Protective effect of ferulic acid on human umbilical vein endothelial cell model of cold stress
Si Huang1, Shengsuo Ma2, Di Zhang2, Bing Yang2, Jianxin Sun2, Meina Huang2, Haoming Lin2, Meijing Xie2, Guoping Zhao2
1 College of Traditional Chinese Medicine, Jinan University, Guangzhou; Nanhai Hospital of Traditional Chinese Medicine, Foshan, Guangdong, China
2 College of Traditional Chinese Medicine, Jinan University, Guangzhou, China
|Date of Submission||22-Dec-2018|
|Date of Decision||18-Jan-2019|
|Date of Web Publication||11-Feb-2020|
527 Room, College of Traditional Chinese Medicine, Jinan University, Guangzhou, Guangdong 510630
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Ferulic acid (FA) is an active principle derived from the traditional Chinese medicine Angelica sinensis, which has been used for the treatment of cardiovascular and cerebrovascular diseases in China for many years. However, a thorough understanding of effects on vascular function by FA has not been investigated. Aims: The aim of the present study was to investigate the potential mechanism of FA by suppressing Transient receptor potential cation channel subfamily M member 8 (TRPM8) channels and regulating endothelial nitric oxide (NO) pathway to ameliorate cold explore injury in human umbilical vascular endothelial cells (HUVECs). Subjects and Methods: The effects of cold exposure and FA on cell viability were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and lactate dehydrogenase (LDH) assay. Quantitative polymerase chain reaction and Western blot were utilized to detect TRPM8, hypoxia-inducible factor-alpha (HIF-1α), endothelin-1 (ET-1), inducible NO synthase (iNOS), endothelial NO synthase (eNOS) messenger RNA, and protein expression in HUVECs. Enzyme-linked immunosorbent assay method was used to detect the concentration of ET-1 in culture supernatants of HUVECs. Results: Cold exposure at 18°C had no significant effect on cell morphology but increased secretion of LDH and ET-1 and the expression of TRPM8, HIF-1α, iNOS, and ET-1. Treatment with FA decreased all of these changes. The levels of NO and eNOS decreased in cold stress model, while FA treatment attenuated the cold-induced decrease of NO and eNOS. Conclusion: Cold stress can cause an increase in vasoconstrictors such as TRPM8 and ET-1 and reducing cell viability, but FA can prevent cold stress-related cardiovascular disease by regulating the expression of these substances in cells.
Keywords: Cold stress, endothelin-1, ferulic acid, transient receptor potential ion channel subfamily M member 8, vascular endothelial cell
|How to cite this article:|
Huang S, Ma S, Zhang D, Yang B, Sun J, Huang M, Lin H, Xie M, Zhao G. Protective effect of ferulic acid on human umbilical vein endothelial cell model of cold stress. Phcog Mag 2020;16:7-12
|How to cite this URL:|
Huang S, Ma S, Zhang D, Yang B, Sun J, Huang M, Lin H, Xie M, Zhao G. Protective effect of ferulic acid on human umbilical vein endothelial cell model of cold stress. Phcog Mag [serial online] 2020 [cited 2021 Jul 25];16:7-12. Available from: http://www.phcog.com/text.asp?2020/16/67/7/278019
- Exposure of human umbilical vascular endothelial cells (HUVECs) to a temperature of 18°C increased transient receptor potential ion channel subfamily M member 8 (TRPM8) messenger RNA and disrupts the balance of the endothelin-nitric oxide (ET-NO) system
- FA treatment restores balance in the ET-NO system by increasing endothelial NO synthase expression and decreasing inducible NO synthase and ET-1 expression in VECs, resulting in vasodilation
- FA tends to restore TRPM8 expression to normal levels after low-temperature exposure
- FA might regulate the endothelium-mediated vasoconstriction and vasodilatation in part through the TRPM8/hypoxia-inducible factor-alpha/ET-1 signaling pathway.
