Antibodies against light chain 3B (LC3B; cat. no. 3868), Caspase-9 (cat. no. 9502), Caspase-3 (cat. no. 9662), poly(ADP-ribose) polymerase (PARP; cat. no. 9532), autophagy related 5 (Atg5; cat. no. 12994), Atg13 (cat. no. 13468), Bach1 (cat. no. 4578) and β-actin (cat. no. 3700) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA), while antibodies against Nrf2 (cat. no. R1312-8), heme oxygenase-1 (HO-1; cat. no. ET1604-45), and Histone H3 (cat. no. ET1601-14) were obtained from HuaBio (Hangzhou, Zhejiang, China). The secondary antibodies used in the present study included goat anti-rabbit horseradish peroxidase (HRP)-conjugated immunoglobulin (Ig)-G and goat anti-mouse HRP-conjugated IgG (cat. nos. BL003A and BL001A; Biosharp, Shanghai, China), as well as FITC goat anti-rabbit IgG (HA1004, HuaBio, Hangzhou, Zhejiang, China). Control (CTL), Nrf2 and HO-1 small interfering (si)-RNAs (cat. nos. sc-37007, sc-37030 and sc-35554, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), while Lipofectamine^® 2000 transfection reagent and dimethyl sulfoxide (DMSO) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). 3-Methyladenine (3-MA), chloroquine (CQ) and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), H₂O₂ was obtained from Tianjin Yongda Chemical Reagent Co., Ltd. (Tianjin, China), while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and Triton X-100 were from Solarbio Science & Technology Co., Ltd. (Beijing, China). The following kits were used in this study: ROS determination kit, total SOD assay kit (hydroxylamine method), MDA kit and reduced GSH assay kit, all purchased from Jiancheng Bioengineering Institute (Nanjing, China); BCA protein concentration determination kit and enhanced chemiluminescence (ECL) system, both from Beyotime Institute of Biotechnology (Shanghai, China); and Annexin V-FITC apoptosis detection kit, DNA Purification kit, and Nuclear and Cytoplasmic Protein Extraction kit, which were obtained from KeyGen Biotech Co., Ltd. (Nanjing, China).

Young leaves of Lindera aggregate (Sims) Kosterm. were collected from the Tiantaishan district (Zhejiang, China) and authenticated by Professor Jingkui Tian in Zhejiang University (Hangzhou, China), where the voucher specimens were stored and evaluated during our previous study (no. LA201301-08) (16). Separation, purification and structural analysis of QI were performed as described in a previous study (16). Briefly, dried plant material (10 kg) was extracted using 70% ethanol under reflux for 2 h, and the process was repeated three times. All the extracts were combined and concentrated under a vacuum. The combined extracts were diluted with H₂O and chromatographed over an AB-8 resin column, prior to eluting with NaOH (1 BV; pH 9.0), H₂O (3 BV) and 50% ethanol (3 BV). Subsequently, the 50% ethanol elution was concentrated and chromatographed on a reverse phase silica gel column (sample amount, 1 g; intermediates, 40 ml column volume; ethanol concentration, 14.25%; elution, 3 BV/h). Finally, an ethanol-water system was used for re-crystallization, and the solution was then set aside for 24 h at room temperature to obtain the QI.

Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Endothelial Cell Medium (ECM; ScienCell Research Laboratories, Inc., San Diego, CA, USA) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution (100 IU/ml penicillin and 100 mg/ml streptomycin). The cells were maintained in a 37°C humidified incubator with 5% CO₂.

The cytotoxicity of QI was evaluated by an MTT assay. Briefly, cells were seeded in 96-well plates at 3×10⁴ cells/well in a final volume of 100 µl. After 16 h, fresh FBS-free medium with various concentrations of QI (0, 62.5, 125, 250 and 500 µM) was added to each well. Following 24 h of incubation, 20 µl MTT (5 mg/ml) per well was added, and the cells were incubated for a further 2 h. Subsequently, DMSO was used to dissolve the formazan crystals, and a microplate reader was used to measure the optical density at 570 nm. Untreated cells were set to 100% viability in order to determine the cytotoxicity of QI.

