Impact of gardenia extract on coagulation function in rats with acute myocardial ischemia model

Abstract

Objective

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This study aims to investigate the effects of gardenia extract (GE) on coagulation function in a rat model of acute myocardial ischemia (AMI).

Methods

Healthy male SD rats were randomly divided into five groups: Sham, AMI, GE-L (low-dose GE), GE-M (medium-dose GE), and GE-H (high-dose GE). Two weeks later, echocardiography was performed to assess cardiac function, including left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic diameter (LVEDD), and left ventricular end-systolic diameter (LVESD). Hematoxylin-eosin (HE) staining and TUNEL staining were implemented to observe myocardial pathological changes and apoptosis, respectively. Enzyme-linked immunosorbent assay (ELISA) was employed to measure serum levels of inflammatory factors and oxidative stress markers. Coagulation function was evaluated using an automatic coagulation analyzer, with parameters including prothrombin time (PT), thrombin time (TT), prothrombin time ratio (PTR), international normalized ratio (INR), and fibrinogen level (FIB).

Results

Compared with the Sham group, the AMI group exhibited myocardial injury, coagulation dysfunction, impaired cardiac function, and significantly increased levels of inflammation, oxidative stress, and PTR. In contrast, all GE dose groups showed elevated TT, PT, FIB, and INR compared to the AMI group.

Conclusion

GE can reverse the cardiac function, inflammatory and oxidative stress indicators, as well as coagulation function in AMI model rats, thereby improving AMI.

Introduction

Coagulation function is a vital physiological process that ensures the formation of blood clots to prevent excessive bleeding and maintain vascular integrity. This intricate process involves a cascade of interactions between endothelial cells, platelets, von Willebrand factor, and coagulation factors [1]. Diagnostic methods for assessing coagulation function include measuring parameters such as prothrombin time (PT), activated partial thromboplastin time, antithrombin activity, fibrinogen, and fibrin degradation product, which provide insights into the body’s clotting ability [2]. Dysregulation of coagulation function can lead to severe complications, which are often associated with conditions like preeclampsia or acute myocardial ischemia (AMI). AMI is a relatively common disease with severe myocardial damage, resulting in an imbalance in oxygen demand between the blood and myocardium, accompanied by significant coagulation dysfunction and thrombosis [3,4,5]. Studies have shown that during the early stages of AMI, platelet activation, fibrin deposition, and excessive release of procoagulant factors all participate in the process of myocardial ischemia-reperfusion injury, further exacerbating myocardial cell necrosis and dysfunction [6,7,8]. Therefore, interventions targeting the coagulation mechanism hold significant importance in the treatment of AMI.

Gardenia extract (GE), derived from gardenia jasminoides, has been widely recognized for its anti-diabetic, anti-depressive, anti-oxidative, and anti-inflammatory properties [9]. These pharmacological effects make GE a promising candidate for mitigating conditions associated with inflammation, oxidative stress, and coagulation dysfunction. Modern research indicates that GE is rich in active ingredients such as flavonoids, iridoids, and organic acids [10, 11]. Some of these components have been confirmed to possess antithrombotic, anticoagulant, and microcirculation-improving functions [11,12,13]. Given the interplay between inflammation, oxidative stress, and coagulation, GE’s potential to improve coagulation function in pathological conditions such as AMI warrants further investigation. GE has also demonstrated strong protection against blue light damage and the prevention of premature aging through its melatonin-like properties [14]. These findings suggest that GE could be a valuable therapeutic agent for conditions involving oxidative stress and inflammation, which are often linked to coagulation dysfunction. This study aims to investigate whether GE, in addition to its myocardial protective effects, can alleviate hypercoagulability by regulating the coagulation/fibrinolytic system in an AMI model, thereby exerting a comprehensive multi-target protective effect.

Materials and methods

Ethical approval

The study was under the approval of the Ethic Committee of Zhuzhou Central Hospital and followed the tenets of the Declaration of Helsinki/in accordance with the Declaration of Helsinki. All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals.

