Epigallocatechin-3-gallate Alleviates Ethanol-Induced Endothelia Cells Injury Partly through Alteration of NF-κB Translocation and Activation of the Nrf2 Signaling Pathway

Abstract

Ethanol (alcohol) is a risk factor that contributes to non-communicable diseases. Chronic abuse of ethanol is toxic to both the heart and overall health, and even results in death. Ethanol and its byproduct acetaldehyde can harm the cardiovascular system by impairing mitochondrial function, causing oxidative damage, and reducing contractile proteins. Endothelial cells are essential components of the cardiovascular system, are highly susceptible to ethanol, either through direct or indirect exposure. Thus, protection against endothelial injury is of great importance for persons who chronic abuse of ethanol. In this study, an in vitro model of endothelial injury was created using ethanol. The findings revealed that a concentration of 20.0 mM of ethanol reduced cell viability and Bcl-2 expression, while increasing cell apoptosis, intracellular reactive oxygen species (ROS) levels, mitochondrial depolarization, and the expression of Bax and cleaved-caspase-3 in endothelial cells. Further study showed that ethanol promoted nuclear translocation of nuclear factor kappa B (NF-κB), increased the secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 in the culture medium, and inhibited nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling pathway. The aforementioned findings suggest that ethanol has a harmful impact on endothelial cells. Nevertheless, the application of epigallocatechin-3-gallate (EGCG) to the cells can effectively mitigate the detrimental effects of ethanol on endothelial cells. In conclusion, EGCG alleviates ethanol-induced endothelial injury partly through alteration of NF-κB translocation and activation of the Nrf2 signaling pathway. Therefore, EGCG holds great potential in safeguarding individuals who chronically abuse ethanol from endothelial dysfunction.

INTRODUCTION

Ethanol (alcohol) is a risk factor that results in non-communicable diseases, and there is estimated to be one in every six people dying from ethanol.^1,2) Ethanol intake is a double-edged sword, depending on the amount consumption.^3,4) Data showed that people who routinely take in reasonably ethanol have fewer cardiovascular diseases than those who never.⁵⁾ Nevertheless, chronic abuse of ethanol is toxic to overall health, and even results in death.^6,7) Excessive alcohol consumption can damage vascular endothelial cells directly, which leads to abnormal structure and function of the cell membrane. This can cause vasoconstriction, increase blood pressure, and elevate the risk of developing various cardiovascular diseases.⁸⁾ Thus, protection against endothelial dysfunction is of great importance for persons who chronically abuse ethanol.

Tea is a popular drink because of its pleasant taste and beneficial to health.⁹⁾ Traditional Chinese medicine has suggested drinking green tea to prevent disease, and is considered to be a beneficial practice. Tea has a lot of bioactive substances, which play a variety of physiological functions, such as antioxidant, antibacterial, anti-tumor, etc.^10,11) The above benefits have been assigned to tea’s high content of biologically active ingredients, such as polyphenols.^12,13) Catechin is a kind of tea polyphenols, which is the highest among the polyphenols in tea.¹⁴⁾ Epigallocatechin-3-gallate (EGCG), a catechin monomers extracted from tea, is the main bioactive and water-soluble ingredient of green tea.¹⁵⁾ Numerous evidences suggest that EGCG acts as a powerful antioxidant, inhibiting cell apoptosis and apoptosis related proteins as well as suppressing nuclear factor kappa B (NF-κB) transcriptional factor activation.^16–18) Administration of EGCG can mediate ethanol-induced oxidative stress injury.¹⁹⁾ However, roles of EGCG in endothelial protection remain poorly defined, especially in ethanol injured endothelial cells. In this study, ethanol was utilized to induce injury in endothelial cells, and various methods were used to observe the protective ability of EGCG.

MATERIALS AND METHODS

Cell Culture and Drug Treatment

The human umbilical vein endothelial cells (HUVECs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Cat. No. 11965092, Gibco, NY, U.S.A.) supplemented with 10% fetal bovine serum (FBS), 100 U/mL streptomycin and 100 U/mL penicillin. The cells were incubated at 37 °C in a 5% CO₂ atmosphere. After reaching sub-confluence, the cells were cultured in specific dishes and serum-starved with DMEM for 12 h. Cells were treated with ethanol to cause injury, while the control group received DMEM. EGCG (Cat. No. E3893, Sigma-Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China) was given as a protective agent.

