S1PR1 knockdown protects endothelial cells from oxidized low-density lipoprotein-induced injury via reducing interleukin-1β and tumor necrosis factor-α expression

Abstract

BACKGROUND

Coronary atherosclerotic heart disease (CAHD) is a prevalent cardiovascular condition. Recent research has uncovered the significant role of sphingosine-1-phosphate receptor 1 (S1PR1) in cardiovascular disorders, including atherosclerosis, acute myocardial infarction, myocardial ischemia, and reperfusion.

AIM

To explore the clinical relevance, diagnostic utility, and molecular mechanisms of S1PR1 in CAHD.

METHODS

The expression of S1PR1 was examined by quantitative real-time polymerase chain reaction. Cytokines in serum were detected by flow cytometry using a twelve-cytokine detection kit. Receiver operating characteristic curves were employed to assess the diagnostic value of S1PR1, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in CAHD. The cell counting kit-8 assay was utilized to determine cell viability, and flow cytometry was used to detect cell apoptosis. Western blot analysis was conducted to detect the protein levels of S1PR1.

RESULTS

S1PR1 was highly expressed in CAHD, with significantly higher levels observed in the high Gensini score group (≥ 40) compared to the low score group (< 40). Compared with the healthy control group, the CAHD group exhibited significantly increased levels of IL-1β and TNF-α, no significant difference in IL-2 and IL-6 levels, and a significant decrease in the levels of other cytokines. Furthermore, a correlation was observed between S1PR1 and both IL-1β and TNF-α in CAHD. The receiver operating characteristic curve analysis demonstrated that the combined detection of S1PR1, IL-1β, and TNF-α exhibited superior diagnostic value for CAHD compared to individual tests. Additionally, in oxidized low-density lipoprotein-treated human umbilical vein endothelial cells, the expressions of S1PR1, IL-1β, and TNF-α were increased. However, knockdown of S1PR1 resulted in decreased expression of IL-1β and TNF-α, accompanied by enhanced cell viability and attenuated apoptosis.

CONCLUSION

S1PR1 could act as a new diagnostic and monitoring biomarker for CAHD. Knockdown of S1PR1 protected human umbilical vein endothelial cells from oxidized low-density lipoprotein-induced injury, which is mediated through regulation of IL-1β and TNF-α expression.

Key Words: Sphingosine-1-phosphate receptor 1; Coronary atherosclerotic heart disease; Oxidized low-density lipoprotein; Interleukin-1β; Tumor necrosis factor-α

Core Tip: In this study, sphingosine-1-phosphate receptor 1 (S1PR1) is upregulated in coronary atherosclerotic heart disease and positively correlates with interleukin-1β and tumor necrosis factor-α. It serves as a promising diagnostic biomarker, and combined detection with inflammatory cytokines enhances diagnostic performance. S1PR1 knockdown alleviates oxidized low-density lipoprotein-induced endothelial cell injury via modulating inflammatory responses, highlighting S1PR1 as a potential target for coronary atherosclerotic heart disease diagnosis and intervention.

INTRODUCTION

Coronary atherosclerotic heart disease (CAHD) results from myocardial ischemia secondary to coronary artery stenosis caused by lipid- and calcium-rich plaque deposition[1]. Despite rising incidence and mortality rates, early diagnosis and risk assessment remain critical for improving patient prognosis. Known risk factors include diabetes, hypertension, hyperlipidemia, smoking, and genetic predisposition[2-4]; however, the pathogenic mechanisms underlying CAHD remain unclear.

