Matrine alleviates coronary microvascular dysfunction in ischemia with non-obstructive coronary artery disease mice induced by advanced glycation end products via inhibition of the reactive oxygen species-mediated endoplasmic reticulum stress in cardiac microvascular endothelial cells

Abstract

OBJECTIVE:

To investigate the protective effect of matrine on coronary microvascular dysfunction (CMD) induced by advanced glycation end products (AGEs) in a mouse model of ischemia with non-obstructive coronary artery disease (INOCA), with a focus on the underlying mechanisms, particularly the endoplasmic reticulum (ER) stress protein kinase R-like ER kinase (PERK)/ nuclear factor of activated T-cells (NFAT) signaling pathway.

METHODS:

An INOCA model was established in mice, and CMD was induced by peritoneal injections of AGEs. Matrine was administered daily via intraperitoneal injections. Coronary microcirculation was evaluated using coronary flow velocity reserve (CFVR), and cardiac microvascular endothelial cells (CMECs) were isolated for assessment of apoptosis, inflammation, oxidative stress, and microthrombosis. Markers of ER stress and the PERK/NFAT pathway were examined through immunoblotting, immunofluorescence, and enzymatic assays. The effect of matrine were further evaluated in CMECs treated with AGEs and the PERK agonist.

RESULTS:

Matrine treatment significantly improved CFVR and reduced CMD in AGEs-exposed INOCA mice. In CMECs, matrine attenuated AGEs-induced apoptosis, inflammation, and microthrombosis. It also suppressed intracellular reactive oxygen species (ROS) generation, ER stress markers, and PERK/NFAT signaling. Matrine's effects were concentration-dependent and partially reversed by the PERK agonist, confirming its action through the ER stress pathway. No significant toxicities were observed with matrine administration.

CONCLUSION:

Matrine attenuates AGEs-induced CMD in INOCA by suppressing the ROS-mediated ER stress PERK/NFAT signaling pathway in CMECs. This study highlights matrine’s potential as a therapeutic agent for CMD in diabetic cardiovascular complications.

1. INTRODUCTION

In patients without significant coronary arterial stenosis, evidence of severe cardiac ischemia can still be observed, presenting a paradoxical clinical condition known as ischemia with non-obstructive coronary artery disease (INOCA). Previous studies suggest that INOCA occurs in 40%-60% of cases and is strongly associated with major adverse cardiac events (MACE).^1-4 In recent years, it has been widely recognized that INOCA is closely linked to coronary microvascular dysfunction (CMD).⁵

The mechanisms underlying INOCA are complex. In acute coronary syndrome, capillary leakage, hemorrhage, microembolization, and microvascular inflammation play critical roles.⁶ In chronic cardiac ischemia, factors such as microthrombosis, microvascular endothelial cell apoptosis, inflammation, and vasomotor dysfunction are key contributors.⁷ Coronary microvascular networks, lined by cardiac microvascular endothelial cells (CMECs), are essential for maintaining cardiac blood flow to meet the metabolic demands of tissue. Microvascular endothelial dysfunction is considered a major factor in CMD.⁵ Previous investigations, including our own, have demonstrated that microvascular endothelial dysfunction significantly contributes to CMD by promoting microvascular apoptosis, inflammation, and microthrombosis.^8,9

Diabetes is a metabolic disease and an independent risk factor for coronary artery disease, with individuals with diabetes experiencing a significantly higher incidence of MACE compared to those without diabetes.¹⁰ Diabetic microvascular complications are closely associated with advanced glycation end products (AGEs), which are characteristic pathological metabolites of diabetes.¹¹ Our previous research demonstrated that AGEs exacerbate CMD in INOCA models by activating the endoplasmic reticulum (ER) stress protein kinase R-like endoplasmic reticulum kinase (PERK)/nuclear factor of activated T-cells (NFAT) pathway in cardiac microvascular endothelial cells (CMECs) via ER stress.⁹

Matrine (C₁₅H₂₄N₂O) is a biologically active alkaloid extracted from the roots of the Chinese medicinal herb Kushen (Radix Sophorae Flavescentis). Evidence has shown its pharmacological effects, including the suppression of inflammation, fibrosis, oxidative stress, and ER stress.^12-14 In cardiovascular diseases, matrine exerts protective effects through various mechanisms. For example, it has been shown to inhibit atherosclerotic plaque formation, reduce myocardial hypertrophy, and suppress cardiac fibrosis.¹⁵ These effects are thought to be mediated through the modulation of multiple signaling pathways involved in inflammation, oxidative stress, and apoptosis.¹⁶ Matrine's derivatives, such as oxymatrine and sophocarpine, have also been studied for their cardiovascular benefits. Oxymatrine has been reported to protect against myocardial ischemia-reperfusion injury and ventricular remodeling.¹⁷ Sophocarpine has demonstrated anti-hypertensive effects and protection against cardiac hypertrophy.¹⁸

Previous studies have shown that matrine can suppress the activation of the PERK pathway, and our research has demonstrated its effectiveness in inhibiting ER stress-mediated NFAT signaling.^19,20 Based on these findings, we propose that matrine may attenuate coronary microvascular dysfunction induced by AGEs by suppressing ER stress-mediated NFAT signaling in CMECs.

