Baicalin regulates macrophages polarization and alleviates myocardial ischaemia/reperfusion injury via inhibiting JAK/STAT pathway.

CONTEXT: Baicalin is an active compound which demonstrates cardioprotection effects against myocardial ischaemia/reperfusion injury (MI/RI).
OBJECTIVE: To investigate how baicalin protects against myocardial injury and to explore its potential mechanism. We hypothesized that baicalin-modulated macrophages change from M1 (pro-inflammatory subset) to M2 (anti-inflammatory subset) under I/R stress.
MATERIALS AND METHODS: We established an ischaemia/reperfusion (I/R) model using Sprague Dawley (SD) rat, then baicalin was intragastric administration (20, 60 or 120 mg/kg) for 24 h. The rats were randomly divided into five groups (n = 10): control, I/R, I/R + baicalin (20 mg/kg), I/R + baicalin (60 mg/kg) and I/R + baicalin (120 mg/kg). Cardiac function was detected by echocardiography, HE staining and ELISA, respectively. Macrophage phenotype was examined by flow cytometry. Furthermore, IHC, qRT-PCR and WB were employed to analyse the related mechanisms.
RESULTS: The study showed that baicalin (20, 60 or 120 mg/kg) significantly improved cardiac function and impeded cardiac apoptosis in rats. In addition, the repair of myocardial morphology (reduced neutrophil infiltration) further confirmed its cardiacprotective effect. Moreover, baicalin effectively decreased iNOS, IL-1β and IL-6, and up-regulated Arg-1, IL-10 and TGF-β via changing the macrophage phenotype (from M1 towards M2). Notably, treatment with baicalin also inhibited the phosphorylation levels of JAK2 and STAT3. Discussion and conclusions: It was confirmed that baicalin alleviated post-I/R myocardial injury and reduced inflammation via JAK/STAT pathway, and baicalin treatment might be recommended as a new approach for myocardial ischaemic complications.

Introduction

Currently, acute myocardial infarction (MI) is a common cardiovascular disease that causes high morbidity and mortality around the world. For MI patients, the most timely and effective treatment is to ameliorate myocardial ischaemia and restrict the size of myocardial infarction. To the best of our knowledge, ischaemia/reperfusion (I/R) has so far been the principal or only strategy for MI treatment, which is clinically achieved by angioplasty or thrombolytic therapy, thereby promptly restoring blood supply (Vincent et al. 2017). Yet, this sudden reperfusion should result in secondary cascade damages, known as myocardial ischaemia/reperfusion injury (MI/RI), which paradoxically deteriorated ischaemic damages and further swelled infarct size (Paelestik et al. 2017). In addition, MI/RI may trigger a large variety of pathological changes, including local acute inflammatory reactions, metabolic disorders and cell apoptosis and/or necrosis, ultimately leading to cardiac dysfunction (Kitano et al. 2014). Simultaneously, the pathogenesis of MI/RI is complex and involves multiple molecular processes such as reactive oxygen species (ROS) accumulation (Ravindran et al. 2017), calcium ion overload (Mozaffari et al. 2013), inflammatory cytokines influx (Badalzadeh et al. 2017), and mitochondrial permeability transition pore (mPTP) opening (Xu et al. 2019). Although there are many basic research reports on MI/RI, the clinical effects are not satisfactory, and we still need further research to find more effective clinical treatments.

The pathological process of MI/RI can trigger injurious inflammatory reaction involving in the infiltration of inflammatory cells (such as neutrophils, macrophages and lymphocytes) (Epelman et al. 2015) and accumulation of pro-inflammatory cytokines, ultimately resulting in substantial cardiac dysfunction. As tissue injury occurs, inflammatory monocytes infiltrate into the surrounding area of injury and differentiate into macrophage populations, thereby regulating tissue injury and repair (Wynn and Vannella 2016). Interestingly, macrophages include two subpopulations, such as pro-inflammatory macrophages (M1-phenotype) and anti-inflammatory macrophages (M2-phenotype). Upon MI, M1 macrophages infiltrate into the injured cardiac tissues, triggering inflammation and releasing pro-inflammatory cytokines, including iNOS, TNF-α, IL-1β and IL-6 (Novak and Koh 2013). Conversely, anti-inflammatory macrophages produce anti-inflammatory cytokines (Arg1, IL-4 and IL-10), and promote the wound healing and scar formation (Nahrendorf et al. 2010). Therefore, switching the dynamic polarization of macrophages M1/M2 in time and transforming macrophages from M1 to M2, has important pathological significance for mitigating inflammation and repairing myocardial function (Mantovani et al. 2013).

