• Users Online: 191
  • Print this page
  • Email this page

 
Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 65  |  Issue : 5  |  Page : 217-225

Study on the role of naringin in attenuating Trimethylamine-N-Oxide-Induced human umbilical vein endothelial cell inflammation, oxidative stress, and endothelial dysfunction


The Second Department of Cardiology, Tianjin Hospital, Tianjin, China

Date of Submission27-Jun-2022
Date of Decision17-Aug-2022
Date of Acceptance06-Sep-2022
Date of Web Publication27-Oct-2022

Correspondence Address:
Dr. Hui Zhao
The Second Department of Cardiology, Tianjin Hospital, No. 406, Jiefang South Road, Hexi, Tianjin 300211
China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0304-4920.359796

Rights and Permissions
  Abstract 


Trimethylamine-N-oxide (TMAO), a phospholipid metabolite, can modulate cholesterol synthesis and promote vascular inflammation and endothelial dysfunction, thereby increasing the risk of atherosclerosis (AS). Previously, it was found that naringin reduced damage to human umbilical vein endothelial cells (HUVECs) triggered by oxidized low-density lipoprotein. This article continues to explore the role and mechanism of naringin in protecting HUVECs from TMAO-induced damage. After the construction of TMAO-induced AS model in HUVECs, inflammation, oxidative stress, and endothelial function were examined by real-time quantitative polymerase chain reaction, Western blotting, nitric oxide (NO), reactive oxygen species (ROS), superoxide dismutase, and malondialdehyde (MDA) kits. Results showed that naringin pretreatment inhibited endothelial inflammation and oxidative stress, promoted NO release, and inhibited the degradation of Zona occludens-2, occludin, and vascular endothelial-cadherin, thereby restoring the functional and structural integrity of the endothelium. Furthermore, the addition of mitogen-activated protein kinase (MAPK) agonist demonstrated that the therapeutic effect of naringin was achieved through inactivating TMAO-stimulated MAPK signaling in HUVECs.

Keywords: Endothelial dysfunction, inflammation, naringin, oxidative stress, trimethylamine-N-oxide


How to cite this article:
Zhao H, Zhao J. Study on the role of naringin in attenuating Trimethylamine-N-Oxide-Induced human umbilical vein endothelial cell inflammation, oxidative stress, and endothelial dysfunction. Chin J Physiol 2022;65:217-25

How to cite this URL:
Zhao H, Zhao J. Study on the role of naringin in attenuating Trimethylamine-N-Oxide-Induced human umbilical vein endothelial cell inflammation, oxidative stress, and endothelial dysfunction. Chin J Physiol [serial online] 2022 [cited 2022 Nov 26];65:217-25. Available from: https://www.cjphysiology.org/text.asp?2022/65/5/217/359796




  Introduction Top


Atherosclerosis (AS) is a condition in which plaques formed by the proliferation of endothelial cells in blood vessels collapse and combine with subendothelial lipid deposition to form atheromatous plaques, gradually causing blockage of the lumen of blood vessels, which can induce severe cardiovascular and cerebrovascular diseases.[1],[2],[3] A recent metabolomics study has identified plasma trimethylamine-N-oxide (TMAO), a choline metabolite, as a novel independent risk factor for AS.[4] TMAO is produced by gut microbiota by metabolizing dietary phosphatidylcholine, choline, and betaine.[5] TMAO promotes endothelial cell inflammation and endothelial dysfunction, and likewise promotes oxidative stress in endothelial cells.[6],[7] Inflammation as well as endothelial dysfunction is the most critical dependent variable in the development of AS.[8],[9] Therefore, effective inhibition of TMAO-induced inflammatory response, oxidative stress, and endothelial dysfunction has an important role in reducing AS.

Naringin is widely available and can be found in fruits and vegetables, such as grapefruit and tomatoes.[10] Naringin has been widely studied for its ability to inhibit the development of AS.[11] Naringin has been observed to have anti-inflammatory properties, inhibit vascular smooth muscle cell proliferation, and be able to ameliorate diet-induced hypercholesterolemic AS.[12],[13],[14] Previous results from our group showed that naringin could reduce oxidized low-density lipoprotein (ox-LDL)-induced endothelial cell inflammation and apoptosis by modulating the YAP pathway,[15] but its ability to attenuate TMAO-induced endothelial cell injury is not known.

Therefore, it was speculated that naringin also played a suppressive role in TMAO-evoked endothelial cell injury and damage. Accordingly, AS was stimulated in vitro by exposing human endothelial cells to TMAO. This work aimed to observe the suppressive effects of naringin on the inflammation, oxidative stress, and functional aspects of cells after TMAO injury, and explore the related mechanism, thus providing experimental support for naringin treatment of AS from different perspectives.