In this study, we investigated the potential of matrine to attenuate AGEs-induced CMD in INOCA models and explored the underlying mechanisms, with a specific focus on the ER stress PERK/NFAT signaling pathway. Our findings contribute to the current understanding of matrine's protective effects on cardiovascular complications related to diabetes and its associated microvascular dysfunction.

2. MATERIALS AND METHODS

2.1. AGES-BOVINE SERUM ALBUMIN (BSA) PREPARATION

The AGEs-BSA preparation was conducted as previously described.^9,21,22 Briefly, BSA (Gibco, Carlsbad, CA, USA) was incubated with 0.1 mmol/L glyceraldehyde (Sigma-Aldrich, Darmstadt, Germany) in 0.2 mmol/L NaPO₄ buffer solution (pH 7.4) for 7 d at 37 ℃ under sterile conditions. Unincorporated sugars were removed via phosphate-buffered saline (PBS) dialysis using a PD-10 desalting column (GE Healthcare, Chicago, IL, USA). Non-glycated BSA was prepared as a control using the same protocol without glyceraldehyde incubation.

2.2. Animal model and treatment

An INOCA mouse model was established based on our and others' previous investigations to simulate CMD.^9,23 Ten-week-old male and female C57BL/6J-lep^ob mice (ob/ob^-/-, Jackson Laboratory, Bar Harbor, ME, USA) were used and acclimatized for 1 week in an artificially controlled environment with a 12-h light/dark cycle, 25 ℃ temperature, and 50% humidity. The mice were fed sterilized water and rodent chow ad libitum. AGEs-BSA or control BSA was administered via peritoneal injections at a dose of 1 mg/d for 20 consecutive days.⁹ Several mice were also co-treated with daily intra-peritoneal injections of matrine at a dose of 200 mg/kg bodyweight.^15,24-26 Their C57BL healthy counterparts were also used for CMEC isolation. We assessed blood biomarkers, including transaminases, creatine kinases, creatinine, and electrolytes. All animal experimental protocols were approved by the Medical Research Ethics Committee of Shaanxi Provincial People's Hospital.

2.3. Coronary microcirculation evaluation

To evaluate coronary microcirculation, we employed coronary flow velocity reserve (CFVR) as previously described.^9,23 Briefly, mice were anesthetized with isoflurane (3% in oxygen) and placed supine on a heated procedure board. Anesthesia was maintained with isoflurane (1% in oxygen) via a nosecone, and flow velocity was assessed using a high-frequency ultrasound imaging system equipped with a 40 MHz transducer (Vivo 2100, Visual Sonics, Toronto, Canada). The baseline profile was recorded after the signal stabilized, and hyperemia was induced by increasing isoflurane to 2.5% in oxygen. After 4 min, the hyperemic profile was recorded, and CFVR was calculated as the hyperemic coronary flow velocity divided by the baseline coronary flow velocity.

2.4. Immunofluorescent stains

Animals were euthanized by CO₂ inhalation, and their hearts were excised and fixed with 4% paraformaldehyde. After embedding and dehydration, 5 μm-thick cardiac sections were prepared. To assess endothelial and inflammatory cell activation, CMECs cultured on cover glass were also fixed with 4% paraformaldehyde. Following washing with PBS and blocking, sections or cells were incubated with primary antibodies against CD42b (1:200, Abcam, Cambridge, UK), CD45 (1:200, Abcam, Cambridge, UK), and nuclear Factor of Activated T-cells cytoplasmic 4 (NFATc4 1:200, Abcam Cambridge, UK) at 4 ℃ for 8 h. Secondary antibodies conjugated to Alexa Fluor 555 (1:500, Invitrogen, Carlsbad, CA, USA) were used to label the sections at 25 ℃ for 1 h. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Abcam Cambridge, UK), and images were captured and analyzed using a fluorescence microscope (Axio Imager 2, Zeiss, Oberkochen, Germany).

2.5. CMECs isolation, culturing and treatments

The isolation protocol for CMECs was performed according to previous methods.^9,27 CMECs were collected from animal models and their healthy counterparts. Briefly, ventricular tissue was harvested, dissected, and washed with Dulbecco’s phosphate-buffered saline (DPBS) five times under sterile conditions. Endocardial and epicardial endothelial cells were devitalized by immersing the tissue in 75% ethanol for 15 s. The tissue was then digested with collagenase Ⅱ (0.2%, Invitrogen, Carlsbad, CA, USA) and trypsin (0.25%, Invitrogen, Carlsbad, CA, USA) at 37 ℃ for 10 min, followed by centrifugation. The dissociated cells were resuspended in Dulbecco's modified eagle medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 15% fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and antibiotics mix (Sigma-Aldrich, Darmstadt, Germany). CMECs were identified using Von Willebrand factor (VWF) antibody staining. CMECs isolated from healthy mice were pre-treated with matrine at 0, 0.25, 0.5 and 1.0 mmol/L for 48 h. Several CMECs were treated with selective PERK activator CCT020312 (MedChemExpress, Monmouth Junction, NJ, USA) at 10 μmol/L for 24 h.²⁸ Several CMECs were exposed to AGEs at 10μmol/L for 24 h.⁹

2.6. CMECs permeability evaluations

The permeability of CMECs was evaluated by fluorescein isothiocyanate (FITC)-dextran clearance assay and transendothelial electrical resistance (TER) assay.