Traditional Chinese medicine has been used for thousands of years in China and has attracted more and more scholars’ attention due to its natural, low cost and extremely low toxicity. Baicalin, a flavonoid active substance derived from the roots of Scutellaria baicalensis Georgi (Lamiaceae), has been used to treat various of diseases such as cancer (I El-Gogary et al. 2019), osteoarthritis (Wang et al. 2018), nephritis and hepatitis (Cai et al. 2008). Increasing reports demonstrate that baicalin possesses antioxidant, anti-inflammatory and anti-apoptotic activity (Luan et al. 2015; Li et al. 2017) in Ical. Previous studies found that baicalin protects rat cardiomyocytes via weakening hypoxia/reoxygenation-induced injury (Woo et al. 2005; Lin et al. 2010). Kong et al. (2014) confirmed that baicalin protected the myocardial damage after reperfusion via the antioxidant and paracrine effects. Luan et al. (2019) revealed that baicalin attenuated myocardial I/R-induced damage, inhibited myocardial apoptosis and inflammation by Akt/NF-κB signalling. Bai et al. (2019) showed that baicalin protected cardiac microvascular endothelial cells (CMECs) in I/R rats by promoting the release of nitric oxide. However, it was still of interest to determine if baicalin could induce macrophages to regulate inflammatory response and protect myocardial injury post-I/R.

The JAK/STAT pathway is involved in many important biological processes such as cell proliferation, differentiation, apoptosis and immune regulation. In recent years, several reports have proposed that JAK/STAT signalling is associated with cardiac dysfunction in MI/RI (Mascareno et al. 2001; Bolli et al. 2003). Moreover, Yang et al. (2013) reported the JAK/STAT pathway was quickly activated after I/R, Zhang et al. (2019) found propofol-alleviated inflammation and tissue injury in MI/RI rats through inhibiting the JAK/STAT pathway. But, the role of baicalin on MI/RI in JAK/STAT signalling pathway is rarely reported.

In this study, we provide the evidence that baicalin mediates cardioprotection against myocardial injury post-I/R via JAK/STAT pathway and confirm the hypothesis that baicalin stimulates macrophages change to alleviate inflammation caused by I/R.

Materials and methods

Main materials and reagents

Baicalin (10 mM × 1 mL in DMSO, molecular formula: C21H18O11, molecular weight: 446.3, purity: 98.01%) was purchased from MedChemExpress (China) LLC (Shanghai, China), adult male Sprague-Dawley (SD) rats (220–250 g) were purchased from Guangdong Medical Laboratory Animal Centre (Foshan, Guangdong, China) and were kept with appropriate environment (23 ± 2 °C, relative humidity 60%, and 12-h light/dark cycle), which had free access to water and food. All antibodies were purchased from Abcam (Shanghai) Trading Co., Ltd. (Shanghai, China) as follows: anti-Ki67 antibody, ab15580; anti-cleaved-caspase-3 antibody, ab2303; anti-Bcl-2 antibody, ab59348; anti-Bax antibody, ab32503; anti-CD86 antibody, ab213044; anti-CD206 antibody, ab64693; anti-iNOS antibody, ab15323; anti-Arg1 antibody, ab91279; anti-JAK2 antibody, ab108596; anti-p-JAK2 antibody, ab32101; anti-STAT3 antibody, ab119352 and anti-p-STAT3 antibody, ab76315. Collagenase B and dispase II were purchased from Sigma-Aldrich (China) (Shanghai, China). AG-490 was purchased from ApexBio Technology (Houston, TX, USA). All ELISA kits (CK-MB ELISA kit, Mb ELISA kit, cTnI ELISA kit) were purchased from Thermo Fisher Scientific (China) LLC (Shanghai, China).

Rat I/R model

All animal experiment steps were conducted in accordance with the National Institutes of Health (NIH) guide for the care and use of laboratory animals (National Academies Press 2011) and approved by the ethical committees of Wuhan Asia Heart Hospital. All SD rats were fasted for 12 h before operation. They were then anaesthetised with 2% isoflurane and fastened on the operating table, and I/R procedures were based on previous descriptions (Zhang et al. 2011). Briefly, open the left chest to expose the heart, peel off the pericardium to find the coronary artery, and ligature the left anterior descending coronary artery (LAD). After 45 min of ligation and 180 min of blood reperfusion, the same procedure was also used for sham but no ligation. Then, blood specimens were centrifuged at 400 g for 10 min. Finally, cardiac tissues and/or serum were immediately collected and stored at −80 °C for further analysis.

Rat administration

Rats were given baicalin (20, 60 or 120 mg/kg) by intragastric administration for 24 h and were randomly grouped (n = 10) as follows: control group, I/R group, I/R + baicalin (20 mg/kg) group, and I/R + baicalin (60 mg/kg) group, I/R + baicalin (120 mg/kg) group. The control operation group did not have I/R but was given normal perfusion at the same time. Rats in the control group and I/R group were given 0.9% saline, yet in I/R + baicalin treatment groups were given different doses of baicalin (once a day for two weeks), followed by I/R surgery.

Echocardiography

Two-hours post-I/R, SD rats were narcotised by intraperitoneal injection of 40 mg/kg pentobarbital sodium and placed in the flat position. The echocardiography measurement of cardiac function was operated by digital ultrasound equipment Vevo 2100 (VisualSonics, Ontario, Canada) and an 18 MHz transducer after reperfusion. Then, the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS) and left ventricular wall thickness (LVWT) were calculated for M-mode recordings. All results were averaged over at least three consecutive cardiac cycles measuring and analysed by a blinded researcher.

