Introduction
Acute myocardial infarction (AMI) is a prevalent and severe form of heart disease, and vascular recanalization therapy is widely employed as a primary treatment for AMI [
1]. However, the occurrence of myocardial ischemia–reperfusion injury (MIRI) significantly hampers the success rate of treating AMI and may even exacerbate the deterioration of myocardial function, posing a grave threat to patient's health and life [
2]. However, there is no recommended treatment specifically for MIRI [
3,
4].
MIRI usually leads to myocardial mitochondrial damage and dysfunction. In the presence of mitochondrial dysfunction, nuclear DNA damage, or bacterial invasion, the resulting DNA fragments are recognized by the cyclic GMP-AMP synthase (cGAS) enzyme in the cytoplasm [
5]. This leads to the activation of cGAS, which generates a molecule called 2′3’-cyclic GMP-AMP (cGAMP) as a second messenger. Subsequently, cGAMP binds to the stimulator of interferon genes (STING) protein in the endoplasmic reticulum, triggering the activation of STING [
6]. STING activates TANK-binding kinase 1 (TBK1), which proceeds to phosphorylate interferon regulatory factor 3 (IRF3) [
7]. Upon translocation from the cytoplasm to the nucleus, IRF 3 interacts with other transcription factors, leading to the upregulation of interferon and other genes associated with immune responses, thereby amplifying the cellular immune response [
8]. The STING signaling pathway plays a critical role in intracellular immune responses [
9,
10].
It reported that the STING signaling pathway in cardiomyocytes becomes excessively activated in cardiovascular diseases [
11,
12]. This abnormal activation may result from mitochondrial dysfunction caused by oxidative stress, which leads to the release of mitochondrial DNA (mtDNA), or result from nuclear DNA damage that allows DNA fragments to enter the cytoplasm [
13,
14]. In either case, this further exacerbates the damage to the cardiomyocytes. Thus, targeting the STING signaling pathway could be a potential strategy for treating MIRI [
13]. Several studies have demonstrated that inhibiting the activity of cGAS or STING can reduce the inflammatory response and damage caused by ischemia–reperfusion injury [
15,
16].
Traditional Chinese medicine (TCM) has accumulated extensive experiences in the treatment of cardiovascular diseases, commonly using the principle of "Replenishing Qi and activating blood" in TCM [
17].
Astragalus membranaceus (Fisch.) Bunge ("Huangqi" in Chinese), representing the "Replenishing Qi" category, and
Salvia miltiorrhiza Bunge("Danshen" in Chinese) representing the "activating blood" category, are well-known traditional Chinese medicines [
18‐
20]. The combined use of
Astragalus membranaceus (Fisch.) Bunge and
Salvia miltiorrhiza Bunge, along with other traditional Chinese medicals, can have a synergistic effect in treating cardiovascular diseases [
21]. Tanshinone IIA (Ta-IIA), the main active ingredient in
Salvia miltiorrhiza Bunge [
19,
22,
23], and Astragaloside IV (As-IV), the main active ingredient in
Astragalus membranaceus (Fisch.) Bunge [
24,
25], have antioxidant, anti-inflammatory properties, and are widely used in the treatment of cardiovascular diseases in clinic in China [
26,
27]. In addition, Ta-IIA can inhibit platelet aggregation and prevent blood clots formation [
19], As-IV is capable of regulating immune function by modulating the polarization of macrophages [
28]. Therefore, Ta-IIA and As-IV play pivotal roles in the treatment of cardiovascular diseases including MIRI, potentially with a synergistic effect. However, further research and clinical validation are necessary to identify for the specific treatment regimens and mechanisms.
The objective of this study is to compare the effects of As-IV, Ta-IIA, and the combination of As-IV and Ta-IIA (Co) in the treatment of MIRI, further elucidate the mechanism underlying the effects of As-IV, Ta-IIA and Co in treating MIRI, and investigate the involvement of the STING signaling pathway in MIRI.