  Materials and Methods Top


Cell culture

Human umbilical vein endothelial cells (HUVECs), American Type Culture Collection were applied throughout the study, and cells were cultured in endothelial cell medium (ScienCell), maintained at 37°C in a 5% CO2 environment. Cells were induced with 300 μM TMAO (Sigma-Aldrich, Merck) for 6 h for model preparation;[6] cells in the naringin (MedChemExpress) group were administrated with 50 μM, 75 μM, and 100 μM naringin for 2 h prior to TMAO induction.[15] TMAO was dissolved in water and naringin was dissolved in dimethyl sulfoxide (DMSO). p38 mitogen-activated protein kinase (MAPK) agonist anisomycin (25 μg/ml, MedChemExpress) was used 30 min prior to naringin pretreatment.[16]

Cell counting kit-8 assay

Cells were treated with DMSO or 50 μM, 75 μM, and 100 μM naringin for 2 h and then cells were further cultured under normal culture conditions for 24 h. Then, 10 μl cell counting kit-8 (CCK-8) solution (Boster) was added into each well and incubated for 2 h. The absorbance was measured at 450 nm using a microplate reader (Bio-Rad).

Real-time quantitative polymerase chain reaction

Cells in each group were collected, mixed with TRIzol (Invitrogen, Thermo Fisher), and centrifuged at 12,000 rpm for 5 min to extract total RNA. Reverse transcription was completed using the First-Strand cDNA Synthesis Kit (Thermo Fisher) according to the manufacturer's instructions. The SYBR Green polymerase chain reaction (PCR) premix (Applied Biosystems; Thermo Fisher) was subsequently mixed with the cDNA obtained by reverse transcription and real-time fluorescent quantitative PCR was performed. The cycle number (Ct value) of target genes out of the peak was compared with the internal reference gene (GAPDH), and the 2−ΔΔCT value of each target gene was calculated.[17] The relative expression level of each target gene in each group was calculated using the blank group as the baseline. Primer information: interleukin-6 (IL-6), F 5'-TAGTGAGGAACAAGCCAGAG-3', R 5'-TACA TTTGCCGAAGAGCC-3'; IL-1β, F 5′-GCCCTAAAC AGATGAAGTGCTC-3′, 5′-GAACCAGCATCTTCC TCAG-3′; tumor necrosis factor-α (TNF-α), F 5'-CTGGCAAT GGCATGGAT-3', R 5'-GGTACAGCCCATCTGCTGGTA-3'; IL-18, F 5'-GCCAACGTCGAACCCAATTC-3', R 5'-CGGG ATAGCTGGTTACAAGTCACAC-3'; Zona occludens-1 (ZO-1), F 5'-GAGAAAGGTGAAACACTGCTGAG-3', R 5'-CGAGGAGTCGGATGATTTTAGAG-3'; occludin F 5'-GTGGAAAGAGTTGACAGT-3', R 5'-CAGCCAT GTACTCTTCACT-3'; VE-cadherin F 5'-CCACATTC AGGGAAATGCTT-3', R 5'-GAACATCTGCCCCTCTC AG-3'; β-actin F 5'-CGTGACATTAAGGAGAAGCTG-3', R 5'-CTAGAAGCATTTGCGGTGGAC-3'.

Western blotting assay

Cells were lysed with radioimmunoprecipitation assay lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor (both from Solarbio) and centrifuged at 4°C for 5 min at 12,000 rpm, and bicinchoninic acid method quantified the protein concentration in the supernatant. The protein was sampled at 30 μg per well and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Beyotime). The proteins were transferred to polyvinylidene fluoride membranes, and the membranes were sealed with 5% bovine serum albumin at room temperature for 1 h. The membranes were then incubated with the corresponding primary antibodies at 4°C overnight. Bands were incubated with HRP-labeled goat anti-rabbit secondary antibody (1:2000, Cell Signaling Technology, 7074S) for 1 h at room temperature and visualized in a gel imaging system (Bio-Rad) by dropwise addition of ultrasensitive ECL chemiluminescent substrate. Quantity One 1-D software (Bio-Rad, California, USA) analyzed the bands. The specific information of primary antibodies was: TNF-α (1:1000, Cell Signaling Technology, 6945), IL-6 (1:1000, Abcam, ab233706), IL-1β (1:1000, Cell Signaling Technology, 12703), IL-18 (1:1000, Abcam, ab243091), NOD-like receptor family pyrin domain containing 3 (NLRP3) (1:500, Cell Signaling Technology, 15101S), the apoptotic speck-like protein containing a caspase recruitment domain (ASC) (1:500, Cell Signaling Technology, 13833S), caspase-1 (1:600, Cell Signaling Technology, 3866S), superoxide dismutase 2 (SOD2) (1:600, Cell Signaling Technology, 13141S), endothelial nitric oxide synthase (eNOS) (1:600, Abcam, ab252439), p-p38 MAPK (1:500, Cell Signaling Technology, 9216S), phosphorylated-extracellular signal-regulated kinase (p-ERK) (1:1000, Cell Signaling Technology, 4370S), phospho of c-Jun N-terminal kinase (p-JNK) (1:1000, Abcam, ab124956), ERK (1:500, Abcam, ab17942), JNK (1:1000, Abcam, ab179461), p38 MAPK (1:600, Cell Signaling Technology, 8690S), GAPDH (1:1000, Abcam, ab9485), and β-actin (1:1000, Abcam, ab8227).

