Norwogonin Attenuates Hypoxia-induced Oxidative Stress And Apoptosis in PC12 Cells

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Abstract

Background: Norwogonin is a natural flavone with three phenolic hydroxyl groups in skeletal structure and has excellent antioxidant activity. However, the neuroprotective effect of norwogonin remains unclear. Here, we investigated the protective capacity of norwogonin against oxidative damage elicited by hypoxia in PC12 cells. Methods: The cell viability and apoptosis were examined by MTT assay and Annexin V-FITC/PI staining, respectively. Reactive oxygen species (ROS) content was measured using DCFH-DA assay. Lactate dehydrogenase (LDH), malondialdehyde (MDA), and antioxidant enzyme levels were determined using commercial kits. The expression of related genes and proteins was measured by real-time quantitative PCR and Western blotting, respectively. Results: We found that norwogonin alleviated hypoxia-induced injury in PC12 cells by increasing the cell viability, reducing LDH release, and ameliorating the changes in cell morphology. Norwogonin also acted as an antioxidant by scavenging ROS, reducing MDA production, maintaining the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and decreasing the expression levels of HIF-1α and VEGF. In addition, wogonin prevented cell apoptosis via inhibiting the expression levels of caspase-3, cytochrome c, and Bax while increasing the expression levels of Bcl-2 and the ratio of Bcl-2/Bax. Conclusions: Norwogonin attenuates hypoxia-induced injury in PC12 cells by quenching ROS, maintaining the activities of antioxidant enzymes, and inhibiting the mitochondrial apoptosis pathway.

Keywords: Norwogonin, Antioxidant activity, Hypoxia, Oxidative stress, Apoptosis

Background

Aerobic organisms need oxygen (O2) for producing energy. Hypoxia is defined as insufficient O2 supply to maintain cellular function in tissue and often occurs in some physiological situations such as high altitude [1], and in many pathological situations such as stroke [2]. The brain is particularly sensitive to hypoxia-induced injury due to its high oxygen consumption, rich in unsaturated fatty acids, and low antioxidant capacity [3]. Increasing evidence has indicated that hypoxia can induce adverse effects on the brain [4–6].

Linlin Jing, Rongmin Gao, Jie Zhang, Dongmei Zhang, Jin Shao, Zhengping Jia, and Huiping Ma

Department of Pharmacy, the 940th Hospital of Joint Logistics Support force of PLA, Lanzhou 730050, Gansu, China

Oxidative stress and apoptosis are considered as two contributing factors in hypoxia-induced injury [7, 8]. Hypoxia exposure has been reported to increase the production of intracellular reactive oxygen species (ROS), which facilitates oxidative stress. Excessive ROS, such as superoxide anion (O2−˙ ), hydrogen peroxide (H2O2), and hydroxyl radical (HO•), leads to structural and functional cellular changes by attacking lipids, membranes, proteins and DNA, and subsequently causes cell damage [9].

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Simultaneously, overproduced ROS also facilitates the opening of mitochondrial permeability transition pore (mPTP) [10] and transferring pro-apoptosis proteins to the outer mitochondrial membrane, which induces depolarization of mitochondrial membranes and releases of cytochrome c [11]. These changes ultimately cause mitochondrial-dependent apoptosis [12]. So, it is believed that antioxidants with the ability to inhibit or eliminate excessive ROS may exert their protective effect via attenuating oxidative stress and apoptosis induced by hypoxia. Lots of studies have proved that antioxidant supplements like vitamin C [13], isoflavone [8], and nitroxide radicals [14], can limit hypoxia-induced injury in vitro and in vivo. Flavonoids are a large and diverse class of ubiquitous plant secondary metabolites. They are always considered as an excellent natural antioxidant with the ability to scavenge free radicals and inhibit lipid peroxidation.

