Vanillic Acid Alleviates Acute Myocardial Hypoxia/Reoxygenation Injury by Inhibiting Oxidative Stress

Oxidative stress is an important factor of myocardial hypoxia/reoxygenation (H/R) injury. Our research focuses on how to reduce the cardiac toxicity caused by oxidative stress through natural plant extracts. Vanillic acid (VA) is a phenolic compound found in edible plants and rich in the roots of Angelica sinensis. Experimental studies have provided evidence for this compound's effectiveness in cardiovascular diseases; however, its mechanism is still unclear. In this study, molecular mechanisms related to the protective effects of VA were investigated in H9c2 cells in the context of H/R injury. The results showed that pretreatment with VA significantly increased cell viability and decreased the percentage of apoptotic cells, as well as lactate dehydrogenase and creatine phosphokinase activity, in the supernatant, accompanied by reduced levels of reactive oxygen species and reduced caspase-3 activity. VA pretreatment also restored mitochondrial membrane potentials. Moreover, preincubation with VA significantly attenuated mitochondrial permeability transition pore activity. VA administration upregulated adenosine monophosphate-activated protein kinase α2 (AMPKα2) protein expression, and interestingly, pretreatment with AMPKα2-siRNA lentivirus effectively attenuated the cardioprotective effects of VA in response to H/R injury.


Introduction
Myocardial hypoxia/reoxygenation (H/R) injury leads to significant morbidity and mortality [1], and oxidative stress is one of the most important factors causing cardiac toxicity. Natural plant extracts have the advantages of few side effects and easy access. Therefore, our research focuses on the use of natural plant extracts to protect the heart against oxidative stress, thereby reducing cardiac toxicity caused by I/R injury. Vanillic acid (VA) is a phenolic compound found in secondary plant products with the molecular formula C 8 H 8 O 4 . VA is widely used in the food industry as flavouring agent, food additive, and preservative. We detected VA in various foods, such as herbs, tea, coffee, wine, and beer [2][3][4][5][6][7]. VA possesses powerful antioxidant functions, antihypotensive effects, cardioprotective effects, hepatoprotective effects, and antiapoptotic activities [8][9][10][11].
Adenosine monophosphate-activated protein kinase (AMPK) is a stress responsive kinase that modulates a number of physiologically and metabolically significant pathways, including apoptosis, energy dynamic balance, and cellular metabolism [12]. AMPK ameliorates cellular antioxidant enzyme systems, such as manganese superoxide dismutase (Mn-SOD) and catalase, consequently reducing oxidant-induced injury [13].
Accordingly, we hypothesized that VA exerts a protective effect against hypoxia/reoxygenation (H/R) injury, which might be related to the AMPK signalling pathway. Therefore, the present study is aimed at addressing the following aims: (1) determine whether VA pretreatment protects H9c2 cells from hypoxia/reoxygenation (H/R) injury and (2) explore the underlying protective mechanisms of VA on hypoxia/reoxygenation (H/R) injury in H9c2 cells.

Cell
Culture. Embryonic rat heart-derived H9c2 cells were purchased from the Chinese Academy of Sciences cell bank. H9c2 cells were incubated at 37°C in a 5% CO 2 incubator (Forma™ 310, Thermo Fisher, USA) with high glucose (4.5 g/l glucose) DMEM (Solarbio, Beijing, China) by adding 13% FBS (foetal bovine serum, WISENT, Canada) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Solarbio, Beijing, China). The hypoxia/reoxygenation (H/R) cell model was set up to imitate an ischaemia/reperfusion (I/R) model in vitro [14][15][16][17]. Briefly, after different pretreatments, H9c2 cells were cultured in hypoxic solution (sodium lactate 40 mM, NaH 2 PO 4 0.9 mM, NaHCO 3 [19]. After adding 200 μM CaCl 2 to the mitochondria, the opening of the mitochondrial permeability transition pore leads to mitochondrial swelling causing a stable decline in mitochondrial optical density. Due to the ability of the mitochondria to dilate, optical density (OD) was recorded using a microplate reader at 520 nm (Bio-Rad 680, USA). Mitochondrial swelling was recorded as the optical density (OD 1 ) at 520 nm, and a second optical density (OD 2 ) was recorded 20 min after induction. The optical density at 520 nm was continuously recorded over 20 min. mPTP activity was calculated as ΔODðOD 1 -OD 2 Þ/ min × 1000.
2.10. Measurement of Caspase-3 Activity. Caspase-3 activity was measured using a caspase-3 activity assay kit (Beyotime, Shanghai, China) following the manufacturer's instructions. H9c2 cells were lysed in lysis buffer on ice for 15 min and then collected and centrifuged at 16,000g for 15 min at 4°C to obtain the supernatant. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). After adding detection buffer and Ac-DEVD-рNA and incubating at 37°C for 18 h, optical density was detected at 405 nm. Caspase-3 activity was measured using a microplate reader (Bio-Rad 680, USA). A.U. is the abbreviation for arbitrary units.
2.11. Analysis of Apoptosis by Flow Cytometry. We previously published methods for measuring apoptosis [20]. Briefly, apoptosis was assessed by flow cytometry using an Annexin V-FITC/PI apoptosis kit (KeyGEN BioTECH, Nanjing, China) following the manufacturer's instructions. Briefly, H9c2 cells were collected and washed three times with ice-cold PBS. Next, collected H9c2 cells were resuspended in 1x Binding Buffer at a final concentration of 5 × 10 5 cells/ml. After the addition of 5 μl Annexin V-FITC and 5 μl PI, H9c2 cells were incubated in the dark for 15 min at room temperature. Cellular fluorescence was analysed by flow cytometry (ex 488 nm; em 530 nm, Beckman Coulter, USA).