Abbreviations used: FA: Ferulic acid; TRPM8: Transient receptor potential ion channel subfamily M member 8; HVECs: Human umbilical vein endothelial cells; ET-1: Endothelin-1; HIF-1α: Hypoxia-inducible factor-1α; eNOS: Endothelial nitric oxide synthase; iNOS: Inducible nitric oxide synthase; NO: Nitric oxide; CHD: Coronary heart disease; LDH: Lactate dehydrogenase.
| Introduction|| |
Cold temperatures are closely linked to the morbidity and mortality of many types of diseases. Numerous studies have suggested that the mortality rate is much higher in winter than in other seasons and that seasonal mortality increases are greater among individuals with prevalent ischemic heart disease.,,,,,, A worldwide population analysis shows that rates of coronary events increased during comparatively cold periods. This is generally believed to be due to hypothermia and low-temperature activation of the body's, leading to severe vasoconstriction, resulting in tissue ischemia and metabolic disorders., Endothelium injury or dysfunction may be a critical and initiating factor in this process by control of vasomotor tone by the release of vasoactive substances such as endothelin-1 (ET-1), hypoxia-inducible factor-alpha (HIF-1α), and NO. Recent studies show that transient receptor potential ion channel subfamily M member 8 is also widely expressed in the endothelium and vascular smooth muscles and on activation causes vasoconstriction or vasodilatation., As the inner cellular lining of blood vessels, the vascular endothelium which regulates vascular tone, proliferation, and permeability of inflammatory inducers or infiltration of leukocytes is emphasized as the central spot of endothelium dysfunction. Because of the similarity between human umbilical vein endothelial cells (HUVECs) and arterial endothelial cells, HUVEC is a common material to construct endothelial injury model., Ferulic acid (FA) is a phenolic acid widely found in natural plants. It has many pharmacological effects such as inhibiting platelet aggregation, releasing serotonin, scavenging free radicals,, fighting infection, and regulating immunity. In China, FA is usually used in cold-related diseases such as coronary heart disease (CHD), Raynaud's syndrome, and vasculitis. Evidence suggests FA and its derivatives exert multiple effects on the formation of CHD, including the inhibition of coronary atherosclerosis, improvement vascular function, and protective effect of the myocardium.,,, In the past research, we have found that FA and other compositions in DangGui SiNi Decoction play a protective role in ischemia/reperfusion heart through regulating the endothelial nitric oxide synthase (eNOS)/NO signal pathway by increasing the PeNOS protein expression and decreasing the expression of inducible NO synthase (iNOS) protein, and FA also can regulate cold-induced vasospasm by inhibiting the expression of transient receptor potential ion channel subfamily M member 8 (TRPM8)., The goals of the study described here were as follows: (1) to explore the hypothesis that cold-mediated activation of the TRPM8 channel is responsible for an imbalance in the ET-NO system and (2) to determine the role of the TRPM8 channel in the molecular mechanisms of FA-mediated cardioprotection.
| Subjects and Methods|| |
Cell viability assay
HUVECs were cultured in 96-well tissue culture plates (5 × 103cells/well) with 10% fetal bovine serum for 24 h and exposed to FA at different temperatures (37 or 18°C) for 5 h. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method according to the manufacturer's instructions (SIGMA Company, Continental, USA).
Determination of lactate dehydrogenase activity and NO and endothelin-1 expression
After exposure to FA at different temperatures (37°C or 18°C), the medium in individual wells was collected and centrifuged to remove dead cells. Lactate dehydrogenase (LDH) activity in the medium was measured using an LDH assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. The NO and ET-1 levels in the supernatant were measured using an NO assay kit (Nanjing Jiancheng Bioengineering Institute, China) and an ET-1 enzyme-linked immunosorbent assay kit (Bioleaf Company, China), respectively, according to the manufacturer's instructions.
RNA isolation, reverse transcription, and real-time polymerase chain reaction analysis
Total RNA was extracted from the cells using the Trizol reagent (Takara, Japan) according to the manufacturer's instructions. Isolated RNA was reverse transcribed into complementary DNA (cDNA) using a high-capacity cDNA reverse transcription kit (Takara, Japan) according to the manufacturer's instructions and incubated in a gradient polymerase chain reaction (PCR) thermal cycler (Bio-Rad, USA). Gene expression of relative messenger RNA (mRNA) was monitored by quantitative real-time PCR using PrimeScript RT Master Mix (Takara, Japan) and a PCR instrument for fluorescence detection (Bio-Rad, USA). The amplification cycles consisted of denaturation at 95°C for 2 min, 40 cycles of denaturation at 95°C for 5 s, annealing at 58°C for 30 s (eNOS/ET-1 is annealing at 60°C for 30 s). The sequences of the specific primers were as follows: TRPM8 (sense: 5'-CAGCACTGGCACCTGAAAAC-3' and antisense: 5'-GGACTGCGCGATGTAGATGA-3'), iNOS (sense: 5'-GAGCATCACCCCCGTGTTTCA-3' and antisense: 5'-TCTTGGGTCTCCGCTTCTCGTC-3'), eNOS (sense: 5'-CGAGTGAAGGCGACAATCCT-3' and antisense: 5'-CGAGGGACACCACGTCATAC-3'), ET-1 (sense: 5'-GAAACCCACTCCCAGTCCAC-3' and antisense: 5'-TCTTGGACCTAGGGCTTCCA-3'), and β-actin (sense: 5'-GGGAAATCGTGCGTGACATTAAGG-3' and antisense: 5'-CAGGAAGGAAGGCTGGAAGAGTG-3').