Preparation of GE

Two kilograms of dried Gardenia flowers, purchased from Yuanchunlin Pharmacy in Zhuhai, China, were added to 12 times their weight of water and decocted twice, each time for one hour. The decoction was filtered while hot, and the filtrates were combined. The combined filtrate was concentrated using a rotary evaporator (Model 170036, Yarong Biochemical Instrument Factory, Co., Ltd., China, Shanghai) to a relative density of approximately 1.05 and then filtered again. The filtrate was passed through HPD-100 macroporous adsorption resin. Initially, the resin was washed with water until the eluate became clear, removing water-soluble impurities. Subsequently, gradient elution was performed using 10%, 30%, 50%, and 75% ethanol. The eluates from each ethanol concentration were collected, and the ethanol was recovered under reduced pressure and concentrated into a thick paste. Finally, the paste was dried under vacuum or freeze-dried to produce the dry extract, completing the preparation of GE [15].

AMI model establishment and drug administration

In this experiment, 60 male Sprague-Dawley (SD) rats aged 8–10 weeks, weighing between 200 ± 20 g, were supplied by SLAC Laboratory Animal Co., Ltd. (China, Shanghai). The rats were housed under controlled conditions with a temperature of 22 ± 1 °C, relative humidity of 55 ± 2%, and a 12-hour light/dark cycle, with free access to food and water. After one week of acclimatization, the rats were randomly divided into five groups (n = 12 per group): the sham group, the AMI group, the high-dose GE group (GE-H), the medium-dose GE group (GE-M), the low-dose GE group (GE-L), and the positive control group (Heparin, 100 IU/kg). For the AMI model establishment, rats in the five groups were anesthetized via intraperitoneal injection of 10% chloral hydrate solution at a dose of 0.3 ml per 100 g body weight. Once anesthetized, a left intercostal incision was made to open the thoracic cavity, exposing the heart. The left anterior descending coronary artery (LAD) was identified between the pulmonary artery cone and the left atrium, and a 0 suture was used to ligate the LAD 3 mm from its origin. After ligation, the heart was quickly repositioned into the thoracic cavity, and the chest was gently compressed to expel residual blood. The thoracic wall was then sutured, successfully establishing the AMI model. For the Sham group, rats underwent the same anesthesia and thoracotomy procedures, but the heart was only exposed and immediately sutured without any further manipulation, simulating the sham operation.

Twenty-four hours after model establishment, rats in the GE-L, GE-M, and GE-H groups were administered GE at doses of 0.14, 0.28, and 0.56 g·kg⁻¹, respectively, via oral gavage. The doses were referenced from the application dose range of GE in rat models in previous literature [16]. Rats in the Sham and AMI groups received an equivalent volume of distilled water. The gavage was continued for two weeks. To ensure the repeatability of the experiment, the GE used in this study was all purchased from a unified source and prepared in the same batch. The doses were calculated based on the weight of the dry extract, and the gavage volume was controlled within 10 mL/kg [17].

Echocardiographic assessment of cardiac function

Twenty-four hours after the completion of oral gavage administration, the rats were anesthetized and placed in a supine position on a temperature-controlled heating platform. Their limbs and head were gently secured with tape, and the platform angle was adjusted to fully expose the chest, facilitating the placement of the ultrasound probe. Using the built-in analysis software of the echocardiography system (Consona AT, Mindray), the endocardial border of the left ventricle was manually traced on the end-diastolic image in the long-axis view. The software automatically calculated the left ventricular end-diastolic dimension (LVEDD). The same procedure was repeated at end-systole to obtain the left ventricular end-systolic dimension (LVESD). The left ventricular fractional shortening (LVFS) was calculated using the formula: LVFS = (LVEDD - LVESD)/LVEDD × 100%. The left ventricular ejection fraction was calculated as: LVEF = (LVEDV - LVESV)/LVEDV × 100%, where LVEDV and LVESV represent the left ventricular end-diastolic volume and end-systolic volume, respectively, automatically computed by the instrument software based on the measured dimensions. Each measurement was repeated three times, and the average value was taken as the final result.

