Alpha-lipoic acid ameliorates H₂O₂-induced human vein endothelial cells injury via suppression of inflammation and oxidative stress

ABSTRACT

The study was aimed to investigate the effect of alpha-lipoic acid (ALA) on human umbilical vein endothelial cells (HUVECs) injury induced by hydrogen peroxide (H₂O₂) and to explore its possible mechanisms. We established the H₂O₂-induced HUVECs injury model and the ALA treatment groups in which HUVECs were co-incubated with H₂O₂ (250 μmol/L) and different final concentrations of ALA (100,200,400 μmol/L) for 48 h. Cell survival rate assay and LDH activity assay were carried out. The levels of related proteins were performed by Western Blot. We observed that H₂O₂ administration resulted in an increase in the LDH activity and a decrease in cell survival rate. The expression levels of Nox4, Bax, NF-κB p65, Caspase-9, Caspase-3, iNOS, VCAM-1 and ICAM-1 were up-regulated, while the expression level of Bcl-2 was down-regulated. All these factors were significantly improved by ALA treatment. In brief, ALA treatment ameliorates H₂O₂-induced HUVECs damage by inhibiting inflammation and oxidative stress.

Graphical abstract

Alpha-lipoic acid attenuates H₂O₂-induced human umbilical vein endothelial cells injury by inhibiting apoptosis and the expression of inflammatory-related factors.

KEYWORDS:

• Alpha-lipoic acid
• oxidative stress
• inflammation
• apoptosis

Atherosclerosis is a progressive inflammatory cardiovascular disorder and the main cause of cardiovascular morbidity and mortality worldwide. The pathogenesis of atherosclerosis is a chronic inflammatory process in which various damaging factors affect vascular endothelial cells, resulting in endothelial cell dysfunction and release of various leukocyte adhesion molecules, which adhesion at the activation site of endothelial cells [1].

Endothelial dysfunction leads to abnormal blood circulation, atherosclerosis, inflammation, stroke and even cancer [2,3]. Endothelial cell apoptosis promotes atherosclerosis and may be further aggravated by oxidative stress. In general, oxidative stress is considered to be a major element causing cell dysfunction of vascular endothelial cells [4] and the apoptosis of endothelial cells may be caused by the accumulation of reactive oxygen species (ROS) [5,6]. In addition, ROS act as signaling molecules that promote the activation of the inflammatory response of vascular endothelial cells, leading to vascular inflammation and endothelial dysfunction [7]. Furthermore, the translocation of NF-κB from cytoplasm to nucleus triggers the expression of inflammatory-related factors such as vascular cell adhesion molecule-1 (VCAM-1) and inter-cellular adhesion molecule-1 (ICAM-1) [8]. Therefore, it has been inferred that drugs that block NF-κB signal transduction and adhesion molecules’ expression in vitro may also have significant effects on the inflammatory response in vivo. It has been shown [9,10] that H₂O₂-induced oxidative stress in endothelial cells upregulates the apoptotic gene, Bax, and the caspase signaling pathway. Hence, it is essential to understand the roles of ROS in vascular endothelial cells’ physiology and pathophysiology. A more elaborate investigation of how H₂O₂ influences endothelial cell apoptosis and inflammation will help formulate a new strategy for treatment or prevention of cardiovascular diseases.

Previous studies have found increased expression of adhesion molecules such as ICAM-1 and VCAM-1 at lesion-prone arterial sites and atherosclerotic lesions [11,12]. The genetic defect of the mouse adhesion molecules are associated with reduction in atherosclerosis [13]. Therefore, the regulation of the interaction between mononuclear cells and endothelial cells may be an important target for the prevention and treatment of atherosclerosis.

In general, abnormal production of proinflammatory mediators such as iNOS plays an important role in the pathogenesis of cardiovascular diseases. The overexpression of inflammatory mediators causes vascular damage which can be alleviated by inhibiting the expression of pro-inflammatory mediators [14,15].

Currently, antioxidants are used to reduce the production of intracellular ROS and limit its harmful effects on the body. α-Lipoic acid (ALA) has been proven to be one of the most important antioxidants that can improve vascular function [16].