Cell Viability Assay

Cell viability was evaluated using a Cell Counting Kit-8 (CCK-8) kit (Cat. No. C0037, Beyotime, Shanghai, China) according to the manufacturer’s instructions. A total of 1.5 × 10³ cells were seeded in a 96-well plate and serum-starved in DMEM for 12 h. Except for the control group, cells were treated with varying concentrations of EGCG with or without different concentrations of ethanol for 12 h. After the designated treatment, 10 µL of CCK-8 solution was added to each well, and the cells were incubated at 37 °C for 4 h. The absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, U.S.A.). Each experiment was repeated six times.

Cellular Apoptosis Assay

The Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (Cat. No. C1067s, Beyotime) was utilized to perform the apoptosis assay. Nunc glass bottom dishes were used to seed 1 × 10⁶ cells/well, which were serum-starved in DMEM for 12 h until they reached sub-confluence. Except for the control group, cells were treated with EGCG at 100.0 µM with or without ethanol at 20.0 mM for 12 h. After treatment, cells were washed with phosphate-buffered saline (PBS) three times. Then, 5 µL of FITC-labeled annexin V and 195 µL of binding buffer were added to each dish. The dishes were incubated in the dark at 25 °C for 10 min. After that, propidium iodide (10 µL) was added to the dishes and they were incubated again in the dark at 4 °C for 20 min. Apoptotic cells were visualized using laser confocal microscopy (Dim8, Leica Microsystems, Wetzlar, Germany) and categorized into three groups: Normal cells with low fluorescence, early-stage apoptotic cells exhibiting green fluorescence, and later-stage apoptotic/necrotic cells stained with both red and green fluorescence.

Western Blot

Approximately 6–8 × 10⁶ cells were seeded in a 6-well plate and subjected them to serum starvation in DMEM until they reached sub-confluency. Except for the control group, cells were treated with EGCG at 100.0 µM in absence or presence of ethanol at 20.0 mM for 12 h. After treatment, the cells were washed three times with ice-cold PBS containing phenylmethylsulfonyl fluoride (PMSF) and then solubilized using 200 µL of ice-cold lysis buffer (Cat. No. P0013C, Beyotime). The supernatants were obtained by centrifuging at 10000 rpm for 10 min at a temperature of 4 °C. To each supernatant, a loading buffer was added and boiled for 8 min. Then, 15 µL of the lysate was electrophoresed on a 12% sodium dodecyl sulfate (SDS) gel and transferred onto nitrocellulose membranes. The membrane was blocked with 5% bovine serum albumin overnight at 4 °C. The blots were treated with antibodies against Bcl-2 (dilution 1 : 8000, Cat. No. AB112, Beyotime), Bax (dilution 1 : 5000, Cat. No. AF0054, Beyotime), cleaved-caspase-3 (dilution 1 : 5000, Cat. No. 9661s, Cell Signaling Tech., MA, U.S.A.), nuclear factor-E2-related factor 2 (Nrf2) (dilution 1 : 5000, Cat. No. YT3189, ImmunoWay Biotechnology, Suzhou, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (dilution 1 : 10000, Cat NO.: AF0006, Beyotime) or β-actin (dilution 1 : 10000; Cat. No. NC011; Zhuangzhi Biotech., China) overnight at a temperature of 4 °C. The blots were then incubated with peroxidase-conjugated secondary antibodies (dilution 1 : 20000) for 2 h at room temperature. Chemiluminescence (Cat. No. WBULS0500, from Merck Life Science U.K. Limited, Gillingham, U.K.) was utilized to amplify the immune complexes and the data was analyzed using Quantity One software (Bio-Rad Laboratories).

Measurement of Mitochondrial Transmembrane Potential

The JC-1 probe (Cat. No. C2006, Beyotime) was utilized to assess mitochondrial depolarization, according to the manufacturer’s instructions. Briefly, cells were seeded at a density of 1.5 × 10⁶ in Nunc glass bottom dishes and subjected to serum starvation in DMEM for 12 h once they reached sub-confluence. Except for the control group, the remaining cells were treated with EGCG at a concentration of 100.0 µM in the presence or absence of ethanol at 20.0 mM for 12 h. The cells were treated and then exposed to 25 µg/mL JC-1 at 37 °C for 25 min. After that, they were washed twice with PBS and transferred to fresh serum-free DMEM. Finally, laser confocal microscopy measured JC-1 emissions to assess mitochondrial membrane potential. A higher green/red fluorescence ratio suggested depolarization.