Materials and methods
Materials
Tanshinone IIA (CAS NO:568-72-9) and Astragaloside IV (CAS NO: 84687-43-4) were purchased from Chengdu Herbpurify Co., Ltd (Chengdu, China). The diABZI STING agonist (Compound 3)(CAS NO: 2138498-18-5) was acquired from Selleck Chemicals (Houston, USA). Other important materials, including reagents, compounds, reagent kits, etc., can be found in Additional file
2: Tables S1 and S2.
Animal management and treatment
In this study, 8-9 week-old male C57BL/6 mice were obtained from Vital River (Beijing Vital River Laboratory Animal Technology Co., Ltd., China). The mice underwent acclimated and were housed in a specific-pathogen-free breeding room located in the animal center of Tongji Medical College, Huazhong University of Science and Technology (HUST). All animal experiments strictly adhered to the guidelines established by the Animal Research Institute Committee, which were approved by the Institutional Animal Care and Use Committee (IACUC) of HUST.
The mice were randomly divided into different groups for the experiment, including the sham group (sham), ischemia/reperfusion (IR) group, IR + As-IV group, IR + Ta-IIA group, and IR + Co group, with each group consisting of six mice. The dosages of As-IV and Ta-IIA were determined based on previous studies [
24,
26], The sham and IR groups were given PBS. In the drug treatment groups, intraperitoneal injections of As-IV (15 mg/kg/day), Ta-IIA (10 mg/kg/day) and the combination therapy were initiated 7 days prior to the IR surgery and continued until 7 days after the surgery. For the combination therapy group, we established three different dose groups, namely low-dose group (Ta-IIA 5 mg/kg/day + As-IV 10 mg/kg/day), medium-dose group (Ta-IIA 10 mg/kg/day + As-IV 15 mg/kg/day) and high-dose group (Ta-IIA 15 mg/kg/day + As-IV 20 mg/kg/day), and selected the most appropriate dose group for further study.
In experiments involving the administration of diABZI compound 3, a STING agonist, the following groups were established: sham group, IR group, IR + As-IV group, IR + Ta-IIA group, IR + Co group, IR + As-IV + diABZI group, IR + Ta-IIA + diABZI group, and IR + Co + diABZI group. DiABZI was administered via the tail vein at a dosage of 3 mg/kg/day, starting immediately after reperfusion surgery and given every other day for a total of 3 doses.
Establishment and administration of MIRI
All the procedures were conducted in accordance with the experimental model of myocardial ischemia and infarction guidelines. The mice were administered pentobarbital (1%, 40 mg/kg) via intraperitoneal injection and positioned supine. The left chest fur of each mouse was shaved using a specialized razor, and the surgical site was sterilized with iodine and a solution containing 75% ethanol. Endotracheal intubation was performed, and a ventilator was applied for air supply. Subsequently, the subcutaneous pectoralis muscle was gently separated, and a 3–4 intercostal space incision was made along the left border of the sternum for each mouse. Under microscopic observation, the left atrial appendage (LAD) was ligated. Myocardial ischemia was induced for a duration of 30 min, following which the slipknot was released, and the thoracic cavity was meticulously closed in layers. The mice were subsequently extubated and allowed to recover naturally.
Detection of serum creatine kinase (CK), creatine kinase isoenzyme MB (CKMB), and lactate dehydrogenase (LDH)
One day after the IR surgery, the mice were anesthetized, and blood samples were collected from the orbital venous plexus. Serum levels of CK, CKMB, and LDH were determined with a mouse CK assay kit, a CKMB isoenzyme assay kit, and an LDH Cytotoxicity Assay Kit (Nanjing Jiancheng Bioengineering Institute. Nanjing, China), respectively. The optical density(OD) of each sample was measured with a microplate reader (Agilent Technologies, California, USA). The CK assay was read at a wavelength of 660 nm, the CKMB assay at 340 nm, and the LDH assay at 490 nm.