It shown that ALA has protective effect on early atherosclerosis in diabetic rats [17]. Many studies have confirmed that ALA improves vascular function and reduces the burden of atherosclerotic plaque [18,19]. ALA meliorates vascular dysfunction by regulating lipid metabolism and activating AMPK in endothelial cells [20]. Jones et al [21] found that ALA/DHLA redox coupling enhances antioxidant defense and human endothelial cells function. The role of ALA in preventing oxidative damage in EA-hy926 cells has been demonstrated [22]. Shen et al proposed that H₂O₂-induced human umbilical vein endothelial cells (HUVECs) can mediate apoptosis through the mitochondrial pathway [23]. However, the mechanism of ALA in repairing endothelial function is still unclear. To our knowledge, little research has been done on the mechanism by which ALA inhibits apoptosis induced by H₂O₂ in HUVECs. To explore ALA’s mechanism of action, its effects on HUVECs induced by H₂O₂ were studied from two aspects: inflammation and apoptosis induced by oxidative stress.

Our study provides a possible mechanism for the anti-apoptosis effect of ALA and provides important insight into the theoretical basis for the prevention of cardiovascular diseases.

Materials and methods

Materials

HUVECs were provided by the Biochemistry and Molecular Biology Laboratory (Hebei College of Traditional Chinese Medicine). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS) and trypsin were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). ALA, H₂O₂, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were from Sigma–Aldrich (St Louis, MO, USA). RIPA lysis buffer was from Beyotime Institute of Biotechnology (Haimen, China). The lactate dehydrogenase (LDH) assay kit was purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Bicinchoninic acid (BCA) protein assay kit was from Beyotime Institute of Biotechnology. Antibodies against Bcl-2 (2876), Bax (2772), cleaved Caspase-3 (9664), cleaved Caspase-9 (9501), NF-κB p65 (4764), GAPDH (5174), β-actin (4967) and Histone H3 (9715) were purchased from Cell Signaling Technology (Danvers, MA, USA); Antibodies against VCAM-1 (sc-1504), ICAM-1 (sc-1511), iNOS (sc-650) and Nox4 (sc-30141) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). All the secondary antibodies were purchased from Beyotime Institute of Biotechnology. All other reagents used in the experiments were of analytical grade.

Cell culture and experimental grouping

HUVECs were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco) and incubated at 37°C in 5% CO₂. After cell monolayers were fused, they were digested with digestion solution containing 2.5 g/L trypsin and 0.2 g/L disodium EDTA (EDTA-Na₂) and seeded in a 6-well cell culture plate at a density of 1 × 10⁴ cells/well. After 24 h of incubation, the supernatant was discarded. The cells were then randomly divided into three groups: (1) control group, in which the cells were incubated in DMEM medium for 48 h; (2) H₂O₂ injury group, in which cells were exposed to H₂O₂ at 50 to 400 μmol/L (final concentration in the wells) for 48 h; the cell viability was analyzed to determine the appropriate H₂O₂ concentration for the H₂O₂ injury model in subsequent experiments; (3) H₂O₂ + ALA group, in which cells were co-incubated with H₂O₂ (250 μmol/L) and different concentrations of ALA (final concentrations 100,200, 400 μmol/L) for 48 h.

MTT assay

The viability of HUVECs was performed by the MTT assay as previously described [24]. In brief, the control group and the experimental groups mentioned above were inoculated into 96-well plates. Each group of experiments was repeated six times. After 48 h of incubation, 20 μL of MTT solution were added into the wells for another 4 h, and then 150 μL of DMSO were added to each well. After gently shaking the plates for 10 min, the absorbance (A) of each well at 490 nm was measured with a microplate reader (Bio-Rad, Hercules, CA, USA). The formula used: cell survival rate (%) = A₄₉₀ (sample wells – blank wells)/A₄₉₀ (control wells – blank wells) * 100

LDH activity assay

To determine the extent of cell damage, the supernatants of the cell cultures were collected and the LDH activity was measured by an LDH kit (Nanjing Jiancheng Bioengineering Institute). The absorbance was read at a wavelength of 440 nm and enzyme activity was expressed as units per L.