Briefly, CMECs were cultured on a transwell insert (Corning, Corning, NY, USA) until a monolayer is formed. After AGEs and/or matrine treatments, the medium in the apical compartment is replaced with a solution containing FITC-dextran (Sigma-Aldrich, Darmstadt, Germany). Following a 2-h incubation period, a sample from the basolateral compartment is collected, and the fluorescence was measured using a spectro-photometer (BioTek Instruments, Winooski, VT, USA). The fluorescence intensity was proportional to the amount of FITC-dextran that has crossed the monolayer, indicating the permeability of the endothelial cell barrier. The TER is then measured using a voltohmmeter (EVOM2, World Precision Instruments, Sarasota, FL, USA) which applies a small current across the monolayer and measures the resistance. This resistance is directly proportional to the tightness of the junctions between the endothelial cells, providing a quantitative measure of barrier integrity.

2.7. Cell apoptosis evaluations

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and flow cytometry were used to evaluate CMEC apoptosis, following previously described protocols.^29-31 Isolated CMECs were fixed in 4% paraformaldehyde, washed with PBS, and then subjected to a TUNEL assay kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. Apoptotic CMECs were detected using DAPI staining and a fluorescence microscope (Axio Imager 2, Zeiss, Oberkochen, Germany). Annexin-V and propidium iodide (PI) double-staining was used for flow cytometry analysis, with CMECs incubated with 5 μL of Annexin V-FITC and 5 μL of PI (BD) in binding buffer in the dark for 15 min. Flow cytometry analysis was performed using a fluorescence-activated cell sorting (FACS) Calibur (BD, Franklin Lakes, NJ, USA) and data were analyzed accordingly.

2.8. Intracellular reactive oxygen species (ROS) detection

Intracellular ROS generation was detected using 2,7-dichlorofluorescein (DCFH-DA) fluorescent staining as previously described26. Isolated CMECs were washed with PBS and incubated with DCFH-DA (Molecular Probes, Eugene, OR, USA) at a concentration of 10 μmol/L in a dark, humidified incubator at 37 ℃ for 30 min. Images were captured using a fluorescence microscope (Axio Imager 2, Zeiss, Oberkochen, Germany) and fluorescent intensities were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA, version 1.53t) to quantify ROS levels.

2.9. Calcineurin (CaN) enzymatic activity determination

The enzymatic activity of CaN was assessed using a Calcineurin Activity Assay kit (Merck, Darmstadt, Germany) following the manufacturer's instructions. A colorimetric method was employed for detection.

2.10. Western blotting

Isolated CMECs were lysed with radioimmune-oprecipitation assay (RIPA) lysis buffer (Santa Cruz, Dallas, TX, USA) supplemented with phenyl-methylsulfonyl fluoride (PMSF). Total and nuclear protein were extracted using the total protein extraction kit and nuclear protein extraction kit (Beyotime, Shanghai, China), respectively. Protein concentrations were quantified using the BCA kit (Invitrogen, Carlsbad, CA, USA). Proteins were separated by 10%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with QuickBlockTM (Beyotime, Shanghai, China), membranes were probed with primary antibodies against glucose regulated protein 78 (GRP78, 1:1000, Abcam, Cambridge, UK), PERK (1:1000, Abcam, Cambridge, UK), p-PERK (p-PERK, 1:1000, Abcam, Cambridge, UK), inositol-requiring enzyme 1 (IRE1, 1:1000, Abcam, Cambridge, UK), p-IRE1 (1:1000, Abcam, Cambridge, UK), X-box binding protein 1 (XBP1, 1:500, Abcam, Cambridge, UK), activating transcription factor 4 (ATF4, 1:800, Abcam, Cambridge, UK), C/EBP-homologous protein (CHOP, 1:500, Abcam, Cambridge, UK), NFATc4 (1:500, Abcam, Cambridge, UK), Fas (1:500, Proteintech, Wuhan, China), FasL (1:500, Proteintech, Wuhan, China), tumor necrosis factor α (TNFα, 1:500, Abcam, Cambridge, UK), interleukin 6 (IL6, 1:500, Abcam, Cambridge, UK), Histone H3 (1:2500, Abcam, Cambridge, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2500, Abcam, Cambridge, UK). Following Tris-buffered saline and tween-20 (TBST) washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and developed using an enhanced chemiluminescence (ECL) kit (Invitrogen, Cambridge, UK). Protein expression was visualized with a Gene Genius System (Syngene, Cambridge, UK).

