Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2025 Feb 19;10(8):8512–8522. doi: 10.1021/acsomega.4c10795

Carvacrol Regulates the Expression of SLC25A6 by Inhibiting VDAC1 to Improve Mitochondrial Function and Reduce LPS-Induced Inflammatory Injury in HMEC-1 Cells

Cuifang Lu †, Bin Yang ‡, Ying Liu §, Wenbo Liu †, Jie Yan †, Chenggong Guo ∥, Tingyu Song ⊥, Xiaofei Wang #,*
PMCID: PMC11886903  PMID: 40060777

Abstract

graphic file with name ao4c10795_0006.jpg

Carvacrol has been demonstrated to possess anti-inflammatory and antioxidant properties. This study aims to further explore the mechanisms by which carvacrol mitigates LPS-induced human microvascular endothelial cells injury by improving mitochondrial function. An inflammatory injury model of human microvascular endothelial cells was established using LPS. The expression levels of inflammatory cytokines (IL-1β, IL-6, IL-18, TNF-α) were measured. Assessment of apoptosis, necrosis, and proliferation was conducted using the YO-PRO-1/PI apoptosis and necrosis detection kit and EdU assay. The evaluation of oxidative stress levels was facilitated by the use of ROS, MDA, and SOD assay kits. Angiogenic capacity and cytoskeletal changes were also examined. Assessment of mitochondrial function and energy metabolism was achieved by measuring mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (mPTP) opening, ROS levels, and ATP production. Western blot analysis was performed to detect the expression of VDAC1 and SLC25A6. The results show that carvacrol significantly reduced LPS-induced expression of IL-1β, IL-6, IL-18, and TNF-α and alleviated the effects of LPS on cell proliferation and apoptosis of HMEC-1. It also decreased oxidative stress levels, inhibited excessive tube formation capacity, and promoted cytoskeletal remodeling. Furthermore, carvacrol has been shown to reduce VDAC1 protein expression, improve mitochondrial function and energy metabolism by regulating MMP, mPTP opening, ROS levels, and ATP production, and increase SLC25A6 protein expression. Importantly, carvacrol and VDAC1 knockdown exhibited similar effects. In the mechanism of inflammatory injury, SLC25A6 may act as a downstream effector of VDAC1. The results of this study demonstrate that carvacrol exerts a protective effect on human microvascular endothelial cells by improving mitochondrial function and alleviating oxidative stress through the inhibition of VDAC1.

Introduction

The development of new therapeutic approaches and drugs has become the most effective means of controlling inflammation. The identification of novel inflammatory mediators and the exploration of the roles of inflammatory factors are of crucial importance for a better understanding of the mechanisms of inflammatory responses and for elucidating the action mechanisms of anti-inflammatory drugs.1 It has been demonstrated by means of rigorous research that lycopene possesses the capacity to mitigate the toxicity of antibiotics by diminishing oxidative stress and inflammation. The mechanism by which this occurs involves the reduction of CAT and SOD, and the significant upregulation of interleukin (IL-18, IL-6 and IL-1β), as well as the activation of ASC, NLRP3, caspase-1, TNF-α and nuclear factor NF-κB.2 Furthermore, the progression of inflammation is associated with mitochondrial dysfunction. Exposure to BPA and low selenium has been demonstrated to result in structural abnormalities, oxidative stress, mitochondrial dysfunction and homeostasis imbalance, cell apoptosis, and mitochondrial phagocytosis in pancreatic tissue. The underlying mechanism of this process involves the activation of the PTEN/PI3K/AKT/mTOR pathway. In the context of multifactorial inflammation, human microvascular endothelial cells exhibit a rapid reaction, initiating a series of cascade events, that culminate in mitochondrial dysfunction, oxidative stress, and the development of various diseases, including atherosclerosis, hypertension, and myocardial infarction.3,4 In recent years, there has been a focus on investigating the ultrastructural changes in endothelial cells during inflammatory injury and actively searching for novel markers in this process, providing a theoretical foundation for the development of new targeted therapies.

The dysfunction of mitochondrial function-related proteins has been demonstrated to promote the progression of inflammation through various mechanisms.5 Voltage-dependent anion channel 1 (VDAC1), a mitochondrial gating protein, has been demonstrated to play a regulatory role in mitochondrial function.6 VDAC1 plays a central role in maintaining mitochondrial energy and metabolic homeostasis by mediating metabolic exchange between the mitochondria and cytoplasm.7 It has been demonstrated that VDAC1 regulates mitochondrial oxidative phosphorylation by controlling the transport of metabolites such as ATP, ADP, phosphate groups, and calcium ions, thereby sustaining mitochondrial function.8 The process of VDAC1 oligomerization has also been implicated in the activation of the NLRP3 inflammasome.9 Ginsenoside Rb1 has been shown to regulate the DUSP-1-TMBIM-6-VDAC1 axis, thereby suppressing the release of pro-inflammatory factors, altering the structure and composition of the gut microbiota, and improving cardiac function.10 The interaction between VDAC1 and solute carrier family 25 (SLC25) transport proteins (such as SLC25A6), as well as BCL2 family proteins, has been identified as a key mechanisms in regulating apoptosis and energy metabolism.11

SLC25A6, alternatively designated ANT3, is classified as a member of the SLC25 family of proteins. It is a pivotal mitochondrial transport protein, situated within the inner mitochondrial membrane. It plays a crucial role in maintaining mitochondrial function and energy metabolism mechanisms.12 In the event of a loss of mitochondrial membrane potential or apoptotic signaling, SLC25A6 regulates ion permeability across the mitochondria, affecting mitochondrial membrane integrity and thereby mediating the process of apoptosis.13,14 SLC25A6 dysfunction has been demonstrated to result in impaired mitochondrial energy production, increased ROS generation, activation of inflammatory signaling pathways (such as the NF-κB pathway), and the release of related inflammatory cytokines, thereby promoting the progression of inflammation.15,16 Consequently, these findings imply that SLC25A6 could be a pivotal target for the treatment of inflammatory progression. Given the critical roles of VDAC1 and SLC25A6 in cellular energy metabolism and apoptosis, the development of targeted drugs regulating VDAC1 and SLC25A6 may provide a novel approach for the treatment of inflammation.