Sphingosine-1-phosphate receptor (S1PR), a G protein-coupled receptor family member, comprises five subtypes (S1PR1-5) with distinct tissue distributions and functions[5]. S1PR1-3 are widely expressed in vivo, while S1PR4 is predominantly distributed in lymphoid tissues and lungs, and S1PR5 is exclusively expressed in the brain and skin. Among these subtypes, S1PR1 was initially identified due to its remarkable ability to promote endothelial cell differentiation and angiogenesis, and was hence designated endothelial differentiation gene-1. Its dysregulation is closely associated with endothelial dysfunction and vascular inflammatory injury, which constitute the core pathogenesis of coronary atherosclerosis and CAHD[6]. Sphingosine-1-phosphate (S1P), its biologically active lipid ligand, regulates angiogenesis, endothelial integrity, and smooth muscle cell proliferation/migration. The myocardium expresses three subtypes of S1PRs, namely S1PR1, S1PR2 and S1PR3, with an expression abundance ranked as follows: S1PR1 > > S1PR3 > S1PR2[7]. Early studies have highlighted its anti-atherosclerotic effects. For instance, high-density lipoprotein (HDL) - the primary carrier of S1P in plasma - forms a complex with S1P, and its binding to S1PR1 induces the assembly of the S1PR1-β-arrestin 2 complex on the cell surface; this signaling cascade inhibits tumor necrosis factor (TNF)-α production, thereby suppressing proinflammatory responses in vascular endothelial and smooth muscle cells[8]. In contrast, emerging evidence has implicated S1PR1 in plaque progression and arterial stenosis, with S1PR1 inhibition shown to reduce the hyperproliferation and migration of smooth muscle cells. This action attenuates atherosclerosis and post-angioplasty restenosis, ultimately improving the clinical outcomes of interventional procedures[9]. Wang et al[10] demonstrated that S1PR1 mediates the anti-apoptotic effect of S1P on bone marrow-derived endothelial progenitor cells, protecting them from hydrogen peroxide-induced injury via activation of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway and thereby playing a critical role in endothelial progenitor cell-mediated endothelial repair. In the present study, we aimed to investigate the expression and clinical significance of S1PR1 in patients with CAHD, and to explore its role and underlying mechanisms in oxidized low-density lipoprotein (ox-LDL)-induced endothelial injury and inflammation, with the goal of identifying a novel diagnostic biomarker and potential therapeutic target for CAHD.

MATERIALS AND METHODS

Clinical samples

Seventy-one patients diagnosed by coronary angiography at Nantong First People’s Hospital from January 2023 to January 2024 formed the CAHD group; 40 healthy (physical examination) individuals during the same period formed the control group. Based on the median Gensini score, 48 and 23 patients were in the low (< 40 points) and high (≥ 40 points) stenosis subgroups. Ethical approval for the study was granted by the hospital’s Medical Ethics Committee (approval No. 2024-KT228-01). The inclusion criteria were as follows: CAHD group: (1) Positive diagnosis via electrocardiogram, coronary angiography, and symptoms; (2) No other cardiovascular diseases; and (3) Informed consent. Control group: (1) No diabetes, hypertension, or hyperlipidemia; and (2) Normal biochemical tests. Exclusion criteria were as follows: Hepatic/renal insufficiency, other cardiovascular diseases, malignancies, infections, or immune disorders.

Collection of clinical data

General data, including gender, age, blood pressure, and smoking/drinking history, were collected from both the CAHD and control groups. Fasting venous blood (5 mL) was drawn from all participants in the morning. After centrifugation at 3000 rpm for 8 minutes, routine biochemical parameters (fasting blood glucose, cholesterol, triglyceride, HDL-cholesterol, low-density lipoprotein-cholesterol, apolipoprotein A1, apolipoprotein B, lipoprotein(a), alanine transaminase, g-glutamyl transferase, and uric acid) were measured using the Beckman AU5800 analyzer. The remaining serum was stored at -80 °C for subsequent quantitative real-time polymerase chain reaction (qRT-PCR).

Cell culture

Human umbilical vein endothelial cells (HUVECs) were obtained from American Type Culture Collection (Manassas, VA, United States) and cultured in RPMI-1640 medium (Gibco, Rockville, MD, United States) with 10% fetal bovine serum (Gibco, Rockville, MD, United States) at 37 °C with 5% CO₂.