2.11. Enzyme linked immunosorbent assay (ELISA)

Serum and culture medium of CMECs were centrifuged at 12 000 × g for 5 min at 4 ℃. The resulting supernatant was collected and subjected to ELISA. Mouse cTnT ELISA kit (Kamiya Biomedical, Seattle, WA, USA) was used to measure the serum concentrations of cardiac troponin T (cTnT), while the concentrations of IL6, TNFα, and thromboxane B2 (TXB2) in cell culture medium were detected with mouse IL6 ELISA kit (Sangon Biotech, Shanghai, China), mouse TNFα ELISA kit (Sangon Biotech, Shanghai, China), and mouse TXB2 ELISA kit (Sangon Biotech, Shanghai, China), respectively. The detection procedures were performed according to the instructions provided by the manufacturers.

2.12. Cardiac tissue AGEs concentration detection

The protocol for measuring cardiac tissue AGEs concentration was adapted from previous studies.^32,33 Briefly, minced cardiac tissue was treated with a chloroform and methanol mixture (2/1, v/v) for 8 h. After washing with methanol and distilled water, the tissue was homogenized with NaOH solution (1 nmol/L) and then centrifuged at 8000 × g at 4 ℃ for 15 min. The AGEs content in the alkali-soluble samples was determined by measuring fluorescence intensities at 460 nm after 360 nm excitation. The concentration of AGEs was expressed in arbitrary units (AU).

2.13. Statistical analysis

The data in this study are presented as mean ± standard deviation ($\overline{x}\pm s$) and the number of independent experiments conducted was denoted as “n”. Differences between two groups were compared using t-test (for parametric variables), Mann-Whitney U test (for non-parametric variables), and χ² test (for categorical variables). Post-hoc analysis was performed using the Student-Newman-Keuls (SNK) test. A P-value less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA).

3. RESULTS

3.1. Matrine treatment improved CMD in AGEs-exposed INOCA mice

An INOCA mouse model was established, and CMD was induced by AGEs exposure. Serum cTnT levels were not significantly affected by INOCA modeling or AGEs exposure, indicating that coronary microvascular function was not compromised by severe coronary ischemia-associated infarction (Table 1). CMD was evaluated by determining CFVR, and no significant differences in CFVR_rest were found among groups (Table 2). AGEs exposure significantly reduced CFVR_hypereamia and CFVR_ratio when compared with control groups, but matrine treatment improved both CFVR_hypereamia and CFVR_ratio in AGEs-exposed INOCA mice (Table 2). Blood biomarkers were not significantly affected by matrine treatment, indicating that it induced no significant in vivo toxicities (Table 3). In the in vitro evaluation of CMECs permeability, as demonstrated in Table 4, AGEs exposure significantly impaired both FITC-dextran clearance and TER in CMECs. The matrine treatment, however, significantly improved both FITC-dextran clearance and TER.

Table 1.

Serum cTNT levels in animals at 0, 10 and 20 d after injections of AGEs-BSA or control BSA ($\overline{x}\pm s$, μg/L)

Group	n	0 d	10 d	20 d
control	6	19.3±2.8	20.6±4.2	20.6±4.2
matrine	6	19.4±1.9a	22.7±5.6a	22.7±5.6a
INOCA	6	17.5±4.0b	17.8±6.1b	17.8±6.1b
INOCA+AGEs	6	18.8±2.9c	20.7±4.1c	20.7±4.1c
INOCA+AGEs+matrine	6	19.9±2.3d	20.6±2.1d	20.6±2.1d

Notes: control: treated with control BSA; matrine: treated with matrine at dosage of 200 mg/kg bodyweight; INOCA: INOCA model (ob/ob^-/- mice); INOCA + AGEs: INOCA model treated with AGEs at 1 mg/d; INOCA + AGEs + matrine: INOCA model co-treated with matrine and AGEs. BSA: bovine serum albumin; cTNT: cardiac troponin T; INOCA: ischemia with non-obstructive coronary artery disease; AGEs: advanced glycation end products. Compared with control, ^aP > 0.05; compared with control, ^bP > 0.05; compared with INOCA, ^cP > 0.05; compared with INOCA + AGEs, ^dP > 0.05. Intergroup comparisons were performed using the t-test, Mann-Whitney U test, or χ² test as appropriate. Post hoc comparisons were conducted using the SNK test.

Table 2.

CFV at rest, during hypereamia and their ratio ($\overline{x}\pm s$)

Group	n	CFVrest (cm/s)	CFVhypereamia (cm/s)	CFVR
Control	6	210.88±13.02	701.44±10.33	3.34±0.23
Matrine	6	208.42±18.54a	695.39±15.19a	3.37±0.33a
INOCA	6	212.64±12.76b	664.36±12.21e	3.14±0.19e
INOCA+AGEs	6	209.44±7.84c	584.45±15.61f	2.79±0.13f
INOCA+AGEs+matrine	6	214.35±7.74d	648.94±11.86g	3.03±0.12g

Notes: control: treated with control BSA; matrine: treated with matrine at dosage of 200 mg/kg bodyweight; INOCA: INOCA model (ob/ob^-/- mice); INOCA + AGEs:INOCA model treated with AGEs at 1 mg/d; INOCA + AGEs + matrine: INOCA model co-treated with matrine and AGEs. BSA: bovine serum albumin; INOCA: ischemia with non-obstructive coronary artery disease; GEs: advanced glycation end products; CFV: coronary flow velocity; CFVR: coronary flow velocity reserve. Intergroup comparisons were performed using the t-test, Mann-Whitney U test, or χ² test as appropriate. Post hoc comparisons were conducted using the SNK test. Compared with control, ^aP > 0.05, ^bP > 0.05, ^eP < 0.01; compared with INOCA, ^cP > 0.05, ^fP < 0.001; compared with INOCA + AGEs, ^dP > 0.05, ^gP < 0.001.