Myocardial infarct area detection
Following a reperfusion period of 3 days, the mice were compassionately sacrificed and secured in a supine position on the surgical table while under anesthesia. Subsequently, an injection of 1 mL Evans blue (3%) was administrated through both the inferior vena cava and the right atrium. After a brief waiting period of 2 min, the hearts were extracted and subjected to freezing. The frozen hearts were dissected into 2.5 mm sections along their longitudinal axis. These heart sections were then immersed in a solution of 2% triphenyltetrazolium chloride (TTC) and incubated for 20 min at a temperature of 37 ℃ in darkness. Following this, the heart sections were transferred to a solution of 4% paraformaldehyde and stored overnight in darkness at a temperature of 4 ℃. In the resultant stained images, the color blue denoted normal myocardial tissue, red indicated the area of risk, and white represented the infarcted region. The areas were precisely quantified using Image Pro Plus 6.0 software (Media Cybernetics, Inc. USA), and the percentage of the infarcted area was expressed as the percentage of the white area of each section of the total heart area.
Echocardiography
To evaluate the cardiac functions of 8–9-week-old mice weighing 23–25 g, M-mode echocardiography was performed using the Vevo2100 Imaging System from VisualSonics (Toronto, Canada). The system was equipped with a 10-MH2 phased-array transducer. A medical ultrasonic couplant from Tianjin Yajie Medical Material Co., Ltd.(Tianjin, China) was then applied to the mice. Two-dimensional targeted M-mode traces were recorded from the parasternal short-axis view at the mid-papillary muscle level and from the parasternal long-axis view just below the papillary muscle. A minimum of six consecutive cardiac cycles were captured and analyzed to determine the left ventricular systolic diameter (LVIDs), left ventricular diastolic diameter (LVIDd), left ventricular end diastolic volume (LVEDV), and left ventricular end systolic volume (LVESV). Finally, the ejection fraction (EF) was calculated using the formula EF = (LVEDV × LVESV)/LVEDV × 100% and the fractional shortening (FS) was determined by (LVIDd‒LVIDs)/LVIDd × 100%. The outcomes were based on three consecutive beat measurements.
Hematoxylin and eosin (HE) staining
The mouse hearts were immersed in ice-cold phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde, subjected to gradient dehydration and paraffin embedding, and then sectioned into 5 μm sections. These sections were then deparaffinized, stained with hematoxylin and eosin, dehydrated, permeabilized, sealed, and photographed for subsequent analysis.
The hearts of mice (n = 6 per group) were homogenized using RIPA lysis buffer supplemented with phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors. The resulting supernatant was collected after centrifugation. Protein quantification was performed using a BCA protein assay kit. The expression of apoptosis-related proteins and STING pathway-related proteins were detected according to the routine procedure of Western Blot. Antibodies used are shown in Table 3 of the Supplement; Important reagents are shown in Additional file
2: Tables S1 and S2.
Frozen sections, with a thickness of 5 μm, were fixed in 4% paraformaldehyde at a temperature of 4 ℃ for a duration of 24 h. Post-fixation, the sections were subjected to a blocking solution for 10 min at room temperature to subdue endogenous peroxidase activity. Subsequently, the sections were exposed to 50 μL of the TUNEL reaction solution for 1 h at 37 ℃, ensuring a light-free environment. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) for 15 min at room temperature, also in the absence of light. Finally, photomicrographs were captured using a fluorescence light scanning microscope.
Bax fluorescent staining of myocardial tissue
Frozen sections of mouse heart were washed with PBS. The tissue samples were incubated with permeabilizate solution (0.1% Triton X-100) at room temperature for 30 min, then blocked at room temperature for 2 h, and further incubated with Bax antibody at 4 ℃ overnight, followed with another incubation with the secondary antibody at room temperature after washing, and DAPI nuclear staining. After washing, Tissue samples were scanned under fluorescence microscopy.