Carvacrol, a naturally occurring phenolic isopropyl monoterpene found primarily in some aromatic plants, has been shown to possess pharmacological activities, including anticancer and antioxidant properties.17 Research has shown that carvacrol exerts significant anti-inflammatory effects by regulating the NF-κB and MAPK signaling pathways, thereby inhibiting the production of inflammatory mediators such as TNF-α, IL-6, and PGE2, and consequently reducing the inflammatory response.18 Additionally, carvacrol has been shown to reduce pro-inflammatory cytokine levels in adipose tissue by blocking TLR2 and TLR4-mediated signaling pathways.19 Furthermore, carvacrol has been shown tu alleviate NLRP3 inflammasome-mediated apoptosis by inhibiting ROS production in H9c2 cells.20

Although the anti-inflammatory activity of carvacrol has been extensively studied, its mechanisms in improving mitochondrial function and energy metabolism remain unclear. This study aims to further explore the protective mechanisms of carvacrol against LPS-induced injury in HMEC-1 cells, involving the release of inflammatory cytokine, mitochondrial dysfunction, and oxidative stress.

Materials and Methods

Cell Source and Culture

The HMEC-1 cell line and its complete growth medium were procured from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (https://www.zqxzbio.com/). The HMEC-1 cells were cultivated in a complete growth medium (MCDB 131 basal medium supplemented with fetal bovine serum and epidermal growth factor) at 37 °C in a 5% CO2 incubator. The lipopolysaccharides (LPS) and carvacrol were dissolved in dimethyl sulfoxide (DMSO), with the final concentration of LPS being 1 μg/mL and the working concentration of carvacrol set at 10 μM. 70 μL of sterile water were respectively added to the vials of recombinant TNF-α (rTNF-α) (Cat: 10602-HNAE), recombinant IL-1β (Cat: 10139-HNAE), recombinant IL-6 (Cat: 10395-HNAE), and recombinant IL-18 (Cat: 10119-HNCE) to prepare a stock solution of 0.15 mg/mL.

Cell Transfection

Small interfering (si)RNA targeting VDAC1 was designed by Sangon Biotech Co., Ltd. to knock down the corresponding gene. The sequence is as follows: siRNA-VDAC1 (sense 5′-GCUUGGUCUAGGACUGGAAUU-3′, antisense 5′-AAUUCCAGUCCUAGACCAAGC-3′), siRNA-NC (sense 5′-CCAGCACUUGUGCCUGUACCAGAAA-3′, antisense 5′-UUUCUGGUACAGGCACAAGUGCUGG-3′). The cells were initially seeded in a 6-well plate (2 × 105 cells/well) and incubated at 37 °C and 5% CO2 for 24 h. Subsequently, 100 pmol siRNA and 4 μL Lipo8000 (Cat no. C0533; Beyotime, China) were added in each well for transfection at room temperature for 24 h. The cells were harvested for subsequent Western blot assays to confirm the efficiency of the transfections. Following successful transfection, the cells were cultivated at 37 °C and 5% CO2 for 24 h prior to subsequent experiments.

Determination of IL-1β, IL-6, TNF-α, MDA and SOD Levels

A conventional biochemical kit (Wuhan Ji Yin Mei Biotechnology Co., LTD, Wuhan, China) was utilized in accordance with the established protocol to ascertain the concentrations of IL-1β (JYM0083Hu), IL-6 (JYM0140Hu), and TNF-α (JYM0110Hu). A conventional biochemical kit (Solarbio, Beijing, China) was used as instructed to determine the contents of MDA (BC0025) and SOD (BC0175).

Detection of Apoptosis and Necrosis

The effects of LPS and carvacrol on the apoptosis and necrosis of HMEC-1 cells were detected using an Apoptosis and Necrosis Detection Kit with YO-PRO-1and PI (C1075S, Beyotime Biotechnology, China). The HMEC-1 cells treated with the drug were seeded in six-well plates and cultured for 24 h. The cells were then washed once with PBS, following which by 1 mL of YP1/PI staining solution was added and the cells were incubated at 37 °C in the dark for 20 min. Following this, the staining was observed under a fluorescence microscope.

Cell Proliferation Assay

The effects of LPS and carvacrol on HMEC-1 cells proliferation were assessed using the BeyoClick EdU Cell Proliferation Kit with Alexa Fluor 594 (C0078S, Beyotime Biotechnology, China), following the manufacturer’s instructions. The HMEC-1 cells were treated with the drug and then cultured on coverslips. The cells were then incubated in a cell incubator at 37 °C with 5% CO2 for 24 h. Thereafter, the cells were then treated with prewarmed 10 μM EdU working solution at 37 °C for 2 h. Following the removal of the medium, the cells were fixed with 4% paraformaldehyde for 15 min. Following the removal of the fixative, the cells were washed three times with PBS containing 3% BSA for 3 min each time. Following this step, the cells were permeabilized with PBS containing 0.3% Triton X-100 for a period of 15 min. The permeabilization solution was then removed, and the cells were washed twice before adding 0.5 mL of Click reaction solution. This was then left to incubate at room temperature in the dark for 30 min. Finally, the coverslips were mounted using DAPI (10 μg/mL), and fluorescence imaging was performed using a fluorescence microscope.

Mitochondrial Membrane Potential Assay

MMP was assessed using the JC-1 fluorescent probe (C2006, Beyotime Biotechnology, China). The HMEC-1 cells that had been treated with the drug were seeded in a 6-well plate (2 × 105 cells/well) and cultured for 24 h at 37 °C with 5% CO2. Following this, the culture medium was removed, and the cells were washed with PBS. Thereafter, 1 mL of cell culture medium and 1 mL of JC-1 staining working solution were added to each well, mixed thoroughly, and incubated for 20 min in the cell culture incubator. Following this, the cell culture medium was removed and the cells were washed twice with JC-1 staining buffer (1X). Finally, 2 mL of cell culture medium was added, and the cells were observed under a fluorescence microscope.

Mitochondrial Permeability Transition Pore Assay

The mPTP Assay Kit (C2009S, Beyotime Biotechnology, China) was utilized to assess the degree of mPTP opening. The subsequent steps were executed in strict accordance with the manufacturer’s instructions: The HMEC-1 cells were treated with the drug and then seeded in a 6-well plate and cultured for 24 h at 37 °C. The culture medium was then removed, and the cells were washed twice with PBS. Thereafter, 1 mL of Calcein AM staining solution, fluorescence quenching solution, or Ionomycin was added. The plate was then gently shaken to ensure even coverage of the cells, followed by incubation at 37 °C in the dark for 30 min. Following this, the staining solution was replaced with fresh, prewarmed culture medium, and the cells were incubated again at 37 °C in the dark for another 30 min. The medium was then discarded, and the cells were washed three times with PBS. Finally, detection buffer was added, and the cells were observed under a fluorescence microscope.