Detection of reactive oxygen species, superoxide dismutase, and malondialdehyde

The reactive oxygen species (ROS) levels of each group were detected by ROS Assay Kit (Beyotime). Detection of ROS was performed using the fluorescent probe DCFH-DA, which can be oxidized by ROS and emit fluorescence. Finally, the fluorescence of each group was observed under a fluorescence leading microscope (Nikon), and images were observed from five random fields of view. The SOD activity and malondialdehyde (MDA) content of each group of cells were measured by the xanthine oxidase method and thiobarbituric acid colorimetric method using Total SOD Assay Kit (Beyotime), Lipid Peroxidation MDA Assay Kit (Beyotime), and enzyme marker (Bio-Rad). The wavelengths were selected as 450 nm and 540 nm, respectively.

Nitric oxide detection

Using the NO content assay kit (Keygen Bio), the NO content in the cells was measured using the nitrate reduction method, and the absorbance measured at 550 nm was read by the enzyme marker, and the NO content was calculated according to the standard curve.

Statistical analysis

A minimum of three replicate wells were used for each group of cells, and each experiment was repeated a minimum of three times. GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA) was applied for statistical analysis of the obtained results. t-test was used for comparison of two separate groups, and one-way analysis of variance was used for comparison of multiple measures. The obtained data were expressed as mean ± standard deviation. P < 0.05 was statistically significant.


  Results Top


Naringin attenuated trimethylamine-N-oxide-induced inflammatory response and oxidative stress in human umbilical vein endothelial cells

To investigate whether DMSO and naringin affected the viability of HUVECs, CCK-8 assay was performed. The result of [Figure 1]a indicated that DMSO and naringin could not affect the viability of HUVECs. To estimate the impacts of naringin on HUVECs under treatment with TMAO, HUVECs were first exposed to TMAO and then inflammation and oxidative stress were measured. Compared to the control group, inflammatory response was significantly noticed in HUVECs upon TMAO exposure, as evidenced by a very exaggerated increase in the expression of the pro-inflammatory factors TNF-α, IL-6, IL-1β, and IL-18 [Figure 1]b and [Figure 1]c, and a significant increase in protein levels of the inflammasome NLRP3, ASC, and caspase-1 in cells following TMAO induction [Figure 1]d. After the administration of increasing concentrations of naringin, there was a concentration-dependently measurable reduction in the expression of pro-inflammatory factors as well as inflammasome-associated proteins.
Figure 1: Naringin attenuated TMAO-induced inflammatory response and oxidative stress in HUVECs. (a) The viability of HUVECs treated by only DMSO or naringin was detected by CCK-8 assay. (b) RT-qPCR and (c) Western blotting were performed to detect the expression levels of TNF-α, IL-6, IL-1β, and IL-18 in TMAO-induced HUVECs treated by naringin. (d) Western blotting was used to analyze the protein levels of NLPR3, ASC, and caspase-1 in TMAO-induced HUVECs treated by naringin. The levels of ROS (e) (×200), SOD (f), SOD2 (g), and MDA (h) were measured by ROS Assay Kit, Total SOD Assay Kit, Western blotting, and Lipid Peroxidation MDA Assay Kit, respectively, in TMAO-induced HUVECs treated by naringin. Naringin treatment for 2 h prior to TMAO induction. GAPDH is an internal control. *P < 0.05, **P < 0.01, ***P < 0.001. N = 3. TMAO: Trimethylamine-N-oxide, HUVEC: Human umbilical vein endothelial cell, CCK: Cell Counting Kit, RT-qPCR: Real-time quantitative polymerase chain reaction, TNF-α: Tumor necrosis factor-α, IL: interleukin, ROS: Reactive oxygen species, SOD: Superoxide dismutase, MDA: Malondialdehyde.

Click here to view


To evaluate oxidative stress, the activities of three indicators SOD, MDA, and ROS were measured. As shown in [Figure 1]e,[Figure 1]f,[Figure 1]g,[Figure 1]h, the cells in the TMAO group showed weaker SOD activity, lower SOD2 protein expression, increased contents of ROS, and MDA, which indicated that TMAO induced oxidative stress. Compared with the TMAO group, different doses of naringin inhibited TMAO-induced oxidative stress, and this effect was gradually prominent with increasing naringin concentrations. It could be seen that naringin was able to reduce TMAO-induced endothelial inflammatory response and oxidative stress in a concentration-dependent manner.