Table 3.

Blood biochemical markers determination in matrine-treated animals ($\overline{x}\pm s$)

Biomarker	Control (n = 6)	Martine treatment (n = 6)
ALT (U/L)	31.10±3.20	30.46±3.39a
AST (U/L)	99.10±5.27	101.08±6.31a
CRE (mmol/L)	26.70±1.76	25.26±4.45a
CK (U/L)	527.12±16.20	519.93±23.39a
CK-MB (U/L)	196.02±14.51	200.32±14.82a
Na+ (mmol/L)	130.71±3.07	129.91±1.51a
K+ (mmol/L)	4.81±0.45	5.14±0.16a
Cl- (mmol/L)	100.47±3.59	102.55±4.19a

Notes: control: treated with control BSA; matrine: treated with matrine at dosage of 200 mg/kg bodyweight. BSA: bovine serum albumin; ALT: glutamic-pyruvic transaminase; AST: glutamic-oxalacetic transaminease; CRE: creatinine; CK: creatine kinase; CK-MB: creatine kinase- muscle/brain isoenzyme. Differences between groups were analyzed using the t-test or Mann-Whitney U test as appropriate. Compared with control, ^aP > 0.05.

Table 4.

FITC-dextran leakage and TER of CEMCs ($\overline{x}\pm s$)

Group	n	FITC-Dextran leakage (fold of control)	TER (Ω·cm²)
Control	6	1.02±0.08	642.14±29.75
Matrine	6	0.97±0.06a	666.73±14.65a
AGEs	6	2.17±0.14b	325.75±9.47b
AGEs+matrine	6	1.43±0.04c	613.74±13.67c

Note: control: untreated CMECs; matrine: CMECs treated with matrine at a concentration of 1.0 mmol/L; AGEs: CMECs treated with AGEs at a concentration of 10 μmol/L; AGEs + matrine: CMECs co-treated with matrine and AGEs. FITC: fluorescein isothiocyanate; CMECs: cardiac microvascular endothelial cells; TER: transendothelial electrical resistance. Differences among groups were analyzed using the t-test or Mann-Whitney U test as appropriate. Compared with control, ^aP > 0.05, ^bP < 0.001; compared with AGEs, ^cP < 0.001.

3.2. Matrine treatment alleviated AGEs- induced coronary microcirculatory apoptosis, inflammation and microthrombosis

In the present study, we found that AGEs-BSA administration significantly increased AGEs content in cardiac tissue (supplementary Figure 1). Matrine treatment did not alter the matrine concentration in AGEs-exposed hearts. Matrine treatment significantly attenuated apoptosis of CMECs isolated from AGEs-exposed INOCA animals, as evidenced by TUNEL assay and flow cytometry (Figures 1A, 1D, supplementary Figures 2, 3A, 3B). Matrine administration also reduced pro-inflammatory lymphocytes infiltration in cardiac tissue (Figures 1B, 1E, supplementary Figure 4) and decreased the production of IL6, TNFα and the thrombogenic factor TXB2 from CMECs extracted from AGEs-exposed INOCA mice (supplementary Figures 5-7). Furthermore, immunofluorescent staining of CD42b-positive cells in cardiac sections (Figures1C, 1F, supplementary Figure 8) demonstrated that matrine treatment significantly reduced the proportion of active platelets in cardiac tissue sampled from AGEs-exposed INOCA mice.

Figure 1. Effect of matrine on AGEs-induced coronary microcirculatory apoptosis, inflammation, and microthrombosis.

A: images of isolated CMECs (×400), with TUNEL-positive cells indicated by red fluorescence in Control (A1), Matrine (A2), INOCA (A3), INOCA + AGEs (A4), INOCA + AGEs + Matrine (A5). Cell nuclei were stained with DAPI; D: apoptotic rate of CMECs calculated from TUNEL staining (n = 6); B: cardiac tissue sections stained for CD45 by immunofluorescence (× 400) in Control (B1), Matrine (B2), INOCA (B3), INOCA + AGEs (B4), INOCA + AGEs + Matrine (B5). Cell nuclei were stained with DAPI; E: mean fluorescence intensities of CD45 staining (n = 6); C: cardiac tissue sections stained for CD42b by immunofluorescence (× 400) in Control (C1), Matrine (C2), INOCA (C3), INOCA + AGEs (C4), INOCA + AGEs + Matrine (C5). Cell nuclei were stained with DAPI; F: mean fluorescence intensities of CD42b staining (n = 6). Model groups: Control: treated with control BSA; matrine: treated with matrine at dosage of 200 mg/kg bodyweight; INOCA: INOCA model (ob/ob^-/- mice); INOCA + AGEs: INOCA model treated with AGEs at 1 mg/d; INOCA + AGEs + matrine: INOCA model co-treated with matrine and AGEs. AGEs: advanced glycation end products; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; DAPI: 4,6-diamidino-2-phenylindole; CMECs: cardiac microvascular endothelial cells; IL6: interleukin 6; TNFα: tumor necrosis factor alpha; TXB2: thromboxane B2. Differences between two groups were compared using t-test. Data are presented as mean±standard deviation. Compared with control, ^aP > 0.05; compared with INOCA, ^bP < 0.001; compared with INOCA + AGEs, ^cP < 0.001.