Currently, more and more attention has been paid to this class of compounds due to their beneficial effects on human health. Flavonoids have been shown to own a wide range of pharmacological actions, such as anti-inflammatory, antinociceptive, and neuroprotective activity, etc., all of which may be attributed to their antioxidant activities [15]. Many studies have indicated that flavonoids exhibit excellent protective effects on hypoxia-induced failure. For example, rutin has a strong neuroprotective effect against retinal ganglion cell death induced by hypoxia [16]. A recent study also demonstrates that rutin can alleviate cobalt chloride-induced hypoxia damage by inhibiting oxidative stress and apoptosis in H9c2 cells [17]. Moreover, Liu et al suggest that nobiletin (3′,4′,5,6, 7,8-hexamethoxy flavone) attenuates myocardial I/R injury via activating of Akt/GSK-3β pathway in H9c2 cell [18]. In addition, acacetin can protect rat cardiomyocytes and H9C2 cardiomyoblasts against hypoxia/reoxygenation-induced injury via AMPK-mediated activation of the Nrf2 signaling pathway [19].

Norwogonin (5,7,8-trihydroxy flavone, Fig. 1) is a pharmacologically active flavone separated from the root of Scutellaria baicalensis Georgi (“Huang Qin” in Chinese), a traditional Chinese herb used to treat influenza and cancer [20, 21]. However, limited studies have been reported on the biological activities of norwogonin due to its low levels in natural plants. In order to address this problem, several synthesis methods of norwogonin are reported [22, 23].

Our previous study also established a simple method for obtaining norwogonin from chrysin in four steps [24]. These researches have positively influenced the further evaluation of norwogonin′s biological activities. Studies have revealed that norwogonin owns antioxidant [25], anticancer [26, 27], antiviral [28], and antimicrobial activities [29] as well as inhibits the cyanidestimulated production of ROS [30]. However, whether norwogonin has protective capacities against hypoxia-induced injury remains unknown. The aim of the present study was to investigate the protective effects of norwogonin against hypoxia-induced oxidative stress and apoptosis in PC12 cells.

Methods 

Materials and reagents 

Norwogonin (purity≥98%) was synthesized according to our previously reported method [24]. Rutin (purity≥96%) was purchased from Ci Yuan Biotechnology Co., Ltd. (Xian, Shannxi, China). Norwogonin and rutin were dissolved in sterile dimethyl sulfoxide (DMSO), stored at − 20 °C, and diluted in the cell culture medium immediately before use. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), streptomycin, and penicillin were purchased from Solarbio co., Ltd. (Beijing, China).

The kits of malondialdehyde (MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). 2′,7′-dichloride-hydrofluorescein diacetate (DCFH-DA) and (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) tetrazolium (MTT) was obtained from Sigma-Aldrich Co (St. Louis, MO, USA). Primary antibodies for hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), B cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), Caspase-3, Cytochrome C, and β-actin were all purchased from Abcam (Cambridge, UK). Secondary antibodies were obtained from ZsBio Company (Beijing, China).

An apoptosis analysis kit was obtained from the Beyotime Institute of Biotechnology (Jiangsu, China). All chemicals and solvents were of analytical grade and were obtained from a commercial supplier in China. Cell culture The PC12 cells were purchased from Cell Bank of the Chinese Academy of Sciences (TCR 9, Shanghai, China) and maintained in DMEM with 10% (v/v) FBS, 100 U/ mL penicillin, and 100 U/mL streptomycins at 37 °C in a humidified incubator containing 5% CO2. To evaluate the cytotoxicity of norwogonin, PC12 cells (passage 4 ~ 6) were pre-incubated with different concentrations (10− 8 , 10− 7, 10− 6, 10− 5, 10− 4 mol/L) of norwogonin for 1 h and then cultured for 24 h. Hypoxia exposure To induce cell hypoxia injury model, PC12 cells were subjected to a hypoxia environment (1% O2, 5% CO2, and 94% N2) at 37 °C for 24 h in a humidified chamber. Normoxic control cells were cultured at 37 °C in a 5% CO2 incubator for 24 h.