Naringin attenuated trimethylamine-N-oxide-induced human umbilical vein endothelial cell dysfunction

To further uncover the influence of naringin on TMAO-triggered HUVEC dysfunction, proteins associated with endothelial function and endothelial junction were measured. Endothelial cells depend on eNOS to synthesize and release NO, which dilates the blood vessels. The content of NO and the amount of eNOS in each group were measured. As shown in [Figure 2]a and [Figure 2]b, TMAO notably inhibited the expression of eNOS and inhibited the release of NO from HUVECs. Naringin significantly increased intracellular eNOS expression and increased NO release in HUVECs after TMAO injury.
Figure 2: Naringin attenuated TMAO-induced HUVEC dysfunction. (a) NO release in TMAO-induced HUVECs treated by naringin was assessed by NO content assay kit. (b) Western blotting was used to analyze the protein levels of eNOS in TMAO-induced HUVECs treated by naringin. (c) RT-qPCR was performed to detect the mRNA levels of ZO-2, occludin, and VE-cadherin in TMAO-induced HUVECs treated by naringin. Naringin treatment for 2 h prior to TMAO induction. **P < 0.01, ***P < 0.001. N = 3. TMAO: Trimethylamine-N-oxide, HUVEC: Human umbilical vein endothelial cell, eNOS: Endothelial nitric oxide synthase, RT-qPCR: Real-time quantitative polymerase chain reaction, ZO: Zona occludens, VE: Vascular endothelial.

Click here to view


Intercellular junctions play an important role in the maintenance of endothelial integrity and function.[18] Therefore, the mRNA levels of ZO-2, occludin, and VE-cadherin in endothelial cells were also examined using real-time quantitative PCR. As shown in [Figure 2]c, quantitative results concluded that naringin substantially increased the mRNA expression of ZO-2, occludin, and VE-cadherin in TMAO-induced HUVECs in a concentration-dependent manner. Thus, it was observed that naringin promoted NO release from endothelial cells, while it coordinated the maintenance of VE integrity.

Naringin suppressed trimethylamine-N-oxide-induced activation of mitogen-activated protein kinase signaling in human umbilical vein endothelial cells

For further investigation into the regulatory mechanism of naringin in protecting against TMAO-evoked HUVEC injury, the involvement of MAPK signaling was explored. The main reason for the selection of MAPK signaling as the focus of this in-depth study is its important role in the development of cardiovascular diseases.[19] Western blotting was to detect MAPK signaling-associated proteins JNK, p38 MAPK, and ERK and their phosphorylation in each group of cells [Figure 3]. There was no apparent difference in the expression of JNK, p38 MAPK, and ERK among the groups, but the expression levels of their corresponding phosphorylated proteins remarkably differed among the groups. TMAO induced a marked increase in the phosphorylation of JNK, p38 MAPK, and ERK in HUVECs compared with the control group, while the expression of p-JNK, p-p38 MAPK, and p-ERK in the naringin group were decreased with the increase of drug concentration, especially after the addition of 100 μM naringin. This confirmed that naringin suppressed TMAO-activated MAPK signaling in HUVECs.
Figure 3: Naringin suppressed TMAO-induced activation of MAPK signaling in HUVECs. Western blotting was used to analyze the expression levels of JNK, p-JNK, p38 MAPK, p-p38 MAPK, ERK, and p-ERK in TMAO-induced HUVECs treated by naringin. Naringin treatment for 2 h prior to TMAO induction. GAPDH is an internal control. ***P < 0.001. N =3. TMAO: Trimethylamine-N-oxide, MAPK: Mitogen-activated protein kinase, HUVEC: Human umbilical vein endothelial cell, p-JNK: phospho of c-Jun N-terminal kinase, p-ERK: Phosphorylated-extracellular signal-regulated kinase.

Click here to view


Naringin protected human umbilical vein endothelial cells from trimethylamine-N-oxide-triggered impairment by inhibiting mitogen-activated protein kinase signaling