3.3. Matrine treatment suppressed activation of ROS-mediated ER stress PERK/CaN/NFAT pathway in CMECs

In Figures 2A, 2B and Supplementary Figure 9, matrine administration effectively inhibited intracellular ROS generation in CMECs isolated from AGEs-exposed INOCA mice. In Figures 2C-2E, matrine treatment significantly decreased expression levels of ER stress markers including GRP78, ATF4, XBP1S and CHOP in CMECs isolated from AGEs-exposed INOCA mice. In Figures 2F-2J, matrine administration inhibited the phosphorylation levels of PERK and IRE1α in CMECs isolated from AGEs-exposed INOCA mice. In Figure 2K, matrine treatment significantly reduced CaN enzymatic activity in CMECs isolated from AGEs-exposed INOCA mice.

Figure 2. Effect of matrine on AGEs-induced ROS-mediated ER signaling in CMECs.

A: images of isolated CMECs (× 400) with DCFH-DA fluorescent staining in Control (A1), Matrine (A2), INOCA (A3), INOCA + AGEs (A4), INOCA + AGEs + Matrine (A5). Cell nuclei were stained with DAPI ; B: mean fluorescence intensities of DCFH-DA in CMECs (n = 6); C: immunoblots of phosphorylated PERK (p-PERK), PERK, phosphorylated IRE1 (p-IRE1), and IRE1 in CMECs isolated from animals; Relative phosphorylation levels of PERK (D) and IRE1 (E) were indicated by the columns (n = 6); F: immunoblots of GRP78, ATF4, XBP1s, CHOP, and GAPDH in CMECs; Columns indicate the relative expression levels of GRP78 (G), ATF4 (H), XBP1s (I), and CHOP (J) in isolated CMECs (n = 6); K: enzymatic activity of calcineurin in isolated CMECs (n = 6). Model groups: control: treated with control BSA; matrine: treated with matrine at dosage of 200 mg/kg bodyweight; INOCA: INOCA model (ob/ob^-/- mice); INOCA+AGEs: INOCA model treated with AGEs at 1 mg/d; INOCA + AGEs + matrine: INOCA model co-treated with matrine and AGEs. AGEs: advanced glycation end products; ROS: reactive oxygen species; DAPI: 4,6-diamidino-2-phenylindole; CMECs: cardiac microvascular endothelial cells; DCFH-DA: 2,7-dichlorofluorescein; PERK: protein kinase R-like endoplasmic reticulum kinase; IRE1: inositol-requiring enzyme 1; GRP78: glucose regulated protein 78; ATF4: activating transcription factor 4; XBP1s: X-box binding protein 1s; CHOP: C/EBP-homologous protein. Differences between two groups were compared using t-test. Data are presented as mean ± standard deviation. Compared with control, ^aP > 0.05; compared with INOCA, ^bP < 0.001; compared with INOCA + AGEs, ^cP < 0.001.

3.4. Matrine treatment inhibited NFAT- mediated pathways in CMECs isolated from AGEs-exposed INOCA mice

The effect of matrine on NFAT-mediated pathways are shown in Figure 3. Matrine treatment significantly inhibited NFATc4 nuclear translocation in CMECs isolated from AGEs-exposed INOCA mice, as shown in Figures 3A-3D and Supplementary Figure 10. Additionally, matrine treat-ment decreased the expression levels of cyclooxygenase 2 (COX2), IL6, Fas, FasL, and TNFα in CMECs isolated from AGEs-exposed INOCA mice (Figures 3E-3J).

Figure 3. Effect of matrine on NFAT-mediated pathways in CMECs.