Fluorescence assay of reactive oxygen species (ROS)
For the detection of ROS fluorescence, dihydroethidium (DHE) (Beyotime Biotech. Inc. Shanghai, China) was employed. myocardial tissue sections were thoroughly washed with PBS and subsequently incubated with a 5 μM concentration of DHE at 37 ℃ for 30 min. The specimens were then scanned with a fluorescence microscope and the intensity of fluorescence was quantitatively analyzed using ImageJ software.
Determination of MDA contents, SOD and GSH activity
MDA contents and the activity of SOD and GSH in heart tissues and cultured cells were quantified using commercially available MDA, SOD, and GSH kits (Nanjing Jiancheng Bioengineering Institute. Nanjing, China). The obtained data was analyzed spectrophotometrically using an Agilent BioTek Gen5 spectrophotometer (Agilent Technologies, California, USA). MDA contents were measured at a wavelength of 532 nm, SOD at 450 nm, and GSH at 405 nm.
Real-time quantitative PCR
Total RNA samples from mouse tissues and cultured cells were extracted using TRIzol reagent (ATG Biotechnology Co., Ltd., Nanjing, China). The RNA samples were used for reverse transcription and real-time quantitative PCR (qRT-PCR) following the protocol provided by TSINGKE (Beijing Qingdao Biotechnology Co., Ltd., China), SynScript®IIIRT SuperMix for qPCR (+ gDNA Remover) and 2 × TSINGKE®Master qPCR Mix (SYBR Green I). The Relative RNA levels were analyzed using the 2
-ΔΔct method, with GAPDH serving as an internal control. The primers sequences utilized for amplification can be found in Additional file
2: Table S4.
Cell culture
The mouse cardiac muscle cell line (HL1) was acquired from Pricella (CL-0605, Procell Life Science &Technology Co., Ltd. China). HL1 cells were cultured in a high-glucose DMEM supplemented with 10% fetal bovine serum and penicillin (100 U/mL), and streptomycin (100 μg/mL) at a temperature of 37 ℃ with 5% CO2.
To establish the cellular model of hypoxia-reoxygenation, HL1 cells were cultivated in a hypoxic chamber comprising 1% O2, 5% CO2, and 94% N2 for a period of 12 h. Subsequently, the cells were reintroduced to normal oxygen conditions and reoxygenated in a complete medium for 6 h. This experimental procedure was utilized to simulate the physiological conditions of hypoxia and subsequent reoxygenation. The cells were divided into the following groups: an NC group, HR group, HR + As-IV group, HR + Ta-IIA group, and HR + Co group. Additionally, a solvent control group was established for both As-IV and Ta-IIA, namely the HR + DMSO group.
To generate an oxidative stress model, cells were stimulated with H
2O
2 (600 μM, a concentration determined from our previous experiments, Additional file
1: Fig. S3A) for a duration of 6 h. The cell samples were then divided into the following groups: NC group, H
2O
2 group, H
2O
2 + As-IV group, H
2O
2 + Ta-IIA group, and H
2O
2 + Co group.
Cell viability analysis
The viability of the cells was determined by a CCK8 assay (Beyotime Biotech. Inc. Shanghai, China). HL1 cells were seeded in 96-well plates at a density of 104 cells per well. Then, 10 μL of the CCK8 reagent was added to each 200 μL DMEM medium per well, and a blank control group was included. The cells were further incubated for an additional 40 min. The absorbance of each well was measured at a wavelength of 450 nm. The optimal concentrations of As-IV and Ta-IIA were optimized for subsequent experiments using cell viability assays. Co is a combination of As-IV and Ta-IIA with optimal concentrations.