ATP Level Detection

The effects of LPS and carvacrol on intracellular ATP levels in individual live cells were monitored in real-time using the ATP fluorescent probe (pCMV-AT1.03) (D2604, Beyotime Biotechnology, China). The HMEC-1 cells (2 × 105 cells/well) were seeded in 12-well plates and cultured at 37 °C with 5% CO2 for 24 h. Thereafter, 1 μg of the ATP fluorescent probe was added to each well, and the cells were further incubated for 24 h. Fluorescence imaging was then performed using a fluorescence microscope.

ROS Detection

Following the administration of the drug, HMEC-1 cells were subjected to staining with 10 μM DCFH-DA (S0033M, Beyotime Biotechnology, China) in a dark environment for 30 min. Subsequent to this, the intracellular ROS were imaged using a fluorescence microscope with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Skeleton Remodelling

The morphology and distribution of actin filaments were detected using Actin-Tracker Red-594 (C2205S, Beyotime Biotechnology, China). HMEC-1 cells were treated with the drug and then cultured on coverslips for 24 h. The cells were then washed twice with PBS. The cells were fixed with 3.7% formaldehyde solution at room temperature for 10 min, followed by three washes with PBS containing 0.1% Triton X-100. Staining solution (200 μL per coverslip) was applied, and the cells were incubated in the dark at room temperature for 30 min. Following this, the cells were washed twice with PBS containing 0.1% Triton X-100 for 5 min each. After air-drying, the coverslips were mounted using DAPI and observed under a fluorescence microscope for imaging.

Angiogenesis Experiment

The angiogenesis experiment was conducted in a 96-well plate. The plate was first filled with 50 μL of Matrigengel (Corning) and incubated at 37 °C for 1 h. Then, 50 μL of cell suspension was added to each well and incubated at 37 °C for 4 h. Images were captured using an inverted microscope.

Western Blot

The protein extraction process was carried out using a Total protein extraction kit (KGP2100; KeyGEN BioTECH, Nanjing, China). An BCA Protein Assay Kit (PC0020; Solarbio, Beijing, China) was used to determine protein concentrations. Protein separation was conducted via 9% SDS-PAGE, followed by transfer to PVDF membranes. The membranes were incubated with blocking solution comprising TBST (TBS with Tween-20) and 5% milk powder for 1 h at room temperature. Following this, the membranes were incubated with primary antibodies VDAC1 (1:1000; Cat no. 55259–1-AP; Proteintech, Wuhan, China), SLC25A6 (1:1000, Cat no. 14841–1-AP; Proteintech, Wuhan, China), Bax (1:1000; bs-0127R; bioss, Beijing, China), BCL2 (1:1000; BA0412; Boster, Wuhan, China), caspase-3 (1:1000; 9662S; Cell Signaling Technology, America), IL-1β (1:1000; bs-6319R; bioss, Beijing, China), IL-18 (1:1000; ab71495, abcam, Shanghai, China), IL-6 (1:1000, bs-0782R, bioss, Beijing, China), TNF-α (1:1000; bs-2081R, bioss, Beijing, China), GAPDH (1:5000; Cat no. 60004–1-Ig; Proteintech, Wuhan, China). Following overnight incubation at 4 °C, the secondary antibody Horseradish Peroxidase (HRP)-conjugated goat-antirabbit IgG (ZB-2301, ZSGB-Bio) or HRP-conjugated goat-antimouse IgG (ZB-2305, ZSGB-Bio) was utilized for an incubation of the PVDF membranes for 1 h at room temperature. Finally, the protein signals were detected with electrochemiluminescence solution.

Statistical Analysis

All data were expressed as mean ± standard error and analyzed with SPSS 27.0 software. Differences between groups were compared with one-way ANOVA followed by Bonferroni post hoc test. A significance level of P < 0.05 indicates a significant difference, whereas greater statistical significance is denoted by ***P < 0.001, **P < 0.01, or *P < 0.05.

Results

Carvacrol Reduces LPS-Induced Oxidative Stress and Inflammation Progression in HMEC-1 Cells

Mitochondrial homeostasis is closely associated with cellular oxidative stress and inflammation. Elevated oxidative stress has been demonstrated to impair mitochondrial function, thereby inhibiting electron transport efficiency and reducing both the structural integrity and functional activity of mitochondria. The present study employed ELISA to assess the effects of LPS and carvacrol on oxidative stress and the release of inflammatory factors in HMEC-1 cells. As demonstrated in Figure 1, compared to the control group, the LPS group exhibited decreased SOD activity and increased levels of MDA and ROS, while carvacrol significantly increased SOD activity and reduced MDA and ROS levels. Furthermore, the levels of TNF-α, IL-1β, IL-6 in HMEC-1 cells were markedly elevated by LPS, while the addition of carvacrol significantly reduced the levels of these inflammatory factors.

Figure 1.

Figure 1

Open in a new tab

Carvacrol significantly reduces the oxidative stress levels and inhibits the release of inflammatory factors induced by LPS in HMEC-1 cells. The content of (A) MDA, (B) SOD, (C) TNF-α, (D) IL-1β, (E) IL-6 in the supernatant of HMEC-1 cells treated with LPS and Carvacrol was assessed using ELISA (n=3). **P < 0.01. ***P <0.001. (F) The fluorescent probe DCFH-DA was used to stain reactive oxygen species (ROS) in HMEC-1 cells treated with LPS and Carvacrol. Rosup was used as a positive control for ROS. Scale bar, 100 μm. (G) The level of ROS in HMEC-1 cells was quantified (n=3). **P < 0.01. ***P < 0.001.

Carvacrol Promotes HMEC-1 Cell Proliferation and Inhibits LPS-Induced Apoptosis

EdU was used to assess cell proliferation, while YO-PRO-1 and Propidium Isodide double fluorescence staining were used to assess apoptosis and necrosis. The experimental results showed that LPS promoted apoptosis and necrosis and inhibited proliferation in HMEC-1 cells compared to the control group. The addition of carvacrol significantly ameliorated apoptosis and necrosis in HMEC-1 cells and promoted proliferation (Figure 2A–D). Western blot analysis showed that LPS significantly increased the protein expression of Bax and Caspase-3, while suppressing the expression of BCL2. In contrast, carvacrol significantly attenuated the effects of LPS (Figure 2E–H).

Figure 2.

Figure 2

Open in a new tab

Carvacrol promotes the proliferation of HMEC-1 cells and inhibits LPS-induced apoptosis. (A) EdU assay for cell proliferation of HMEC-1 cells treated with LPS or Carvacrol. Scale bar, 200 μm. (B) Percentage of EdU-positive cells was recorded (n = 3). **P < 0.01. ***P < 0.001. (C) HMEC-1 cells treated with LPS or Carvacrol were stained with both YO-PRO-1 and PI dye, apoptotic cells showed green fluorescence, and necrotic cells showed both red and green fluorescence. Scale bar, 200 μm. (D) Percentage of apoptotic and necrotic cells were recorded (n = 3). ***P < 0.001. (E) Western blot analysis of Bax, Caspase-3 and BCL2 expression in HMEC-1 cells treated with LPS or Carvacrol. (F–H) The gray blots were analyzed with ImageJ software (n = 3). *P < 0.05; ***P < 0.001.

