The Antioxidant and Anti-Inflammatory Effects of Flavonoids from Propolis via Nrf2 and NF-κB Pathways

Accumulating evidence shows that oxidative stress and inflammation contribute to the development of cardiovascular disease. It has been suggested that propolis possesses antioxidant and anti-inflammatory activities. In this study, the antioxidant and anti-inflammatory effects of the main flavonoids of propolis (chrysin, pinocembrin, galangin, and pinobanksin) and propolis extract were researched. The results showed that the cellular ROS (Reactive oxygen species) levels, antioxidant enzymes, Nrf2 (Nuclear factor erythroid 2-related factor 2) nuclear translocation, and the expression of NQO1 (NAD(P)H:quinone oxidoreductase 1) and HO-1 (heme oxygenase 1) were regulated by different concentrations of individual flavonoids and propolis extract, which showed good antioxidant and pro-oxidant effects. For example, ROS levels were decreased; SOD and CAT activities were increased; and the expression of HO-1 protein was increased by chrysin. The results demonstrated that NO (Nitric Oxide), NOS (Nitric Oxide Synthase), and the activation of the NF-κB signaling pathway were inhibited in a dose-dependent manner by different concentrations of individual flavonoids and propolis extract. Moreover, the results revealed that the phytochemicals presented antioxidant effects at lower concentrations but pro-oxidant effects and stronger anti-inflammatory effects at higher concentrations. To maintain the balance of antioxidant and anti-inflammatory effects, it is possible that phytochemicals activate the Nrf2 pathway and inhibited the NF-κB (Nuclear factor kappa B) pathway.


Background
Cardiovascular disorders such as myocardial infarction are considered to be among the leading causes of mortality. Cardiovascular disorders produce excessive oxygen free radicals in the pathological process, and the disorders of free-radical metabolism in the state of oxidative stress are important triggers of myocardial damage [1]. One of the essential and indispensable immune-defense mechanisms of the human body is inflammation. However, persistent chronic inflammation can damage the visceral function and cause immune-system dysregulation, which can lead to a variety of chronic metabolic diseases or cancers [2,3].
The preparation of propolis extract was performed as follows: Propolis was de-mixed and frozen at −18 • C for 24 h; then, it was crushed with a pulverizer and sieved through a 120-mesh sieve, and the finished powder was stored at −80 • C. Propolis powder was immersed in a ratio of 1:30 (w/v) in ethanol-water solution (80%, v/v) and ultrasonicated (GA92-II DA Ultrasonic cell grinder, China) at the power of 100 W and 20 kHz. The mixture was centrifuged (Heal Force Neofuge 15R high-speed freezing centrifuge; China) at 4200× g for 5 min. Subsequently, the supernatant was gathered, and the extraction of the residue was repeated four times at least under the same conditions as before. After that, the supernatants obtained in multiple extraction experiments were mixed together, condensed under vacuum at 37 • C, and finally lyophilized for further study.

Coupling of HPLC-ESI-QTOF-MS/MS
The liquid chromatography analyses were performed on an Agilent 1260 HPLC system (Agilent, Santa Clara, CA, USA). Chromatographic separation was implemented on an Agilent Eclipse XDB C18 (Agilent, Santa Clara, CA, USA) column with detection being carried out at 280 nm with the operating temperatures being kept at 35 • C. The analyses were completed with a gradient elution of methol (A) and 0.1% formic acid in purified water (B). The gradient protocol was: 22-36% A, 0-5 min; 36-52% A, 5-30 min; 52-63% A, 30-65 min; 63-70% A, 65-95 min; 70-80% A, 95-120 min; 80-22% A, 120-122 min. The injected sample volume was 5 µL, and the flow rate was 0.6 mL/min. An Agilent 1260 HPLC (quaternary pump) system and AB Sciex TripleTOF™ 5600 mass spectrometer were used in the ESI-QTOF-MS/MS system. An electrospray ionization source was used to drive the TOF mass spectrometer. The capillary voltage was set to 4 kV; the collision voltage was set to 135 V; the drying-gas temperature was set to 350 • C; the dryinggas flow rate was set to 10 L/min; the nebulizer pressure was set to 40 psi; the collision gas was nitrogen; the collision energy was set to 30 eV; the full ionic scan mode was used; and the scan range was set to m/z = 50-2000.

Cell Culture and Treatment
H9c2 cells were plated into DMEM containing inactivated 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The cells were incubated in an incubator at 37 • C with 5% CO 2 . Cells were seeded into appropriate dishes or plates for 24 h before being subjected to various treatments. Chrysin, pinobanksin, galagin, and pinocembrin were dissolved in DMSO as reserve solutions (10 mM). All reserve solutions were stored at −80 • C and diluted into DMEM at various ratios prior to cellular incubating.