Western blot analysis
After exposure to different stimulation conditions for 5 h, the cells were laced with an equal volume of radioimmunoprecipitation assay buffer. The protein concentration was determined using the bicinchoninic acid assay. Total cellular protein (30 μg) was subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with Tris-buffered saline with Tween-20 containing 5% nonfat milk for 1 h, the membrane was probed with primary antibodies against TRPM8 (1:1000; Abcam, #ab104569), HIF-1α (1:1000; Cell Signaling, #3716), iNOS (1:500; Novus Company, #NBP2-22119), eNOS (1:500; Cell Signaling, #5880S), ET-1 (1:1000; Novus Company, #NB300-526), and β-actin (1:200; Boster Company, #BM0627) overnight at 4°C. After incubation with the appropriate secondary antibody for 1 h, BeyoECL Plus (Beyotime Biotechnology) was used for detection.
All data are shown as the means ± standard deviations. Data analyses were finished with SPSS version 19.0 software (IBM, USA). Measurement data in multiple groups were compared with two-way ANOVA followed by the Student–Newman–Keuls or Tamhane's post hoc test to assess the significance of differences between groups. P <0.05 was considered statistically significant.
| Results|| |
The temperature-mediated effects on cellular morphology are presented in [Figure 1]. HUVECs in the blank control group maintained a paved stone-like structure with closely packed cells and serrated edges that fit next to each other. Similar cell morphology was observed in the model-alone group cultured at 18°C.
The effects of different temperatures and drug on LDH activity in the growth medium are presented in [Figure 2]. Compared to the blank control group, the model-alone groups exhibited significant increases in LDH activity (P < 0.05). Lower temperatures produced greater increases in LDH activity. Compared to the model-alone group at 18°C, the medium- and high-dose FA groups exhibited significant decreases in LDH activity (P < 0.05).
|Figure 2: Influence of 18°C, ferulic acid, and transient receptor potential ion channel subfamily M member 8 to lactate dehydrogenase activity in the medium; data were shown as means ± standard deviations (n = 4). Δ:P < 0.05 versus untreated control, #:P < 0.05 versus 18°C group|
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The effects of different temperatures and drug treatment on cell viability are presented in [Figure 3]. No differences in cell viability were observed in any experimental group cultured at 18°C.
|Figure 3: Influence of 18°C, ferulic acid, and transient receptor potential ion channel subfamily M member 8 to cell viability in human umbilical vascular endothelial cells; data were shown as means ± standard deviation s (n = 5). Δ:P < 0.05 versus untreated control, #:P < 0.05 versus 18°C group|
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The effects of different temperatures and drug treatments on ET-1 and NO secretion levels are presented in [Figure 4]. Compared to the blank control group, the ET-1 secretion of the model-alone group at 18°C was significantly increased (P < 0.05). Compared to the model-alone group at 18°C, the ET-1 and NO secretion levels of the FA were significantly decreased (P < 0.05). Compared to the blank control group, the NO secretion of the model-alone group at 18°C was significantly decreased (P < 0.05). Compared to the model-alone group at 18°C, the high-dose FA groups exhibited significantly increased NO secretion (P < 0.05).
|Figure 4: Influence of 18°C and ferulic acid and transient receptor potential ion channel subfamily M member 8 to endothelin-1 and nitric oxide secretions in the medium. Data were shown as means ± standard deviations. Δ:P < 0.05 versus untreated control, #:P < 0.05 versus 18°C group, *:P < 0.05 versus 18°C + WS-12 20-μM group. (a) Nitric oxide secretions of different groups (n = 3); (b) Endothelin-1 secretions of different groups (n = 4)|
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The effects of different temperatures and FA treatment on the gene and protein expression levels of TRPM8 are presented in [Figure 5]. Compared to the blank control group, the model-alone group at 18°C exhibited significantly increased TRPM8 expression (P < 0.05). Compared to the model-alone group at 18°C, TRPM8 expression in the medium- and high-dose FA groups was significantly decreased (P < 0.05).
|Figure 5: Influence of 18°C, ferulic acid to transient receptor potential ion channel subfamily M member 8 expressions. Data were shown as means ± standard deviations (n = 3). Δ:P < 0.05 versus untreated control. (a) Transient receptor potential ion channel subfamily M member 8 mRNA expressions were determined by quantitative polymerase chain reaction. (b) Transient receptor potential ion channel subfamily M member 8 protein expressions were determined by Western blot|
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The effects of different temperatures and drug treatments on the expression levels of genes and proteins related to endothelial function are illustrated in [Figure 6] and [Figure 7]. Compared to the blank control group, the expression of iNOS, ET-1, and HIF-1α in the model-alone group at 18°C was significantly increased (P < 0.05), whereas the expression of eNOS in the model-alone group at 18°C was significantly decreased (P < 0.05). Compared to the model-alone group at 18°C, the expression levels of HIF-1α and ET-1 in the FA groups were significantly decreased (P < 0.05).
|Figure 6: Influence of 18°C and ferulic acid to hypoxia-inducible factor-alpha, inducible nitric oxide synthase, endothelial nitric oxide synthase, and endothelin-1 expressions. Data were shown as means ± standard deviations (n = 3). Δ:P < 0.05 versus untreated control, #:P < 0.05 versus 18°C group. (a) Inducible nitric oxide synthase, endothelial nitric oxide synthase, and endothelin-1 messenger RNA expressions were determined by quantitative polymerase chain reaction. (b) Hypoxia-inducible factor-alpha, inducible nitric oxide synthase, endothelial nitric oxide synthase, and endothelin-1 proteins expressions were determined by Western blot|
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|Figure 7: Influence of transient receptor potential ion channel subfamily M member 8 agonist WS-12 to hypoxia-inducible factor-alpha, endothelin-1, and inducible nitric oxide synthase, all proteins levels were determined by Western blot. Data were shown as means ± standard deviations (n = 3). Δ:P < 0.05 versus untreated control, #:P < 0.05 versus 18°C group|
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| Discussion|| |
Considerable interest has been raised in the causes and mechanisms of exacerbation of CHD because such exacerbations are important causes of morbidity and mortality in CHD patients. Low ambient temperatures might contribute to CHD exacerbations through the induction of vascular contraction, leading to coronary spasm stenosis and myocardial ischemia. These conditions may cause angina pectoris or even acute coronary syndrome. Low ambient temperatures are combined with an increased frequency of exacerbations in CHD patients.
TRPM8 channels are present in mammal VECs  and play an important role in the pathogenesis of numerous cold-related diseases. Low temperatures and chemical cooling agents increase bronchial epithelial cell TRPM8 expression which induces airway responsiveness. TRPM8 channels expressed in VECs may act as sensory receptors and participate in endothelium-mediated vasoconstriction induced by low ambient temperatures. Here, we have found that exposure of HUVECs to a temperature of 18°C increased TRPM8 mRNA and protein expression levels, and this increase was inhibited in a dose-dependent manner by treatment with FA.
VECs regulate vasoconstriction mainly through the ET-NO system which is also associated with inflammation, thrombosis, and vascular elasticity. ET-1 concentrations in the blood of rats increase during the first few days of cold exposure. Increased ET-1 expression plays an important role in cold-induced diseases. Excessive ET-1 secretion causes local disturbances of the microcirculation, ischemia, and hypoxia, leading to inflammation and cell necrosis. NO is generated by NOS in mammals. The major NOSs in VECs consist of iNOS and eNOS. NO, which is secreted by eNOS, can not only vasodilate vessels but also inhibit platelet aggregation, ET-1 synthesis, and thrombosis. iNOS, which is mildly expressed under normal physiological conditions, increases in expression following cellular injury. Excessive iNOS production leads to cytotoxicity, which induces inflammation, oxidative stress, and cell necrosis. Our research has shown that low ambient temperatures upregulate iNOS and ET-1 expression, downregulate eNOS expression, decrease NO secretion, increase ET-1 secretion, and lead to vasoconstriction. FA treatment restores balance in the ET-NO system by increasing eNOS expression and decreasing iNOS and ET-1 expression in VECs, resulting in vasodilation.
Low ambient temperatures disrupt the balance of the ET-NO system. We determined whether the TRPM8 channel is responsible for imbalances in the ET-NO system. Calmodulin-dependent kinase (CaMK), which is regulated by intracellular Ca 2+ concentrations, regulates the expression of many proteins, including iNOS. Reducing intracellular Ca 2+ concentrations inhibits the activity of CaMK and downregulates iNOS expression. WS-12, a derivative of menthol and a TRPM8-specific agonist, activated TRPM8 channels to induce Ca 2+ influx, leading to dowregulation of iNOS expression and secretion of NO. The increased iNOS expression induced in culture at 18°C was not mediated by TRPM8.
HIF-1α is the active subunit of HIF-1, which is a heterodimer consisting of HIF-1α and HIF-1β. Normally, HIF-1α would be degraded by ubiquitination as soon as translation is initiated. HIF-1 activity could be regulated by an O2-independent but Ca 2+-dependent mechanism involving HIF-1α interaction with a scaffolding protein, RACK1.,,,, Activated HIF-1 enters the nucleus to initiate mRNA transcription of downstream genes such as eNOS, iNOS, and ET-1. It has been shown that overexpressed TRPM8 suppresses RACK-1 dimerization, promoting activation of HIF-1 in prostate cancer cells. In sickle cell anemia model mice, cold allodynia was related to acute VASO occlusion and caused pain; besides, the transcription levels of pain genes such as TRPM8 and ET-1 were significantly increased. The expressions of HIF-1α in mammals were correlated with temperature, and it was found that HIF-1α involved in regulation of bitter pain sensitivity and continuous in mice by gene knockout technique. Our research showed simultaneous induction of TRPM8, HIF-1α, and ET-1 expression induced by culture at 18°C and FA treatment. We speculated that TRPM8 channels regulate cold-induced ET-1 expression through HIF-1α. FA might regulate the endothelium mediated vasoconstriction and vasodilatation in part through the TRPM8/HIF-1α/ET-1 signaling pathway, as shown in [Figure 8].
|Figure 8: Sketch map of synthesis of endothelin-1 mediated by transient receptor potential ion channel subfamily M member 8. Under the low temperature situation, activated TRPM8 channels induce Ca2+ influx which suppresses the expression of RACK-1 and promote activation of HIF-1. Activated HIF-1 enters the nucleus to initiate mRNA transcription of ET-1|
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Very low ambient temperatures disrupt cell membrane integrity and intracellular proteins, which significantly inhibit the normal cellular metabolism. The cell membrane has a large surface area and is highly water permeable. Low ambient temperatures lead to severe cellular dehydration and cell shrinkage. The increased concentration of intracellular electrolytes induced by dehydration damages cell membranes and intracellular proteins. The cell membrane transforms from liquid crystals to gel molecules as dehydration progresses. Direct structural changes to the cell membrane lead to increasing cell membrane permeability, loss control over exchange of internal and external material, and leakage of soluble substances into the cell, which cause cell damage. However, electrolyte concentrations deactivate some intracellular damage-activated proteins, especially key enzymes. The LDH activity in the medium could be used to estimate the membrane permeability, and our results showed that culture at 18°C damaged cell membranes. Further study has responsibility for determining whether the increase in NO secretion and inhibition of TRPM8 expressions related to membrane damage.
| Conclusion|| |
In summary, our study indicated that FA exerted protective effects on HUVECs model of cold stress through the TRPM8/HIF-1α/ET-1 signaling pathway. It raises the possibility that FA is a promising candidate for the development of anticardiovascular disease agents.
The authors would like to thank the Natural Science Foundation of China and the School of Traditional Chinese Medicine of Jinan University for their support of this research.
Financial support and sponsorship
This work was financially supported by the National Natural Science Foundation of China Grant (8187-4404).
Conflicts of interest
There are no conflicts of interest.
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