Carvacrol Attenuates LPS-Induced Mitochondrial Dysfunction and Restores Mitochondrial Energy Production

We evaluated the effects of LPS and carvacrol on mitochondrial function by assessing MMP and mPTP opening. The results showed that LPS significantly reduced the mitochondrial membrane potential in HMEC-1 cells, induced a sustained opening of the mPTP (Figure 3A,B). In contrast, carvacrol significantly increased the mitochondrial membrane potential and inhibited excessive mPTP opening (Figure 3C,D). In addition, mitochondria represent the core of cellular energy metabolism and the primary site for ATP production. In this study, a mitochondrial ATP fluorescent probe was utilized to monitor real-time changes in mitochondrial ATP concentration. As shown in Figure 3, compared to the control, LPS inhibited mitochondrial ATP production, while carvacrol significantly increased ATP production (Figure 3E,F). These results indicate that carvacrol alleviates LPS-induced mitochondrial dysfunction, promotes mitochondrial ATP generation and improves mitochondrial energy metabolism.

Figure 3.

Figure 3

Open in a new tab

Carvacrol alleviates LPS-induced mitochondrial dysfunction and enhances ATP production. (A) The level of mitochondrial membrane potential in HMEC-1 cells treated with LPS or Carvacrol was detected by staining with JC-1 fluorescent probe. Scale bar, 100 μm. The relative ratio of red and green fluorescence was used to measure the proportion of mitochondria depolarized. (B) Mitochondrial membrane potential of cells was quantified (n = 3). ***P < 0.001. (C) The mPTP opening of HMEC-1 cells treated with LPS or carvacrol was detected by Calcein AM staining. Scale bar, 100 μm. (D) The mPTP opening of HMEC-1 cells was quantified (n = 3). *P < 0.05; ***P < 0.001. (E) ATP levels of HMEC-1 cells treated with LPS or carvacrol was detected by ATP fluorescent probe. Scale bar, 20 μm. (F) ATP levels of HMEC-1 cells was quantified (n = 3). **P < 0.01. ***P < 0.001.

Carvacrol Induces Cytoskeletal Remodeling in HMEC-1 Cells and Inhibits Their Angiogenic Capacity

The cytoskeleton plays a crucial role in mitochondrial dysfunction and apoptosis. The effects of LPS on the F-actin skeleton were observed using Phalloidin staining. The results demonstrated that in the control group, F-actin was distributed as green filaments throughout the cell with intense green fluorescence. However, following induction with LPS, a significant decrease in fluorescence intensity was observed, accompanied by substantial contraction and disorganization, suggesting a disruption in the cytoskeletal integrity. However, following the administration of carvacrol, a significant increase in the fluorescence intensity of F-actin was observed (Figure 4A,C). These findings suggest that carvacrol can reverse LPS-induced cytoskeletal damage and promote cytoskeletal remodelling. Abnormal angiogenesis has been linked to inflammation, vascular diseases, and tumor progression. The present study investigated the effects of LPS and carvacrol on angiogenesis at the in vitro level (Figure 4B,D,E). The results obtained confirmed that, in comparison with the control group, LPS induced angiogenesis, while carvacrol significantly inhibited it.

Figure 4.

Figure 4

Open in a new tab

Carvacrol induces cytoskeletal remodeling in HMEC-1 cells and inhibits their capacity for tube formation. (A) Phalloidin staining of F-actin in HMEC-1 cells transfected with LPS or carvacrol. Scale bar, 50 μm. (C) Relative expression of F-actin in HMEC-1 cells was quantified (n = 3). ***P < 0.001. (B) Capacity for tube formation of HMEC-1 cells treated with LPS and carvacrol for 4 h. Scale bar, 500 μm (D,E) Quantitative analysis of the number of length and area of capillary-like tubes formed by HMEC-1 cells treated with LPS and carvacrol (n = 3). ***P < 0.001.

Carvacrol Inhibits the Expression of VDAC1 Maintaining Mitochondrial Function, Thereby Affecting SLC25A6 and Alleviating LPS-Induced Damage

The results of Western blot analysis demonstrated that LPS increased the expression levels of inflammatory factors (IL-1β, IL-6, IL-18, TNF-α) and VDAC1, while reducing SLC25A6 expression Conversely, carvacrol exerted a substantial inhibitory effect on the expression of inflammatory factors and VDAC1, while concomitantly inducing an increase in SLC25A6 expression (Figure 5C–I). To further identify the inflammatory factor most significantly affecting VDAC1, recombinant proteins for IL-1β, IL-6, IL-18, and TNF-α were used for intervention. The results indicated that recombinant TNF-α markedly increased VDAC1 protein expression (Figure 5J,K).

Figure 5.

Figure 5

Open in a new tab

Carvacrol regulates the expression of SLC25A6 by inhibiting VDAC1. (A) Western blot for verifying the transfection effect of VDAC1 small interfering RNA on HMEC-1 cells. (B) The gray blots were analyzed with ImageJ software (n = 3). ***P < 0.001. (C) Western blot analysis of VDAC1, SLC25A6, TNF-α, IL-1β, IL-6, IL-18 expression in HMEC-1 cells treated with LPS or carvacrol. (D–I) The gray blots were analyzed with ImageJ software (n = 3). ***P < 0.001. (J) Western blot analysis of VDAC1 expression in HMEC-1 cells treated with recombinant IL-1β (rIL-1β), recombinant IL-6 (rIL-6), recombinant IL-18 (rIL-18) and recombinant TNF-α(rTNF-α). (K) The gray blots were analyzed with ImageJ software (n = 3). ***P < 0.001. (L) Western blot analysis of VDAC1 and SLC25A6 expression in HMEC-1 cells treated with rTNF-α, rTNF-α plus VDAC1 knockdown or rTNF-α plus Carvacrol. (M,N) The gray blots were analyzed with ImageJ software (n = 3). ***P < 0.001.

Furthermore, it was observed that on the basis of TNF-α recombinant protein, carvacrol and VDAC1 knockdown downregulated VDAC1 protein expression and increased SLC25A6 protein expression. Collectively, these observations suggest a regulatory relationship between VDAC1 and SLC25A6, and we hypothesize that SLC25A6 may be a downstream regulatory gene of VDAC1 (Figure 5L–N). The present study proposes that carvacrol may improve mitochondrial function and energy metabolism, maintain endothelial cell integrity, and reduce the progression of vascular inflammation by regulating SLC25A6 expression through the inhibition of VDAC1.