A: immunofluorescent staining of NFATc4 in isolated CMECs in Control (A1), Matrine (A2), INOCA (A3), INOCA + AGEs (A4), INOCA + AGEs + Matrine (A5). Cell nuclei were stained with DAPI; B: NFATc4 nuclear translocation rate (n = 6); C: immunoblots of NFATc4 and histone H3 in nuclear protein extracted from CMECs; D: relative nuclear translocation of NFATc4 (n = 6); E: immunoblots of COX2, IL6, TNFα, Fas, FasL, and GAPDH in isolated CMECs; F-J: relative expression levels of COX2 (F), FAS (G), FASL (H), IL6 (I), and TNFα (J) (n = 6). Model groups: Control: treated with control BSA; matrine: treated with matrine at dosage of 200 mg/kg bodyweight; INOCA: INOCA model (ob/ob^-/- mice); INOCA + AGEs: INOCA model treated with AGEs at 1 mg/d; INOCA + AGEs + matrine: INOCA model co-treated with matrine and AGEs. AGEs: advanced glycation end products; DAPI: 4,6-diamidino-2-phenylindole; CMECs: cardiac microvascular endothelial cells; NFAT: nuclear factor of activated T-cells; COX2: cyclooxygenase 2; IL6: interleukin 6; TNFα: tumor necrosis factor alpha; FASL: FAS ligand. Differences between two groups were compared using t-test. Data are presented as mean ± standard deviation. Compared with control, ^aP > 0.05; compared with INOCA, ^bP < 0.001; compared with INOCA + AGEs, ^cP < 0.001; compared with INOCA + AGEs, ^dP < 0.01.

3.5. Concentration- dependent suppression of AGEs-induced PERK/NFAT signaling by matrine in primary CMECs

Primary CMECs were isolated from healthy mice and subsequently subjected to exposure to AGEs. In parallel, these cells were treated with varying concentrations of matrine. Additionally, we employed a PERK agonist, CC020312, to further probe the cellular responses. The efficacy of CC020312 in promoting PERK phosphorylation in CMECs was confirmed (Figures 4A, 4B). Results depicted in Figures 4C-4K, revealed a significant upregulation in the expression of GRP78, ATF4, CHOP, and the nuclear translocation of NFATc4, along with an increase in the phosphorylation of PERK and IRE1α in CMECs upon AGEs exposure. Interestingly, matrine treatment effectively suppressed these AGEs-induced changes, including PERK phosphorylation, nuclear translocation of NFATc4, and the expression of ATF4 and CHOP, in a concentration-dependent manner. Furthermore, our data suggest that the administration of CC020312 significantly reversed the suppressive effect of matrine on PERK phosphorylation, nuclear translocation of NFATc4, and the expression of ATF4 and CHOP in CMECs exposed to AGEs (Figures 4C-4K).

Figure 4. Effect of matrine on AGEs-induced PERK/NFAT signaling in primary CMECs.

A: immunoblots of p-PERK, PERK, and GAPDH in primary CMECs treated with CCT020312; B: relative phosphorylation levels of PERK (n = 3). Compared with control, ^aP < 0.05; C: immunoblots of NFATc4 and histone H3 in AGEs-exposed primary CMECs treated with matrine and/or CCT020312; D: relative nuclear levels of NFATc4 (n = 3); E: immunoblots of GRP78, p-PERK, PERK, p-IRE1α, IRE1α, XBP1s, ATF4, CHOP, and GAPDH in AGEs-exposed primary CMECs treated with matrine and/or CCT020312; F: relative expression level of GRP78 (n = 3); G: relative phosphorylation level of IRE1α (n = 3); H: relative expression level of XBP1s (n = 3); I: relative phosphorylation level of PERK (n = 3); J: relative expression level of ATF4 (n = 3); K: relative expression level of CHOP (n = 3). Model groups: control: untreated primary CMECs; primary CMECs treated with CCT020312 at 10 μmol/L. AGEs: advanced glycation end products; CMECs: cardiac microvascular endothelial cells; NFAT: nuclear factor of activated T-cells; PERK: protein kinase R-like endoplasmic reticulum kinase; IRE1: inositol-requiring enzyme 1; GRP78: glucose regulated protein 78; ATF4: activating transcription factor 4; XBP1s: X-box binding protein 1s; CHOP: C/EBP-homologous protein. Differences between two groups were compared using t-test. Data are presented as mean ± standard deviation. Compared with control CMECs, ^aP > 0.05; compared with CMECs treated with matrine at 1.0 mmol/L, ^bP < 0.05; compared with CMECs treated with AGEs, ^cP < 0.05; compared with CMECs co-treated with matrine at 0.25 mmol/L and AGEs, ^dP < 0.05; compared with CMECs co-treated with matrine at 0.5 mmol/L and AGEs, ^eP < 0.05; CMECs co-treated with matrine at 1.0 mmol/L and AGEs, ^fP < 0.05.

4. DISCUSSION

Coronary angiography often reveals slow or no flow during acute coronary syndrome, and severe cardiac ischemia can occur in individuals without significant coronary artery stenosis in chronic ischemic heart disease, a condition known as INOCA. In this study, we used a previously established INOCA animal model and induced CMD by exposing animals to AGEs, as demonstrated by CFVR. Importantly, we found that matrine treatment significantly improved AGEs-induced CMD in INOCA animals and AGEs-exposed CMECs, primarily through the suppression of ER stress PERK signaling as one of the underlying molecular mechanisms.