Detection of Intracellular Reactive Oxygen Species (ROS)

The ROS levels were measured using the probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). This probe can easily enter cells and is converted by esterases to non-fluorescent 2′,7′-dichlorodihydrofluorescein (DCFH), which reacts with intracellular ROS to produce fluorescent dichlorofluorescein (DCF). Assay performed with 1.5 × 10⁶ cells cultured in glass bottom dishes and serum-starved for 12 h in DMEM until sub-confluence was reached. Cells, excluding the control group, were subjected to treatment with EGCG at a concentration of 100 µM for 4 h. The treatment was carried out in the presence or absence of ethanol at a concentration of 20 mM. After treatment, the culture medium was removed and cells were washed three times with PBS. DCFH-DA diluted into DMEM at a concentration of 10 µM was added to cultures and incubated for an additional 20 min at 37 °C. The fluorescence was read using laser confocal microscopy with excitation at 485 nm and emission at 530 nm.

Assay of the Nuclear Translocation of NF-κB

NF-κB activation was evaluated using the NF-κB activation nuclear translocation assay kit (Cat. No. SN368, Beyotime). Cells were cultured at a density of 1 × 10⁶ in Nunc glass bottom dishes and serum-starved in DMEM for 12 h until they got sub-confluence. Cells, excluding the control group, were subjected to EGCG treatment at a concentration of 100.0 µM for 12 h in the absence or presence of ethanol at 20.0 mM. After, the cells were rinsed with PBS and then preserved by fixing them with 4% paraformaldehyde. The cells were blocked with 5% bovine serum albumin at room temperature for 1.5 h. The samples were left to incubate with primary rabbit anti-NF-κB p65 antibody overnight at 4 °C, and then exposed to Cy3-labeled secondary antibody for two hours at 25 °C. Following each step, the sample was subjected to a triple wash lasting 5 min each time. Finally, the movement or activation of NF-κB towards the nucleus was monitored using laser confocal microscopy.

Measurements of Protein Levels of Tumor Necrosis Factor (TNF)-α, Interleukin (IL)-1β and IL-6 by Enzyme-Linked Immunosorbent Assay (ELISA)

Approximately 1–1.5 × 10⁵ cells were seeded in a 96-well plate and subjected them to serum starvation in DMEM until they reached sub-confluency. Except for the control group, cells were treated with EGCG at 100.0 µM in absence or presence of ethanol at 20.0 mM for 12 h. Protein levels of TNF-α, IL-1β and IL-6 in the medium were determined using human TNF-α (Cat. No. E-EL-H0109), IL-1β (Cat. No. E-EL-H0149) and IL-6 (Cat. No. E-EL-H6156) ELISA kits (Elabscience, Wuhan, China), according to the manufacturer’s instructions.

Statistical Analysis

The data were analyzed using SPSS version 17.0 (SPSS Inc., Chicago, IL, U.S.A.) and expressed as means ± standard error of the mean (S.E.M.). The t-test was used to assess data distribution. If there were more than two sets of data, one-way analysis of variance (ANOVA) was used. Normality and homogeneity were checked before analysis. Statistical significance was set at p < 0.05.

RESULTS

EGCG Increases Ethanol-Decreased Cell Viability

To test the suitable concentration of ethanol and EGCG, the cells were treated with different concentrations of ethanol or with/without EGCG. The findings in Fig. 1a suggest that the viability of cells decreased as the concentration of ethanol increased (with a low OD value after the treatment of ethanol). However, Fig. 1b demonstrates that treatment with 100  and 200 µM EGCG was able to counteract the negative effects of ethanol on cell viability. Based on these results, 100 µM EGCG and 20 mM ethanol were selected for further experimentation.