To verify whether naringin exerted its protective effect on TMAO-induced HUVECs via inhibition of MAPK signaling pathway, 100 μM naringin and MAPK agonist anisomycin were used in the following experiments. As expected, anisomycin apparently restored naringin-inhibited expression of inflammatory factors TNF-α, IL-6, IL-1β, and IL-18 [Figure 4]a and [Figure 4]b. Furthermore, the downregulated intracellular levels of NLPR3, ASC, and caspase-1 imposed by naringin in TMAO-treated HUVECs were again significantly increased by anisomycin [Figure 4]c. To be concluded, MAPK agonist inhibited the anti-inflammatory effect of naringin on TMAO-insulted HUVECs.
Figure 4: Naringin attenuated TMAO-induced inflammatory response and oxidative stress in HUVECs via inhibition of MAPK signaling. (a) RT-qPCR and (b) Western blotting were performed to detect the expression levels of TNF-α, IL-6, IL-1β, and IL-18 in TMAO-induced HUVECs treated by naringin and anisomycin. (c) Western blotting was used to analyze the protein levels of NLPR3, ASC, and caspase-1 in TMAO-induced HUVECs treated by naringin and anisomycin. The levels of ROS (d) (×200), SOD (e), SOD2 (f), and MDA (g) were measured by ROS Assay Kit, Total SOD Assay Kit, Western blotting, and Lipid Peroxidation MDA Assay Kit, respectively, in TMAO-induced HUVECs treated by naringin and anisomycin. Naringin treatment for 2 h prior to TMAO induction. Anisomycin treatment for 30 min prior to naringin pretreatment. GAPDH is an internal control. *P < 0.05, **P < 0.01, ***P < 0.001. N =3. TMAO: Trimethylamine-N-oxide, HUVEC: Human umbilical vein endothelial cell, MAPK: Mitogen-activated protein kinase, RT-qPCR: Real-time quantitative polymerase chain reaction, TNF-α: Tumor necrosis factor-α, IL: interleukin, ROS: Reactive oxygen species, SOD: Superoxide dismutase, MDA: Malondialdehyde.

Click here to view


The similar results were obtained in the detection of cellular oxidative stress indicators. Anisomycin addition increased the contents of ROS, MDA, and inhibited SOD activity, SOD2 expression in naringin-protected endothelial cells following TMAO injury. Likewise, anisomycin reversed the anti-oxidative stress activities of naringin [Figure 4]d,[Figure 4]e,[Figure 4]f,[Figure 4]g. The supplementation of anisomycin led to a decrease in eNOS expression as well as NO release and significantly lower mRNA levels of ZO-2, occludin, and VE-cadherin in naringin-treated HUVECs under the condition of TMAO injury [Figure 5]a,[Figure 5]b,[Figure 5]c. Taken together, it could be concluded that the protective effect exerted by naringin against TMAO-evoked HUVEC damage was accomplished by inhibiting MAPK signaling.
Figure 5: Naringin attenuated TMAO-induced HUVEC dysfunction via inhibition of MAPK signaling. (a) NO release was assessed by NO content assay kit in TMAO-induced HUVECs treated by naringin and anisomycin. (b) Western blotting was used to analyze the protein levels of eNOS in TMAO-induced HUVECs treated by naringin and anisomycin. (c) RT-qPCR was performed to detect the mRNA levels of ZO-2, occludin, and VE-cadherin in TMAO-induced HUVECs treated by naringin and anisomycin. Anisomycin treatment for 30 min prior to naringin pretreatment. *P < 0.05, **P < 0.01, ***P < 0.001. N =3. TMAO: Trimethylamine-N-oxide, HUVEC: Human umbilical vein endothelial cell, MAPK: Mitogen-activated protein kinase, eNOS: Endothelial nitric oxide synthase, RT-qPCR: Real-time quantitative polymerase chain reaction, ZO: Zona occludens, VE: Vascular endothelial.

Click here to view



  Discussion Top


After dietary choline and L-carnitine reach the intestine, they are metabolized by the microbiota and produce an intermediate compound called trimethylamine, which is absorbed sequentially through the portal circulation to the liver, where it is oxidized by flavin-containing monooxygenase to TMAO.[20],[21] High expression of TMAO in the circulation may increase the risk of cardiovascular diseases, AS is included.[22],[23] It has been suggested that TMAO accelerates the course of AS by promoting foam cell formation.[22],[24] TMAO can promote the expression of inflammatory cytokines, enhance platelet hyperreactivity, and induce endothelial cell dysfunction, and so on.[6],[25] These findings indicate that TMAO is an essential factor affecting cardiovascular diseases. Thus, finding new methods to target TMAO is of great importance for AS therapy. We have previously investigated the role of naringin in inhibiting ox-LDL-induced inflammation and oxidative stress, reducing endothelial damage, and thus alleviating AS. In this article, we again elucidated the function of naringin on the treatment of AS in terms of its ability to protect endothelial cells from TMAO-induced injury, thereby providing new insights into naringin for the treatment of cardiovascular diseases.