LDH activity (U/L) = (OD_test – OD_blank)/(OD_standard – OD_blank) * standard concentration * 1000 * dilution ratio of sample

Flow cytometry analysis of apoptosis

Cell apoptosis was analyzed by flow cytometry. The treated cells and the control cells were collected separately after digestion with trypsin and centrifugation to remove the medium. The cells were washed once with phosphate-buffered saline (PBS) and fixed in 70% pre-chilled ethanol overnight at 4°C. After centrifugation, cells were washed with ice-cold PBS and incubated with 100 µg/mL RNase A for 30 min at 37°C. Then the cells were stained with 50 μg/mL propidium iodide (PI) solution according to a method described previously [25]. After incubation for 30 min at 4°C in the dark, the apoptosis of HUVECs was detected by flow cytometry (Beckman Coulter, Inc., USA). The experiment was performed in triplicate.

Western blot analysis

After the treatments, cells were collected, washed three times with ice-cold PBS and lysed in RIPA lysis buffer with 1% PMSF and protease inhibitors (Merk) to obtain protein extracts. The protein concentration of the lysates was quantified by a BCA kit (Beyotime) according to the manufacturer’s instructions. Equal amounts of protein lysates were subjected to SDS-PAGE and transferred to PVDF membranes (Merck Millipore Corporation, Darmstadt, Germany). The membranes were blocked with 5% nonfat dry milk for 2 h, and then incubated with primary antibodies overnight at 4°C, followed by three washes with Tris-buffered saline containing Tween 20. The blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; Beyotime) for 1 h at room temperature. Finally, the protein bands were detected by the enhanced chemiluminescent (ECL) system. The protein levels were quantified by Image J software.

Statistical analysis

Statistical analysis was conducted with SPSS 21.0 software (IBM Corporation, New York, USA). The values are expressed as the mean ± SEM. Data were analyzed by one-way ANOVA. P < 0.05 was considered statistically significant.

Results

Establishment of the H₂O₂-induced HUVECs injury model

Oxidative stress is an important factor that causes cardiovascular diseases and excessive ROS may lead to endothelial cell dysfunction [26]. Therefore, HUVECs induced by H₂O₂ were selected as the model of oxidative stress injury to examine the protective effects of ALA on vascular endothelial cells.

To determine the appropriate H₂O₂ concentration for the H₂O₂-induced HUVECs injury model, the cells were divided into seven groups (control and different concentrations of H₂O₂). The MTT assay showed that the cell survival rate gradually decreased with the increase in H₂O₂ concentration, and it decreased to about 44.26% ± 5.83% (P < 0.01) after 48 h of exposure to 250 μmol/L H₂O₂, indicating an obvious oxidative stress injury to HUVECs caused by H₂O₂ treatment. Therefore, 250 μmol/L H₂O₂ was designed for the injury model in the subsequent experiments (Figure 1(a)).

Figure 1. Effects of H₂O₂ and ALA on HUVECs cell viability. (a) Effects of H₂O₂ on the viability of HUVECs. HUVECs were either not treated (control) or treated with different concentrations of H₂O₂ for 48 h. Cell viability was evaluated by the MTT. Data are mean ± SEM. N = 6. ^##P < 0.01 vs control. (b) Effects of ALA on survival rate of injuried cells induced by H₂O₂. In H₂O₂ injury model, HUVECs were treated with H₂O₂ at 250 μmol/L for 48 h. In ALA groups, HUVECs were co-incubated with H₂O₂ (250 μmol/L) and ALA at 100 μmol/L, 200 μmol/L and 400 μmol/L for 48 h. Cell viability was evaluated by the MTT assay. Data are mean ± SEM. N = 6. ^## P < 0.01 vs control, *P < 0.05 vs model, **P < 0.01 vs model.