As typical toxic metabolites of diabetes, AGEs stimulate ER stress. Our previous studies, along with others, have demonstrated a strong association between AGEs and diabetic cardiovascular complications.³⁴ Specifically, circulating AGEs interact with the receptor for AGEs (RAGE) in individuals with diabetes, inducing ROS generation by activating mitochondrial NADPH oxidase (NOX).³⁵ In the current study, we found that AGEs exposure led to intracellular ROS generation in CMECs.⁹ We also observed a significant upregulation of key markers of ER stress, including p-IRE1, p-PERK, XBP1s, ATF4, and CHOP, indicating that ROS-mediated ER stress through the PERK signaling pathway was significantly activated in CMECs isolated from INOCA animals exposed to AGEs.

As a downstream effector of PERK, the enzymatic activity of CaN increases upon direct interaction with phosphorylated PERK.³⁶ Activated CaN further promotes NFATc4 nuclear translocation to regulate the expression of its target genes related to apoptosis, inflammation, and thrombogenesis.^37-39 NFATc4 induces cell apoptosis via the formation of the Fas/FasL complex, which recruits Fas-associated death domain proteins (FADD).⁴⁰ The CaN/NFATc4 signaling pathway promotes the production of several inflammatory cytokines, such as ILs and TNFs.⁴¹ Clearance of these cytokines has been shown to improve cell function and alleviate remodeling.^42,43 Furthermore, NFATc4 can increase COX2 expression, facilitating platelet activation and thrombosis by generating TXA2 during the processing of arachidonic acid (AA).⁴⁴ Previous studies have suggested that endothelial NOX-induced ROS generation could modulate CD45+ lymphocyte infiltration into the microvascular network, thereby exacerbating local inflammation.^45,46 In this study, we found that AGEs exposure mediated ROS-induced CD45+ immune cell infiltration into the CMECs. Therefore, AGEs exposure significantly induced apoptosis, inflammation, and thrombogenesis in CMECs, exacerbating CMD in INOCA animals.

Matrine (C₁₅H₂₄N₂O) is a bioactive component extracted from the Chinese medical herb, Kushen (Radix Sophorae Flavescentis). Our previous studies have demonstrated the therapeutic effect of matrine on diabetes-associated cardiovascular diseases, such as heart failure, cardiac fibrosis, and atherosclerosis. Our recent investigation has also shown that matrine exerts an anti-oxidative stress effect by suppressing DNA methylation of the promoter of glutathione peroxidase 1, a critical antioxidant.⁴⁷ In the current study, we found that matrine significantly reduced intracellular ROS generation in CMECs isolated from AGEs-exposed INOCA animals. Matrine also inhibits oxidative stress-mediated ER stress PERK signaling, which in turn suppressed activation of the CaN/NFATc4 pathway.^19,48 As a result, apoptosis and pro-inflammatory cytokines production in CMECs were effectively attenuated, as well as pro-inflammatory lymphocyte infiltration. Matrine treatment was also shown to decrease the activation of platelets in cardiac sections, as indicated by the decreased expression of CD42 and lowered TXB2 levels, both markers for platelets activation.⁴⁹ Notably, no serious side effect of matrine were observed in this study. As a potential drug demonstrating therapeutic potential in various diseases including cancer, where matrine induces cell toxicity against cancer cells.⁵⁰ While matrine's benefits are significant, its use is also associated with certain toxic effects, and further research is needed to fully understand its toxicity profile and develop strategies for minimizing potential side effects.⁵¹

In comparison with previous studies, our research contributes to the growing body of literature on the therapeutic effect of matrine in cardiovascular diseases. Our study extends these findings by demonstrating the potential of matrine to alleviate AGEs-induced CMD in INOCA, a condition that has not been extensively explored in the context of matrine treatment. Furthermore, our findings align with the existing literature on the antioxidative stress effect of matrine. However, it is important to note that our study focused on a specific signaling pathway (ERS PERK/CaN/ NFATc4), and further research is needed to fully understand the mechanisms underlying matrine's therapeutic effects. In conclusion, our study provides a novel perspective on the therapeutic potential of matrine in treating AGEs-induced CMD in INOCA. Future studies should aim to further elucidate the mechanisms underlying matrine's therapeutic effects and explore its potential in other cardiovascular diseases.

Despite the promising findings, our study has several limitations. First, it was primarily conducted in a mouse model, and the results may not fully translate to human disease due to species differences. Second, while we focused on the ER stress PERK/CaN/NFATc4 signaling pathway, other pathways may also contribute to the therapeutic effect of matrine on CMD in INOCA. Future studies should investigate these additional mechanisms. Third, the long-term effects and potential side effect of matrine treatment require further exploration. This study addresses an important gap by examining the effect of matrine on AGEs-induced CMD in INOCA, a topic that has been relatively underexplored. However, additional research, particularly human clinical trials, is needed to confirm and expand upon these findings.

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.com.

Funding Statement

Supported by National Natural Scientific Foundation of China: Mechanisms of Macrophage-Mediated Vascular Smooth Muscle Cells Phenotypic Conversion in Advanced Glycation End Products-induced Atherosclerosis and Therapeutic Effects of Targeted Gene Silencing (82070858);Youth Scientific Research and Innovation Team Program of Shaanxi Province: Diabetes-Related Atherosclerosis Basic Research and Application Research Team (2022-SLRH-LJ-014)