qRT-PCR

Total RNA was extracted from serum using a spin-column RNA extraction kit for blood (liquid samples) and reverse transcribed into cDNA. The reaction system included 5 × buffer (4 μL), deoxynucleoside triphosphate (2 μL), oligo-dT (1 μL), reverse transcriptase (reverse transcriptase, RNase inhibitor, 1 μL each), diethylpyrocarbonate water (1 μL), and total RNA (10 μL), incubated at 42 °C for 60 minutes and then 72 °C for 5 minutes. Products were stored at -20 °C. qRT-PCR primers for S1PR1 (5’-TGTCGGGTGTTGGTGGGTA-3’ upstream, 5’-CAATGCTAGACCTTTGGCTCAGT-3’ downstream) and β-actin (5’-TCAAGATCATTGCTCCTCCTGAG-3’ upstream, 5’-ACATCTGCTGGAAGGTGGACA-3’ downstream) were used with SYBR Green. The PCR system included Mix (10 μL), primers (1 μL each), diethylpyrocarbonate water (5 μL), and cDNA (2 μL), with the following cycling conditions: 95 °C for 5 minutes (pre-denaturation), followed by 35 cycles of 95 °C for 15 seconds (denaturation), 60 °C (annealing), and 72 °C for 30 seconds (extension). Relative S1PR1 expression was calculated using the 2^-△△Ct method.

Cytokine assay

Serum cytokines were detected using a twelve cytokines flow cytometry immunoassay kit purchased from Shenzhen Wellgrow Biotech Co., Ltd. (Shenzhen, China). Fluorescence signals were acquired, and standard curves were used to calculate cytokine concentrations.

Cell viability assay

The cell counting kit-8 assay evaluated HUVEC viability. Cells (5 × 10³/well) were seeded in 96-well plates, incubated overnight, and then exposed to 25 μg/mL ox-LDL for 24 hours. After adding 10 μL cell counting kit-8 solution and 2 hours incubation, the absorbance at 490 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, United States). Experiments were repeated three times.

Apoptosis assay

HUVEC apoptosis was assessed by flow cytometry using an Annexin V-FITC/propidium iodide kit (BD Biosciences, San Jose, CA, United States). Cells (2 × 10⁵/well) were seeded in six-well plates, incubated overnight, and then exposed to 25 μg/mL ox-LDL for 24 hours. After washing with phosphate-buffered saline, cells were stained with 10 μL Annexin V-FITC and 5 μL propidium iodide, incubated at room temperature for 20 minutes, and analyzed by flow cytometry using FlowJo 7.6.

Cell transfection

Negative controls and S1PR1 siRNA were synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cells (2 × 10⁵/well) were transfected into six-well plates using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, United States). Transfection efficiency was assessed by qRT-PCR and western blotting after 24 hours.

Western blotting

Total protein was extracted from HUVECs using RIPA buffer with 1 mmol/L phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Shanghai, China). Protein concentrations were measured using the BCA assay (Pierce Biotechnology, Rockford, IL, United States). Samples were mixed with loading buffer and electrophoresed on 10% SDS-PAGE (NCM Biotech, Suzhou, China), then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States). Membranes were blocked with 5% non-fat milk (2 hours, room temperature), incubated with primary antibodies (β-actin 1:50000, S1PR1 1:10000; Abcam, Cambridge, United Kingdom) overnight at 4 °C, and with secondary antibodies (2 hours, room temperature). After washing with Tris-buffered saline Tween, bands were detected using a chemiluminescence system (BioRad Laboratories, Hercules, CA, United States), with β-actin as the loading control. Experiments were repeated three times.

Statistical analysis

Data were analyzed using SPSS 25.0 and GraphPad 5. Enumeration data are presented as frequencies (χ² test). Measurement data were tested for normality: Normally distributed data (mean ± SD, t test); non-normally distributed data [median (interquartile range), Mann-Whitney U test]. Receiver operating characteristic (ROC) curves assessed the diagnostic value of S1PR1, IL-1β, and TNF-α for CAHD. P < 0.05 was considered statistically significant.

RESULTS

Comparison of general clinical data

According to general information analysis, there were no significant difference in gender, systolic blood pressure, diastolic blood pressure, triglycerides, alanine transaminase, and g-glutamyl transferase between the CAHD and control groups (P > 0.05). HDL-cholesterol and apolipoprotein A levels in the CAHD group were significantly lower than those in the control group (P < 0.05), while the other biochemical indicators, age, smoking history, and drinking history, were significantly higher than those in the control group (P < 0.05). Based on the coronary artery stenosis Gensini scoring criteria, the CAHD group was further divided into a low score group (< 40 points) and a high score group (≥ 40 points). With the exception of cholesterol and lipoprotein(a) (P < 0.05), no significant differences were detected in other biochemical indicators between the two groups. These data are shown in Tables 1 and 2.