Discussion

In order to investigate the mechanism of carvacrol in oxidative stress and inflammatory injury in human microvascular endothelial cells, an initial examination of its antioxidant effects was conducted. The intracellular activity of MDA and SOD is indicative of the extent of free radical damage. It is well established that excessive production of oxidative free radicals and ROS leads to oxidative stress and inflammation, resulting in cellular damage.21−23 Research has demonstrated that carvacrol augments the body’s antioxidant capacity through two primary mechanisms: first, as a natural antioxidant, carvacrol can directly react with free radicals (such as hydroxyl radicals and superoxide anion radicals) and neutralize them. The phenolic hydroxyl group (−OH) in carvacrol can donate hydrogen atoms, stabilizing the free radicals.24 Additionally, carvacrol has been observed to regulate the activity of endogenous antioxidant enzymes, thereby exerting indirect antioxidant effects.25 In a study by Gore Karaali et al., it was demonstrated that carvacrol increases the activities of SOD, GPx, and CAT while reducing MDA levels, thereby inhibiting free radical synthesis and enhancing the body’s antioxidant capacity.26 Furthermore, carvacrol has also been shown to enhance the activities of enzyme such as SOD, GSH, and CAT, while concomitantly reducing lipid peroxidation (MDA) in murine kidneys, thereby enhancing antioxidant status.27 In the present study, carvacrol was found to modulate LPS-induced production of MDA and SOD in HMEC-1 cells, indicating that carvacrol enhances the ability to scavenge superoxide anions and reduces the degree of lipid peroxidation in cell membranes. These findings are consistent with those of previous studies, which have confirmed that carvacrol possesses antioxidant activity.

Cerrah et al. reported that pretreatment with carvacrol can reduce the levels of TNF-α, IL-1β, and NF-κB in acrylamide-induced hepatic inflammation in rats, decrease the inflammatory response, and thus exert a significant protective effect on the livers of rats.28 In a separate study, Riaz et al. reported that carvacrol can inhibit the ROS/NLRP3 pathway in vivo, thereby reducing NF-κB expression and alleviating the body’s inflammatory response.29 Consequently, the anti-inflammatory mechanism of carvacrol may be associated with its ability to inhibit the release and expression of inflammatory factors and to block the activation of the NF-κB pathway. The present study demonstrates that carvacrol significantly reduces the secretion of inflammatory cytokines TNF-α, IL-1β, and IL-6 in LPS-stimulated HMEC-1 cells. A substantial body of research has utilized umbilical vein endothelial cells as research models to elucidate the pathological changes in endothelial inflammation from a venous perspective.30 However, human microvascular endothelial cells possess characteristics of immortalization and stability. Consequently, we selected HMEC-1 cells for our experimental investigations to elucidate the pathological changes in microvascular inflammation.

Angiogenesis is commonly associated with the promotion of inflammation progression. Abnormal angiogenesis is typically driven by pro-inflammatory factors such as VEGF, TNF-α and IL-1β.31,32 These factors stimulate the formation of new blood vessels and activate the release of additional pro-inflammatory cytokines, leading to the persistence and exacerbation of inflammation. In chronic inflammatory diseases, angiogenesis not only sustains inflammation but also contributes to its prolonged presence of inflammation by providing a persistent blood supply to the inflammatory sites.33 The present study demonstrates that carvacrol effectively inhibits LPS-induced angiogenesis, thus highlighting its potential therapeutic value in both acute and chronic inflammation.

Following the confirmation of the antioxidant and anti-inflammatory properties of carvacrol, a further investigation was conducted into its impact on cell proliferation and apoptosis. Arsenite exposure increases BAX/Bcl-2 levels, leading to apoptosis in fish spleen cells through both endogenous and extrinsic pathways.34 Bax is a key pro-apoptotic gene, while BCL2 is an antiapoptotic protein located on the outer mitochondrial membrane, preventing apoptosis by inhibiting mitochondrial disruption, cytochrome C release, and caspase activation.35 Studies have shown that damaged mitochondrial membranes can release cytochrome C into the cytoplasm, triggering apoptosis via caspase-dependent signaling pathways.36 The present study corroborates these findings by demonstrating that carvacrol inhibits apoptosis by suppressing Caspase-3 activity and upregulating the expression of the antiapoptotic protein BCL2. During the process of apoptosis, cells undergo characteristic morphological changes, in which the cytoskeleton plays an active role.37 Previous studies have demonstrated that the process of apoptosis in numerous cell types is accompanied by disruptions to the actin microfilament structure and a decrease in F-actin expression levels.38 In particular, during the early phases of apoptosis, significant changes to the cytoskeleton are evident, including cell contraction, microfilament reorganization, and microtubule depolymerization. These observations suggest that alterations to the cytoskeleton may function as early signals that trigger the initiation of apoptosis.39 In accordance with previous findings, this study demonstrates that LPS stimulation results in significant cytoskeletal contraction, manifesting as disorganized morphology and compromised cytoskeletal integrity. However, pretreatment with carvacrol restores F-actin morphology and induces cytoskeletal remodeling. These results suggest that carvacrol can alleviate LPS-induced oxidative stress, inflammation, and apoptosis in human microvascular endothelial cells. Furthermore, it was determined that carvacrol treatment resulted in an increase in mitochondrial membrane potential and an inhibition in the opening of the mitochondrial permeability transition pore. This suggests that the mechanism by which carvacrol improves cell survival may be related to the regulation of mitochondrial function.