Flow cytometry analysis of annexin V-PI staining
Apoptosis was quantified using an Annexin V-APC/PI Apoptosis Kit (MULTISCIENCES, Hangzhou, China). Briefly, the cells were stained with Annexin V and propidium iodide (PI) after washed with PBS. Flow cytometry was performed using a CytoFLEX™ flow cytometer (Beckman Coulter, Inc., California, USA). Annexin V–PI − , Annexin V–PI + , Annexin V + PI − , and Annexin V + PI + staining represented viable cells, necrotic cells, early apoptotic cells, and late apoptotic cells, respectively.
Analysis of ROS by flow cytometry
ROS production in cells was measured using an ROS assay kit (Beyotime Biotech. Inc. Shanghai, China). HL1 cells were washed with PBS for 3 times, and then incubated with the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (10 μM) at 37 ℃ for 30 min. Stained cells were then acquired and analyzed with CytoFLEX™ flow cytometry to calculate mean fluorescence intensity and fluorescence curves.
Binding affinity between Ta-IIA, As-IV, and STING by molecular docking
The canonical 2D structures of As-IV and Ta-IIA were retrieved from the PubChem database (
https://pubchem.ncbi.nlm.nih.gov/) and converted into a 3D structures using ChemBio3D Ultra 14.0 (Cambridgesoft Inc. Cambridge, Massachusetts, United States). The 3D structures were then energetically minimized and saved in the MOL2 format. Additionally, the 3D structures of STING were generated using the PDB database (
https://www.rcsb.org/). PyMOL 2.5.2 (
http://autodock.scripps.edu/) was used to add hydrogen atoms and remove water molecules from the 3D structures, while the formats of As-IV, Ta-IIA, and STING were converted to a pdbqt file by AutoDock Tools 1.5.7 software (
http://autodock.scripps.edu/). Finally, AutoDock Vina v.1.2.0 software (
http://autodock.scripps.edu/) was used for molecular docking. The lower the binding energy, the more stable the ligand–protein binding conformation.
Phosphorylated STING (p-STING) fluorescence staining of HL1 cells
The HL1 cells were seeded in 6-well plates with appropriate replicates and grouped accordingly. Following the assigned treatment, the cells were immobilized using 4% paraformaldehyde. Subsequently, they were incubated with a permeabilization solution (0.1% Triton X-100) for 30 min at room temperature. Afterwards, a blocking step was performed at room temperature for 2 h. The cells were then incubated overnight at 4 ℃ with the primary antibody against p-STING. After thorough washing, the cells were incubated at room temperature for 1 h with appropriate secondary antibodies. Meanwhile, DAPI was used to achieve nuclear staining. After blocking and washing, the cell samples were photographed and scanned with a fluorescence microscope.
STING siRNA of HL1 cells
The HL1 cells were cultured at a density that ensured a confluence of 70%–80% at the time of transfection. The culture medium was replaced with serum-free medium. The siRNA and lipofectamine 3000 were diluted with Opti-MEM, respectively, incubated for 5 min, followed by thorough mixing in the same tube and incubation for 15 min. The prepared transfection solution was added to the cell culture plate drop by drop and gently shaken. After 6 h of siRNA transfection, the serum-free medium was replaced with complete medium. The transfection efficiency was confirmed after 24 h by performing Western blotting and qPCR to detect the levels of STING protein and gene expression respectively.
Following transfection, the experimental groups were set up as follows: negative control (NC) group, hypoxia-reoxygenation (HR) group, HR + STING siRNA group, HR + STING siRNA + As-IV group, HR + STING siRNA + Ta-IIA group, and HR + STING iRNA + Co group.
Statistical analysis
GraphPad Prism v 8.2.1 was used for statistical analysis. The data were presented as mean (M) ± standard error of the mean (SEM). The comparison between the two groups was performed by unpaired t test. The significance level was set at p < 0.05.