ALA improves the viability of H₂O₂-induced HUVECs

The selected experimental concentrations for the low, medium and high-dose ALA groups were 100, 200 and 400 μmol/L, respectively. After co-incubation with 250 μmol/L H₂O₂ and different concentrations of ALA for 48 h, the MTT assay was performed to assess cell viability. As shown in Figure 1(b), the cell survival rate of the H₂O₂ injury group was significantly reduced (P < 0.01) compared with the control group. However, after co-incubation with ALA, the cell survival rate increased from 46.75% ± 6.31% to 57.09% ± 6.37%, 68.17% ± 7.28% and 78.10% ± 6.58%, respectively. The differences among the ALA treatment groups were significant (P < 0.05). These results confirmed that ALA protected HUVECs from injury induced by H₂O₂ (Figure 1(b)).

ALA reduces LDH release from H₂O₂-induced cells

LDH is considered to be a biomarker for assessing cellular injury [27,28]. To further investigate ALA’s protective effects on H₂O₂-induced HUVECs, LDH release to the culture medium was measured. As expected, the LDH activity in the model group was significantly higher than that of the control group (P < 0.01). However, after co-incubation with ALA (100, 200 or 400 μmol/L) for 48 h, the release of LDH decreased in a dose-dependent manner compared with the model group (P < 0.05; Figure 2).

Figure 2. Effects of different concentrations of ALA on the LDH activity in H₂O₂-induced HUVECs. The LDH activity was measured by LDH kit. In H₂O₂ injury model, HUVECs were treated with H₂O₂ at 250 μmol/L for 48 h. In ALA groups, HUVECs were co-incubated with H₂O₂ (250 μmol/L) and ALA at 100 μmol/L, 200 μmol/L and 400 μmol/L for 48 h. Values are expressed as mean ± SEM, n = 6 independent experiments. ^##P < 0.01 vs control, *P < 0.05 vs model, **P < 0.01 vs model.

ALA downregulates the expression of Nox4

In HUVECs, ROS is produced mainly by Nox4 [29]. In this paper, ALA’s effect on Nox4 protein expression was measured. The Nox4 level in the H₂O₂ model group was remarkably high compared with the control group. However, co-incubation with ALA resulted in obvious downregulation of Nox4 protein expression in a dose-dependent manner (P < 0.05; Figure 3).

Figure 3. The expression of Nox4 protein in different treatment groups. HUVECs were exposed to H₂O₂ (250 μmol/L) while treated with or without ALA (100, 200, 400 μmol/L) for 48 h. (a) Western blotting analysis of Nox4 protein. (b) The mean ± SEM relative protein levels of Nox4 were normalized to GADPH (internal control). All the experiments were executed three times independently. ^##P < 0.01 vs control, **P < 0.01 vs model.

ALA inhibits H₂O₂-induced activation of the NF-κB signaling pathway

NF-κB as a master transcription factor induces expression of many inflammation-related genes. It has been shown that NF-κB is involved in oxidative stress-mediated endothelial cell dysfunction [30].

The protein levels of NF-κB p65 from HUVECs were analyzed to assess their translocation from cytoplasm to nucleus. By western blotting, we observed that the expression of nuclear p65 was remarkably increased after stimulation with H₂O₂. In a parallel comparison, the cytoplasmic levels of p65 decreased. More importantly, we noticed that the H₂O₂-enhanced nuclear translocation of p65 was alleviated by treatment with different concentrations of ALA (Figure 4).

Figure 4. Effects of ALA on NF-κB activation in HUVECs. HUVECs were exposed to H₂O₂ (250 μmol/L) while treated with or without ALA (100, 200, 400 μmol/L) for 48 h. (a) Western blotting analysis of NF-κB p65 in the cytoplasm and nucleus. (b) Mean ± SEM relative protein levels of cytoplasmic NF-κB p65 were normalized to β-actin. (c) Mean ± SEM relative protein levels of nuclear NF-κB p65 were normalized to Histone H3. All the experiments were executed three times independently. ^##P < 0.01 vs control, **P < 0.01 vs model.

The results suggest that ALA could suppress the activation of NF-κB signal pathway.

