpressing hippocampal neurons (Figure 5).

Figure 5.

Effect of CTRP3 overexpression on the mRNA expression levels of HO-1, NQO-1, and GPx-3 in hippocampal neurons under OGD/R condition. Hippocampal neurons were transfected with pcDNA3.0-CTRP3 or pcDNA3.0 for 48 h, and then exposed to OGD/R injury. (a to c) The mRNA expression levels of HO-1, NQO-1, and GPx-3 were detected using qRT-PCR. n = 3. Statistical analysis was carried out with one-way ANOVA followed by Tukey post hoc test. **p < 0.01 versus control hippocampal neurons. ^## p < 0.01 versus OGD/R-induced hippocampal neurons.

AMPK inhibition partially reversed CTRP3-mediated Nrf2/ARE activation and the neuroprotective effect in OGD/R-exposed neurons

To inhibit the AMPK signaling, the neurons were treated with compound C, which is a specific inhibitor of AMPK. We found that treatment with compound C reversed the neuroprotective effect of CTRP3, as proved by decreased cell viability (p = 0.014), increased ROS level (p = 0.001), increased caspase-3 activity (p = 0.002; Figure 6(a) to (c)).

Figure 6.

Effect of compound C on CTRP3-mediated Nrf2/ARE activation and the neuroprotective effect in OGD/R-exposed neurons. Hippocampal neurons were transfected with pcDNA3.0-CTRP3 for 48 h in the presence of compound C (10 µM), and then they were subjected to OGD/R injury. (a) Cell ability was assessed by CCK-8 assay. n = 4. (b) ROS production was measured using the ROS-sensitive dye H₂DCFDA. n = 5. (c) Caspase-3 activity was measured using a Caspase-3 ELISA kit. n = 5. Statistical analysis was carried out with one-way ANOVA followed by Tukey post hoc test. *p < 0.05; **p < 0.01.

Discussion

In the present study, we provided convincing evidence that CTRP3 exerted a crucial role in protecting the hippocampal neurons from OGD/R-induced injury. Our results demonstrated that CTRP3 expression was regulated by OGD/R exposure. Overexpression of CTRP3 improved the cell viability and inhibited oxidative stress and apoptosis in OGD/R-induced hippocampal neurons. Furthermore, the protective effects of CTRP3 were mediated by AMPK/Nrf2/ARE signaling pathway.

Several lines of evidences indicate that CTRP3 possesses anti-oxidative and anti-inflammatory effects. CTRP3 inhibits high glucose-stimulated oxidative stress in retinal pigment epithelial cells with decreased levels of ROS and malondialdehyde (MDA), and increased SOD activity.²⁰ CTRP3 attenuates cardiac dysfunction, inflammation, oxidative stress and cell death in streptozotocin (STZ)-induced diabetic cardiomyopathy in rats.²³ Moreover, CTRP3 has been demonstrated to have neuroprotective effect. CTRP3 reduces brain edema and disruption of blood-brain barrier, and improves functional outcomes after ICH, indicating that CTRP3 has neurological protective effect.¹² CTRP3 was found to inhibit oxidative stress, which is critical pathogenesis of cerebral injury after ICH.¹³ In the current study, we investigated the role CTRP3 in I/R injury. The results showed that CTRP3 expression was significantly upregulated in hippocampal neuronal cells exposed to OGD/R. Overexpression of CTRP3 attenuated the OGD/R-caused reduction in cell viability and increase in oxidative stress and apoptosis in hippocampal neurons. Knockdown of CTRP3 exhibited opposite effect with the CTRP3 overexpression. Collectively, CTRP3 exerted protective effect on OGD/R-induced cerebral injury.

According to the previous studies, CTRP3 executed its roles via multiple mechanisms, such as regulation of p38 MAPK,²⁴ c-Jun NH-terminal kinase (JNK),²⁴ extracellular signal-regulated kinase (ERK1/2),²⁵ Nrf2/HO-1,²⁰ AKT/mTOR,²⁶ Notch,²⁷ and AMPK.¹⁹ Among these signaling pathways, AMPK is a heterotrimeric complex that plays an important role in maintaining energy homeostasis in eukaryotic cells. It has been demonstrated that AMPK serves as a potential target for therapeutic intervention in human diseases, including I/R injury.²⁸ Punicalagin pretreatment attenuates myocardial I/R injury through activating AMPK.²⁹ Barbaloin protects cardiomyocytes from I/R-induced oxidative stress and myocardial injury through activation of AMPK.³⁰ Baicalin attenuates hyperglycemia-exacerbated I/R injury by regulating mitochondrial function in a manner dependent on AMPK.³¹ In the current study, CTRP3 was found to induce the phosphorylation of AMPK in hippocampal neurons under OGD/R condition, suggesting that CTRP3 activates the AMPK pathway.

A number of studies have reported that AMPK phosphorylates multiple downstream targets, such as Nrf2 transcription factor. AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at Serine 550 for ARE-driven gene transactivation.³² Hou et al.³³ reported that galanthamine improves myocardial I/R-induced cardiac dysfunction, endoplasmic reticulum stress (ERS)-related apoptosis and myocardial fibrosis by suppressing AMPK/Nrf2 pathway in rats. Compound 13 activates AMPK/Nrf2 signaling to protect neuronal cells from OGD-R.³⁴ These findings suggested that AMPK/Nrf2 signaling pathway play crucial roles in the I/R injury. Our results showed that CTRP3 overexpression significantly upregulated the expression level of Nrf2 and induced the activation of ARE in OGD-R-induced hippocampal neurons. Moreover, CTRP3 upregulated the expression levels of Nrf2/ARE downstream genes including HO-1, NQO-1 and GPx-3. The data indicated that CTRP3 caused significant activation of Nrf2/ARE signaling pathway. Additionally, treatment with the inhibitor of AMPK partially reversed the neuroprotective effect of CTRP3 in OGD/R-exposed neurons. Collectively, the role of CTRP3 in OGD-R-induced hippocampal neurons was regulated by AMPK/Nrf2/ARE signaling pathway.

There existed several limitations in this study. Firstly, we only evaluated the effect of CTRP3 on OGD/R-induced hippocampal neurons in vitro. An in vivo animal study will be considered in the following studies. Secondly, the distribution of CTRP3 protein in brain tissue after cerebral I/R operation will require further experiments.

In conclusion, our present study showed that CTRP3 protected hippocampal neurons from OGD-R-induced oxidative damage and apoptosis, which might be mediated by AMPK/Nrf2/ARE signaling pathway. Thus, targeting CTRP3 might be a novel management of cerebral I/R injury.

Supplemental material

Supplemental Material, sj-tif-1-het-10.1177_0960327121989412 - CTRP3 protects hippocampal neurons from oxygen-glucose deprivation-induced injury through the AMPK/Nrf2/ARE pathway

Supplemental Material, sj-tif-1-het-10.1177_0960327121989412 for CTRP3 protects hippocampal neurons from oxygen-glucose deprivation-induced injury through the AMPK/Nrf2/ARE pathway by H Ding, Z Wang and W Song in Human & Experimental Toxicology

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2018JM7110).

ORCID iD

W Song

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

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