Chronic inflammation of blood vessels is a typical feature of AS.[26] TMAO can elicit the release of the inflammatory cytokines IL-6 and TNF-α to promote the inflammatory process where macrophages may migrate to and accumulate in blood vessels, so as to form foam cells that eventually induce AS.[27] The surge in TMAO levels in vivo activates Nuclear factor kappa B (NF-κB) signaling as well as MAPK signaling, stimulating the expression of inflammatory factors.[28] NLRP3 inflammasome is a complex of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1. The activation of NLRP3 inflammasome induces pyroptosis and increases the levels of IL-18 and IL-1β, further enhancing the inflammatory response;[29],[30] it also stimulates macrophages and thus regulates the formation of atheromatous plaques.[31] It has been shown that TMAO activates the NLRP3 inflammasome to initiate inflammatory response and trigger endothelial damage.[7] We also observed that TMAO promoted strong endothelial inflammation and naringin treatment alleviated endothelial inflammation by reducing the expression of pro-inflammatory factors and NLRP3 inflammasome in a concentration-dependent way [Figure 1]a,[Figure 1]b,[Figure 1]c.

The imbalance between ROS production and clearance leads to oxidative stress in the body. Oxidative stress has been shown to cause abnormalities in cardiovascular structure and function.[32] It has been reported that TMAO drives the release of large amounts of ROS from endothelial cells, stimulates thioredoxin-reducing protein-NLRP3 signaling, activates the inflammasome, and mediates more severe inflammation and oxidative stress.[6] We also obtained consistent results with the generation of oxidative stress in endothelial cells under TMAO induction; naringin had a pronounced ability to counteract the oxidative stress generated by TMAO in endothelial cells, as evidenced by the finding that naringin significantly upregulated SOD activity in a concentration-dependent manner and decreased ROS and MDA levels [Figure 1]d,[Figure 1]e,[Figure 1]f,[Figure 1]g.

VE cells maintain the stability of the intravascular environment though synthesizing eNOS and releasing NO, thereby inhibiting platelet adhesion and aggregation in blood vessels.[33] In this report, TMAO reduced eNOS production and suppressed NO release [Figure 2]a and [Figure 2]b, which was similar to previous studies. Meanwhile, we examined the tight junction proteins ZO-2, occludin, and VE-cadherin. ZO binds to tight junction proteins such as occludin, which bridges it to transmembrane proteins and maintains cytoskeletal stability.[34] Reduced expression of ZO and occludin increases vascular permeability.[35] Inhibition of VE-cadherin diminishes intercellular adhesion, thereby reducing endothelial barrier function.[18] We noticed a remarkable decline in the expression of ZO-2, occludin, and VE-cadherin in HUVECs exposed to TMAO [Figure 2]c, suggesting that TMAO contributed to the destruction and degradation of connexins, increased the permeability of the vascular wall, disrupted the vascular wall barrier, and promoted the development of cardiovascular diseases. However, the upregulation on connexin expression was observed after the addition of naringin treatment. In conclusion, naringin could protect endothelial cells from TMAO-triggered injury and partially restore endothelial function.

MAPK signaling is closely associated with cardiovascular diseases. Activated MAPK signaling, which induces apoptosis in HUVECs, induces the secretion of large amounts of inflammatory factors, induces oxidative stress, stimulates endothelial disorders, and contributes to neutrophil activation and inflammatory cell aggregation.[36],[37] An investigation has indicated that TMAO induces foam cell formation and promotes AS in apolipoprotein E-deficient mice via activation of the CD36/MAPK/JNK pathway.[38] Inhibition of the MAPK/NF-κB pathway could inhibit AS by suppressing the inflammation.[39],[40] Ginsenoside F1 suppressed inflammation in endothelial cells and inhibited AS by inactivating NF-κB Signaling.[41] We found that naringin also inhibited the inflammation and oxidative stress in TMAO-induced HUVECs via inhibition of MAPK signaling pathway [Figure 3]. In addition, the anti-oxidative stress and anti-apoptotic effects of naringin were similarly inhibited when MAPK agonist was used [Figure 4] and [Figure 5]. The augmented endothelial injury by MAPK agonist also suggested that TMAO positively induced vascular injury by activating MAPK, whereas naringin protected endothelial cells from TMAO injury by inhibiting MAPK in vitro. In addition, NF-κB signaling pathway is also an important inflammatory regulator in the development of AS, which may imply that naringin regulates NF-κB signaling to mediate the inflammatory response, which will be explored in our future study.


  Conclusion Top


Altogether, we present the first report that naringin attenuates inflammation, oxidative stress, and endothelial dysfunction caused by TMAO, which may be at least dependent on the inhibition of MAPK signaling. Compared with the existing studies, this article initially introduces the protective role of naringin in TMAO-elicited endothelial injury and dysfunction and clarifies the underlying regulatory mechanism. Meanwhile, we will continue to conduct animal studies to fully demonstrate the effects of naringin. This brings new insights into the treatment of TMAO-involved diseases and provides experimental support for naringin treatment of AS.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Abd Alamir M, Goyfman M, Johnson D, Liu Y, Dabbous F, Chaus A, et al. The relationship between endothelial function and aortic valve calcification: Multi-Ethnic study of atherosclerosis. Atherosclerosis 2019;280:155-65.  Back to cited text no. 1
    