Table 1 General clinical data analysis of coronary atherosclerotic heart disease group and control group, n (%)/mean ± SD/median (interquartile range).

Clinical data	CAHD (n = 71)	Control (n = 40)	χ2/t/U value	P value
Gender (male/female)	49/22	33/7	2.411	0.121
Age (years)	67.31 ± 1.30	57.03 ± 1.40	5.09	< 0.0001
Systolic pressure (mmHg)	134 (123, 147)	128 (123, 136)	1123	0.068
Diastolic pressure (mmHg)	81 (74, 88)	80 (74, 85.75)	1325	0.561
Smoking history	35 (49.30)	9 (22.5)	7.678	0.006
Drinking history	36 (50.70)	12 (30)	4.469	0.035
FBG (mmol/L)	5.75 ± 0.32	4.69 ± 0.17	2.364	0.020
Cholesterol (mmol/L)	5.07 (4.46,5.07)	3.94 (3.16, 4.74)	723.5	< 0.0001
Triglyceride (mmol/L)	1.58 ± 0.09	1.53 ± 0.12	0.34	0.74
High-density lipoprotein-cholesterol (mmol/L)	1.17 (0.98, 1.33)	1.36 (1.2, 1.72)	812.5	0.0002
Low-density lipoprotein-cholesterol (mmol/L)	2.90 ± 0.11	2.34 ± 0.11	3.43	0.0009
Apolipoprotein A1 (g/L)	1.18 ± 0.03	1.31 ± 0.04	2.53	0.013
Apolipoprotein B (g/L)	0.8 (0.66, 0.95)	0.72 (0.63, 0.78)	1037	0.012
Lipoprotein(a) (mg/L)	207 (110, 422)	118 (64.5, 254)	974	0.006
Alanine transaminase (U/L)	23 (15, 32)	18.5 (15, 30.5)	1249	0.29
G-glutamyl transferase (U/L)	26 (18, 39)	21.5 (17, 35.25)	1247	0.29
Uric acid (μmol/L)	345.3 ± 10.11	312.0 ± 15.76	2.114	0.037

Normally distributed continuous data are expressed as mean ± SD and compared using the independent samples t-test. Non-normally distributed continuous data are expressed as median (interquartile range) and compared using the Mann-Whitney U test. Categorical data are expressed as n (%) and compared using the χ² test. CAHD: Coronary atherosclerotic heart disease; FBG: Fasting blood glucose.

Table 2 General clinical data analysis of low score group and high score group, n (%)/mean ± SD/median (interquartile range).

Clinical data	Low score (n = 48)	High score (n = 23)	χ2/t/U value	P value
Gender (male/female)	33/15	16/7	0.005	0.945
Age (years)	66.46 ± 1.64	69.03 ± 2.08	0.947	0.347
Systolic pressure (mmHg)	134 (123, 150)	135 (124, 145)	540.5	0.893
Diastolic pressure (mmHg)	83.5 (75, 88)	77 (71, 86)	425.0	0.120
Smoking history	20 (41.6)	15 (65.22)	3.450	0.063
Drinking history	23 (47.92)	13 (56.52)	0.461	0.497
FBG (mmol/L)	5.51 ± 0.35	6.24 ± 0.68	1.050	0.298
Cholesterol (mmol/L)	4.72 ± 0.14	5.30 ± 0.24	2.29	0.028
Triglyceride (mmol/L)	1.53 ± 0.10	1.69 ± 0.17	0.89	0.380
High-density lipoprotein-cholesterol (mmol/L)	1.26 (1.04, 1.45)	1.16 (0.98, 1.31)	446.0	0.195
Low-density lipoprotein-cholesterol (mmol/L)	2.88 ± 0.11	2.97 ± 0.14	0.49	0.630
Apolipoprotein A1 (g/L)	1.22 ± 0.05	1.16 ± 0.04	0.88	0.383
Apolipoprotein B (g/L)	0.84 ± 0.04	0.81 ± 0.06	0.37	0.712
Lipoprotein(a) (mg/L)	183 (100, 329)	309 (147, 608)	391	0.049
Alanine transaminase (U/L)	23.5 (16.25, 32)	20 (13, 35)	492	0.460
G-glutamyl transferase (U/L)	26 (18, 38.5)	26 (17, 41)	542.5	0.912
Uric acid (μmol/L)	342.0 ± 11.98	352.2 ± 19.02	0.470	0.640

FBG: Fasting blood glucose.