The multifunctional protein VDAC1 plays a critical role in regulating mitochondrial function and energy metabolism. Indeed, studies have demonstrated that it exerts significant regulatory effects in oxidative stress and inflammation.40 In rat brain models, studies have demonstrated that VDAC1 interacts with Bax, thereby enlarging the associated pore and increasing mitochondrial permeability, thus promoting apoptosis.41 It has been reported that VBIT-3, VBIT-4, and VBIT-12 interact with VDAC1 by disrupting its oligomerization, thereby modulating ROS levels and alleviating mitochondrial dysfunction associated with apoptosis and inflammation, which contributes to the mitigation of diseases such as type 2 diabetes, lupus, and ulcerative colitis.42 In addition, VDAC1 has been observed to form multiprotein complexes with Ca2+ channels in other organelles, such as the IP3R-VDAC1-GRP75-DJ-1 complex in the endoplasmic reticulum and the RyR2-VDAC1 complex in the sarcoplasmic reticulum, facilitating Ca2+ transfer to the mitochondria.43 In summary, VDAC1 indirectly participates in inflammatory responses through its diverse functional roles, and targeting the VDAC1 channel may offer novel therapeutic strategies for treating inflammatory diseases. The present study demonstrates that LPS exerts a direct effect on VDAC1 expression, and that VDAC1 knockdown impedes the release of LPS-induced inflammatory factors. Notably, the construction of recombinant IL-1β, IL-6, IL-18, and TNF-α proteins revealed that the TNF-α recombinant protein significantly increased VDAC1 expression. Consequently, we hypothesize that TNF-α, released by LPS, is the pivotal inflammatory factor that activates VDAC1, thus providing a foundation for further investigation into VDAC1’s role in inflammation progression. Furthermore, VDAC1 and SLC25A6 are recognized as components of the mPTP.44 VDAC1 and SLC25A6 collaborate in energy metabolism and apoptosis to regulate mitochondrial membrane permeability, ATP/ADP exchange, and the mechanisms of cell survival and death.45 The present study demonstrates that carvacrol significantly inhibits the decrease in MMP and excessive mPTP opening induced by LPS. At the protein expression level, carvacrol mitigated LPS-induced mitochondrial dysfunction by modulating the expression of VDAC1 and SLC25A6. Of particular note is the observation that, in the presence of the TNF-α recombinant protein, carvacrol and VDAC1 knockdown exhibited similar effects. Consequently, we hypothesize that carvacrol enhances mitochondrial function and energy metabolism by inhibiting VDAC1 and regulating SLC25A6 expression, thereby reducing LPS-induced oxidative stress and inflammatory damage in human microvascular endothelial cells.

Conclusion

In conclusion, the present study demonstrates that carvacrol exerts a protective effect by improving mitochondrial function and energy metabolism. The mechanism of action of carvacrol may be related to the regulation of VDAC1 and SLC25A6 protein expression, which further supports the potential application of carvacrol in the treatment of microvascular inflammation.

Glossary

Abbreviations

CAT

catalase

ASC

apoptotic speck-containing protein with a card

caspase-1

cysteinyl aspartate specific proteinase-1

BPA

bisphenol A

LPS

lipopolysaccharide

IL-1β

interleukin-1beta

IL-6

interleukin-6

IL-18

interleukin-18

TNF-α

tumour necrosis factor alpha

MMP

mitochondrial membrane potential

mPTP

mitochondrial permeability transition pore

HMEC

human microvascular endothelial cells

VDAC1

voltage-dependent anion channel 1

SLC25

solute carrier family 25

ROS

reactive oxygen species

ANT3

adenine nucleotide translocase 3

NF-κB

nuclear factor-kappaB

MAPK

mitogen-activated protein kinases

PGE2

prostaglandin E2

SOD

superoxide dismutase

TLR2

toll-like receptor 2

TLR4

toll-like receptor 4

NLRP3

NOD-like receptor family pyrin domain containing 3

VEGF

vascular endothelial growth factor

rTNF-α

recombinant TNF-α

rIL-1β

recombinant IL-1β

rIL-6

recombinant IL-6

rIL-18

recombinant IL-18

MDA

malondialdehyde

Data Availability Statement

Due to privacy restrictions and other issues, this study does not disclose the research data on which the article is based at the time of publication.

Author Contributions

Cuifang Lu: Conceptualization, Methodology; Bin Yang: Data curation, Formal analysis; Ying Liu: Resources, Software; Wenbo Liu: Visualization, Investigation; Jie Yan: Project administration, Supervision; Chenggong Guo: Validation; Tingyu Song: Writing-review and editing; Xiaofei Wang: Funding acquisition, Writing-original draft.

This study was supported by Hebei Province Medical Science Research Project Plan (20242167).

The authors declare no competing financial interest.