Hematoxylin-eosin (HE) staining

After completing echocardiography, three rats from each group were selected and deeply anesthetized with chloral hydrate. The hearts were excised, and rinsed with PBS buffer at 4 °C, and the anterior wall of the left ventricle below the ligation line was collected. The tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 μm slices. The sections were sequentially dewaxed in xylene I and II, rehydrated through a graded ethanol series (100%, 95%, 80%, 75%) and distilled water, stained with hematoxylin for 5 min, rinsed with distilled water, and then stained with eosin for 2 min. Following dehydration through a graded ethanol series and clearing, the sections were mounted with neutral balsam and allowed to air-dry overnight. Microscopic examination and imaging were performed using a microscope (Carl Zeiss, Germany) [15].

TUNEL staining

After echocardiography was performed, three rats from each group were selected and deeply anesthetized with chloral hydrate. The hearts were excised, rinsed with PBS buffer at 4 °C, and the anterior wall of the left ventricle below the ligation line was collected. The tissue was fixed in 4% paraformaldehyde and then preserved in 70% ethanol. The TUNEL kit (11684817910, Roche) was prepared. The tissue was embedded in paraffin, sectioned, dewaxed, subjected to antigen retrieval, and permeabilized. It was then sequentially incubated with equilibration buffer and TUNEL reaction mixture, washed with PBS, blocked, and stained with DAB. The tissue was dyed with DAPI for 15 min under dark conditions. After that, apoptosis was observed under a fluorescence microscope (Leica, Germany). For each sample, five separate observation areas were selected. Cells with clearly marked nuclei were defined as TUNEL-positive cells. The number of TUNEL-positive nuclei and total nuclei was counted, and the apoptosis index was defined as the ratio of TUNEL-positive nuclei to the total number of nuclei [18, 19].

Enzyme-linked immunosorbent assay (ELISA) detection

Following the completion of echocardiography, the rats (the aforementioned six rats used for heart collection) were anesthetized, and blood was drawn by inserting a syringe needle into the cardiac region of the rats. The blood volume collected was controlled to approximately 3–5 milliliters. The collected blood samples were placed in clean centrifuge tubes and allowed to stand at room temperature for 30 min. After complete coagulation, the samples were centrifuged at 3000 revolutions per minute for 15 min to separate the serum. Operations were carried out in accordance with the instructions of the antithrombin III (AT-III) ELISA kit (Elabscience, E-EL-R0386c), the D-dimer (DD) ELISA kit (Jiangsu Meimian, MM-31262R), the platelet activation marker CD62P (P-selectin) ELISA kit (CUSABIO, CSB-E08404r), the tumor necrosis factor-α (TNF-α) ELISA kit (E-CL-R0019), Interleukin-6 (IL-6) ELISA kit (E-CL-R0015), and IL-1β ELISA kit (E-CL-R0012 (all from ARP American Research Products, Inc., USA). The absorbance was ultimately measured at a wavelength of 450 nm using a microplate reader, and the concentrations of the inflammatory factors IL-6, TNF-α, and IL-1β in the serum were calculated based on the standard curve.

The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) were measured according to the instructions provided by the respective assay kits: SOD (BYS-11927B, Boyan Biotechnology Co., Ltd., China, Zhenjiang), MDA (SHYY-2487, Yiyan Biotechnology Co., Ltd., China, Shanghai), and GSH-Px (CSB-E12146r, Shunyuan Biotechnology Co., Ltd., China, Shanghai). The detection wavelengths for SOD, MDA, and GSH-Px were typically set at 550 nm, 532 nm, and 412 nm, respectively [20].

Measurement of coagulation parameters

Following the completion of echocardiography, the remaining six rats in each group were anesthetized. A syringe needle was inserted into the cardiac region of the rats to collect blood, with the volume controlled at approximately 3–5 milliliters. The collected blood samples were immediately transferred into chilled plastic tubes containing EDTA and stored at 4 °C for 30 min. Subsequently, the samples were centrifuged using a refrigerated centrifuge at 3000 rpm for 15 min at 4 °C to separate the plasma. The processed rat plasma samples were then placed on the sample platform of a fully automated coagulation analyzer (CS-5100, Sysmex Corporation, Japan). Following the manufacturer’s instructions, the following coagulation parameters were measured: PT, thrombin time (TT), prothrombin time ratio (PTR), international normalized ratio (INR), and fibrinogen level (FIB).