Cell Counting Kit-8 Assay for Cell Viability
H9c2 cells (1 × 10 5 cells per well) were seeded in a 96-well plate and allowed to complete from 80% to 90% confluence before being treated. Subsequently, the cells were exposed to various different treatments. Then, CCK-8 reagent (10 µL) was combined with 90 µL of DMEM to generate a working solution. After that, 100 µL of CCK-8 working solution was transferred towards each well, and the cells were additionally cultured for 1 h. Ultimately, the absorption at 450 nm was monitored with a microplate reader.

Production of Reactive Oxygen Species (ROS)
H9c2 cells (1 × 10 6 cells per well) were seeded inside a six-well plate and allowed to complete from 80% to 90% confluence before being treated. Cells were treated with standards and propolis extract for 12 h, followed by inducing with 150 µM H 2 O 2 for 1 h, and then incubated with 5 mM DCF-DA (fluorescent probe) in DMEM at 37 • C for 15-20 min in the dark. Cells were digested with 0.02% EDTA (ratios of commonly used cell lysis solutions that are recommended in flow cytometry) and then centrifuged at 1500 rpm for 10 min to remove the supernatant. The pellets were re-suspended in 200 µL of cold PBS. Fluorescence intensity was monitored via flow cytometry (BD FACS, Becton Dickinson Co., Franklin Lakes, NJ, USA).

SOD and CAT Assays
H9c2 cells (1 × 10 6 cells per well) were seeded inside a six-well plate and allowed to complete from 80% to 90% confluence before being treated. After various treatments, upon lysis with a tissue lysis solution, the supernatant of H9c2 cells was collected to measure the SOD and CAT activities. The supernatant was then added to the corresponding 96-well plate in a volume of 50 µL. The intracellular SOD and CAT activities were determined according to the procedures of Total Superoxide Dismutase Assay Kit and Catalase Assay Kit. Finally, the absorbance at 560 nm (SOD) or 520 nm (CAT) was measured with a microplate reader (BioTek Instruments, Santa Clara, CA, USA). The supernatant was used to determine the protein concentration in the BCA protein-concentration assay kit.

NO and NOS Assays
H9c2 cells (1 × 10 6 cells per well) were seeded inside a six-well plate and allowed to complete from 80% to 90% confluence before being treated. After various treatments, Griess Reagent was used to detect the release of NO. Then, we took a 96-well plate and placed 50 µL of cell culture medium into the corresponding wells. A total of 50 µL of Griess Reagent I and 50 µL of Griess Reagent II were added to the corresponding wells. Nitric Oxide Synthase Assay Kit was allowed to equilibrate at 25 • C for 20 min. The treated 96-well plate was aspirated out of the culture solution, and 100 µL of NOS Assay Buffer was added. Another 100 µL of Assay Reaction Solution was added and gently mixed. Finally, the absorbance at 540 nm was measured with a microplate reader (BioTek Instruments, Santa Clara, CA, USA).

Extraction of Whole-Cell Protein, Cytosolic Protein, and Nuclear Protein
H9c2 cells (1 × 10 6 cells per well) were cultured in culture dishes 3 cm in diameter and were allowed to reach from 80% to 90% of confluence before treatment. After various treatments, cells were cleaned three times with cold PBS. Whole-cell proteins were extracted using a protein extraction kit (Shanghai, China). Briefly, after adding 200 µL of lysate/dish, the culture dishes were placed in an ice-box for 30 min and then centrifuged at 12,000× g for 10 min; finally, the supernatant was obtained as whole-cell proteins.
Cytoplasmic Protein Extraction Reagent was included in Nucleoprotein and Cytoplasmic Protein Extraction Kit. We scraped off the treated cells with a cell scraper, centrifuged the cells, poured off the supernatant, and added 200 µL of PMSF-added Cytoplasmic Protein Extraction Reagent A per 20 µL of cell sediment. To completely suspend and distribute the cell sediment, it was violently vortexed for 5 s; then, it was subjected to an ice bath for 10-15 min. Then, 10 µL of Cell Plasma Protein Extraction Reagent B was added. It was vigorously vortexed for 5 s; then, it was subjected to an ice bath for 1 min. It was centrifuged at 12,000× g for 5 min at 4 • C after being violently vortexed for 5 s. The resulting supernatant was the cell pulp protein obtained via extraction. For the remaining precipitate, we completely aspirated the residual supernatant and added 50 µL of PMSF-added cell nuclear-protein extraction reagent. It was vigorously vortexed for 15-30 s to completely suspend and disperse the cell precipitate; then, it was returned to the ice bath and vigorously vortexed for another 15-30 s every 1-2 min for 30 min. Finally, it was centrifuged at 12,000× g for 10 min at 4 • C. The nucleoproteins recovered from the cells were observed in the supernatant.