ALA protects HUVECs from H₂O₂-induced apoptosis

It has been revealed that injury and apoptosis in the vascular endothelium play a critical role in atherosclerotic plaque formation [31], In our study, the apoptosis rate was detected by flow cytometry to investigate whether ALA has an effect on apoptosis induced by H₂O₂. As shown in Figure 5, compared with the control group (24.83% ± 2.27%), there was an obvious increase in the apoptosis rate in the H₂O₂ model group (72.20% ± 5.79%, P < 0.01). However, after treatment with different concentrations of ALA, this rate reduced to 59.00% ± 3.95%, 52.73% ± 5.01% and 41.10% ± 4.37%, respectively. The difference between the H₂O₂ model group and the ALA groups was statistically significant (P < 0.01). These results illustrated that ALA significantly reduced the H₂O₂-induced apoptosis rate.

Figure 5. Cell apoptotic rate evaluation by flow cytometry. HUVECs were exposed to H₂O₂ (250 μmol/L) while treated with or without ALA (100, 200, 400 μmol/L) for 48 h. The apoptosis rate was detected by flow cytometry. (a) Control group. (b) H₂O₂ (250 μmol/L) group. (c) ALA (100 μmol/L) group. (d) ALA (200 μmol/L) group. © ALA (400 μmol/L) group. (f) The mean ± SEM percentage of apoptotic cells of three individual experiments. ^##P < 0.01 vs control, **P < 0.01 vs model.

Effects of ALA on the protein expression of Bcl-2, Bax, cleaved caspase-3 and caspase-9

To study the potential mechanism by which ALA regulates H₂O₂-induced apoptosis, the expression changes in apoptosis-related proteins in HUVECs were evaluated by western blotting.

Figure 6(b and c) shows that in comparison to the control group, the expression level of Bax was significantly higher (P < 0.01) and that of Bcl-2 was obviously decreased in the H₂O₂ group (P < 0.01). In contrast, when the cells were co-incubated with ALA, Bax expression was downregulated in a dose-dependent manner (P < 0.01) and Bcl-2 expression was upregulated (P < 0.01). As previously reported, the ratio between Bcl-2 and Bax protein expression is a key factor in apoptosis regulation [32–34]. Statistical analysis showed that the Bcl-2/Bax ratio increased in association with the ALA dose (Figure 6(d)).

Figure 6. Effects of ALA on the expression of apoptosis-related proteins in HUVECs. (a) Western blotting analysis of Bax, Bcl-2 and the cleaved caspase-3 and caspase-9. The protein expression levels of Bax (b), Bcl-2 (c), cleaved caspase-3 © and cleaved caspase-9 (f) were analyzed, respectively. GAPDH was used to normalize the data. The Bcl-2/Bax ration (d) was calculated. Results are expressed as mean ± SEM (n = 3 per group). ^##P < 0.01 vs control, **P < 0.01 vs model.

We also found that the expression of cleaved caspase-3 and caspase-9 in HUVECs was significantly elevated (P < 0.01) after H₂O₂ stimulation alone. On the contrary, ALA co-incubation with H₂O₂ markedly reduced the expression levels of these caspases (P < 0.01; Figure 6(e and f)).

These results suggest that H₂O₂ stimulation led to obvious apoptosis, whereas ALA reversed this effect. The anti-apoptotic effect of ALA may be mediated by the regulation of Bcl-2 and Bax protein expression and/or the inhibition of the activation of caspase-3 and caspase-9.

ALA suppresses the upregulation of iNOS, VCAM-1 and ICAM-1 in H₂O₂-induced HUVECs

Inflammation usually occurs with the development of oxidative stress. To assess whether ALA has an anti-inflammatory effect on H₂O₂-stimulated HUVECs, we examined the expression of iNOS, which is closely linked to the inflammatory response [35]. In addition, we examined the changes in the protein expression of VCAM-1 and ICAM-1 (Figure 7(a)). Western blot analysis revealed that compared with the control group, the protein expression levels of iNOS, VCAM-1 and ICAM-1 in the injury model group were significantly upregulated (P < 0.01). Notably, co-incubation with ALA attenuated this upregulation. The differences between the groups were significant (P < 0.05; Figure 7(b-d)).