Assessment of Apoptosis by TUNEL Staining. Apoptotic
H9c2 cells were analysed by optical microscopy using the terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) staining method, which was performed using a TUNEL Apoptosis Detection kit (Promega, USA) [21]. Apoptotic H9c2 cells are stained brown, identifying them as TUNEL positive. Then, apoptotic H9c2 cells were subsequently observed on a microscope (Olympus, Japan). The number of TUNEL-positive H9c2 cells/the total number of H9c2 cells represents the apoptotic index.
2.13. Statistical Analysis. All values are presented as the mean ± SEM. ANOVA was applied to measure the significance of examined data across different groups, followed by post hoc analysis to determine individual differences. p values ≤ 0.05 were considered statistically significant.

Pretreatment with VA Ameliorates the Viability of H9c2
Cells and Reduces Levels of LDH and CPK in H9c2 Cells after H/R. To measure the effects of VA on H9c2 cells undergoing hypoxia/reoxygenation (H/R), MTS assay was performed on H9c2 cells that were pretreated with different concentrations of VA 24 h prior to H/R. H9c2 cell viability decreased markedly in response to H/R (Figure 1(a), p < 0:01 vs. control group). We demonstrated that VA significantly increased H9c2 cell viability. Pretreatment with different concentrations of VA progressively increased viability of H9c2 cell in a concentration-dependent manner. H9c2 cell viability peaked at 1.00 mM VA (p < 0:05 vs. 0.50 mM VA+H/R group). However, H9c2 cells pretreated with concentrations of VA higher than 2.00 mM exhibited a significant reduction in cell viability, illustrating that higher VA concentrations cause toxicity. Thus, 1.00 mM VA was selected as the optimal pretreatment concentration for subsequent experiments. In addition, in Figure 1(b), our results show that AMPKα2-siRNA caused significantly reduced cell viability (p < 0:05 vs. VA+H/R group). Thus, we hypothesized that VA protects H9c2 cells from H/R injury through the AMPK signalling pathway.
To measure injury to H9c2 cell membranes in response to H/R, we evaluated levels of LDH and CPK in the supernatant after H/R treatment. The levels of both LDH and CPK were increased in the H/R group (p < 0:01 vs. control group) and markedly decreased in the VA+H/R group (p < 0:01 vs. H/R group). Furthermore, the VA+AMPKα2-siRNA+H/R group significantly increased the levels of LDH and CPK (p < 0:01 vs. VA+H/R group) (Figure 1(c)).

Pretreatment with VA Upregulates AMPKα2
Protein Levels in H9c2 Cells Undergoing H/R. To verify whether the AMPKα2 protein was associated with observed cardioprotective activity and optimal pretreatment concentration of VA, we assessed protein levels of AMPKα2 by western blot. We pretreated H9c2 cells with different concentrations (0.25, 3 Oxidative Medicine and Cellular Longevity 0.50, 1.00, 2.00, 4.00, and 8.00 mM) of VA for 24 h before H/R. In Figure 2(a), most of VA-pretreated groups exhibited upregulated levels of AMPKα2 protein relative to those of control and H/R groups, and the 1.00 mM VA+H/R group exhibited the highest levels of AMPKα2 protein (p < 0:05 vs. 0.50 mM VA+H/R group). The optimal pretreatment concentration was consistent with that observed by MTS. However, levels of AMPKα2 protein were markedly decreased in the VA+AMPKα2-siRNA+H/R group relative to those in the VA+H/R group (p < 0:01) (Figure 2(b)). Thus, our data indicate that VA exhibits cardioprotective effects against H/R by upregulating AMPKα2 protein levels.   (Figure 3).

Pretreatment with VA Restores Δψ m in H9c2 Cells
Exposed to H/R. To determine Δψ m in H9c2 cells after H/R injury, a JC-1 fluorescent probe assay was performed. We used the fluorescence ratio of the upper right quadrant and lower right quadrant to verify Δψ m . The results demon-strated that Δψ m decreased in response to H/R (p < 0:01 vs. control group) ( Figure 4). However, pretreatment with VA markedly reduced the loss of Δψ m in the VA+H/R group (p < 0:01 vs. H/R group). In contrast, a disruption in Δψ m was observed when the AMPKα2 gene was knocked down (p < 0:01 vs. VA+H/R group), demonstrating that AMPKα2-siRNA effectively abrogates the cardioprotective effects of VA against H/R injury.