References

  1. Arulselvan P.; Fard M. T.; Tan W. S.; Gothai S.; Fakurazi S.; Norhaizan M. E.; Kumar S. S. Role of Antioxidants and Natural Products in Inflammation. Oxid. Med. Cell. Longev. 2016, 2016, 5276130. 10.1155/2016/5276130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Lu H.; Su H.; Liu Y.; Yin K.; Wang D.; Li B.; Wang Y.; Xing M. NLRP3 inflammasome is involved in the mechanism of the mitigative effect of lycopene on sulfamethoxazole-induced inflammatory damage in grass carp kidneys. Fish Shellfish Immunol. 2022, 123, 348–357. 10.1016/j.fsi.2022.03.018. [DOI] [PubMed] [Google Scholar]
  3. Uldry E.; Faes S.; Demartines N.; Dormond O. Fine-Tuning Tumor Endothelial Cells to Selectively Kill Cancer. Int. J. Mol. Sci. 2017, 18, 1401. 10.3390/ijms18071401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hellenthal K. E. M.; Brabenec L.; Wagner N. M. Regulation and Dysregulation of Endothelial Permeability during Systemic Inflammation. Cells 2022, 11, 1935. 10.3390/cells11121935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dabravolski S. A.; Nikiforov N. G.; Eid A. H.; Nedosugova L. V.; Starodubova A. V.; Popkova T. V.; Bezsonov E. E.; Orekhov A. N. Mitochondrial Dysfunction and Chronic Inflammation in Polycystic Ovary Syndrome. Int. J. Mol. Sci. 2021, 22, 3923. 10.3390/ijms22083923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Shoshan-Barmatz V.; Pittala S.; Mizrachi D. VDAC1 and the TSPO: Expression, Interactions, and Associated Functions in Health and Disease States. Int. J. Mol. Sci. 2019, 20, 3348. 10.3390/ijms20133348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Pandey S. K.; Machlof-Cohen R.; Santhanam M.; Shteinfer-Kuzmine A.; Shoshan-Barmatz V. Silencing VDAC1 to Treat Mesothelioma Cancer: Tumor Reprograming and Altering Tumor Hallmarks. Biomolecules 2022, 12, 895. 10.3390/biom12070895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brahimi-Horn M. C.; Giuliano S.; Saland E.; Lacas-Gervais S.; Sheiko T.; Pelletier J.; Bourget I.; Bost F.; Féral C.; Boulter E.; Tauc M.; Ivan M.; Garmy-Susini B.; Popa A.; Mari B.; Sarry J. E.; Craigen W. J.; Pouysségur J.; Mazure N. M. Knockout of Vdac1 activates hypoxia-inducible factor through reactive oxygen species generation and induces tumor growth by promoting metabolic reprogramming and inflammation. Cancer Metabol. 2015, 3, 8. 10.1186/s40170-015-0133-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ma Y.; Yang W.; Liang P.; Feng R.; Qiu T.; Zhang J.; Sun X.; Li Q.; Yang G.; Yao X. The VDAC1 oligomerization regulated by ATP5B leads to the NLRP3 inflammasome activation in the liver cells under PFOS exposure. Ecotoxicol. Environ. Saf. 2024, 281, 116647. 10.1016/j.ecoenv.2024.116647. [DOI] [PubMed] [Google Scholar]
  10. Pu X.; Zhang Q.; Liu J.; Wang Y.; Guan X.; Wu Q.; Liu Z.; Liu R.; Chang X. Ginsenoside Rb1 ameliorates heart failure through DUSP-1-TMBIM-6-mediated mitochondrial quality control and gut flora interactions. Phytomedicine 2024, 132, 155880. 10.1016/j.phymed.2024.155880. [DOI] [PubMed] [Google Scholar]
  11. Shoshan-Barmatz V.; Shteinfer-Kuzmine A.; Verma A. VDAC1 at the Intersection of Cell Metabolism, Apoptosis, and Diseases. Biomolecules 2020, 10, 1485. 10.3390/biom10111485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Skakkebæk A.; Kjær-Sørensen K.; Matchkov V. V.; Christensen L. L.; Just J.; Cömert C.; Andersen N. H.; Oxvig C.; Gravholt C. H. Dosage of the pseudoautosomal gene SLC25A6 is implicated in QTc interval duration. Sci. Rep. 2023, 13, 12089. 10.1038/s41598-023-38867-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Shen X.; Ma M.; Mi R.; Zhuang J.; Song Y.; Yang W.; Li H.; Lu Y.; Yang B.; Liu Y.; Wu Y.; Shen H. EFHD1 promotes osteosarcoma proliferation and drug resistance by inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP) by binding to ANT3. CMLS Cell. Mol. Life Sci. 2024, 81, 236. 10.1007/s00018-024-05254-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Zamora M.; Granell M.; Mampel T.; Viñas O. Adenine nucleotide translocase 3 (ANT3) overexpression induces apoptosis in cultured cells. FEBS Lett. 2004, 563, 155–160. 10.1016/S0014-5793(04)00293-5. [DOI] [PubMed] [Google Scholar]
  15. Sharaf el dein O.; Gallerne C.; Brenner C.; Lemaire C. Increased expression of VDAC1 sensitizes carcinoma cells to apoptosis induced by DNA cross-linking agents. Biochem. Pharmacol. 2012, 83, 1172–1182. 10.1016/j.bcp.2012.01.017. [DOI] [PubMed] [Google Scholar]
  16. Jang J. Y.; Lee C. E. IL-4-induced upregulation of adenine nucleotide translocase 3 and its role in Th cell survival from apoptosis. Cell. Immunol. 2006, 241, 14–25. 10.1016/j.cellimm.2006.07.006. [DOI] [PubMed] [Google Scholar]
  17. Singh J.; Luqman S.; Meena A. Carvacrol as a Prospective Regulator of Cancer Targets/Signalling Pathways. Curr. Mol. Pharmacol. 2023, 16, 542–558. 10.2174/1874467215666220705142954. [DOI] [PubMed] [Google Scholar]
  18. Gunes-Bayir A.; Guler E. M.; Bilgin M. G.; Ergun I. S.; Kocyigit A.; Dadak A. Anti-Inflammatory and Antioxidant Effects of Carvacrol on N-Methyl-N’-Nitro-N-Nitrosoguanidine (MNNG) Induced Gastric Carcinogenesis in Wistar Rats. Nutrients 2022, 14, 2848. 10.3390/nu14142848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Cacciatore I.; Spalletta S.; Di Rienzo A.; Flati V.; Fornasari E.; Pierdomenico L.; Del Boccio P.; Valentinuzzi S.; Costantini E.; Toniato E.; Martinotti S.; Conte C.; Di Stefano A.; Robuffo I. Anti-Obesity and Anti-Inflammatory Effects of Novel Carvacrol Derivatives on 3T3-L1 and WJ-MSCs Cells. Pharmaceuticals 2023, 16, 340. 10.3390/ph16030340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Joshi S.; Kundu S.; Priya V. V.; Kulhari U.; Mugale M. N.; Sahu B. D. Anti-inflammatory activity of carvacrol protects the heart from lipopolysaccharide-induced cardiac dysfunction by inhibiting pyroptosis via NLRP3/Caspase1/Gasdermin D signaling axis. Life Sci. 2023, 324, 121743. 10.1016/j.lfs.2023.121743. [DOI] [PubMed] [Google Scholar]
  21. Xiao Z.; Yu X.; Zhang S.; Liang A. The Expression Levels and Significance of GSH, MDA, SOD, and 8-OHdG in Osteochondral Defects of Rabbit Knee Joints. BioMed Res. Int. 2022, 2022, 6916179. 10.1155/2022/6916179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Celik Topkara K.; Kilinc E.; Cetinkaya A.; Saylan A.; Demir S. Therapeutic effects of carvacrol on beta-amyloid-induced impairments in in vitro and in vivo models of Alzheimer’s disease. Eur. J. Neurosci. 2022, 56, 5714–5726. 10.1111/ejn.15565. [DOI] [PubMed] [Google Scholar]