Figure 7. Effects of ALA on the expression of iNOS, VCAM-1 and ICAM-1 in HUVECs. (a) The protein levels of iNOS, VCAM-1 and ICAM-1 in different treatment groups were determined by Western blot analysis. (b), (c), (d) The relative levels of iNOS, VCAM-1 and ICAM-1 were normalized to GAPDH. Values are presented as mean ± SEM from 3 independent experiments. ^##P < 0.01 vs control, **P < 0.01 vs model.

Discussion

In the present study, our results demonstrated that ALA regulates the cardiovascular system by inhibiting apoptosis and reducing the release of inflammatory-related factors to protect against H₂O₂ mediated HUVECs injury.

Under normal conditions, the proliferation rate and apoptosis rate of endothelial cells are very low. ROS produced in the cells during metabolism causes oxidative damage to the vascular endothelium and leads to apoptosis. HUVECs apoptosis induced by oxidative stress plays an important role in the pathogenesis of cardiovascular diseases, thus eliminating excessive ROS and inhibiting apoptosis may provide beneficial intervention in the treatment of cardiovascular diseases [36]. Lipid peroxidation is one of the main events in free radical-mediated cell damage [37]. H₂O₂ causes lipid peroxidation, markedly increasing cell permeability and LDH leakage. Therefore, an in vitro model of H₂O₂-induced endothelial cells injury has been applied to mimic the oxidative stress in endothelial cells. Previous reports have suggested that H₂O₂ increases cell permeability, impairs the cellular antioxidant defense and induces endothelial cell apoptosis [38]. Our results showed that H₂O₂ damaged cell, decreased cell viability, increased the ratio of apoptotic cells and LDH activity. Furthermore, we also shown that ALA significantly reduced H₂O₂-induced HUVECs injury in a dose-dependent manner (Figures 1(b) and 2). Namely, ALA protected HUVECs against oxidative damage induced by H₂O₂, as assessed by cell viability and LDH activity. The results of the current study are consistent with previous reports that ALA significantly reduced LDH release from HUVECs induced by hypoxia/reoxygenation or ox-LDL [39,40]. The present results suggest that ALA can suppress LDH release from HUVECs induced by H₂O₂ in a dose-dependent manner. It seems that ALA can promote HUVECs repair and protect the endothelial cell. The experimental results showed that ALA appears to have the potential to reduce excessive ROS production, thereby protecting HUVECs from peroxide toxicity.

There is evidence that the expression of Nox4 in endothelial cells is significantly higher than that of other Nox members and that Nox4 plays a crucial role in endothelial redox reactions and in functional regulation of endothelial cells physiology [41]. H₂O₂ inhibit the proliferation of vascular endothelial cells, activate the NADPH oxidase pathway, and cause the cells to be oxidized stress, NADPH oxidase can transfer NADPH electrons to molecular oxygen to produce superoxide anions, indicating that ROS production is excessive, the balance of the oxidative stress system is disturbed, and eventually damages cells or tissues. In our study, ALA reduced Nox4-mediated ROS generation in HUVECs and further inhibited the NF-κB signaling pathway. Furthermore, ALA downregulated the expression of Nox4 induced by H₂O₂ in HUVECs (Figures 3(b) and 4(b)). Thus, we propose that this pathway is one of the mechanisms by which ALA reduces oxidative stress in endothelial cells. So, ALA may exert its antioxidant effects by regulating Nox4 protein expression and play a protective role in cardiovascular disease.

Oxidative stress mediates mitochondrial apoptosis in endothelial cells [42]. The mitochondrial pathway regulated by the Bcl-2 family is one of the most important pathways [43]. Bcl-2 and Bax have opposing functions and play important roles in the mitochondrial pathway of apoptosis. Previous study has reported that H₂O₂ downregulated Bcl-2 protein expression [38], which is consistent with our research. In contrast, the present study indicated that ALA promoted Bcl-2 protein expression and inhibited Bax expression in the H₂O₂-induced HUVECs, showing the anti-apoptotic effects of ALA on H₂O₂-induced HUVECs (Figure 6(b and c)). These results suggest that ALA regulates the balance between the antiapoptotic protein, Bcl-2, and the proapoptotic protein, Bax.