The antioxidant properties of QI on cell viability were also assessed using MTT assay. Briefly, cells or transfected cells were treated with DMSO, H₂O₂ only or H₂O₂ (1,400 µM) and QI (62.5, 125, 250 and 500 µM) combined for 3 h. As described earlier, the medium was removed and replaced by 20 µl MTT (5 mg/ml). After 2 h, the supernatants were removed from the wells, and MTT dye was solubilized in DMSO at 200 µl/well. The absorbance at 570 nm was determined on a microplate reader.

HUVECs were seeded into 96-well plates at 3×10⁴ cells/well in a final volume of 100 µl. When completely adherent, cells were treated with 25 µM 3-MA (an autophagy inhibitor) for 1 h at 37°C, followed by QI treatment as aforementioned. Following 3 h, the medium was replaced with fresh FBS-free ECM, and 20 µl MTT (5 mg/ml) was added to each well for a further 2 h incubation. The crystals were then dissolved in DMSO and the optical density at 570 nm was detected using a microplate reader following high-speed vibration for 3 min. The cell viability was calculated by considering untreated cells as 100% viable.

Cells were transfected with plasmids encoding red fluorescent protein (RFP; Addgene, Inc., Cambridge, MA, USA) using the VigeneFection (VGF) plasmid transfection reagent (Vigene Biosciences, Inc., Rockville, MD, USA), according to the manufacturer’s protocol. Briefly, cells were treated with 0.5 µg plasmid DNA and 2 µl VGF in each well of a 24-well plate. Then HUVECs were seeded in confocal dishes following 24 h of stable transfection. The expression of RFP in HUVECs was confirmed by fluorescence detection. When 80-90% confluence was reached, treatments were initiated. Confocal images were obtained with an Olympus FV1000 confocal microscope (Olympus Corp., Tokyo, Japan).

Cells were seeded in 6-well plates at 5×10⁵ cells/well and then transfected with the CTL siRNA (17), Nrf2 siRNA (18) or HO-1 siRNA (19) using Lipofectamine 2000™. Briefly, 3 µg of siRNA was added to each well with 6 µl Lipofectamine 2000™. Treatments were initiated following 24 h transfection. The expression of the Nrf2 and HO-1 proteins was confirmed by western blot analysis.

HUVECs were cultured at a density of 2×10⁵ per well in 6-well plates and cultured overnight and then treated with H₂O₂ (1,400 µM) and QI (62.5, 125, 250 and 500 µM) for 3 h. Then cells were collected and assay kits (Jiancheng Bioengineering Institute) were used to measure the activity of MDA, GSH and SOD.

The role of QI in protecting HUVECs from H₂O₂-induced apoptosis was assessed by flow cytometry using an Annexin V-FITC apoptosis detection kit. Briefly, cells were harvested by non-enzymatic cell dissociation and centrifuged (500 × g for 5 min at room temperature) to remove the medium. The cells were then washed twice with phosphate-buffered saline (PBS) prior to re-suspending in 500 µl binding buffer. Next, the cells were stained with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI). After 15-min incubation without exposure to light, the Annexin V and PI emissions were detected in the FL1-H and FL2-H channels, respectively, with an excitation wavelength of 488 nm and emission wavelength of 530 nm on a FACS-Vantage flow cytometer (Cytomics FC 500; Beckman Coulter, Inc., Brea, CA, USA).

HUVECs were seeded in 6-well plates at a concentration of 7.0×10⁵ cells/well. At 16 h after planting, the cells were treated with H₂O₂ with or without QI. Following the treatment, DNA was isolated with a DNA Purification kit and electrophoretically analyzed on 1.5% agarose gel.

Cells were seeded into 6-well plates at 5×10⁵ cells/well and treated with H₂O₂ and QI at a range of concentrations for 3 h. The medium was removed and replaced with 1 ml PBS containing 1 µM CM-H₂DCFDA (Jiancheng Bioengineering Institute), and cells were then incubated in a 5% CO₂ incubator at 37°C for 30 min in the dark. Next, the cells were collected in 15 ml centrifuge tubes. Subsequent to washing with PBS three times, the ROS level for each treatment group was determined using a flow cytometer (Cytomics FC 500) and analyzed using FlowJo software (version 7.6; FlowJo LLC, Ashland, OR, USA).