Peripheral blood complete blood count (CBC) detection

After the experiment, fresh peripheral blood samples (approximately 0.5 mL) were collected from rats in each group by orbital blood collection. The samples were immediately added to blood collection tubes containing EDTA-K2 anticoagulant and gently mixed to prevent coagulation. After blood collection, the samples were tested within 1 h using an automatic blood cell analyzer (such as Mindray BC-5000Vet, Shenzhen Mindray Bio-Medical Electronics Co., Ltd.).

The detection parameters included: white blood cell count (WBC, ×10⁹/L), red blood cell count (RBC, ×10¹²/L), hemoglobin (HGB, g/L), hematocrit (HCT, %), mean corpuscular volume (MCV, fL), platelet count (PLT, ×10⁹/L), etc.

Statistical analysis

All data were expressed as mean ± standard deviation. The t-test was used for statistical analysis between two groups, and One-Way ANOVA, followed by Tukey’s post hoc analysis was employed to compare more than two groups. All experiments were repeated three times. A P-value < 0.05 was considered statistically significant. All tests were performed using GraphPad software version 8.0.

Results

GE improves cardiac function in AMI rats

Echocardiography was used to evaluate the left ventricular function of rats. The results showed that compared with the Sham group, the LVEF and LVFS in the AMI group decreased, suggesting impaired cardiac systolic function (Fig. 1A, B). Meanwhile, the LVEDD and LVESD significantly increased (Fig. 1C, D), indicating ventricular dilatation and structural remodeling. Compared with the AMI group, the LVEF and LVFS in the high-, medium-, and low-dose GE groups and the heparin positive control group showed a dose-dependent increasing trend, while the LVEDD and LVESD showed a dose-dependent decreasing trend. This suggests that GE has the potential to improve cardiac function and alleviate myocardial injury and left ventricular remodeling induced by AMI.

Fig. 1

GE improves cardiac function in AMI rats. A–D. Echocardiographic assessment of LVEF (A), LVFS (B), LVEDD (C), and LVESD (D). ns no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001

GE ameliorates myocardial pathological conditions and apoptosis in AMI rats

To further evaluate the protective effect of GE on myocardial tissue injury in AMI rats, HE staining and TUNEL staining were used to analyze the pathological changes and apoptosis of myocardial tissue.

HE staining results showed (Fig. 2A): In the Sham group, the myocardial muscle bundles were arranged regularly, and no inflammatory cell infiltration or vascular dilation was observed. In the AMI group, the myocardial tissue structure was damaged, with disordered arrangement of muscle bundles, partial myofiber rupture, accompanied by significant inflammatory cell infiltration, vascular dilation, interstitial edema, and myocardial fibrosis. Although the GE-L group still showed disordered arrangement of muscle bundles and inflammatory cell infiltration, the degree of myocardial fibrosis and edema was significantly reduced compared with the AMI group. The pathological changes in the GE-M group were further improved, with reduced inflammatory response and structural disorder. The myocardial tissue structure in the GE-H group and the heparin group basically returned to normal, with relatively neat arrangement of muscle bundles and only mild vascular dilation and slight fibrosis, suggesting that both high-dose GE and heparin could effectively alleviate myocardial pathological damage induced by AMI.

Fig. 2

GE ameliorates myocardial pathological conditions and apoptosis in AMI rats. A: HE staining to detect myocardial pathological conditions in rats; B: TUNEL staining to detect myocardial cell apoptosis in rats; C: Quantification of the apoptosis index. *P < 0.05, **P < 0.01, ***P < 0.001

TUNEL staining results further verified the inhibitory effect of GE on myocardial cell apoptosis (Fig. 2B, C). Compared with the Sham group, myocardial cell apoptosis in rats in the AMI group significantly increased. In the GE intervention groups with different doses, the number of apoptotic cells showed a dose-dependent decrease, especially in the GE-H group, which was similar to the effect of the heparin group. This suggests that GE can effectively inhibit myocardial cell apoptosis induced by AMI and play a tissue-protective role.