Serum S1PR1 expression in different groups

Serum S1PR1 expression in the CAHD and control groups was detected by qRT-PCR. Compared to the control group, S1PR1 level in the CAHD group was significantly increased (P < 0.001) (Figure 1A). Furthermore, the expression of S1PR1 in the low score group was markedly lower than in the high score group (P < 0.01) (Figure 1B).

Figure 1 Sphingosine-1-phosphate receptor 1 expression in different groups. A: Sphingosine-1-phosphate receptor 1 expression in control group (71 cases) and coronary atherosclerotic heart disease group (40 cases); B: Sphingosine-1-phosphate receptor 1 expression in low Gensini score group (48 cases) and high Gensini score group (23 cases). ^aP < 0.001 vs control, ^bP < 0.01 vs low score. S1PR1: Sphingosine-1-phosphate receptor 1; CAHD: Coronary atherosclerotic heart disease.

Cytokine levels in the CAHD and control groups

The levels of 12 cytokines were detected by flow cytometry in the CAHD and control groups. Compared with the control group, the CAHD group exhibited significantly increased levels of IL-1β and TNF-α, whereas the levels of IL-4, IL-5, IL-8, IL-10, IL-12, IL-17, interferon (IFN)-α, and IFN-γ were significantly decreased. The levels of IL-2 and IL-6 were not significantly affected. These data are shown in Table 3.

Table 3 Cytokine expressions in the coronary atherosclerotic heart disease group and control group, median (interquartile range).

Cytokine (pg/mL)	CAHD (n = 71)	Control (n = 40)	U value	P value
IL-1β	2.81 (0.60,4.68)	0.77 (0.12,1.04)	638.5	< 0.0001
IL-2	1.91 (0.68, 4.03)	2.71 (1.63, 3.48)	1137.0	0.08
IL-4	2.87 (0.39, 5.24)	7.89 (6.36, 9.71)	428.0	< 0.0001
IL-5	0.86 (0.09, 1.68)	5.28 (4.08, 6.51)	209.0	< 0.0001
IL-6	16.78 (8.56, 24.09)	12.69 (10.59, 15.46)	1125.0	0.07
IL-8	9.79 (5.31, 26.87)	24.32 (16.59, 32.41)	757.0	< 0.0001
IL-10	3.08 (1.8, 4.38)	6.12 (5.09, 7.33)	440.0	< 0.0001
IL-12	0.59 (0.08, 1.46)	2.79 (1.33, 4.02)	581.0	< 0.0001
IL-17	3.8 (1.76, 6.85)	9.89 (8.0, 12.61)	424.5	< 0.0001
TNF-α	4.12 (3.23, 5.26)	2.91 (2.47, 3.65)	678.5	< 0.0001
Interferon-α	0.3 (0.06, 0.89)	2.09 (1.68, 3.10)	383.0	< 0.0001
Interferon-γ	3.16 (1.79, 5.48)	10.23 (9.61, 12.78)	190.0	< 0.0001

CAHD: Coronary atherosclerotic heart disease; IL: Interleukin.

Correlation between S1PR1 and 12 cytokines in CAHD

The correlation between S1PR1 and 12 cytokines was analyzed in the CAHD group. The results indicated a positive correlation between S1PR1 and IL-1β as well as TNF-α in the CAHD group (P < 0.05), while there was no significant correlation between S1PR1 and IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IFN-α, or IFN-γ (Figure 2).