Fig. 1. EGCG Improves the Cell Viability of HUVECs in the Presence of Ethanol

(a) The effect of ethanol on the viability of HUVECs. The cells were exposed to varying concentrations of ethanol (1.0, 10.0, 20.0, or 40.0 mM) for a duration of 12 h. (b) The protective effect of EGCG on HUVECs damaged by ethanol. The cells were treated with varying concentrations of EGCG (100.0 or 200.0 µM) and co-incubated with 20.0 mM ethanol for a duration of 12 h. Cell viability was evaluated using CCK-8 and the results are presented as mean ± S.E.M. of six independent experiments (** p < 0.01).

EGCG Inhibits Ethanol Induced Cell Apoptosis

To further identify the role of EGCG in apoptosis of endothelial cells after ethanol stimulation, the staining of annexin V-FITC/propidium iodide (PI) were conducted. The results indicated that after 12 h of ethanol treatment, there was a significant increase in early apoptotic and necrotic cells. In contrast, only a few cells in the control group were stained green or red, indicating low levels of early and late apoptosis. Treatment with EGCG significantly reduced cell apoptosis, indicating a protective effect against ethanol-induced cell damage, as shown in Fig. 2.

Fig. 2. EGCG Prevents Ethanol-Induced Apoptosis in HUVECs

The cells were either exposed to 20.0 mM of ethanol for 12 h or treated with EGCG at a concentration of 100.0 µM and co-incubated with ethanol (20.0 mM) for the same duration. (a) Laser confocal microscopy (scale bar = 75 µm) was used to observe cell apoptosis, where live cells exhibited low fluorescence levels, apoptotic cells in the early stages displayed green fluorescence, and necrotic and advanced stage apoptotic cells showed both red and green fluorescence. (b) The cell count of both early stages and necrotic/advanced apoptotic cells (** p < 0.01, *** p < 0.001).

EGCG Decreases Ethanol-Increased the Expression of Bax and Cleaved-caspase-3, Increases the Level of Bcl-2

To identify the role of EGCG in Bcl-2/Bax/cleaved-caspase-3 levels, we analyzed these proteins via Western blotting. Based on the results presented in Fig. 3, it is evident that treatment with ethanol for 12 h led to a significant increase in the expression of Bax and cleaved-caspase-3 proteins, indicating cell apoptosis. However, co-treatment with EGCG (100.0 µM) effectively inhibited the ethanol-induced increase in Bax and cleaved-caspase-3 protein expression. Moreover, the expression of Bcl-2 was found to be increased following EGCG treatment, further indicating its protective effect against ethanol-induced cell apoptosis.

Fig. 3. EGCG Reduces the Expression of Bax and Cleaved-caspase-3, Which Are Increased by Ethanol, While Increasing the Level of Bcl-2

The cells were either incubated with 20.0 mM ethanol for 12 h or co-incubated with EGCG at 100.0 µM and 20.0 mM ethanol for 12 h. Protein levels were measured using Western blot analysis. The data presented here represent the mean ± S.E.M. of six independent experiments (* p < 0.05).

EGCG Decreases Ethanol Increased ROS Formation

Based on the findings that EGCG can reverse the damage caused by ethanol-induced cell apoptosis, conducted additional tests was conducted to evaluate the anti-oxidative properties of EGCG by measuring intracellular ROS levels. After exposing cells to ethanol for 4 h, observed an increase in fluorescence intensity, indicating the formation of ROS. However, pretreatment with EGCG at a concentration of 100.0 µM resulted in a significant reduction in fluorescence intensity as shown in Fig. 4, indicating the antioxidative effects of EGCG.

Fig. 4. EGCG Reduces the Production of ROS in HUVECs Induced by Ethanol

The cells were either exposed to 20.0 mM ethanol for 4 h or treated with EGCG at a concentration of 100.0 µM and co-exposed to 20.0 mM ethanol for 4 h. (a) Intracellular ROS levels were visualized using laser confocal microscopy (scale bar = 50 µm), cells observed under bright light (left panels) and ROS observed under fluorescence microscopy (right panels). (b) The fluorescence intensity. The data presented here represent the mean ± S.E.M. of six independent experiments (**** p < 0.0001).