Cells treated with QI and H₂O₂, or pretreated with 3-MA (25 µM) were washed with cold PBS and fixed with methanol (pre-chilled at −20°C) for 5 min at room temperature. Next, cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min and then blocked with 1% bovine serum albumin (BSA) in PBS for 30 min. Following washing with PBS, the cells were incubated overnight with an anti-Nrf2 antibody in 1% BSA at 4°C, followed by incubation with FITC goat anti-rabbit IgG in 1% BSA for 2 h at room temperature. Nuclei were stained with DAPI in Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA). Confocal images were obtained using the 63x oil immersion lens on a confocal microscope (LSM 780; Carl Zeiss AG, Oberkochen, Germany).

Drug-treated cells were collected by scraping, followed by centrifugation (500 × g for 5 min at 4°C). Nuclear and cytoplasmic proteins were extracted according to the protocol of the Nuclear and Cytoplasmic Protein Extraction kit. The extraction of total protein was performed as follows: Cells were washed once with PBS and then lysed in a lysis buffer consisting of 1% sodium dodecyl sulfate (SDS), 10 mM ethylene diamine tetra acetic acid (EDTA) and 50 mM Tris-HCl (pH 8.1) in the presence of a 1% protease inhibitor mixture (Sigma-Aldrich; Merck KGaA). Lysates were sonicated (15 sec) to shear genomic DNA and then centrifuged at 13,000 × g for 10 min at 4°C. The protein concentrations in the supernatants were determined using the Micro BCA Protein Assay kit. Equal protein amounts were resolved on an SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membrane was then blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 30 min at room temperature, and then incubated with the LC3B (1:1,000), Capase-9 (1:500), Caspase-3 (1:500), PARP (1:1,000), Atg13 (1:500), Atg5 (1:500), Nrf2 (1:500), HO-1 (1:500), Bach-1 (1:500), Histone H3 (1:1,000) and β-Actin (1:1,000) antibodies in TBST at 4°C overnight. The membrane was then washed three times with TBST for a total of 30 min, followed by incubation with goat secondary antibodies against rabbit or mouse IgG conjugated to HRP (1:5,000 dilution) for 2 h at room temperature. Subsequent to washing three times with TBST for a total of 30 min, ECL was used to develop the images and the immunoblots.

HUVECs were seeded into 6-well-plates, and then separately treated with CQ (10 µM) or QI (250 µM), or co-treated with CQ and QI for 3 h. Cells were subsequently lysed to determine LC3B and β-actin levels by western blot analysis. Furthermore, cells that were separately treated with QI (0, 62.5, 125 and 250 µM) and NAC (5 mM) only or NAC pretreatment for 1 h with QI (62.5, 125 and 250 µM) were also lysed to determine LC3B and β-actin levels by western blot analysis.