2.
Lacey M, Baribault C, Ehrlich KC, Ehrlich M. Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers. Atherosclerosis 2019;280:183-91.  Back to cited text no. 2
    
3.
Santos-Gallego CG, Picatoste B, Badimón JJ. Pathophysiology of acute coronary syndrome. Curr Atheroscler Rep 2014;16:401.  Back to cited text no. 3
    
4.
Tang WH, Hazen SL. The contributory role of gut microbiota in cardiovascular disease. J Clin Invest 2014;124:4204-11.  Back to cited text no. 4
    
5.
Jiang S, Shui Y, Cui Y, Tang C, Wang X, Qiu X, et al. Gut microbiota dependent trimethylamine N-oxide aggravates angiotensin II-induced hypertension. Redox Biol 2021;46:102115.  Back to cited text no. 5
    
6.
Sun X, Jiao X, Ma Y, Liu Y, Zhang L, He Y, et al. Trimethylamine N-oxide induces inflammation and endothelial dysfunction in human umbilical vein endothelial cells via activating ROS-TXNIP-NLRP3 inflammasome. Biochem Biophys Res Commun 2016;481:63-70.  Back to cited text no. 6
    
7.
Chen ML, Zhu XH, Ran L, Lang HD, Yi L, Mi MT. Trimethylamine-N-Oxide induces vascular inflammation by activating the NLRP3 inflammasome through the SIRT3-SOD2-mtROS signaling pathway. J Am Heart Assoc 2017;6:e006347.  Back to cited text no. 7
    
8.
Higashi Y, Noma K, Yoshizumi M, Kihara Y. Endothelial function and oxidative stress in cardiovascular diseases. Circ J 2009;73:411-8.  Back to cited text no. 8
    
9.
Tousoulis D, Andreou I, Antoniades C, Tentolouris C, Stefanadis C. Role of inflammation and oxidative stress in endothelial progenitor cell function and mobilization: Therapeutic implications for cardiovascular diseases. Atherosclerosis 2008;201:236-47.  Back to cited text no. 9
    
10.
Chen R, Qi QL, Wang MT, Li QY. Therapeutic potential of naringin: An overview. Pharm Biol 2016;54:3203-10.  Back to cited text no. 10
    
11.
Heidary Moghaddam R, Samimi Z, Moradi SZ, Little PJ, Xu S, Farzaei MH. Naringenin and naringin in cardiovascular disease prevention: A preclinical review. Eur J Pharmacol 2020;887:173535.  Back to cited text no. 11
    
12.
Chao CL, Weng CS, Chang NC, Lin JS, Kao ST, Ho FM. Naringenin more effectively inhibits inducible nitric oxide synthase and cyclooxygenase-2 expression in macrophages than in microglia. Nutr Res 2010;30:858-64.  Back to cited text no. 12
    
13.
Lee EJ, Moon GS, Choi WS, Kim WJ, Moon SK. Naringin-induced p21WAF1-mediated G(1)-phase cell cycle arrest via activation of the Ras/Raf/ERK signaling pathway in vascular smooth muscle cells. Food Chem Toxicol 2008;46:3800-7.  Back to cited text no. 13
    
14.
Lee CH, Jeong TS, Choi YK, Hyun BH, Oh GT, Kim EH, et al. Anti-atherogenic effect of citrus flavonoids, naringin and naringenin, associated with hepatic ACAT and aortic VCAM-1 and MCP-1 in high cholesterol-fed rabbits. Biochem Biophys Res Commun 2001;284:681-8.  Back to cited text no. 14
    
15.
Zhao H, Liu M, Liu H, Suo R, Lu C. Naringin protects endothelial cells from apoptosis and inflammation by regulating the Hippo-YAP Pathway. Biosci Rep 2020;40:BSR20193431.  Back to cited text no. 15
    
16.
Huang Y, Li S, Chen H, Feng L, Yuan W, Han T. Butorphanol reduces the neuronal inflammatory response and apoptosis via inhibition of p38/JNK/ATF2/p53 signaling. Exp Ther Med 2022;23:229.  Back to cited text no. 16
    
17.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-8.  Back to cited text no. 17
    
18.
Carmeliet P, Lampugnani MG, Moons L, Breviario F, Compernolle V, Bono F, et al. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 1999;98:147-57.  Back to cited text no. 18
    
19.
Muslin AJ. MAPK signalling in cardiovascular health and disease: Molecular mechanisms and therapeutic targets. Clin Sci (Lond) 2008;115:203-18.  Back to cited text no. 19
    
20.
Wilson A, McLean C, Kim RB. Trimethylamine-N-oxide: A link between the gut microbiome, bile acid metabolism, and atherosclerosis. Curr Opin Lipidol 2016;27:148-54.  Back to cited text no. 20
    