EGCG Increases Ethanol-Decreased Mitochondrial Transmembrane Potential

EGCG’s protective effect against ethanol-induced cell injury was confirmed by assessing mitochondrial depolarization. Control cells displayed hyperpolarized mitochondria, emitting strong red fluorescence and weak green fluorescence (Fig. 5). In contrast, cells treated with ethanol showed depolarized mitochondria, with increased green fluorescence and decreased red fluorescence. However, treatment with EGCG alleviated the ethanol-induced mitochondrial depolarization, as evidenced by the shift in fluorescent color from green to red.

Fig. 5. EGCG Increases the Mitochondrial Transmembrane Potential

The cells were either incubated with 20.0 mM of ethanol for 12 h or co-incubated with 100.0 µM of EGCG and 20.0 mM of ethanol for 12 h. Laser confocal microscopy was used to measure the mitochondrial membrane potential. (a) Left panels showed cells observed under bright light while right panels displayed a merged field of red fluorescence (JC-1 aggregates) and green fluorescence (JC-1 monomers). (b) The red fluorescence intensity/green fluorescence (scale bar = 50 µm). The data presented here represent the mean ± S.E.M. of six independent experiments (* p < 0.05, ** p < 0.01).

EGCG Inhibits Ethanol-Promoted Nuclear Translocation of NF-κB, the Secretion of TNF-α, IL-1β, IL-6, and Increases Expression of Nrf2

As NF-κB and Nrf2 plays a critical role in endothelial cell dysfunction, we then examined the effect of EGCG on levels of NF-κB and Nrf2 in cytoplasmic. The p65 subunit of NF-κB was detected by anti-p65 antibodies that were recognized by Cy3-labeled secondary antibodies, resulting in red fluorescence. Additionally, cellular DNA was stained with 4′-6-diamidino-2-phenylindole (DAPI) to serve as an indicator and appeared blue under fluorescence microscopy. The immunofluorescence staining revealed that ethanol promoted NF-κB translocation into nuclei of HUVECs. As shown in Fig. 6a, the p65 subunit was observed to be translocated to the nucleus. However, treatment with EGCG provided protection against ethanol-induced cell injury, as evidenced by the faint intensity of red fluorescence, especially in the nucleus.

Fig. 6. EGCG Inhibits Ethanol-Promoted Nuclear Translocation of NF-κB, the Secretion of TNF-α, IL-1β, IL-6, and Increases Expression of Nrf2

HUVECs were exposed to either 20.0 mM ethanol for 12 h or co-exposed to 100.0 µM EGCG and 20.0 mM ethanol for 12 h. (a) Immunofluorescence staining was used to detect the level of NF-κB in the nuclei of HUVECs (scale bar = 50 µm), with red indicating p65 subunit and blue representing cell nucleus, and the merged field of these colors can be seen in the right panels. (b–d) Protein levels of TNF-α (b, n = 6), IL-1β (c, n = 6) and IL-6 (d, n = 6) in the medium were determined using related ELISA kits. (e) The Western blot was used to detect the expression of Nrf2. The data presented here represent the mean ± S.E.M. of three independent experiments unless noted (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Further study showed that EGCG decreases the secretion of TNF-α, IL-1β, IL-6 in the culture medium (Figs. 6b–d), and increases ethanol-inhibited expression of Nrf2 (Fig. 6e). The results suggest that EGCG has a protective effect on ethanol-induced cell injury by inhibiting NF-κB transduction and increasing expression of Nrf2.

DISCUSSION

The cardiovascular system is one of the most easily affected systems by ethanol toxicity.^20,21) High dose or chronic intake ethanol can increase the atherosclerosis process with peripheral vascular involvement, coronary, and cerebral, cause progressive myocardial damage as well as induction of arrhythmias, and increase arterial hypertension.^5,22–24) Some studies have indicated that chronic or heavy ethanol abuse in humans can lead to an increased risk of cardiovascular disease,²¹⁾ liver²⁵⁾ or nervous system diseases.²⁶⁾ Endothelial cells are key component of the cardiovascular system, is a highly metabolically active organ, involved in many physio-pathological processes, and also the direct or indirect target sites ethanol.²⁷⁾

The current research employed HUVECs, a human endothelial cell line, as an in vitro model to investigate the injury caused by ethanol. The results indicated that exposure to 20.0 mM ethanol significantly reduced cell viability, increased intracellular levels of ROS, induced cell apoptosis, caused mitochondrial depolarization, and promoted the translocation of NF-κB into the nucleus, and increase the levels of TNF-α, IL-1β, IL-6 in the culture medium. Additionally, ethanol inhibited the Nrf2 signaling pathway. These findings collectively suggest that the HUVECs model is a reliable in vitro system for studying endothelial injury caused by ethanol.