Statistical Analysis
The SPSS 26.0 statistical software tool (SPSS Inc., Chicago, IL, USA) was used to analyze the data, which were given as mean standard errors of the mean (SEMs). Duncan's tests were used to assess differences after a single-factor analysis of variance (ANOVA), and p < 0.05 was used to denote significant differences. The data were statistically analyzed using GraphPad Prism 8 software (San Diego, CA, USA).

Effects of H2O2 on Cell Viability and Production of ROS
To induce oxidative stress, H9c2 cells were exposed to multiple H2O2 concentrations (0, 50, 100, 150, 200, and 250 μM) for 1 h. Compared with the control group, cell viability was 60.24% ( Figure S1) when the concentration of H2O2 reached 150 μM. In addition, the level of intracellular ROS was significantly increased to 292.20% when the H2O2 concentration was 150 μM. Compared with the control group, ROS levels were gradually decreased to 205.12% ( Figure S2) when the concentration of H2O2 reached 250 μM. ROS levels were gradually increased when the concentration of H2O2 was in the range of 0-150

Effects of H2O2 on Cell Viability and Production of ROS
To induce oxidative stress, H9c2 cells were exposed to multiple H2O2 concentrations (0, 50, 100, 150, 200, and 250 μM) for 1 h. Compared with the control group, cell viability was 60.24% ( Figure S1) when the concentration of H2O2 reached 150 μM. In addition, the level of intracellular ROS was significantly increased to 292.20% when the H2O2 concentration was 150 μM. Compared with the control group, ROS levels were gradually decreased to 205.12% ( Figure S2) when the concentration of H2O2 reached 250 μM. ROS levels were gradually increased when the concentration of H2O2 was in the range of 0-150

Effects of H 2 O 2 on Cell Viability and Production of ROS
To induce oxidative stress, H9c2 cells were exposed to multiple H 2 O 2 concentrations (0, 50, 100, 150, 200, and 250 µM) for 1 h. Compared with the control group, cell viability was 60.24% ( Figure S1) when the concentration of H 2 O 2 reached 150 µM. In addition, the level of intracellular ROS was significantly increased to 292.20% when the H 2 O 2 concentration was 150 µM. Compared with the control group, ROS levels were gradually decreased to 205.12% ( Figure S2) when the concentration of H 2 O 2 reached 250 µM. ROS levels were gradually increased when the concentration of H 2 O 2 was in the range of 0-150 µM. ROS levels were gradually decreased when the concentration was above 150 µM (p < 0.05). The concentration with the highest intracellular ROS level was used in subsequent experiments. Cells were in a state of oxidative stress when the viability was 50-70%, which does not immediately cause death and can be recovered using antioxidants. Oxidation significantly harms cells and does irreparable damage when cell viability falls below 40%. Therefore, 150 µM H 2 O 2 was used to induce cellular oxidative damage in H9c2 cells.

Effects of LPSs on Cell Viability and Expression of Pro-Inflammatory Cytokine Proteins
The viability of H9c2 cells was unaffected by LPS concentrations of 5,10,15,20,25,30,35, and 40 µg/mL ( Figure S3). Compared with the control group, when the concentration of LPSs reached 10 µg/mL, the expression of VCAM and IL-6 pro-inflammatory cytokine proteins was significantly increased to 150.76% and 181.05%, respectively ( Figure S4). The protein expression of VCAM and IL-6 was maximized when the concentrations of LPSs reached 10 µg/mL. Therefore, 10 µg/mL LPSs were used to model the cellular inflammatory damage.

Effects of Chrysin, Pinocembrin, Galangin, Pinobanksin, and Propolis Extract on Cell Viability
The CCK-8 assay was used to assess the potential cytotoxicity of chrysin, pinocembrin, galangin, pinobanksin, and propolis extract in H9c2 cells. When the concentration of chrysin was higher than 30 µM, that of galangin higher than 60 µM, and that of propolis extract higher than 100 µg/mL, cell viability was below 95%. However, pinocembrin below 300 µM and pinobanksin below 300 µM did not inhibit the proliferation of H9c2 cells ( Figure S5). In order to avoid toxicity, the concentration of chrysin below 30 µM, that of pinocembrin below 80 µM, that of galangin below 60 µM, that of pinobanksin below 80 µM, and that of propolis extract below 100 µg /mL were chosen for further study.