Caspases play an important role in apoptosis induced by different stimuli [44]. Caspases can selectively cleave certain proteins and break down intracellular related substrate proteins, resulting in changes in cell structure and metabolism, thus causing apoptosis. It is the condensation point of multiple apoptotic pathways and the final route of apoptosis. Caspase-3 mediates the apoptosis of exogenous and endogenous pathways, during which it is cleaved and activated. It has been reported that Bcl-2 is an antiapoptotic protein that inhibits apoptosis induced by the mitochondria-dependent caspase-9 pathway [43,45]. In the present study, ALA co-treatment reduced the expression of cleaved caspase-3 and inhibited apoptosis compared with the untreated HUVECs induced by H₂O₂. Consistent with the results that ALA suppressed the H₂O₂-induced expression of Bax/Bcl-2 and caspase-3,9, we observed that ALA protected HUVECs from oxidative damage and significantly decreased the number of apoptotic cells (Figure 6(d-f)). This suggests that ALA protects vascular endothelial cells by inhibiting or blocking the mitochondrial apoptosis pathway.

In oxidative stress-induced cell damage, researchers are concerned about the apoptotic pathway of mitochondrial damage, such as decreased cell membrane potential, caspase-3 activation, cytochrome c release, and Bax and Bcl-2 expression. Studies have shown that oxidative stress may be involved in cell damage via the common central NF-κB [46,47]. NF-κB regulates the expression of cell adhesion molecules such as VCAM-1, ICAM-1 and pro-inflammatory mediator iNOS. In this study, the expression of NF-κB p65 and iNOS was significantly upregulated during oxidative stress, but this expression was significantly downregulated when the cells were co-treated with ALA, indicating that ALA reduced the expression of NF-κB p65 and iNOS, and finally the inflammation of HUVECs induced by oxidative stress was suppressed. We also found that ALA can significantly reduce the expression of VCAM-1, ICAM-1 in H₂O₂-induced HUVECs (Figure 7(b-d)). This suggests that ALA can inhibit the expression of VCAM-1, ICAM-1 by interfering with mononuclear cell adhesion. Thus, we believe that the protective effects of ALA on HUVECs are achieved by inhibiting NF-κB activation and reducing the release of inflammatory-related factors. ALA is a powerful antioxidant that removes ROS [48]. Therefore, the activation of the NF-κB pathway caused by oxidative stress is inhibited by ROS scavenging, and then the release of inflammatory-related factors such as VCAM-1, ICAM-1 and pro-inflammatory mediator iNOS are reduced.

In summary, HUVECs from oxidative stress can cause metabolic dysfunction and oxidative damage to proteins, carbohydrates, DNA, RNA, mtDNA and lipids in the cell membrane, thereby activating the apoptotic pathway. On the basis of the changes in protein expression and the results of this study, we propose the following mechanism of ALA protection against H₂O₂-induced apoptosis in HUVECs: H₂O₂ stimulation induces the binding of Bax and Bcl-2, which activate caspase-9 that finally activates the apoptotic executive molecule, caspase-3. Cell opening programmed death mode. Co-treatment with ALA inhibits the apoptosis pathway in HUVECs induced by H₂O₂ mainly through the mitochondrial pathway. ALA suppresses inflammation induced by oxidative stress by downregulating the NF-κB pathway. Furthermore, ALA inhibits the expression of ICAM-1 and VCAM-1, affects monocyte adhesion and ameliorates atherosclerosis (Figure 8).

Figure 8. Potential molecular signaling pathways that ALA protects against H₂O₂-induced HUVECs damage.

Authors contributions

LAY mainly designed the research. WW and ALP performed the research and analyzed the data. LYF, AR and BZ modified the research. LWZ and SQH help us in writing and manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the the Key subjects of Hebei Province Health Department [ZL20140249]; Science Foundation for Youths of the Higher Education Institutions of Hebei Province [QN2016137]; Projects of Hebei Provincial Administration of Traditional Chinese Medicine [2016003].