Discussion
MIRI exerts a significant impact on the development and prognosis of cardiovascular disease. Implementing protective measures targeted towards MIRI can effectively mitigate cardiomyocyte mortality, restore cardiac function, and ultimately enhance the therapeutic outcomes and prognosis of cardiovascular disease [
31]. The utilization of Ta-IIA and As-IV has demonstrated notable improvements in cardiovascular disease, including MIRI treatment [
21,
32]. However, the efficacy and underlying mechanism of the combined therapy involving As-IV and Ta-IIA for the treatment of MIRI have yet to be investigated. Within our study, we sought to examine the therapeutic effectiveness of co-application of these two drugs and found that this approach yielded greater efficacy in mitigating MIRI compared to treatment with either As-IV or Ta-IIA alone. Additionally, this combination exhibited enhanced potency in suppressing STING phosphorylation and its downstream STING signaling pathway, consequently yielding stronger anti-apoptotic, antioxidant and anti-inflammatory effects.
In the realm of traditional Chinese medicine, the principle of "Replenishing Qi and activating blood" is one of the important treatment principles for the treatment of cardiovascular diseases [
33]. The utilization of therapeutic medications embodying the "Replenishing Qi and activating blood" effect ameliorates cardiovascular symptoms [
34]. Numerous studies have embarked on exploring the mechanism of "Replenishing Qi and activating blood" in treating cardiovascular and cerebrovascular diseases [
35‐
37]. However, these studies mainly focus on the overall study of prescriptions, and neither investigate specific small molecule drugs, nor the effect differences between small molecule drugs [
38]. In order to further study the specific mechanism of "Replenishing Qi and activating blood" in the treatment of MIRI, the As-IV (the main pharmaceutical ingredient in the representative drug
Astragalus membranaceus (Fisch.) Bunge of "Replenishing Qi" category [
24]) and Ta-IIA (the main pharmaceutical ingredient in the representative drug
Salvia miltiorrhiza Bunge of the "Activating blood" category [
22]) were selected in our study.
Previous studies have reported that the combined application of Ta-IIA and As-IV can promote the angiogenesis of endothelial cell-like cells [
39], attenuate atherosclerotic plaque vulnerability [
40], and reduce hypoxia-induced cardiomyocytes injury [
32]. Our study was primarily focused on the effect of Co on MIRI and found that compared to As-IV or Ta-IIA alone, Co exhibited superior ability in reducing the area of myocardial infarction, reducing myocardial enzymes levels, and restoring myocardial contractility.
During the process of reperfusion, the abrupt reintroduction of oxygen to the mitochondria generates ROS, which exacerbates mitochondrial damage and triggers an inflammatory response, ultimately resulting in cell death [
4,
41]. Mitochondrial dysfunction plays a crucial role in the development of MIRI [
42]. Numerous cardioprotective measures focus on mitochondria to reduce oxidative stress, inflammatory responses, and alleviate apoptosis [
43‐
45]. Both Ta-IIA and As-IV have demonstrated protective effects in MIRI by reducing cardiomyocyte apoptosis [
25,
46], oxidative stress [
25,
47], and inflammation [
27,
48]. Our experiments demonstrated that As-IV and Ta-IIA effectively decrease apoptosis, oxidative stress, and inflammation in MIRI cardiomyocytes. What's more, we have discovered that Co possesses a more potent anti-apoptotic, antioxidant, and anti-inflammatory effect compared to using As-IV or Ta-IIA individually.
Molecular docking analysis of As-IV, Ta-IIA, and STING were performed to forecast the interaction patterns and binding affinities. The results revealed that both As-IV and Ta-IIA exhibit strong binding affinity to the STING protein, whether docked individually or simultaneously. In the context of simultaneous molecule docking, the binding location and interaction site of the two drugs with the STING protein have undergone alterations when juxtaposed with individual molecule docking. During simultaneous docking, the binding sites of As-IV to STING have changed from G166, R232, Y167, S241, S243, Y261, E260, T263, Y163, and S162 to S162, R238, N242, Y261, Q266, and T267. Meanwhile, Ta-IIA has transformed from R232, R238, S241, T263, T267, and S162 to S243, N211, and Y245. Therefore, it appears to remain within a substantial structural domain, albeit with alterations to the loci of interaction. Among these residues, R232, R238, S162, E260, and T263 are common sites for STING activation [
9]. Additionally, STING can be phosphorylated starting from residue S243 [
49].