Figure 2 Correlation between sphingosine-1-phosphate receptor 1 and cytokines in coronary atherosclerotic heart disease. A-L: The correlation between sphingosine-1-phosphate receptor 1 and 12 cytokines [interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, tumor necrosis factor-α, interferon-α and interferon-γ] in coronary atherosclerotic heart disease. S1PR1: Sphingosine-1-phosphate receptor 1; IL: Interleukin; TNF-α: Tumor necrosis factor-α; IFN: Interferon.

Diagnostic efficacy of S1PR1, IL-1β, and TNF-α for CAHD

Based on ROC curve analysis, the area under the curve, sensitivity, specificity, positive predictive value, and negative predictive value of serum S1PR1, IL-1β, and TNF-α, and their combination in diagnosing CAHD were all higher than those of each individual marker. This indicated that the combination of these three markers could improve the diagnostic efficacy of CAHD (Figure 3 and Table 4).

Figure 3 Receiver operating characteristic curve for prediction of coronary atherosclerotic heart disease based on a combination of sphingosine-1-phosphate receptor 1, interleukin-1β and tumor necrosis factor-α levels, the area under the curve was 0.892 (95% confidence interval: 0.834-0.949). S1PR1: Sphingosine-1-phosphate receptor 1; IL: Interleukin; TNF-α: Tumor necrosis factor-α; AUC: Area under the receiver operating characteristics curve.

Table 4 Diagnostic value of sphingosine-1-phosphate receptor 1, interleukin-1β, tumor necrosis factor-α, and their combination in coronary atherosclerotic heart disease.

	Area under the curve	SEN (%)	SPE (%)	AC (%)	PPV (%)	NPV (%)
S1PR1	0.779	73.2	75	73.87	73.24	75
IL-1β	0.775	66.2	80	71.17	66.20	80
TNF-α	0.761	74.6	70	72.97	74.65	70
S1PR1 + IL-1β + TNF-α	0.892	84.5	80	82.88	84.51	80

S1PR1: Sphingosine-1-phosphate receptor 1; IL: Interleukin; TNF-α: Tumor necrosis factor-α; SEN: Sensitivity; SPE: Specificity; AC: Diagnostic accuracy; PPV: Positive predictive value; NPV: Negative predictive value.

ox-LDL-induced HUVEC injury and S1PR1 expression

HUVECs were exposed to ox-LDL at concentrations of 0, 10, 25, and 50 μg/mL for 24 hours. To assess the temporal effects, HUVECs were treated with 25 μg/mL ox-LDL for 0, 12, 24, and 48 hours. ox-LDL stimulation downregulated the viability of HUVECs in a dose- and time-dependent manner, while the apoptosis rate was upregulated (Figure 4A-D). After treating HUVECs with ox-LDL, expression of S1PR1 also increased in a dose- and time-dependent manner (Figure 4E-H). We chose HUVECs treated with 25 μg/mL ox-LDL for 24 hours for subsequent experiments.

Figure 4 Oxidized low-density lipoprotein-induced human umbilical vein endothelial cells injury and sphingosine-1-phosphate receptor 1 expression. A: The viability of human umbilical vein endothelial cells (HUVECs) decreased with the concentration of added oxidized low-density lipoprotein (ox-LDL); B: The apoptosis rate of HUVECs increased with the concentration of added ox-LDL; C: The viability of HUVECs decreased over time after the addition of ox-LDL; D: The apoptosis rate of HUVECs increased over time after the addition of ox-LDL; E: The quantitative real-time polymerase chain reaction results showed that sphingosine-1-phosphate receptor 1 (S1PR1) expression in HUVECs gradually increased with increasing concentrations of ox-LDL after treatment; F: The quantitative real-time polymerase chain reaction results showed that S1PR1 expression in HUVECs gradually increased over time after treatment with ox-LDL; G: The western blot results showed that S1PR1 expression in HUVECs gradually increased with increasing concentrations of ox-LDL after treatment; H: The western blot results showed that S1PR1 expression in HUVECs gradually increased over time after treatment with ox-LDL. ^aP < 0.01 vs 0 μg/mL, ^bP < 0.001 vs 0 μg/mL, ^cP < 0.05 vs 0 μg/mL, ^dP < 0.05 vs 0 hour, ^eP < 0.01 vs 0 hour, ^fP < 0.001 vs 0 hour. S1PR1: Sphingosine-1-phosphate receptor 1.