Effects of Chrysin, Pinocembrin, Galangin, Pinobanksin, and Propolis Extract on Cell Production of ROS
ROS are among the major intracellular oxidation products and important participants in cell signaling. Their accumulation probably causes macrophages to undergo more apoptosis or autophagy [39]. Pretreatment with different sample concentrations dramatically reduced H 2 O 2 -induced ROS generation (p < 0.05), and significant differences were found among doses that showed a trend of first decreasing and then remaining constant or increasing. On the one hand, after chrysin, pinocembrin, and propolis-extract pretreatment, the antioxidant effects tended to first increase and subsequently decrease. On the other hand, after galangin and pinobanksin pretreatment, the antioxidant effects tended to first increase and subsequently stabilize.

SOD and CAT Activities
SOD plays an essential role in the conversion of superoxide to hydrogen peroxide, while CAT converts hydrogen peroxide to water [40]. To explore the effect of the antioxidant defense of chrysin, pinocembrin, galangin, pinobanksin, and propolis extract, SOD ( Figure 4) and CAT ( Figure 5) activities were evaluated. Compared with the control group, these enzyme activities were significantly decreased in the H 2 O 2 -induced group (p < 0.05). Furthermore, after pretreatment with different sample concentrations, SOD and CAT levels were increased in H9c2 cells induced by H 2 O 2 (p < 0.05). For example, as shown in Figures 4A and 5A

Effects of Chrysin, Pinocembrin, Galangin, Pinobanksin, and Propolis Extract on the Expression of Proteins Encoded by Antioxidant Genes Downstream of Nrf2
A number of antioxidant genes are activated by nuclear factor erythroid 2-related factor 2 (Nrf2) to protect the body from ROS damage [41,42]. Upon exposure to ROS, Nrf2 is translocated to the nucleus and conjugated to antioxidant response elements (ARE); thereby, the transcription of cellular-defense-related genes is up-regulated, including antioxidant proteins, antitoxic enzymes, and drug metabolism [42,43]. Therefore, the expression of antioxidant proteins downstream of Nrf2, HO-1 and NQO-1, was assayed via Western blot. Compared with the control group, the expression of HO-1 and NQO-1 proteins was significantly decreased in H 2 O 2 -induced H9c2 cells (p < 0.05). In addition, the expression of HO-1 and NQO-1 proteins was dramatically increased after pretreatment with different sample concentrations in H 2 O 2 -induced H9c2 cells (p < 0.05; Figure 6).   On the one hand, after pretreatment with chrysin, pinocembrin, and propolis extract, the expression of the HO-1 protein tended to first increase and subsequently decrease. For example, with chrysin ( Figure 6A), compared with the H 2 O 2 -induced group (80.73 ± 0.88%), the expression of HO-1 was significantly increased, and the antioxidant effect increased in the concentration range from 5 µM (98.85 ± 3.22%) to 15 µM (95.31 ± 1.02%), while the expression of HO-1 was gradually decreased and showed pro-oxidant effects in the concentration range from 20 µM (78.69 ± 9.44%) to 25 µM (62.59 ± 0.06%). On the other hand, after galangin and pinobanksin pretreatment, the expression of the HO-1 protein tended to first increase and subsequently stabilize. For example, with pinobanksin ( Figure 6D), compared with the H 2 O 2 -induced group (46.7 ± 0.74%), the expression of HO-1 was significantly increased, and the antioxidant effect increased in the concentration range from 5 µM (75.13 ± 0.83%) to 40 µM (96.85 ± 3.82%), while the expression of HO-1 was no longer consistently elevated and showed pro-oxidant effects in the concentration range from 40 µM to 80 µM (105.95 ± 4%).
Compared to the H 2 O 2 -induced group, there were no significant differences in the expression of NQO1 after chrysin pretreatments (p > 0.05). However, after galangin, pinobanksin, and propolis-extract pretreatments, the expression of the NQO1 protein tended to first increase and then stabilize. For example, with propolis extract (Figure 6E), compared with the H 2 O 2 -induced group (60.36 ± 0.38%), the expression of NQO1 was significantly increased, and the antioxidant effect increased in the concentration range from 5 µM (90.88 ± 4.4%) to 40 µM (106.87 ± 7.5%), while the expression of the NQO1 protein was no longer consistently elevated and showed pro-oxidant effects in the concentration range from 40 µM to 100 µM (114.55 ± 1.97%). In addition, with pinocembrin (( Figure 6B), compared with H 2 O 2 -induced group (82.59 ± 0.84%), in the concentration range from 5 µM (90.50 ± 2.3%) to 80 µM (107.87 ± 0.37%), the expression of the NQO1 protein was significantly increased. On the one hand, after pretreatment with different concentrations of chrysin, pinocembrin, and propolis extract, the accumulation of Nrf2 in the nucleus tended to first increase and subsequently decrease as the concentration was gradually increased. For example, with chrysin ( Figure 7A), compared with the H 2 O 2 -induced group, the translocation of Nrf2 from the cytoplasm to the nucleus was gradually increased and showed anti-oxidant effects in the concentration range from 5 to 10 µM, while the translocation of Nrf2 from the cytoplasm to the nucleus was gradually decreased in a dose-dependent manner and showed pro-oxidant effects in the concentration range from 15 µM to 25 µM.
On the other hand, after pretreatment with different concentrations of galangin and pinobanksin, the accumulation of Nrf2 in the nucleus tended to first increase and subsequently stabilize as the concentration was gradually increased. For example, with galangin, compared with the H 2 O 2 -induced group, the translocation of Nrf2 from the cytoplasm to the nucleus was increased in a dose-dependent manner and showed anti-oxidant effects in the concentration range from 10 µM to 50 µM, while the translocation of Nrf2 from the cytoplasm to the nucleus reached saturation and showed pro-oxidant effects in the concentration range from 50 µM to 60 µM.
It is worth noting that the increase in cytoplasmic Nrf2 was always accompanied by the decrease in nuclear Nrf2. Additionally, the expression of the nuclear translocation of Nrf2 followed a tendency similar to that of the expression of the HO-1 protein. According to these results, the modification of Nrf2 nuclear translocation was the mechanism via which chrysin, pinocembrin, galangin, pinobanksin, and propolis extract showed antioxidant activity. It was found that Nrf2 translocation from the cytoplasm to the nucleus was up-regulated (chrysin range of 5 µM-10 µM, pinocembrin range of 5 µM-40 µM, and propolis-extract range of 5 µg/mL-40 µg/mL) and then down-regulated (chrysin range of 15 µM-25 µM, pinocembrin range of 40 µM-60 µM, and propolis-extract range of 40 µg/mL-100 µg/mL) following treatments with chrysin, pinocembrin, and propolis extract. It was also found that Nrf2 translocation from the cytoplasm to the nucleus was up-regulated and then held relatively constant following treatments with 10-60 µM galangin and 5-80 µM pinobanksin.