Briefly, HUVECs were seeded into 96-well plates at 3×10⁴ cells/well. Following treatment of cells with QI, the medium was removed and cells were washed with 50 µl cold PBS. For the extraction of cellular RNAs, the ExCellenCT Lysis kit (Applied Biological Materials, Inc., Richmond, BC, Canada) was used according to the manufacturer’s protocol. A total of 1 µl protease and 50 µl lysis solution were added to each well, mixed and incubated for 10 min in an incubator. To end the reaction, 1 µl protease inhibitor and 5 µl stop solution were added and incubated for 20 min at room temperature. Next, the RNA concentration was measured with a microplate reader. Reverse transcription was conducted in an RNase-free environment using 5X All-In-One RT MasterMix (Applied Biological Materials, Inc.) according to the manufacturer’s protocol. The reverse transcription system contained 2 µl 5X All-In-One RT MasterMix, 13 µl nuclease-free water and 5 µl total RNA (2 µg). The reverse transcription conditions were as follows: 25°C for 15 min, 42°C for 30 min, 85°C for 5 min, and then held at 4°C. Subsequent to reverse transcription, EvaGreen 2X qPCR MasterMix (Applied Biological Materials, Inc.) was applied in a reaction system containing 0.3 µl forward primer (10 µM), 0.3 µl reverse primer (10 µM), 0.6 µl cDNA (10 ng/µl), 5 µl EvaGreen and 3.8 µl H₂O. The qPCR reaction conditions (25°C for 15 min, 42°C for 30 min, 85°C for 5 min, and then held at 4°C) were similar to those of reverse transcription. The primers used in the present study were as follows: β-actin forward, 5′-CGG GAA ATC GTG CGT GAC AT-3′, and reverse, 5′-GAA CTT TGG GGG ATG CTC GC-3′; Nrf2 forward, 5′-GAT TCT GAC TCC GGC ATT TC-3′, and reverse, 5′-TCC CCA GAA GAA TGT ACT GG-3′; and HO-1 forward, 5′-GCC CTG CCC TTC AGC AT-3′, and reverse, 5′-CTG CAT TTG AGG CTG A GC C-3′. The relative quantification 2^−ΔΔCq method (20) was used to evaluate quantitative variation between treatments.

In the present study, 50 specific-pathogen-free female rats of the Sprague-Dawley strain (>18 weeks; 500-550 g; JOINN Laboratories, Suzhou, China) were housed in groups under a 12-h light/dark cycle, at a constant temperature of 24°C and humidity of 40%; all animals were provided with sterilized food and water ad libitum. Following 7 days of acclimatization, the animals were randomly distributed into five groups, each containing 10 animals of similar average body weight. Animals in the five groups received the following treatments: i) Vehicle (20% polyethylene glycol 2000 and 10% polyethylene glycol 400 in saline); ii) 4.0 mg/kg breviscapine, a Chinese medicine used to treat cardiovascular and cerebrovascular diseases (21), serving as a positive control; iii) QI at a dose of 4.0 mg/kg; iv) QI at a dose of 8.0 mg/kg; and v) QI at a dose of 16.0 mg/kg. All treatments were administered by tail vein injection once daily for 2 weeks. All experimental procedures using live animals were conducted in accordance with protocols approved by the Ethics Review Committee for the Use of Animal Subjects of Zhejiang University (Zhejiang, China).

Animals underwent a 12-h fast from food prior to sacrifice, and then hearts were collected. Tissue homogenates were prepared by grinding 0.1 g of the tissue in 1 ml saline. Following centrifugation at 1,500 × g for 15 min at 4°C, the supernatant was removed and kept at −20°C until further use.

Blood was collected by orbital puncture from the rats, following anesthesia by ethyl ether. The blood samples were then allowed to settle for 30 min prior to centrifugation at 2,500 × g for 10 min at 4°C. Sera and heart tissue homogenates were used for detecting biochemical parameters associated with oxidative stress, including SOD, GSH and MDA, according to the protocols of the corresponding kits.

Data are presented as the mean ± standard deviation. One-way analysis of variance and least significant difference tests were used for statistical analysis in all experiments. Differences were considered to be statistically significant at P<0.05. Statistical analyses were performed using the SPSS statistical software (version 17.0; SPSS, Inc., Chicago, IL, USA).

The MTT cell proliferation assay was used to assess QI toxicity. QI promoted the growth of HUVECs at all the investigated concentrations (62.5-500 µM), and significantly accelerated cell proliferation at the concentrations of 62.5 and 125 µM (Fig. 1B). Treatment with 62.5, 125, 250 and 500 µM QI for 24 h increased cell viability by 5.75, 3.14, 1.44 and 0.49%, respectively, compared to the control (Fig. 1B). These findings suggest that, at concentrations ranging between 62.5 and 500 µM, QI does not damage HUVECs.