Pretreatment with VA Prevents Opening of the mPTP
Induced by H/R. Ca 2+ -induced mitochondria swelling was used to examine the effects of VA on mPTP opening. We determined the degree of mPTP opening by recording varied optical density values at 520 nm/min (OD/min) over a period of time. As shown in Figure 5, there was a significant increase in ΔOD/min in the H/R group (p < 0:01 vs. control group); however, pretreatment of H9c2 cells with VA delayed mPTP 3.6. Pretreatment with VA Attenuates Caspase-3 Activity in response to H/R. To examine the cardioprotective effects of VA on H/R-induced injury, levels of caspase-3 activity were estimated using a colorimetric method. Figure 6 shows a significant increase in caspase-3 activity in the H/R group (p < 0:01 vs. control group), yet VA pretreatment strikingly decreased caspase-3 activity (p < 0:01 vs. H/R group). Furthermore, in line with previous results, a significant increase in caspase-3 activity was observed in the VA+AMPKα2-siRNA+H/R group (p < 0:01 vs. VA+H/R group).

Pretreatment with VA Decreases H9c2
Cell Apoptosis in response to H/R. Apoptosis was assessed by flow cytometry using an Annexin V-FITC/PI apoptosis kit. As shown in Figure 7, H9c2 cell apoptotic rates increased markedly in the H/R group (p < 0:01 vs. control group), while progressively decreasing in the VA+H/R group (p < 0:01 vs. H/R group). Moreover, H9c2 cell apoptotic rates increased again in the VA+AMPKα2-siRNA+H/R group (p < 0:01 vs. VA+H/R group).
To further confirm the VA-induced cardioprotective effect against H/R injury, we scored the number of TUNEL-positive H9c2 cells by optical microscopy. As shown in Figure 8, H/R caused a significant increase in the number of TUNEL-positive H9c2 cells in the H/R group (p < 0:01 vs. control group). In contrast, the pretreatment of H9c2 cells with VA resulted in significantly reduced TUNEL-positive H9c2 cells (p < 0:01 vs. H/R group). In agreement with previous results, the cardioprotective effects of VA were abrogated after AMPKα2 gene knockdown using the AMPKα2-siRNA lentivirus.

Discussion
Vanillic acid (VA) is a phenolic compound in edible plants that is enriched in the roots of Angelica sinensis. VA exhibits powerful antioxidant functions and possesses cardioprotective, antihypotensive, antiapoptotic, and hepatoprotective activities [8][9][10][11]. Experimental studies have provided evidence of this compound's efficacy in cardiac toxicity.
In this study, we found that H9c2 cells pretreated with 1.00 mM VA 24 h prior to H/R significantly increased the viability of H9c2 cells and reduced the levels of LDH and CPK. And our data further confirmed VA pretreatment The cardioprotective effect of VA was extensively investigated, and the potential mechanisms might be associated with countering oxidative stress caused by energy metabolism imbalance, but the mechanism remains to be fully elucidated. AMPK plays an important role in the balance of energy dynamics and is implicated in various diseases [22]. Studies including those from our group have shown that AMPK may be an oxidative stress sensor and redox regulator in addition to its traditional role as an energy sensor and regulator [23,24]. Moreover, AMPK activation is believed to enhance cellular antioxidant capacity [25,26]. These findings all led us to hypothesize that VA protects H9c2 cells against   Oxidative Medicine and Cellular Longevity H/R injury through antioxidant stress mediated by the AMPK signalling pathway. Therefore, we attempted to detect the expression level of AMPKα2 in the treatment of H9c2 anti-H/R injury with different concentrations of vanillic acid and found that the expression level trend of AMPKα2 was consistent with the concentration trend of vanillic acid against H/R injury, as shown in Figures 1(a) and 2(a). H9c2 cells pretreated with 1.00 mM VA 24 h before H/R exhibited the highest AMPKα2 protein levels. AMPKα2 is known to be sensitive to hypoxia and can be activated by increases in AMP. Once H9c2 cells are subjected to H/R damage, they inevitably increase the expression of the AMPKα2 protein as an emergency response. However, the increase in AMPKα2 protein levels induced by H/R damage is very limited and does not have a protective effect. Our results showed that vanillic acid induces H9c2 cells to produce large quantities of AMPKα2 protein and plays a protective role against H/R injury (Figures 1 and 2). So in the follow-ing studies, we focused on whether the protective effect of VA against H/R injury was associated with AMPKα2.
To verify whether the AMPKα2 protein was associated with the cardioprotective activity induced by vanillic acid, we used knockdown of AMPKα2 by its specific siRNA. In the recent years, a number of siRNAs have been successfully used in experimental models [27][28][29]. We specifically suppressed AMPKα2 expression using RNA interference, which selectively silenced gene expression by delivering double-stranded RNA molecules into cells, and western blot analysis showed that the expression of target protein AMPKα2 could be reduced, as shown in Figure 2(b). Once the expression of AMPKα2 was inhibited, the protective effect of vanillic acid pretreatment against H/R injury in H9c2 cells was eliminated. These findings confirmed that VA-induced cardioprotective effects on H/R are associated with the AMPKα2 signalling pathway.
Electron transport chain injury results in the production of mitochondrial ROS and oxidative stress damage due to