NO and NOS Levels
NO is catalyzed by NOS in cells, and it can trigger tissue damage and ultimately lead to pain and inflammation [44,45]. The secretion of NO was significantly reduced by chrysin, pinocembrin, galangin, pinobanksin, and propolis extract (p < 0.05; Figure 8). For example, compared with the control group (2.95 ± 0.1 µM), the levels of NO (4.98 ± 0.13 µM) were significantly increased in H9c2 cells in the LPS-induced group. Moreover, the levels of NO were significantly reduced (1.866 ± 0.03 µM-4.69 ± 0.03 µM) by 5-25 µM chrysin. On the one hand, after pretreatment with different concentrations of chrysin, pinocembrin, and propolis extract, the accumulation of Nrf2 in the nucleus tended to first increase and subsequently decrease as the concentration was gradually increased. For example, with chrysin ( Figure 7A), compared with the H2O2-induced group, the translocation of Nrf2 from the cytoplasm to the nucleus was gradually increased and showed antioxidant effects in the concentration range from 5 to 10 μM, while the translocation of Nrf2 from the cytoplasm to the nucleus was gradually decreased in a dose-dependent manner and showed pro-oxidant effects in the concentration range from 15 μM to 25 μM.
On the other hand, after pretreatment with different concentrations of galangin and pinobanksin, the accumulation of Nrf2 in the nucleus tended to first increase and subsequently stabilize as the concentration was gradually increased. For example, with galangin, compared with the H2O2-induced group, the translocation of Nrf2 from the cytoplasm to the nucleus was increased in a dose-dependent manner and showed anti-oxidant effects in the concentration range from 10 μM to 50 μM, while the translocation of Nrf2 from the cytoplasm to the nucleus reached saturation and showed pro-oxidant effects in the concentration range from 50 μM to 60 μM. In addition, NOS generates NO, which is an inflammatory mediator [46]. The levels of NOS were considerably higher in the treatment group than that in the control group in H9c2 cells induced with 10 µg/mL LPS, and the levels of NOS were significantly attenuated by chrysin, pinocembrin, galangin, pinobanksin, and propolis extract in a dose-dependent manner (p < 0.05; Figure 9). For example, compared with the control group (100%), the level of NO (120 ± 14.65%) was significantly increased in H9c2 cells induced with 10 µg/mL LPS. Moreover, the level of NO was significantly decreased (82.13 ± 2.7%-90.09 ± 0.6%) after 5-25 µM chrysin pretreatment in H9c2 cells induced with 10 µg/mL LPS.