  23. Fatima K.; Luqman S.; Meena A. Carvacrol Arrests the Proliferation of Hypopharyngeal Carcinoma Cells by Suppressing Ornithine Decarboxylase and Hyaluronidase Activities. Front. Nutr. 2022, 9, 857256. 10.3389/fnut.2022.857256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sharifi-Rad M.; Varoni E. M.; Iriti M.; Martorell M.; Setzer W. N.; Del Mar Contreras M.; Salehi B.; Soltani-Nejad A.; Rajabi S.; Tajbakhsh M.; Sharifi-Rad J. Carvacrol and human health: A comprehensive review. Phytother. Res. 2018, 32, 1675–1687. 10.1002/ptr.6103. [DOI] [PubMed] [Google Scholar]
  25. Ismail M.; Srivastava V.; Marimani M.; Ahmad A. Carvacrol modulates the expression and activity of antioxidant enzymes in Candida auris. Res. Microbiol. 2022, 173, 103916. 10.1016/j.resmic.2021.103916. [DOI] [PubMed] [Google Scholar]
  26. Gore Karaali M.; Karaali S.; Demir D.; Yazıcı G. N.; Coban A.; Mammadov R.; Suleyman B.; Suleyman H. The protective effect of carvacrol on bevacizumab-related skin injury in rats: a biochemical and histopathological evaluation. Cutan. Ocul. Toxicol. 2022, 41, 285–290. 10.1080/15569527.2022.2124413. [DOI] [PubMed] [Google Scholar]
  27. Gunes S.; Ayhanci A.; Sahinturk V.; Altay D. U.; Uyar R. Carvacrol attenuates cyclophosphamide-induced oxidative stress in rat kidney. Can. J. Physiol. Pharmacol. 2017, 95, 844–849. 10.1139/cjpp-2016-0450. [DOI] [PubMed] [Google Scholar]
  28. Cerrah S.; Ozcicek F.; Gundogdu B.; Cicek B.; Coban T. A.; Suleyman B.; Altuner D.; Bulut S.; Suleyman H. Carvacrol prevents acrylamide-induced oxidative and inflammatory liver damage and dysfunction in rats. Front. Pharmacol. 2023, 14, 1161448. 10.3389/fphar.2023.1161448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Riaz M.; Al Kury L. T.; Atzaz N.; Alattar A.; Alshaman R.; Shah F. A.; Li S. Carvacrol Alleviates Hyperuricemia-Induced Oxidative Stress and Inflammation by Modulating the NLRP3/NF-κB Pathwayt. Drug Des. Dev. Ther. 2022, 16, 1159–1170. 10.2147/DDDT.S343978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Geng L.; Qin Z.; Han T. L.; Zhou Y.; Zhong X.; Zhang G.; Dong X. Human Amniotic Epithelial Cells Improve Uterine Spiral Artery Remodeling to Ameliorate Preeclampsia in a Rat Model. Biol. Reprod. 2024, 111, 906–918. 10.1093/biolre/ioae113. [DOI] [PubMed] [Google Scholar]
  31. Lee H. W.; Xu Y.; He L.; Choi W.; Gonzalez D.; Jin S. W.; Simons M. Role of Venous Endothelial Cells in Developmental and Pathologic Angiogenesis. Circulation 2021, 144, 1308–1322. 10.1161/CIRCULATIONAHA.121.054071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lee H. J.; Hong Y. J.; Kim M. Angiogenesis in Chronic Inflammatory Skin Disorders. Int. J. Mol. Sci. 2021, 22, 12035. 10.3390/ijms222112035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lu E.; Li C.; Wang J.; Zhang C. Inflammation and angiogenesis in the corpus luteum. J. Obstet. Gynaecol. Res. 2019, 45, 1967–1974. 10.1111/jog.14076. [DOI] [PubMed] [Google Scholar]
  34. Wang Y.; Zhao H.; Mu M.; Guo M.; Xing M. Zinc offers splenic protection through suppressing PERK/IRE1-driven apoptosis pathway in common carp (Cyprinus carpio) under arsenic stress. Ecotoxicol. Environ. Saf. 2021, 208, 111473. 10.1016/j.ecoenv.2020.111473. [DOI] [PubMed] [Google Scholar]
  35. Desagher S.; Martinou J. C. Mitochondria as the central control point of apoptosis. Trends Cell Biol. 2000, 10, 369–377. 10.1016/S0962-8924(00)01803-1. [DOI] [PubMed] [Google Scholar]
  36. Bock F. J.; Tait S. W. G. Mitochondria as multifaceted regulators of cell death. Nat. Rev. Mol. Cell Biol. 2020, 21, 85–100. 10.1038/s41580-019-0173-8. [DOI] [PubMed] [Google Scholar]
  37. Povea-Cabello S.; Oropesa-Ávila M.; de la Cruz-Ojeda P.; Villanueva-Paz M.; de la Mata M.; Suárez-Rivero J. M.; Álvarez-Córdoba M.; Villalón-García I.; Cotán D.; Ybot-González P.; Sánchez-Alcázar J. A. Dynamic Reorganization of the Cytoskeleton during Apoptosis: The Two Coffins Hypothesis. Int. J. Mol. Sci. 2017, 18, 2393. 10.3390/ijms18112393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Chen Q.; Lu G.; Wang Y.; Xu Y.; Zheng Y.; Yan L.; Jiang Z.; Yang L.; Zhan J.; Wu Y.; Zhou J. Cytoskeleton disorganization during apoptosis induced by curcumin in A549 lung adenocarcinoma cells. Planta Med. 2009, 75, 808–813. 10.1055/s-0029-1185399. [DOI] [PubMed] [Google Scholar]
  39. Croft D. R.; Coleman M. L.; Li S.; Robertson D.; Sullivan T.; Stewart C. L.; Olson M. F. Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration. J. Cell Biol. 2005, 168, 245–255. 10.1083/jcb.200409049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wang H.; Li Y.; Liu C.; Lu T.; Zhai Q.; Wang H.; Zhang J. Inhibition of VDAC1 prevents oxidative stress and apoptosis induced by bisphenol A in spermatogonia via AMPK/mTOR signaling pathway. J. Toxicol. Sci. 2023, 48, 109–119. 10.2131/jts.48.109. [DOI] [PubMed] [Google Scholar]
  41. Banerjee J.; Ghosh S. Bax increases the pore size of rat brain mitochondrial voltage-dependent anion channel in the presence of tBid. Biochem. Biophys. Res. Commun. 2004, 323, 310–314. 10.1016/j.bbrc.2004.08.094. [DOI] [PubMed] [Google Scholar]
  42. Ben-Hail D.; Begas-Shvartz R.; Shalev M.; Shteinfer-Kuzmine A.; Gruzman A.; Reina S.; De Pinto V.; Shoshan-Barmatz V. Novel Compounds Targeting the Mitochondrial Protein VDAC1 Inhibit Apoptosis and Protect against Mitochondrial Dysfunction. J. Biol. Chem. 2016, 291, 24986–25003. 10.1074/jbc.M116.744284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rosencrans W. M.; Rajendran M.; Bezrukov S. M.; Rostovtseva T. K. VDAC regulation of mitochondrial calcium flux: From channel biophysics to disease. Cell Calcium 2021, 94, 102356. 10.1016/j.ceca.2021.102356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Gutiérrez-Aguilar M.; Baines C. P. Structural mechanisms of cyclophilin D-dependent control of the mitochondrial permeability transition pore. Biochim. Biophys. Acta 2015, 1850, 2041–2047. 10.1016/j.bbagen.2014.11.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Allouche M.; Pertuiset C.; Robert J. L.; Martel C.; Veneziano R.; Henry C.; dein O. S.; Saint N.; Brenner C.; Chopineau J. ANT-VDAC1 interaction is direct and depends on ANT isoform conformation in vitro. Biochem. Biophys. Res. Commun. 2012, 429, 12–17. 10.1016/j.bbrc.2012.10.108. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Due to privacy restrictions and other issues, this study does not disclose the research data on which the article is based at the time of publication.

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES