Ursolic acid protects against anoxic injury in cardiac microvascular endothelial cells by regulating intercellular adhesion molecule-1 and toll-like receptor 4/MyD88/NF-κB pathway

Abstract

Background

Cardiac microvascular endothelial cells (CMECs) are rapidly damaged after myocardial ischemia or hypoxia. In this study, we intend to explore whether ursolic acid (UA) can protect CMECs against hypoxia/reoxygenation (H/R) injury and to detect related molecular mechanism.

Methods

CMECs were subjected to H/R condition in the absence or presence of UA. Cell behaviors were measured by Cell Counting Kit-8, transwell, ELISA and western blot assays. siRNA was applied to reduce ICAM1 expression, then the effect of co-treatment of UA and si-ICAM1 on CMECs has been detected by biological experiments.

Results

Under H/R stimulation, the proliferation and migration of CMECs were inhibited, as well as the inflammation and oxidative stress were enhanced. UA treatment obviously reversed these H/R-induced injuries and reduced ICAM1 expression. Moreover, knockdown of ICAM1 could alleviate the H/R-induced injuries and strengthen the protective effect of UA on CMECs under H/R condition. Additionally, the protein levels of TLR4, MyD88 and p-P65 NF-κB were obviously increased after H/R stimulation, whereas the addition of UA could alter the phenomena by reducing TLR4, MyD88, and p-P65 NF-κB expression.

Conclusions

Our results insinuated that UA could alleviate H/R-induced injuries in CMECs by regulating ICAM1 and TLR4/MyD88/NF-κB pathway.

Introduction

Myocardial ischemia/reperfusion (I/R) injury is the most common cardiovascular disease with high morbidity and mortality. ¹ It starts with myocardial ischemia, then an augment cell loss and infarct size were appeared successively after reperfusion. ² Compared with other myocardial tissue injury, the injury caused by the original I/R injury is more terrible. ³ Therefore, prevention of hypoxia-reoxygenation (H/R) injury is the key to improve the cure rate of myocardial I/R injury.

Ursolic acid (UA, 3 β-hydroxy-urs-12-en-28-oic acid) is a pentacyclic triterpenoid extracted from berries, fruits of medicinal plants, leaves, and flowers, ⁴ and has been reported to have anti-inflammatory, antibacterial, anti-oxidation, anti-cancer, antihypertensive and cardioprotective properties.^5,6 Cardiovascular disease is the leading cause of mortality and morbidity worldwide. The effect of UA in cardiovascular disease has been reported, for example, Senthil and colleagues have demonstrated that UA (60 mg/kg) decreased lipid peroxide level by scavenging free radicals and improving lipid profiles after 7 days of treatment, ⁶ in addition, a previous research from Lv et al. ⁷ illustrated that UA administration significantly suppressed the growth of human umbilical vein endothelial cells induced by interleukin 6 (IL-6), furthermore, UA contributed to the restoration of cardioprotective enzyme activity to its normal level in rats, which insinuated that it protected against myocardial ischemia. ⁸ UA can be used as an alternative medicine for the prevention and treatment of numerous diseases, including cardiovascular disease. ⁹ However, the effect of UA on cardiac microvascular endothelial cells (CMECs) behaviors has not been clearly elucidated. CMECs hold an obligatory role in modulating and maintaining cardiac function. ¹⁰ The response of CMECs to reactive oxygen species (ROS) affects cardiac function through changes in endothelial barrier function. Therefore, elucidating the function of UA on CMECs is essential for the treatment of myocardial I/R injury.

Intercellular adhesion molecule-1 (ICAM1) is a member of the immunoglobulin (Ig) superfamily, which is essential for the firm block and transduction of leukocytes from blood vessels to tissues. ¹¹ ICAM1 is present in endothelial cells, but its expression is increased by the release of pro-inflammatory cytokines. ¹² Analysis from Comparative Toxicogenomics Database (CTD, http://ctdbase.org/) website showed that ICAM1 was predicted as an underlying target of UA and that ICAM1 expression could be reduced by UA. In our study, we intend to detect the effect of UA/ICAM1 on CMECs and whether the effect of UA on CMECs is related to ICAM1.

Materials and methods

Comparative toxicogenomics database

The public Comparative Toxicogenomics Database (CTD, http://ctdbase.org/) is an innovative website that relates toxicological information for chemicals, genes, phenotypes, diseases, and exposures to promote understanding of human health. ¹³ In our study, CTD website was applied to detect the potential target of UA in myocardial infarction.

Cell culture and stimulation

Human CMECs were obtained from ScienCell Research Laboratories (San Diego, CA) and maintained in complete Endothelial Cell Medium (ECM) including 5% fetal bovine serum (FBS), 1% endothelial cell growth supplement, and 1% penicillin/streptomycin at 37°C with 5% CO₂. Thereafter, cells were exposed to hypoxia conditions (an air-tight chamber with pure N₂) at 37°C for 6 h. Then the hypoxia media was removed by repeated washes with phosphate-buffered saline (PBS) and replaced with fresh oxygenated culture medium. The cells were then transferred into a normoxic incubator (95% air plus 5% CO₂) for 12 h for reoxygenation to mimic the I/R injury.

siRNA transfection

CMECs were implanted in a six-well plate and cultured overnight. Next, ICAM1 siRNA (si-ICAM1) and negative control (si-con) were purchased from RiboBio (Guangzhou, China) and transfected into CMECs with the assistance of Lipofectamine 2000 (Invitrogen, China). After 24 h of culture, the cells were collected for the following experiments.

Cell proliferation detection

The Cell Counting Kit-8 (CCK-8) assay was carried out based on the supplier’s specification. In briefly, the treated CMECs were implanted in a 96-well plate (5000 cells/well). Then, the CMECs were incubated with 10 μL CCK-8 solution and cultured for another 1.5 h at 37°C. Finally, the optical density (OD) values were detected at 450 nm wavelength by using a microplate reader.

Transwell assay

The migration ability of CMECs was analyzed basing on Transwell chamber (8 mm pore size, Corning). After treatment, CMECs were suspended in culture medium excluding FBS, and then 100 μL cell suspension solution was loaded into the upper chamber. Next, a total of 600 μL complete culture medium containing 10% FBS was put into the lower chamber as induction factor. After 24 h incubation, the remained cells on the upper side of the membrane were removed by cotton swab, the cells on the lower side of the membrane were fixed and stained. Finally, the number of cells was counted under a microscope.

Wound healing assay

Firstly, the treated CMECs were seeded in a 24-well plate, and a straight scratch was gently made with a 10 μL pipette tip after the cells reached about 80% confluency. Then, the plate was slowly rinsed to eliminate detached CMECs. The scratch widths were recorded at 0 h and 48 h after cell culture.

qPCR

TRIzol reagent (Invitrogen, USA) was applied to extract the total RNA from CMECs based on the supplier’s specification. The concentration of the total RNA was measured by a microplate reader. Then, 1 μg RNA was applied to synthesize cDNA with the assistance of Reverse Transcript Kit (TaKaRa, Japan). The mRNA expression was carried out with real-time polymerase chain reaction by using SYBR Green qPCR Master Mix kit with GAPDH as the internal standard. The procedures of qPCR were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 30 s and extension at 72°C for 30 s. The primer sequences were ICAM1 forward: 5^′-GTAGCAGCCGCAGTCATAATGG-3^′ and ICAM1 reverse: 5^′-ATGCTGTTGTATCTGACTGAGG-3^′, GAPDH forward: 5^′-GGTGAAGGTCGGTGTGAACG-3^′ and GAPDH reverse: 5^′-CTCGCTCCTGGAAGATGGTG-3’. The relative gene expression was calculated with the assistance of 2^−ΔΔCt method. All the experiments were performed in triplicate.

Western blot assay

Briefly, cells were harvested and lysed with lysis buffer including 0.5 mM PMSF (Sigma, USA). The protein concentration was measured by the BCA protein assay kit. Equal amount (25 μg) proteins were electrophoresed on SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Next, the membranes were sealed with 5% skimmed milk in PBS for 2 h and then incubated with the primary antibodies against IL-1β (Abcam, ab254360, 1:1000), IL-6 (Abcam, ab214429, 1:1000), TNF-α (Abcam, ab183218, 1:1000), SOD (Abcam, ab51254, 1:50,000), catalase (CAT, Abcam, ab209211, 1:2000), glutathione peroxidase (GSH-Px, Abcam, ab108427, 1:5000), ICAM1 (Abcam, ab282575, 1:1000) and GAPDH (Abcam, ab181602, 1:10,000) overnight at 4°C. After washing, the membranes were incubated with the corresponding secondary antibodies for 2 h at ambient temperature. The target proteins were visualized by using enhanced chemiluminescent kit, and the protein bands were quantified by Image J software.

Statistical analysis

The whole data were achieved from three independent replicates and presented as mean ± standard deviation (SD). Student’s t test was applied to compare the difference between two groups, whereas the comparison among multiple groups was analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. A p value less than 0.05 was considered to be statistically significant.

Results

The addition of UA could alleviate the injuries of CMECs induced by H/R stimulation

First, the concentration of UA used in this study was determined. As presented in Figure 1A, the data from CCK-8 showed that the treatment of 0–6 μmol/L UA has no obvious effect on CMECs viability from 0 h to 48 h, however, the addition of 9 μmol/L UA for 48 h significantly reduced the viability of CMECs (p < 0.05). Therefore, 6 μmol/L UA was applied in the following experiments. Under H/R condition, we observed that the viability of CMECs was obviously reduced, and the number of migrating CMECs and the relative migratory area of CMECs were also significantly decreased. However, the addition of UA reversed these phenomena by increasing cell viability, as well as elevating number and area of CMECs migration (Figure 1B–D, p < 0.01). Moreover, the inflammatory response was strengthened under H/R stimulation. We observed that the protein levels of IL-1β, IL-6 and TNF-α were obviously increased after H/R treatment, however, the addition of UA suppressed the inflammatory response by reducing IL-1β, IL-6 and TNF-α under H/R condition (Figure 2A, p < 0.01). Additionally, we observed that the oxidative stress related markers have been altered under H/R condition. The results from Figure 2B presented that the protein levels of SOD, CAT and GSH-Px were obviously reduced under H/R condition, whereas the administration of UA could reverse these phenomena by increasing SOD, CAT, and GSH-Px under H/R condition. All of the above findings insinuated that under H/R stimulation, the viability and migration of CMECs were obviously reduced, the protein levels of IL-1β, IL-6, and TNF-α were obviously increased, as well as the protein levels of SOD, CAT, and GSH-Px were obviously reduced; however, the treatment of UA could alleviate these above phenomena by increasing cell viability and migration, as well as suppressing inflammatory response and oxidative stress.

Figure 1.

The treatment of UA could alleviate the injuries of CMECs induced by H/R. A. The effects of different concentrations of UA on CMECs viability were analyzed, **p < 0.01 vs 0 μmol/L UA. B. Under H/R condition, the viability of CMECs was significantly reduced, whereas the addition of UA could alleviate the damage of H/R on CMECs viability. C. The number of migrating CMECs was obviously decreased under H/R stimulation, whereas the administration of UA could attenuate the inhibiting effect of H/R on CMECs migration. D. The migratory area of CMECs was remarkably decreased under H/R stimulation, whereas the administration of UA could attenuate the inhibiting effect of H/R on CMECs migratory area. **p < 0.01 vs. control, #p < 0.05, ##p < 0.01 vs. H/R. Bar = 100 μm.

Figure 2.

The administration of UA could reduce inflammation and oxidative stress of CMECs induced by H/R. A. The protein levels of IL-1β, IL-6 and TNF-α were obviously increased in CMECs under H/R stimulation, whereas the addition of UA could suppress the phenomena. B. The protein levels of SOD, CAT and GSH-Px were obviously increased in CMECs under H/R stimulation, whereas the addition of UA could suppress the phenomena. C-D. The mRNA and protein levels of ICAM1 were obviously increased in CMECs under H/R stimulation, whereas the administration of UA could alleviate the promoting effect of H/R on ICAM1 expression. **p < 0.01 vs. control, #p < 0.05, ##p < 0.01 vs. H/R.

ICAM1 was identified as a target of UA and contributed to the beneficial effect of UA on CMECs under H/R condition

Analysis from CTD website showed that ICAM1 was predicted as a potential target of UA, and UA could suppress ICAM1 expression. To further verify the hypothesis, we detected ICAM1 expression in CMECs after H/R stimulation. The results from Figure 2C and D showed that ICAM1 expression was significantly increased in CMECs under H/R stimulation, whereas the addition of UA suppressed the phenomenon by reducing ICAM1 expression at both mRNA and protein levels (p < 0.01). Subsequently, we knockdown of ICAM1 to detect the effect of ICAM1 on CMECs under H/R condition, as well as its relationship with UA. The interfering efficiency of si-ICAM1 was presented in Figure 3A and B. We observed that the mRNA and protein levels of ICAM1 were obviously reduced after si-ICAM1 treatment than those in si-con group (p < 0.01). Then, we used si-ICAM1 to transfect CMECs to detect its function. The results showed that under H/R condition, the viability and migration of CMECs were significantly increased after ICAM1 knockdown (Figure 3C and D), as well as the levels of IL-1β, IL-6, and TNF-α were reduced, and the levels of SOD, CAT, and GSH-Px were increased (Figure 4A and B). Furthermore, the effect of co-treatment of UA and si-ICAM1 on CMECs was also detected. The results showed that under H/R stimulation, the co-treatment of UA and si-ICAM1 increased the viability and migration of CMECs compared with just treated with UA or si-ICAM1 (Figure 3C and D), as well as reduced the levels of IL-1β, IL-6, and TNF-α, and increased the levels of SOD, CAT and GSH-Px (Figure 4A and B). Therefore, the above results insinuated that UA may play a beneficial effect on CMECs under H/R condition by decreasing ICAM1 expression.

Figure 3.

Knockdown of ICAM1 strengthened the beneficial effects of UA on CMECs growth and migration under H/R stimulation. A–B. The interfering efficiency of si-ICAM1 was demonstrated at both mRNA and protein levels, **p < 0.01 vs. si-con. C. Depletion of ICAM1 improved the beneficial effect of UA on CMECs viability under H/R stimulation. D-E. Under H/R condition, the migratory ability of CMECs was elevated after UA treatment, which was further improved after ICAM1 knockdown. **p < 0.01 vs. control, #p < 0.05, ##p < 0.01 vs. H/R, $p < 0.05, $$p < 0.01 vs. H/R+UA, @p < 0.05, @@p < 0.01 vs. H/R+si-ICAM1. Bar = 100 μm.

Figure 4.

Depletion of ICAM1 enhanced the beneficial effects of UA on CMECs inflammation and oxidative stress under H/R stimulation, and the beneficial effect of UA on CMECs was related to TLR4/MyD88/NF-κB pathway. A. Under H/R condition, the protein levels of IL-1β, IL-6, and TNF-α in CMECs were reduced after UA stimulation, whereas the phenomena were strengthened after ICAM1 knockdown. B. Under H/R condition, the protein levels of SOD, CAT, and GSH-Px in CMECs were increased after UA stimulation, whereas the phenomena were strengthened after ICAM1 knockdown. C. Under H/R condition, the protein levels of ICAM1, TLR4, MyD88, p-P65 NF-κB were obviously increased, whereas the phenomena were reversed after UA stimulation. **p < 0.01 vs. control, ##p < 0.01 vs. H/R, $$p < 0.01 vs. H/R+UA, @@p < 0.01 vs. H/R+si-ICAM1.

The beneficial effect of UA on CMECs under H/R condition may be related to TLR4/MyD88/NF-κB pathway

ICAM1 is critical for the firm arrest and transmigration of leukocytes of blood vessels and into tissues, and ICAM1 expression is increased in endothelial cells after proinflammatory cytokines stimulation. ¹¹ TLR4 has been reported to mediate the inflammatory response in the myocardium, additionally, its mediated inflammatory signaling pathway plays an important role in myocarditis, myocardial infarction, and ischemia-reperfusion injury.^14–16 Several reports have demonstrated that TLR4/MyD88/NF-κB signaling controls the generation of pro-inflammatory factors and induces the inflammatory response in myocardial tissues, which is the main inducement of myocardial tissue injury.^17,18 All the backgrounds underscored the importance of ICAM1 and TLR4/MyD88/NF-κB in myocardial ischemia/reperfusion injury. The results from western blot assay showed that the protein levels of ICAM1, TLR4, MyD88, and p-P65 NF-κB were obviously increased in MCECs after H/R stimulation, whereas the addition of UA altered the phenomena by reducing ICAM1, TLR4, MyD88, and p-P65 NF-κB levels (Figure 4C). Therefore, our results insinuated that the beneficial effect of UA on CMECs under H/R might be partially related to TLR4/MyD88/NF-κB pathway.

Discussion

In this study, we used a model of CMEC ischemia/reperfusion injury to illustrate the changes in cellular oxidative stress and its regulation by UA after H/R. Our findings demonstrated that H/R treatment significantly reduced the growth and migration, as well as strengthened inflammation and oxidative stress of CMECs, while UA administration reversed the above phenomena. The underlying mechanism might be related with regulation of ICAM1 and TLR4/MyD88/NF-κB signaling pathway.

Recently, the therapeutic effects of herbal medicine on cardiovascular disease have been identified by researchers. ¹⁹ UA has been illustrated to possess potential cardioprotective effect in recent years. ²⁰ For example, a previous study stated that UA appeared to suppress resistin-induced atherosclerosis, indicating that UA might perform a cardioprotective role in obesity-induced cardiovascular diseases. ²¹ In our study, CMECs was applied to simulate an in vitro I/R model after H/R stimulation. After H/R treatment, we observed that the proliferation and migration abilities of CMECs were significantly reduced, whereas the administration of UA limited these behaviors. Meanwhile, the inflammatory response and oxidative stress of CMECs were obviously strengthened after H/R treatment, whilst the addition of UA abolished these phenomena.

ICAM1 is a cell surface glycoprotein that is expressed at low levels in immune, endothelia, and epithelia cells, but its expression is up-regulated under inflammatory stimulation. ²² Additionally, functional studies have identified several novel roles for ICAM1, including innate and adaptive immune response in inflammation, and tumorigenesis. ²³ Functionally, ICAM1 has become a major regulator of numerous basic tissue functions, both at the onset of disease and during the resolution of pathologic conditions. ¹² Since ICAM1 is induced in numerous cell types during inflammatory responses, it is not surprising that ICAM1 is implicated in numerous physiological and pathological processes. In our study, we discovered that ICAM1 was highly expressed in CMECs under H/R stimulation, however, the addition of UA attenuated the expression of ICAM1 in CMECs induced by H/R. Previously, a study indicated that UA could suppress the adhesion of U937 cells to human umbilical vein endothelial cells and downregulate the expression of ICAM1, which is similar to our finding that UA can reduce the expression of ICAM1. ²¹ Additionally, we also discovered that knockdown of ICAM1 attenuated the H/R induced CMECs damage by increasing cell proliferation and migration, as well as decreasing cell inflammation and oxidative stress.

Inadequate oxygen supplement will hinder the energy metabolism in cardiomyocytes, resulting in damage to cardiomyocytes that cannot support the normal function of heart. ²⁴ Oxidative stress has been identified to be associated with the pathophysiology of myocardial ischemia. ²⁵ Increased content of reactive oxygen species, decreased content of GSH and the activity of SOD are indicators of oxidative stress. Emerging evidence suggests that I/R injury is associated with oxidative stress. ²⁶ Several drugs, such as dioxin, alpha-tocopherol, and irisin, have protective effects on I/R injury through oxidative stress.^27,28 A large number of studies have shown that oxidative stress injury of microcirculation, especially oxidative stress of CMECs during reoxygenation, is the main factor regulating the incidence of myocardial ischemia. ²⁹ Interestingly, in our study, we discovered that UA alleviated the damage of H/R induced-CMECs by regulating the oxidative stress, that is, the protein levels of SOD, CAT, and GSH-Px were down-regulated in CMECs stimulated by H/R, which was suppressed by UA or/and si-ICAM1 treatment.

Toll-like receptor 4 (TLR4) has been considered to be a mediator of inflammation and organ damage in some I/R models including myocardial I/R. ³⁰ During ischemia, activating TLR4 promotes NF-κB activity through MyD88-dependent pathway, which induces the expression of pro-inflammatory factors such as IL-1, IL-6, and TNF-α, resulting in serious tissue injury. ³¹ In our study, we discovered that the expression levels of TLR4, MyD88, and p-P65 NF-κB were all significantly increased in H/R-induced CMECs. A previous study revealed that UA exhibited anti-inflammatory action through blocking TLR4-MyD88 pathway. ³² Analogously, our study stated that the increased levels of TLR4, MyD88, and p-P65 NF-κB in H/R-induced CMECs were obviously attenuated by UA addition.

Some deficiencies in the study need to be pointed out. First, our finding was only confirmed in vitro in CMECs, and further validation in vivo is needed. Second, we only observed that the addition of UA reduced the expression of ICAM1 in H/R induced CMECs, but whether UA directly or indirectly affected ICAM1 needs further study.

Taken together, our study indicated that the addition of UA may alleviate the damage of CMECs induced by H/R through downregulating ICAM1 and TLR4/MyD88/NF-κB pathway.