Cytokine expression in HUVECs stimulated by ox-LDL

IL-1β and TNF-α released by HUVECs treated with 25 μg/mL ox-LDL for 24 hours were assessed by flow cytometry. Compared to the control group, expression of IL-1β and TNF-α released by HUVECs treated with ox-LDL increased (Figure 5).

Figure 5 Cytokine levels released by human umbilical vein endothelial cells treated with oxidized low-density lipoprotein. A: The interleukin-1β levels released by human umbilical vein endothelial cells treated with 25 μg/mL of oxidized low-density lipoprotein for 24 hours; B: The tumor necrosis factor-α levels released by human umbilical vein endothelial cells treated with 25 μg/mL of oxidized low-density lipoprotein for 24 hours. ^aP < 0.01 vs 0 μg/mL. IL: Interleukin; TNF-α: Tumor necrosis factor-α.

S1PR1 knockdown attenuated ox-LDL-induced injury of HUVECs

To explore the role of S1PR1 in modulating CAHD events, siRNA specifically designed to target S1PR1 was used in a loss-of-function assay. The knockdown efficacy of this siRNA was initially verified in HUVECs through qRT-PCR and Western blotting. The introduction of S1PR1 siRNA restored the elevated expression of S1PR1 in ox-LDL-induced HUVECs (Figure 6A and B). Inhibition of cell viability and promotion of apoptosis were also attenuated (Figure 6C and D). Our observations revealed that administration of S1PR1 siRNA reversed the ox-LDL-induced upregulated expression of IL-1β and TNF-α (Figure 6E and F). This implied that knockdown of S1PR1 in HUVECs conferred protection against ox-LDL-induced injury by regulating expression of IL-1β and TNF-α.

Figure 6 Sphingosine-1-phosphate receptor 1 expression was detected after transfected with siRNA-sphingosine-1-phosphate receptor 1 and treatment with oxidized low-density lipoprotein. A and B: The expression of sphingosine-1-phosphate receptor 1 (S1PR1) was detected by quantitative real-time polymerase chain reaction and western blot after transfected with siRNA-S1PR1 and treatment with 25 μg/mL oxidized low-density lipoprotein (ox-LDL); C: The cell viability was detected by cell counting kit-8 assay after transfected with siRNA-S1PR1 and treatment with 25 μg/mL ox-LDL; D: The apoptosis ratio was detected by flow cytometry after transfected with siRNA-S1PR1 and treatment with 25 μg/mL ox-LDL; E: The interleukin-1β levels were detected by flow cytometry after transfected with siRNA-S1PR1 and treatment with 25μg/mL ox-LDL; F: The tumor necrosis factor-α levels were detected by flow cytometry after transfected with siRNA-S1PR1 and treatment with 25 μg/mL ox-LDL. ^aP < 0.01 vs siRNA-negative control, ^bP < 0.05 vs siRNA-negative control. S1PR1: Sphingosine-1-phosphate receptor 1; ox-LDL: Oxidized low-density lipoprotein; NC: Negative control.

DISCUSSION

CAHD is characterized by lipid deposition in the coronary artery, which gradually forms plaques, resulting in stenosis of the vascular lumen and poor blood flow. CAHD can seriously damage heart function, and increase the risk of myocardial ischemia, angina pectoris, and even myocardial infarction. Patients may experience a series of symptoms such as chest pain, difficulty breathing, and palpitations; all of which can be life-threatening[11]. Therefore, early diagnosis and timely treatment are crucial for preventing the occurrence of adverse cardiac events.