Research has indicated that ethanol and its byproduct acetaldehyde can have negative effects on the cardiovascular system by interfering with the proper functioning of mitochondria, resulting in damage caused by oxidative stress, and contributing to the depletion of contractile proteins.^28,29) Mitochondria are the primary source of ROS production, and an imbalance in ROS generation can lead to the disruption of mitochondrial membrane potential.³⁰⁾ This disruption can result in cellular damage and dysfunction.³¹⁾ When mitochondrial membrane potential is disrupted, intermembrane proteins, including cytochrome c, are released into the cytosol. This triggers a cascade of events that ultimately leads to the activation of caspase-3, a key enzyme involved in programmed cell apoptosis.^32,33) Once caspase-3 is activated, it initiates a series of biochemical reactions that ultimately lead to the fragmentation of DNA, condensation of nuclear chromatin, and ultimately, the programmed cell death known as apoptosis.^34,35) Bcl-2 and Bax are both located upstream of the mitochondria in the apoptotic pathway. Bcl-2 has an anti-apoptotic function and can inhibit the depolarization of mitochondria and the production of ROS.³⁶⁾ On the other hand, Bax has a pro-apoptotic function and can promote the depolarization of mitochondria and the production of ROS.^37,38) Furthermore, excessive production of ROS can trigger the dissociation of the Nrf2/Keap1 complex, leading to the phosphorylation and subsequent translocation of Nrf2 into the nucleus.³⁹⁾ When Nrf2 translocates into the nucleus, it binds to the antioxidant response element (ARE) and activates the transcription of various antioxidant enzymes, including heme oxygenase-1 (HO-1) and superoxide dismutase (SOD). This results in an increase in the cellular antioxidant defense system, which helps to neutralize the excess ROS and prevent oxidative damage.⁴⁰⁾ The findings are in agreement with previous studies, which demonstrate that ethanol exposure leads to an elevation in intracellular ROS levels, mitochondrial depolarization, and upregulation of cleaved-caspase-3 and Bax expression, while downregulating the expression of Nrf2 and Bcl-2. However, the results indicate that treatment with EGCG can mitigate the detrimental effects of ethanol by reducing the expression of Bax and cleaved-caspase-3, preventing mitochondrial depolarization, and increasing the expression of Nrf2 and Bcl-2.

NF-κB is known to activate cell injury processes. In its inactive state, NF-κB is bound to inhibitor of kappaB (IκB) in the cytosol. However, when stimulated by ethanol, NF-κB p65 becomes phosphorylated on serine-536, allowing it to become active. Once active, NF-κB can enter the nucleus and regulate the transcription of various pro-inflammatory genes. Thus, in turn, leads to the secretion of pro-inflammatory cytokines, which further amplifies the activation of NF-κB.⁴¹⁾ The study found that the exposure to ethanol resulted in a significant increase in the translocation of NF-κB into the nucleus of HUVECs cells, and increase the levels of TNF-α, IL-1β, IL-6 in the culture medium. However, the presence of EGCG was observed to restrain the nuclear translocation of NF-κB, and decrease the secretion of TNF-α, IL-1β, IL-6 in the culture medium.

CONCLUSION

In summary, the results of this study suggest that EGCG has a protective effect against ethanol-induced endothelial injury. This effect is likely due to EGCG’s ability to improve mitochondrial function, activate the Nrf2 signaling pathway, decrease NF-κB nuclear translocation. The study has indicated that EGCG can mitigate the damage caused by ethanol to the endothelial cells by modifying the translocation of NF-κB and activating the Nrf2 signaling pathway.

Acknowledgments

This work was supported by several funding sources, including the National Natural Science Foundation of China (Grant Nos. 82100488, 82105016), Scientific Research Fund Project of Shaanxi Province Department of Education (Grant Nos. 21JK0597, 21JS012), Key Research and Development Program Project of Shaanxi Province (Grant Nos. 2024SF-YBXM-471, 2022SF-357).

Conflict of Interest

The authors declare no conflict of interest.

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