To evaluate the protective effect of norwogonin against hypoxia-induced injury, PC12 cells were pre-incubated with different concentrations (10− 8, 10− 7, 10− 6, 10− 5 mol/L) of norwogonin for 1 h before hypoxia treatment. Cell viability The cells viability was measured by MTT assay as previously described [31]. In brief, PC12 cells (1 × 105 cells/ mL) were seeded in 96 well culture plates. Then different concentrations of norwogonin were added to the wells. An equal volume of DMSO was added to control wells. The final concentration of DMSO in the cell culture medium is 0.1%. After incubation at normoxic or hypoxia conditions, 10 μL of MTT (5.0 mg/mL) was added to each well, followed by incubation at 37 °C for 4 h. Then, the supernatant with MTT was removed and the formazan product was dissolved in 100 μL DMSO. The absorbance was measured on a SpectraMax i3 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm. The results were expressed as the relative percentage of the control group. Hematoxylin and eosin (HE) staining PC12 cells seeded on glass coverslips were incubated for 24 h before they were treated with norwogonin in the same way as described above.

The medium was removed and the glass coverslips were washed with cold PBS, followed by fixation with methanol for 10 min at room temperature, and then washed with cold PBS three times for 5 min. Finally, the cells were stained according to the HE staining protocol [32]. The analyses of the cell were performed using an OLYMPUS IX73 microscope (100×) in order to verify cell morphological changes. Digital images were obtained using the DXM 1200 C digital camera (Nikon) associated with the microscope. ROS content Intracellular ROS level in PC12 cells was determined using DCFH-DA assay [33]. Briefly, PC12 cells (1 × 105 cells/mL) were seeded in 6-well plates. After hypoxia treatment, PC12 cells were washed with PBS and then were incubated in the culture medium containing 10 μM DCFH-DA for 30 min in the dark at 37 °C. The cells were observed with Olympus inverted fluorescence microscope (Tokyo, Japan) and were analyzed by a Becton Dickinson FACScan flow cytometer (BD Biosciences, CA, USA) with an excitation wavelength of 488 nm and emission wavelength of 525 nm. The ROS level was expressed as a relative percentage of control. LDH leakage, MDA content, and antioxidant enzyme activity PC12 cells (1 × 105 cells/mL) were seeded in a 90 mm dish. After hypoxia treatment as described above, 50 μL culture supernatant from each dish was collected, and LDH activity in the medium was detected using commercial assay kits (Jiancheng Institute of Biotechnology, Nanjing, China) and was expressed as U/mL. The PC12 cells were harvested and homogenized after washing two times with cold PBS. The concentration of total protein was measured by the BCA protein assay kit. The MDA content and antioxidant enzyme activity were determined using commercial assay kits (Jiancheng Institute of Biotechnology, Nanjing, China).

The content of MDA was presented as nmol/mg protein. The activities of SOD, CAT, and GPx were presented as U/mg protein. Cell apoptosis(Annexin-V/PI staining) After hypoxia treatment, PC12 cells were harvested, washed two times with cold PBS, and then suspended with binding buffer. The cells were treated with the Annexin V-FITC and PI solution following the protocol of the manufacturer (Beyotime, Shanghai, China). Data collections were performed using the Becton Dickinson FACScan flow cytometer (BD Biosciences, CA, USA). Quantitative real-time PCR analysis The total RNA of PC12 cells was extracted using Trizol reagent (Takara, Dalian, China) and converted to cDNA using the PrimeScript TM RT reagent Kit (AK4301,

Takara, Dalian, China). The cDNA encoding HIF-1α, VEGF, Bcl-2, Bax, caspase-3, cytochrome C, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified by quantitative real-time PCR using a 7300 real-time detection System (Applied Biosystems, CA, USA). The primers used were shown in Table 1. The PCR cycling conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 31 s. The mRNA levels were calculated using the 2-ΔΔCt method and normalized to GAPDH, which is the reference gene. Western blot PC12 cells were harvested and homogenized in RIPA agents. The concentration of total proteins was quantified using the BCA protein assay kit. 30 μg of samples were resolved on 12% SDS-PAGE electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA).

The membranes were blocked with 5% non-fat dry milk in TBST buffer for 1 h at room temperature and incubated with primary antibodies: anti-HIF-1α (1:300, ab179483, Abcam, UK), anti-VEGF (1:1000, ab46154, Abcam, UK), anti-Bcl-2 (1:1000, ab59348, Abcam, UK), anti-Bax (1:500, ab32503, Abcam, UK), anticaspase-3 (1:300, ab44976, Abcam, UK), anticytochrome C (1:1000, ab13575, Abcam, UK) and anti-β-actin (1:2000, ab8227, Abcam, UK) at 4 °C overnight. Then, the membranes were washed and incubated with secondary antibodies (1:2000, ZsBio, Beijing, China;) for 1 h at room temperature. The immunoreactive bands were visualized using enhanced chemiluminescence (ECL) reagents. The relative intensities of bands were normalized to the β-actin internal control and analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA).