GE inhibits inflammation and oxidative stress levels in AMI rats

Studies have shown that AMI is closely related to inflammatory response and oxidative stress, and the two factors interact as both cause and effect in the occurrence and development of AMI, forming a vicious cycle [21]. To further investigate the intervention effect of GE on the inflammation and oxidative stress status induced by AMI, this study used the ELISA method to detect the levels of typical inflammatory factors (TNF-α, IL-1β, IL-6) and oxidative stress-related indicators (MDA, SOD, GSH-Px) in rat serum.

The results showed (Fig. 3A, B): Compared with the Sham group, the levels of TNF-α, IL-1β, IL-6, and MDA in the serum of rats in the AMI group increased, while the levels of SOD and GSH-Px decreased, suggesting that myocardial ischemia induced systemic inflammatory response and oxidative damage. After GE intervention, all dose groups showed good anti-inflammatory and antioxidant effects, with the high-dose GE group showing the most significant effect, which could significantly down-regulate the levels of TNF-α, IL-1β, IL-6, and MDA, and significantly increase the activities of SOD and GSH-Px. The heparin group also showed a similar trend, further supporting the protective potential of GE in regulating inflammation and oxidative stress.

Fig. 3

GE inhibits inflammation and oxidative stress levels in AMI rats. A: Expression of inflammatory factors TNF-α, IL-1β, and IL-6 in rat serum was detected using ELISA kits. B: Levels of oxidative stress markers MDA, SOD, and GSH-Px in rat serum were measured using kits. *P < 0.05, **P < 0.01, ***P < 0.001

GE improves coagulation function in AMI rats

Finally, we explored the effect of GE on the coagulation function of AMI rats. We found that compared with the Sham group, AMI rats showed an increase in PTR and a hypercoagulable state. After GE administration, TT, PT, INR, and FIB significantly increased in the medium- and high-dose groups, suggesting that GE has a delaying effect on the coagulation cascade and can improve the hypercoagulable state. The effect of the heparin group was more obvious, with all coagulation parameters showing a significant trend towards anticoagulation (Fig. 4A–E).

Fig. 4

GE improves coagulation function in AMI rats. A: TT measured by an automated coagulation analyzer; B: PT measured by an automated coagulation analyzer; C: PTR measured by an automated coagulation analyzer; D: INR was measured using a fully automated coagulation analyzer; E: FIB was measured using a fully automated coagulation analyzer; F: Expression level of CD62P; G: Level of DD; H: Expression level of the platelet activation marker AT-III. ***P < 0.001 vs. Sham group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AMI group

To evaluate the regulatory effect of GE on hematological indicators in rats with AMI, we analyzed the peripheral blood CBC indicators of the Sham group, AMI group, GE-H group, GE-M group, GE-L group, and heparin positive control group. Compared with the Sham group, the RBC, HGB, and HCT in the AMI group slightly increased, although the difference was not statistically significant, suggesting a possible compensatory response to the hypoxic state. Notably, the PLT and mean platelet volume in the AMI group significantly increased, suggesting that AMI induced platelet activation and a potential hypercoagulable state. After GE intervention, especially in the medium- and high-dose groups, the levels of PLT and MPV significantly decreased and approached the levels in the Sham group, showing a good anti-platelet aggregation effect. The heparin group also showed a significant platelet-lowering effect, verifying the results of this study as a positive control. In addition, the changes in MCV, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration among the groups were not significant, indicating that GE and heparin had little effect on red blood cell parameters (Table 1).

Table 1 Peripheral blood complete blood count indicators of rats in different treatment groups

To further verify the effect of GE on the coagulation status of rats with the AMI model, we detected the expression levels of AT-III, DD, and the platelet activation marker CD62P in peripheral blood by the ELISA method. The results showed that compared with the Sham group, the plasma level of AT-III in rats in the AMI group significantly decreased, suggesting a decrease in coagulation inhibition ability. Meanwhile, the level of DD significantly increased, reflecting that the body was in a state of continuous fibrinolysis and thrombosis. In addition, the level of CD62P also significantly increased, indicating that AMI induced significant platelet activation. After GE intervention, all indicators showed a significant improvement trend. Especially in the medium- and high-dose GE groups, the level of AT-III significantly increased, while the levels of DD and CD62P significantly decreased, showing that GE has certain anticoagulant and anti-platelet activation abilities. Among them, the high-dose GE group showed the most significant improvement effect, close to the performance of the heparin positive control group (Fig. 4F–H).