Chrysin, Pinocembrin, Galangin, Pinobanksin, and Propolis Extract Down-Regulated the Expression of Pro-Inflammatory Cytokines
To explore the effects of chrysin, pinocembrin, galangin, pinobanksin, and propolis extract on the inflammatory response of LPS-induced H9c2 cells, the expression of proinflammatory cytokines was measured. Compared with the control group, the expression of IL-6 (109.63-230.75%) and VACM proteins (p < 0.05; 167.29-245.79%) was significantly upregulated in the LPS-induced group. After pretreatment with the samples, the expression of IL-6 and VCAM proteins was significantly inhibited in LPS-induced H9c2 cells in a dose-dependent manner (p < 0.05; Figure 10). In addition, NOS generates NO, which is an inflammatory mediator [46]. The levels of NOS were considerably higher in the treatment group than that in the control group in H9c2 cells induced with 10 μg/mL LPS, and the levels of NOS were significantly attenuated by chrysin, pinocembrin, galangin, pinobanksin, and propolis extract in a dose- dependent manner (p < 0.05; Figure 9). For example, compared with the control group (100%), the level of NO (120 ± 14.65%) was significantly increased in H9c2 cells induced with 10 μg/mL LPS. Moreover, the level of NO was significantly decreased (82.13 ± 2.7%-90.09 ± 0.6%) after 5-25 μM chrysin pretreatment in H9c2 cells induced with 10 μg/mL LPS.  inflammatory cytokines was measured. Compared with the control group, the expr of IL-6 (109.63-230.75%) and VACM proteins (p < 0.05; 167.29-245.79%) was signifi up-regulated in the LPS-induced group. After pretreatment with the samples, the e sion of IL-6 and VCAM proteins was significantly inhibited in LPS-induced H9c2 c a dose-dependent manner (p < 0.05; Figure 10).

Inhibition of NF-κB Signaling Pathway
The NF-κB signaling pathway can be activated by the up-regulation of pro-inflammatory cytokines [47]. After pretreatment with different concentrations of chrysin, pinocembrin, galangin, pinobanksin, and propolis extract, the expression of the phosphorylation of the NF-κB p65 protein was examined to explore the possible mechanism of inhibition of the expression of VCAM1 and IL-6 as well as to confirm the effects on the NF-κB signaling pathway. Compared with the control group, the expression of the phosphorylation of NF-κB p65 was increased in the LPS-induced group. However, after pretreatment with the samples, the expression of the phosphorylation of NF-κB p65 was significantly attenuated in a dosedependent manner (p < 0.05; Figure 11). For example, with chrysin ( Figure 11A), compared with the LPS-induced group (138.49 ± 10.2%), the expression of the p65 protein was decreased to 212.11 ± 27.49% when the concentration of chrysin was 5 µM. The expression of the p65 protein gradually decreased to 121.05 ± 18.9% when the concentration of chrysin was gradually increased to 25 µM.
The results indicated that chrysin, pinocembrin, galangin, pinobanksin, and propolis extract markedly suppressed the phosphorylation of NF-κB p65 induced by LPS.

Interpretation of Antioxidant and Pro-Oxidant Effects
In terms of structure, flavonoid compounds provide hydrogen atoms to bind to ox gen radicals and prevent the formation of free radicals. Additionally, the signaling pa ways associated with antioxidant defense systems are regulated by flavonoid compoun This could be how flavonoids interact to exert their antioxidant effects.
Firstly, the structure of a flavonoid indicates whether it has pro-or antioxidant fects. The structures of chrysin, pinocembrin, galangin, and pinobanksin are shown Figure 2. It can be observed that chrysin, pinocembrin, and pinobanksin are flavonoi whereas galangin is a flavonol compound. Because of the C2-C3 double bond and the C hydroxyl group, flavonols (e.g., galangin) have a stronger oxidative activity, while flav noids have a lower oxidative activity [48,49]. It is worth emphasizing that the C2=C3 do ble bond plays an important role in the antioxidant activity of flavonoids. Chrysin a galangin both feature C2=C3 double bonds, but galangin differs in that it has a C-3 h droxyl group that promotes the oxidation to benzoquinone intermediates. However, t oxidation of galangin requires the consumption of free radicals in the system. Due to t lack of a B-ring ortho-hydroxyl group, the pro-oxidation effect of galangin is weaker th