Moreover, it has been demonstrated that As-IV alleviates immunosuppression by modulating the STING signaling pathway in disease infected by virus [
50]. In cardiovascular disease, mitochondrial dysfunction leads to the release of mtDNA into the cytoplasm, triggering cGAS, subsequently activating the STING signaling pathway and eliciting the release of inflammatory mediators that exacerbate cellular damage and inflammatory response [
11,
13].
In our experiments, the expression of cGAS, p-STING, and downstream p-TBK1 and p-IRF3 proteins exhibited an increase in both IR mice and HR-induced HL1 cells. This demonstrated that the activation of STING signaling was also initiated in MIRI. We observed that As-IV, Ta-IIA, and Co significantly reduced the expression of phosphorylated STING, TBK1, and IRF3, but not the expression of cGAS. This indicated that these drugs effectively inhibit the phosphorylation of STING protein and, in turn, STING signaling pathway activation. Additionally, we also found that Co exhibits a stronger inhibition on p-STING and STING signaling pathways compared to As-IV and Ta-IIA. This suggested that Co possesses superior therapeutic efficacy, enhancing the suppressive effect of As-IV and Ta-IIA on STING, thereby augmenting the protection against MIRI.
On the other hand, the efficacy of As-IV, Ta-IIA, and Co in improving myocardial infarction, myocardial enzymes, and restoring cardiac contractile function was diminished upon the addition of STING agonist (diABZI) in in vivo experiments. The data provided further evidence that As-IV, Ta-IIA and Co exert their cardioprotective effects against MIRI by suppressing the STING pathway.
The activation of the STING pathway also induces a plethora of ROS, which can damage the DNA, proteins and lipids of cells, leading to oxidative stress [
51]. In our study, we found a tight link between increased oxidative stress and activation of the STING pathway, which is important in the development and progression of MIRI. However, the application of As-IV, Ta-IIA and Co efficiently inhibited oxidative stress in myocardial caused by STING phosphorylation. Furthermore, the STING agonist (diABZI) can counteract the anti-oxidative stress effects of As-IV, Ta-IIA and Co.
The STING pathway induces the release of interferon, which promotes apoptosis by inhibiting the cell cycle, increasing the permeability of the mitochondrial membrane, activating key proteins in apoptosis pathway, and activating the DNA damage response [
52]. In our study, we have observed a significant increase in STING pathway-associated proteins and cell apoptosis in MIRI-induced myocardium and HR-induced HL1 cells. However, the effect was reduced with As-IV, Ta-IIA and Co treatment, whereas it was reverted with diABZI.
The activated STING pathway produces signaling molecules such as interferon and NF-κB, which in turn activates immune cells to participate in the inflammatory reactions, promotes the release of inflammatory factors, and triggers the inflammatory response [
9]. In our investigation, the inflammatory cytokines in MIRI or HR-induced HL1 cells were increased along with the activation of the STING pathway, but were decreased upon As-IV, Ta-IIA and Co treatment. Nevertheless, diABZI reversed the anti-inflammatory effect of As-IV, Ta-IIA and Co.
Taken together, our results demonstrated that As-IV, Ta-IIA, and Co exerts anti-apoptotic, antioxidant, and anti-inflammatory effect, and inhibit the STING signaling pathway, ultimately suppressed MIRI. However, there exist certain limitations in our study. First, we did not elucidate how the combination of As-IV and Ta-IIA enhances the inhibitory effect on STING phosphorylation. Second, the combination of As-IV and Ta-IIA may potentially affect myocardial cells through other type of cells present in the myocardial tissue, which is a possible aspect that needs to be further investigated.
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