In summary, GE can alleviate the hypercoagulable state induced by AMI from multiple aspects by increasing the expression of anticoagulant proteins, reducing fibrin degradation products and platelet activation levels, and has potential antithrombotic and cardiovascular protective effects.

Discussion

AMI has become one of the key reasons for the increase in morbidity and mortality globally [5]. Previous research has established that abnormal coagulation parameters strongly correlate with poor prognosis in cardiovascular diseases. The complex interactions between platelets and coagulation factors are fundamental to maintaining hemostasis, where disruptions can lead to severe complications [1]. Studies have shown that disseminated intravascular coagulation appears in most deaths related to cardiovascular conditions [22]. Our study investigated the therapeutic potential of GE on coagulation function in AMI rats.

The results of this study not only validate previous findings regarding the pharmacological activity of GE but also provide important evidence for its use as a novel natural drug for anticoagulant therapy in AMI. At the molecular level, GE exerts multi-target anticoagulant effects through the following pathways: (1) significantly downregulating PLT and MPV, inhibiting platelet activation and aggregation, with effects comparable to those of heparin; (2) regulating coagulation parameters (TT, PT, FIB, and INR) to improve the balance of the coagulation-fibrinolytic system; (3) increasing the expression of anticoagulant proteins and reducing fibrin degradation products. This multi-pathway synergistic effect enables GE to effectively alleviate the hypercoagulable state induced by AMI, with a mechanism of action distinct from traditional anticoagulant drugs (e.g., heparin primarily acts on AT-III [23], while aspirin selectively inhibits platelet cyclooxygenase [24, 25], demonstrating more comprehensive coagulation regulatory properties.

Moreover, GE’s anti-inflammatory effects parallel earlier research demonstrating gardenia’s capacity to modulate inflammatory responses [26]. This is particularly significant given the well-established link between myocardial inflammation, cell apoptosis, fibrosis, neurogenesis and tryptophan [27]. In AMI, the inflammatory cascade triggered by myocardial injury exacerbates coagulation dysfunction, creating a vicious cycle that worsens outcomes. It is particularly noteworthy that while exerting anticoagulant effects, GE also breaks the vicious cycle of “coagulation-inflammation-oxidative stress” by reducing oxidative stress (lowering MDA levels and increasing SOD levels) and inhibiting the release of inflammatory factors. This multi-effect protective action gives GE unique advantages in the treatment of AMI: it can avoid the increased bleeding risk associated with drugs like heparin while improving the tendency towards thrombosis exacerbated by inflammation and oxidative damage. Experimental results showed that high-dose GE approached the efficacy of the heparin positive control group in improving coagulation function and reducing myocardial injury. Some studies emphasize the potent antioxidant properties of GE [28] and its ability to inhibit inflammation [29]. Furthermore, the main active ingredients in GE, such as geniposide and genipin, have been reported to possess vascular protective and antiplatelet aggregation effects in addition to anti-inflammatory and antioxidant properties, suggesting that GE has potential as a candidate drug for anti-myocardial ischemia with a composite mechanism [30].

In conclusion, our research demonstrates that GE can reverse cardiac function, inflammation, oxidative stress markers, and coagulation function in AMI model rats, thereby improving AMI. These findings provide new insights into the development of natural product-based antithrombotic strategies. Through molecular mechanisms such as regulating the fibrin network structure, inhibiting platelet activation, and modulating the expression of coagulation-related genes, GE has the potential to become a candidate for adjuvant therapy in AMI, particularly for patient populations requiring long-term anticoagulant therapy but at high risk of bleeding. Future research directions should encompass detailed investigations of the mechanisms underlying GE’s effects. Additionally, clinical trials validating these findings in human subjects are essential to establish the safety, efficacy, and optimal dosing of GE in AMI patients. Exploration of potential synergistic effects with conventional treatments could further enhance its therapeutic utility. These findings establish a foundation for developing natural compound-based therapeutic strategies for cardiovascular diseases, particularly finding a potential therapeutic target in AMI.