Interpretation of Antioxidant and Pro-Oxidant Effects
In terms of structure, flavonoid compounds provide hydrogen atoms to bind to oxygen radicals and prevent the formation of free radicals. Additionally, the signaling pathways associated with antioxidant defense systems are regulated by flavonoid compounds. This could be how flavonoids interact to exert their antioxidant effects.
Firstly, the structure of a flavonoid indicates whether it has pro-or antioxidant effects. The structures of chrysin, pinocembrin, galangin, and pinobanksin are shown in Figure 2. It can be observed that chrysin, pinocembrin, and pinobanksin are flavonoids, whereas galangin is a flavonol compound. Because of the C 2 -C 3 double bond and the C-3 hydroxyl group, flavonols (e.g., galangin) have a stronger oxidative activity, while flavonoids have a lower oxidative activity [48,49]. It is worth emphasizing that the C 2 =C 3 double bond plays an important role in the antioxidant activity of flavonoids. Chrysin and galangin both feature C 2 =C 3 double bonds, but galangin differs in that it has a C-3 hydroxyl group that promotes the oxidation to benzoquinone intermediates. However, the oxidation of galangin requires the consumption of free radicals in the system. Due to the lack of a B-ring ortho-hydroxyl group, the pro-oxidation effect of galangin is weaker than that of chrysin [50]. Therefore, a higher concentration of galangin does not show pro-oxidation effects.
Secondly, chrysin, pinocembrin, galangin, pinobanksin, and propolis extract exhibited antioxidant and pro-oxidant effects in a dose-dependent manner. The occurrence of prooxidant effects may be due to the auto-oxidation of flavonoid compounds, because as the concentration of these flavonoids increases, so does the synthesis of lipid peroxidation products and the development of superoxide anion radicals [51]. Moreover, once flavonoids reach a higher concentration, they may be oxidized to intermediates with pro-oxidant effects such as phenoxy radicals, semi-quinones, and quinone structures [52]. Therefore, high concentrations of chrysin, pinocembrin, and propolis extract showed pro-oxidant effects. Similarly, to confirm the pro-oxidant effect of high doses of genistein (200 µM) in primary muscle cells, Chen et al. [53] analyzed cellular lipid peroxidation, redox homeostasis, and ROS production. Furthermore, Galati et al. [54] indicated that 3 mM epigallocatechin and 2 mM epicatechin-3-gallate were found to have pro-oxidant effects on the mitochondrialmembrane potential and ROS levels in hepatocytes.