21.
Spector R. New insight into the dietary cause of atherosclerosis: Implications for pharmacology. J Pharmacol Exp Ther 2016;358:103-8.  Back to cited text no. 21
    
22.
Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 2011;472:57-63.  Back to cited text no. 22
    
23.
Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 2013;368:1575-84.  Back to cited text no. 23
    
24.
Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med 2013;19:576-85.  Back to cited text no. 24
    
25.
Zhu W, Gregory JC, Org E, Buffa JA, Gupta N, Wang Z, et al. Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk. Cell 2016;165:111-24.  Back to cited text no. 25
    
26.
Ross R. The pathogenesis of atherosclerosis – An update. N Engl J Med 1986;314:488-500.  Back to cited text no. 26
    
27.
Yu ST, Sun BH, Ge JN, Shi JL, Zhu MS, Wei ZG, et al. CRLF1-MYH9 interaction regulates proliferation and metastasis of papillary thyroid carcinoma through the ERK/ETV4 axis. Front Endocrinol (Lausanne) 2020;11:535.  Back to cited text no. 27
    
28.
Seldin MM, Meng Y, Qi H, Zhu W, Wang Z, Hazen SL, et al. Trimethylamine N-Oxide promotes vascular inflammation through signaling of mitogen-activated protein kinase and nuclear factor-κB. J Am Heart Assoc 2016;5:e002767.  Back to cited text no. 28
    
29.
Janoudi A, Shamoun FE, Kalavakunta JK, Abela GS. Cholesterol crystal induced arterial inflammation and destabilization of atherosclerotic plaque. Eur Heart J 2016;37:1959-67.  Back to cited text no. 29
    
30.
Dai W, Wang X, Teng H, Li C, Wang B, Wang J. Celastrol inhibits microglial pyroptosis and attenuates inflammatory reaction in acute spinal cord injury rats. Int Immunopharmacol 2019;66:215-23.  Back to cited text no. 30
    
31.
Chen L, Yao Q, Xu S, Wang H, Qu P. Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux. Biochem Biophys Res Commun 2018;495:382-7.  Back to cited text no. 31
    
32.
Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 2005;25:29-38.  Back to cited text no. 32
    
33.
Hong FF, Liang XY, Liu W, Lv S, He SJ, Kuang HB, et al. Roles of eNOS in atherosclerosis treatment. Inflamm Res 2019;68:429-41.  Back to cited text no. 33
    
34.
Buckley A, Turner JR. Cell biology of tight junction barrier regulation and mucosal disease. Cold Spring Harb Perspect Biol 2018;10:a029314.  Back to cited text no. 34
    
35.
Hashimoto Y, Campbell M. Tight junction modulation at the blood-brain barrier: Current and future perspectives. Biochim Biophys Acta Biomembr 2020;1862:183298.  Back to cited text no. 35
    
36.
Wan Q, Liu Z, Yang Y, Cui X. Suppressive effects of berberine on atherosclerosis via downregulating visfatin expression and attenuating visfatin-induced endothelial dysfunction. Int J Mol Med 2018;41:1939-48.  Back to cited text no. 36
    
37.
Choi H, Nguyen HN, Lamb FS. Inhibition of endocytosis exacerbates TNF-α-induced endothelial dysfunction via enhanced JNK and p38 activation. Am J Physiol Heart Circ Physiol 2014;306:H1154-63.  Back to cited text no. 37
    
38.
Geng J, Yang C, Wang B, Zhang X, Hu T, Gu Y, et al. Trimethylamine N-oxide promotes atherosclerosis via CD36-dependent MAPK/JNK pathway. Biomed Pharmacother 2018;97:941-7.  Back to cited text no. 38
    
39.
Du H, Zhang H, Yang R, Qiao L, Shao H, Zhang X. Small interfering RNA-induced silencing lncRNA PVT1 inhibits atherosclerosis via inactivating the MAPK/NF-κB pathway. Aging (Albany NY) 2021;13:24449-63.  Back to cited text no. 39
    
40.
Li R, Zhou Y, Liu W, Li Y, Qin Y, Yu L, et al. Rare earth element lanthanum protects against atherosclerosis induced by high-fat diet via down-regulating MAPK and NF-κB pathways. Ecotoxicol Environ Saf 2021;207:111195.  Back to cited text no. 40
    
41.
Qin M, Luo Y, Lu S, Sun J, Yang K, Sun G, et al. Ginsenoside F1 ameliorates endothelial cell inflammatory injury and prevents atherosclerosis in mice through A20-mediated suppression of NF-kB signaling. Front Pharmacol 2017;8:953.  Back to cited text no. 41
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Figures

 Article Access Statistics
    Viewed952    
    Printed6    
    Emailed0    
    PDF Downloaded147    
    Comments [Add]    

Recommend this journal