S1PR1 is a key member of the G protein-coupled receptor family and one of the most extensively studied subtypes of the S1PR family. It regulates a variety of biological processes, including cell proliferation, migration, survival, and inflammatory responses, through binding to its endogenous ligand, S1P. S1PR1 exhibits highly selective expression among different tissues, predominantly in immune cells, vascular endothelial cells, and smooth muscle cells[12-14]. Activation of its signaling pathway depends on the concentration gradient of S1P and the dynamic balance among receptor subtypes[15]. S1PR1 forms ligand-dependent complexes with scavenger receptor type B1 in cultured primary macrophages and mouse atherosclerotic plaques, jointly mediating HDL signaling and exerting antiatherosclerotic effects[16]. In contrast, studies utilizing immunostaining and radioligand methods have revealed upregulated expression of S1PR1 in human and mouse atherosclerotic plaques[17]. Notably, the expression of S1PR1 in atherosclerotic plaques exhibits cell type specificity: In vascular smooth muscle cells, S1PR1 activation promotes neointimal hyperplasia[9], whereas in macrophages, S1PR1 signaling exerts anti-atherosclerotic effects by facilitating cholesterol efflux and the resolution of inflammation[16,18]. This bidirectional regulatory characteristic suggests that S1PR1 may play distinct roles at different stages of atherosclerosis. Our study found that expression of S1PR1 in the CAHD group was significantly higher than that in the healthy control group, and it was correlated with the degree of coronary artery stenosis. In clinical practice, the Gensini score is commonly used to quantitatively assess the severity of coronary artery stenosis, with a higher Gensini score indicating a more severe degree of coronary artery disease. In this study, the CAHD group was further divided into a low-score group (< 40 points) and a high-score group (≥ 40 points). Expression of S1PR1 in the low-score group was significantly lower than in the high-score group, suggesting that S1PR1 is involved in the progression of CAHD, and its level may reflect the degree of coronary artery stenosis.

CAHD is a complex, multifactorial disorder with an incompletely elucidated pathogenesis, whose risk factors include diabetes, hypertension, hyperlipidemia, smoking and family history. Beyond lipid metabolism disorders, inflammatory responses play a pivotal role in its development: Proinflammatory factors such as C-reactive protein, IL and TNF are closely associated with the formation, rupture of coronary atherosclerotic plaques and coronary artery spasm, and they drive atherosclerotic initiation and progression by mediating endothelial dysfunction, vascular smooth muscle cell proliferation and migration, and foam cell formation[19-21]. Our study found that levels of the proinflammatory cytokines IL-1β and TNF-α were significantly elevated in patients with CAHD and positively correlated with S1PR1 expression, while levels of anti-inflammatory cytokines (such as IL-10) and Th1/Th2-related cytokines (such as IL-4 and IL-5) decreased. These findings suggest that S1PR1 is involved in the progression of CAHD by regulating the imbalance of the inflammatory microenvironment. Additionally, an ox-LDL-induced HUVECs model confirmed that upregulation of S1PR1 was closely associated with increased release of IL-1β and TNF-α, and knockdown of S1PR1 reversed this effect, suggesting that S1PR1 mediates endothelial damage by directly regulating the secretion of inflammatory cytokines. ROC curve analysis revealed that the combination of S1PR1, IL-1β, and TNF-α significantly improved the diagnostic efficiency of CAHD, supporting the potential of multimarker combined detection in clinical early screening.

The current study had some limitations. First, the sample size was small, and a larger cohort is needed to validate the generalizability of the biomarkers. Second, the specific molecular mechanisms by which S1PR1 regulates inflammatory cytokines were not fully elucidated. Third, animal models are lacking to verify the in vivo efficacy of S1PR1 inhibitors.

In summary, S1PR1 may play a central role in CAHD through the inflammation-endothelial damage-plaque instability axis, and targeted inhibition of S1PR1 could emerge as a novel strategy to alleviate progression of atherosclerosis. Future research should integrate single-cell sequencing technology to dissect the dynamic regulatory network of S1PR1 in specific cellular subpopulations (such as macrophage subtypes) and explore its interaction with metabolic reprogramming (such as lipid accumulation), with the aim of providing a basis for precision therapy.

CONCLUSION

S1PR1 is a potential diagnostic/monitoring biomarker for CAHD, notably in high Gensini score patients, correlating with IL-1β and TNF-α. S1PR1 knockdown protects endothelial cells from ox-LDL injury, likely via cytokine regulation.

ACKNOWLEDGEMENTS

We sincerely thank all members of the research team for their dedicated efforts, professional support and contributions, as well as colleagues who participated in related experiments and data analysis, for their assistance in the successful completion of this study.