Activation of Nrf2 Signaling Pathway and Inhibition of NF-κB Signaling Pathway
The individual flavonoids (chrysin, pinocembrin, galangin, and pinobanksin) and propolis extract exerted strong antioxidant effects as inducers of the Nrf2/HO-1 axis, and they were potent activators of Nrf2 nuclear translocation in H9c2 cells. The phytochemicals showed antioxidant effects at low concentrations and showed pro-oxidant effects at high concentrations, possibly by activating Nrf2. Once activated by oxidative stress, Nrf2 is translocated to the nucleus and binds to antioxidant transcription elements in the promoter region of phase 2 to increase the expression of certain antioxidant and detoxification genes, ultimately leading to cellular resistance to oxidative stress [55]. HO-1 and NQO-1 are well-characterized Nrf2-dependent antioxidant defense genes. It was speculated that transcription factor Nrf2, which was associated with the degree of SOD and CAT activation of cellular antioxidant responses, was enhanced or lowered by chrysin, pinocembrin, and propolis extract in the nucleus of H9c2 cells. Intriguingly, the levels of antioxidant enzymes were typically associated with amplification during the formation of ROS. SOD levels represent a fundamental defense mechanism against excessive ROS [56]. Similar results were observed in quercetin that pretreating with low concentrations of flavonoid stimulated cell proliferation and enhanced the total antioxidant capacity of cells. Moreover, higher concentrations of the flavonoid diminished cell viability and total antioxidant capacity, as well as the activities of catalase, superoxide dismutase, and glutathione S-transferase [57].
Oxidative stress and inflammatory responses are important components in the pathogenesis of cardiovascular stent disease. Accumulating evidence suggests that oxidative stress is inseparable from the inflammatory response. Currently, exploring the relationship between antioxidant and anti-inflammatory agents is a potential target for the prevention or mitigation of cardiovascular disease. Therefore, our study focused on the changes in the Nrf2 and NF-κB pathways involved in oxidative stress and inflammatory processes.
According to the datas of ROS, NOS, and the expression of pro-inflammatory protein, chrysin showed anti-oxidant effects in the concentration range from 5 µM to 10 µM, while it showed pro-oxidant effects in the concentration range from 15 µM to 25 µM, and it showed anti-inflammatory effects in the concentration range from 5 µM to 25 µM. Pinocembrin showed anti-oxidant effects in the concentration range from 5 µM to 40 µM, while it showed pro-oxidant effects in the concentration range from 60 µM to 80 µM, and it showed antiinflammatory effects in the concentration range from 5 µM to 80 µM. Galangin showed anti-oxidant effects in the concentration range from 10 µM to 50 µM, while it showed pro-oxidant effects in the concentration range from 50 µM to 60 µM, and it showed antiinflammatory effects in the concentration range from 10 µM to 60 µM. Pinobanksin showed anti-oxidant effects in the concentration range from 5 µM to 60 µM, while it showed pro-oxidant effects in the concentration range from 60 µM to 80 µM, and it showed antiinflammatory effects in the concentration range from 5 µM to 80 µM. Propolis extract showed anti-oxidant effects in the concentration range from 5 µg/mL to 40 µg/mL, while it showed pro-oxidant effects in the concentration range from 60 µg/mL to 100 µg/mL, and it showed anti-inflammatory effects in the concentration range from 5 µg/mL to 100 µg/mL. Additionally, the higher the concentration of a compound was, the stronger the anti-inflammatory effect was.
The inhibition of the production of NOS induced by flavonoids is caused by their antioxidant properties, and these compounds can exert anti-inflammatory effects by scavenging ROS [58]. Furthermore, flavonoids are used as inhibitors of lipopolysaccharidesignaling molecules to decrease inflammation.
NF-κB is a classical inflammatory signaling pathway that regulates the expression of immune genes encoding cytokines, such as IL-1β, TNF-α, and IL-6 [47]. Previous reports showed that propolis inhibited cytokine production in various cardiovascular injuries [59]. Likewise, it was also observed that propolis inhibited the expression of VCAM1 and IL-6 in our findings. It was documented that NOS, VCAM, and IL-6 were pivotal for NF-κB signaling [60]. The anti-inflammatory effects were enhanced by chrysin, pinocembrin, galangin, pinobanksin, and propolis-extract pretreatment with the increase in concentration. Moreover, the inhibition of the NF-kB signaling pathway was also enhanced. It was speculated that the pro-oxidant effects were produced by high concentrations of phytochemicals triggering a series of inflammatory responses and exerting anti-inflammatory effects by subsequently inhibiting the NF-κB signaling pathway. It was in accordance with the trend of progressively lower expression of inflammatory proteins VCAM and IL-6 downstream of the NF-κB signaling pathway in the results.
Research demonstrates that a functional interaction and crosstalk between Nrf2 and NF-κB pathways exists to maintain balance or regulate oxidative stress and inflammation [15]. Even though many phytochemicals are reported to modulate NF-κB and Nrf2 activities [61], the mechanism of crosstalk remains unclear. Therefore, it is possible that the activation of the Nrf2 pathway dominates at low phytochemical concentrations and that the inhibition of the NF-κB pathway dominates at high phytochemical concentrations.
The bioavailability values of galangin and chrysin in propolis extracts were determined in a study, and they were at 7.8% and 7.5%, respectively [62]. Moreover, propolis extract has a higher bioavailability than single-flavonoid standards [32]. In addition, galangin is frequently used with popular pharmaceuticals. So, it has the potential to improve the bioavailability and chemoprevention of oral drugs and to reverse multidrug resistance [63]. Furthermore, there are numerous ways to improve bioavailability, which sparked renewed interest in propolis research. The above findings make propolis extract a promising antioxidant for use as a food supplement.

Conclusions
The major flavonoids in propolis were identified as chrysin, pinocembrin, galangin, and pinobanksin. It was revealed that flavonoids from propolis mainly presented antioxidant effects at lower concentrations and presented pro-oxidant as well as stronger anti-inflammatory effects at higher concentrations. We observed anti-oxidant effects of chrysin in the concentration range from 5 µM to 10 µM, of pinocembrin in the concentration range from 5 µM to 40 µM, of galangin in the concentration range from 10 µM to 50 µM, of pinobanksin in the concentration range from 5 µM to 60 µM, and of propolis extract in the concentration range from 5 µg/mL to 40 µg/mL, while we observed pro-oxidant effects of chrysin in the concentration range from 15 µM to 25 µM, of pinocembrin in the concentration range from 60 µM to 80 µM, of galangin in the concentration range from 50 µM to 60 µM, of pinobanksin in the concentration range from 60 µM to 80 µM, and of propolis extract in the concentration range from 60 µg/mL to 100 µg/mL. In addition, we observed anti-inflammatory effects of chrysin in the concentration range from 5 µM to 25 µM, of pinocembrin in the concentration range from 5 µM to 80 µM, of galangin in the concentration range from 10 µM to 60 µM, of pinobanksin in the concentration range from 5 µM to 80 µM, and of propolis extract in the concentration range from 5 µg/mL to 100 µg/mL. Additionally, the higher the concentration of the compound was, the stronger the anti-inflammatory activity was. Flavonoids from propolis could probably activate the Nrf2 pathway and inhibit the NF-κB pathway to maintain the balance of antioxidant and anti-inflammatory effects. In the future, it is important to focus on the link between the Nrf2 and NF-κB pathways to explore the anti-oxidative mechanism of low-concentration flavonoids and the anti-inflammatory mechanism of high-concentration flavonoids.