The study next investigated the effect of QI on the growth of HUVECs following H₂O₂-induced oxidative damage. The HUVECs ultimately demonstrated apoptosis-like changes in their morphology, including plasma rounding and cell shrinkage, as a result of oxidative injury in the H₂O₂-treated group (Fig. 2A). By contrast, fewer apoptotic-like cells were observed in the cell groups co-treated with various concentrations of QI and H₂O₂ (Fig. 2B). Co-treatment with 62.5, 125, 250 or 500 µM QI resulted in significant dose-dependent protection against cell death induced by 1,400 µM H₂O₂ (Fig. 2C).

Incubation of HUVECs with H₂O₂ for 3 h significantly increased MDA levels, and decreased SOD and GSH activities as compared with those in the control group. However, co-treatment of HUVECs with QI significantly reduced the H₂O₂-induced MDA levels and restored the SOD and GSH activities compared with the group treated with H₂O₂ alone, in a concentration-dependent manner (Fig. 2D-F). In comparison with the H₂O₂-treated group, cells treated with 62.5, 125, 250 and 500 µM QI displayed increased SOD levels by 89.10, 138.61, 181.19 and 198.02%, respectively. A similar improvement was observed in GSH activity following QI treatment in HUVECs, with a significant increase detected (Fig. 2E). Compared with the H₂O₂-only group, the cell groups treated with 62.5, 125, 250 and 500 µM QI displayed a markedly higher content of GSH by 13.89, 43.18, 76.26 and 102.02%, respectively. By contrast, MDA was reduced by 29.31, 43.10, 51.72 and 67.24% in the 62.5, 125, 250 and 500 µM QI-treated cells, respectively, as compared with the H₂O₂ treatment alone.

Staining with Annexin V-FITC/PI and flow cytometry were performed to identify the apoptotic cells. The results revealed that cells exposed to H₂O₂ alone exhibited a higher apoptotic rate (46.7±3.3%) as compared with the control cells (1.3±0.1%). A two-dimensional diagram also revealed that QI treatment at concentrations of 62.5, 125, 250 and 500 µM decreased the proportion of apoptotic cells to 30.3±1.8, 26.9±1.7, 19.3±1.4 and 18.4±0.8%, respectively, as compared with that observed in cells that suffered H₂O₂ injury (Fig. 3A and B).

The present study further investigated whether H₂O₂ caused DNA fragmentation, which is another hallmark of apoptosis, in HUVECs. Agarose gel electrophoresis revealed that HUVECs treated with H₂O₂ exhibited DNA fragmentation, as indicated by the typical DNA laddering pattern, whereas QI-treated cells demonstrated significantly reduced DNA laddering (Fig. 3C). Additionally, treatment with QI resulted in a dose-dependent decrease in cleaved Caspase-3 and PARP levels, as well as activation of Caspase-9, compared with the H₂O₂ treatment alone (Fig. 3D). Collectively, these results indicate that QI protects against cell death by inhibiting H₂O₂-induced apoptosis.

Oxidative stress inhibition was further corroborated by the fact that QI mediated decreases in H₂O₂-induced ROS production in the HUVECs. The rate of ROS-positive cells was decreased from 94.2±1.1% in the 62.5 µM QI-treated group to 73.2±2.7% in the 500 µM QI-treated group, as compared with the ROS rate of 97.1±2.3% in the H₂O₂ treatment alone group (Fig. 4A and B). These results indicate that QI provides resistance to ROS in a dose-dependent manner.

In light of the aforementioned findings, the pathways involved in QI-mediated protection against oxidative stress were examined in HUVECs by immunofluorescence and western blot assays. Nrf2 is essential for cytoprotection against oxidative stress (22). The present study revealed that nuclear Nrf2 levels were increased subsequent to QI treatment, as indicated by both the immunofluorescence and western blot assay results; Histone H3 was used as a nuclear internal reference (Fig. 5A–C). Furthermore, Bach1 nuclear expression was decreased in QI-treated cells, but was increased in the cytoplasm, indicating that QI promoted the transfer of Bach1 from the nucleus to the cytoplasm, thus releasing its inhibitory effect on Nrf2 (Fig. 5C). The expression of HO-1 protein, which acts downstream of Nrf2, was also increased following QI treatment (Fig. 5D), suggesting that the antioxidant effect of QI was regulated by the Nrf2 antioxidant pathway.

qPCR was used to further investigate the antioxidative mechanism of QI in vitro. The data demonstrated that the expression levels of Nrf2 and HO-1 genes increased following QI treatment (Fig. 6A). Taken together, these results revealed that QI affects both the protein levels of Nrf2 and HO-1, as well as their transcriptional levels.