Statistical analysis 

The results were expressed as mean ± SD derived from at least three independent experiments. The difference between groups was analyzed using one-way analysis of variance (ANOVA) followed by the Student–Newman– Keuls post hoc test. A P-value of < 0.05 was regarded as statistically significant.

Results

Norwogonin protective PC12 cells against hypoxia-induced injury

First, to preclude the proliferative activity of norwogonin, its cytotoxicity on normal PC12 cells was determined using an MTT assay. As seen in Fig. 2a, cellular proliferation was not significantly changed following treatment with norwogonin at concentrations of 1 × 10− 8 -1 × 10− 5 mol/L (P > 0.05). However, cell viability significantly decreased when the concentration of norwogonin was increased to 1 × 10− 4 mol/L (P < 0.05). The results indicated that norwogonin did not exhibit toxicity or proliferative activity on PC12 cells at the concentrations of 1 × 10− 8 -1 × 10− 5 mol/L.

Then we examined the protective effect of norwogonin against hypoxia-induced PC12 cells injury. As shown in Fig. 2b, compared with the control group, the cell viability in the hypoxia group was decreased to 58.71% (P < 0.01). Compared with the hypoxia treatment, pretreated with 1 × 10− 8 , 1 × 10− 7 , and 1 × 10− 6 mol/L norwogonin dose-dependently protected PC12 cells against hypoxia-induced injury, recovering the cell viability from 58.71 to 62.79% (P < 0.05), 66.68% (P < 0.01) and 69.88% (P < 0.01), respectively. Pretreatment with 1 × 10− 6 mol/L rutin also exhibited a protective effect, significantly increasing the cell viability to 63.78% compared to the hypoxia treatment. The viability pretreated with 1 × 10− 5 mol/L norwogonin was decreased to 63.43%, which is still significantly higher than that in the hypoxia group (P < 0.05). These results demonstrated that norwogonin showed significant cytoprotection at the concentrations of 1 × 10− 8 -1 × 10− 5 mol/L and the most effective concentration is 1 × 10− 6 mol/L. Then this dose was used as the optimal dose in the following experiments.

The protective capacity of norwogonin was also verified by morphological alterations. As shown in Fig. 2c, PC12 cells without hypoxia treatment grew well with regular shapes (fusiform), uniform sizes. After hypoxia exposure, PC12 cells exhibited shrinkage, rounded shape, desquamation, and reduced cell density. The cells pretreated with norwogonin or rutin before hypoxia exposure grew better, the number of desquamation cells decreased, and cell shape recovered normally. Besides, the protective ability of norwogonin was confirmed by the LDH leakage, which is associated with the loss of cell membrane integrity. As shown in Fig. 2d, the LDH activity in the culture medium was notably increased following hypoxia exposure (P < 0.01).

Pretreatment with norwogonin or rutin dramatically decreased the LDH leakage, suggesting norwogonin and rutin restored the cell membrane integrity. Norwogonin inhibits hypoxia-induced oxidant stress in PC12 cells ROS and MDA are two important indicators of cellular oxidant stress induced by hypoxia. As shown in Fig. 3a and b, a significantly increased content of ROS and MDA was observed in PC12 cells following hypoxia exposure. Norwogonin or rutin pretreatment significantly inhibited the production of ROS and MDA.

Antioxidant enzymes, such as SOD, CAT, and GPx, are regarded as the main defense system against oxidative stress in cells. As shown in Fig. 3c-e, hypoxia exposure significantly inhibited the activities of SOD, CAT, and GPx in PC12 cells. Treatment with norwogonin or rutin reversed these changes and restored the activities of antioxidant enzymes. All these results indicated that norwogonin protected the PC12 cells against oxidative stress-induced by hypoxia.