As mentioned earlier, nuclear localization of Nrf2 was found to be increased, while Nrf2 gene expression was upregulated by QI under oxidative stress conditions (Figs. 5 and 6A). Therefore, the study further examined whether reducing Nrf2 expression using siRNA transfection affects antioxidants. Nrf2 is known to activate the transcription of numerous cytoprotective enzymes, such as HO-1. The current study results revealed that Nrf2 siRNA transfection suppressed the effect of QI on increased HO-1 expression under oxidative stress conditions (Fig. 6B).

Next, the study examined whether Nrf2 siRNA or HO-1 siRNA were able to suppress the QI-induced cytoprotection in HUVECs by assessing the cell viability using an MTT assay following siRNA transfection. QI significantly reduced the H₂O₂-induced cytotoxicity of HUVECs transfected with CTL siRNA, with a cell viability of 55.1% observed in the H₂O₂ group and 75.2% in the H₂O₂+QI group (P<0.05; Fig. 6C). By contrast, QI treatment did not markedly reduce the H₂O₂-induced cytotoxicity of HUVECs transfected with Nrf2 siRNA (H₂O₂, 53.2%; H₂O₂+QI, 58.3%; P>0.05). However, in cells transfected with HO-1 siRNA, QI was able to reduce the H₂O₂-induced cytotoxicity, with a cell viability of 50.3 and 70.2% in the H₂O₂ and H₂O₂+QI groups, respectively (P<0.05; Fig. 6C). Fig. 6D presents the blots confirming that the transfection of Nrf2 siRNA and HO-1 siRNA resulted in successful knockdown. These results suggest that QI-induced Nrf2 nuclear transcription is critical for cytoprotection against oxidative stress.

Autophagy detection was also performed in the present study. The expression levels of Atg5, Atg13 and the autophagic marker LC3B-II were increased following QI treatment (Fig. 7A and B). When combined with autophagy flux detection, treatment with both QI and CQ resulted in an evident improvement in LC3B-II levels as compared with that observed in cells with QI or CQ treatment alone (Fig. 7C), suggesting that QI promotes autophagy. To further assess whether ROS or QI induced autophagy, the ROS inhibitor NAC was used. The results found that addition of NAC had no effect on the QI-induced increase of LC3B-II levels (Fig. 7D), further demonstrating that QI can induce autophagy. Notably, the transfer of Nrf2 from the cytoplasm to the nucleus was inhibited by 3-MA, an autophagy inhibitor (Fig. 7E). To further test the hypothesis that autophagy is involved, the cell viability was measured following 3-MA pre-treatment. It was observed that the protective effect of QI was diminished by the addition of 3-MA (Fig. 7F), indicating that QI serves its antioxidant role in HUVECs by inducing autophagy.

Next, rats were used to assess the antioxidant ability of QI in vivo(Fig. 8A). Our preliminary experimental results demonstrated that QI has no toxic or side effects on rats when administered at a dose of 0-100 mg/kg once daily for 2 weeks by tail vein injection (data not shown). In addition, the pre-experimental SOD vitality test results revealed that the lowest effective dose was 4 mg/kg (data not shown); therefore, the concentrations of 4, 8 and 16 mg/kg were selected for subsequent experiments. QI treatment displayed a dose-dependent reduction in MDA levels and increase in SOD activity. This effect reached a maximum level at the highest tested dosage (16 mg/kg) in the serum and heart tissue of rats (Fig. 8B and C). In the serum, QI at doses of 4, 8 and 16 mg/kg significantly enhanced GSH content, whereas only the 16 mg/kg dose was able to increase the GSH content in the heart tissue. These results indicate that QI achieves an antioxidant capacity in aging rats primarily through improving SOD, scavenging MDA and moderately increasing GSH.