Measurement of pressure-volume data parameters

After reperfusion, the pressure-volume tracheal catheter (SPR839, Millar Instruments Inc, USA) was inserted into the right common carotid artery, and then, the left ventricle was inserted using a pressure-volume conductance instrument (MPVS-300, Millar Instruments Inc, USA). Signals were recorded and calculated by a PVAN data analysis software (Millar Instruments Inc, USA). Haemodynamics parameters such as heart rate (HR), left ventricular systolic pressure (LVSP) and LVEF were measured.

Haematoxylin–eosin (HE) staining

In brief, the rat heart tissues were fixed in 10% formalin buffered. After paraffin embedding, the tissues sections were chipped to 4 µm thickness, and then stained with haematoxylin–eosin (HE) liquid for the histopathological examination.

ELISA assay

Serum samples were centrifuged and supernatants were collected, the serum levels of CK-MB, Mb and cTnI were detected using a ELISA kits, according to the manufacturer’s protocols (Thermo Fisher, Waltham, MA, USA).

Immunohistochemistry (IHC) assay

Rat heart tissues were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at a thickness of 4 μm according to standard procedures. Next, the deparaffinized sections were hydrated. Furthermore, endogenous peroxidase activity was blocked by Hydrogen Peroxide Block for 15 min. After antigen retrieval in 10 mM heated citrate buffer for 10 min, the sections were incubated with primary antibody: anti-Ki67 antibody (dilution rate, 1:600) and anti-caspase-3 antibody (dilution rate, 1:600) overnight at 4 °C. Subsequently, corresponding secondary antibody was incubated for 1 h at room temperature. Finally, the specimens were stained with DAB kit (Boster Biological Technology Co. Ltd, Wuhan, China). Then, the specimens were washed with PBS and images were captured under a confocal microscopy (Leica Microsystems CMS GmbH, Wetzlar, Germany).

Flow cytometry screening

Collected cardiac tissues post-reperfusion were quickly removed and put into the ice-filled PBS. Cardiac tissues were minced and digested (Collagenase B and Dispase II), the digestion reaction was stopped by cold flow cytometry staining buffer, the mixture was percolated, centrifuged at 400 g for 5 min at 4 °C, then the liquid supernatant was cleared and the cells were resuspended in 1 mL of ice staining buffer. Cells from individual heart tissue were divided into three parts according to the cell count so that each part involved approximately 106 living (Trypan blue negative) cells for flow cytometry (Al-Darraji et al. 2018). Next, the isolated cells were cultivated rapidly on ice for 20-30 min with coupled primary antibodies (APC-CY5.5-coupled CD86, APC-CY7-coupled CD206 and R-PE-CY5-coupled F4/80) for the macrophage phenotype sorting. Next, after 20–30 min cultivation, the cells samples were washed two times continuity and then analysed by an Attune NxT flow cytometer equipment (ThermoFisher Scientific, Waltham, MA, USA). Macrophage phenotypes were divided into pro-inflammatory (F4-80+/CD86+) and ant-inflammatory (F4-80+/CD206+). Finally, manual or automatic calibration and compensation were performed for experiments using unstained and single fluorescence controls.

Quantitative real-time PCR (qRT-PCR)

Making a brief description according to the laboratorial guides. Total RNA of heart tissues were extracted with Trizol reagent (Life Technologies, CA, USA) and reversed to cDNAs using iScript cDNA Synthesis kit (Bio-Rad, CA, USA) according to the manufacturer’s protocols. Quantitative PCR amplification was then achieved with a Takara Taq PCR kit (Takara Bio, Japan). The primer sequences are listed as follows:

iNOS-F: 5′-CAGCTGGGCTGTACAAACCTT-3′,

iNOS-R: 5′-CATTGGAAGTGAAGCGTTTCG-3′;

IL-1β-F: 5′-CAACCAACAAGTGATATTCTCCATG-3′,

IL-1β-R: 5′-GATCCACACTCTCCAGCTGCA-3′;

IL-6-F: 5′-CTTCCATCCAGTTGCCTTCTTG-3′,

IL-6-R: 5′-AATTAAGCCTCCGACTTGTGAAG-3′;

Arg-1-F: 5′-AACACTCCCCTG ACAACCA-3′,

Arg-1-R: 5′-CATCACCTTGCCAATCCC-3′;

IL-10-F: 5′-GCTCT TACTGACTGGCATGAG-3′,

IL-10-R: 5′-CGCAGCTCTAGGAGCATGTG-3′;

TGF-β-F: 5′-CGGAGAGCCCTGGATACCACCTA-3′,

TGF-β-R: 5′-GCCGCACACAGCAGTTCTTCTCT-3′;

β-actin-F: 5′-GGGAAATCGTGCGTGAC-3′,

β-actin-R: 5′-AGGCTGGAAAAGAGCCT-3′.

All processes were performed at least in triplicate for each independent amplification.

Western blot (WB) analysis

Total protein samples were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein lysates were quantified using a Pierce™ BCA protein assay kit (23227, Thermo Fisher, Waltham, MA, USA), according to the manufacturer’s protocols. About 50 μg of proteins loaded onto 12% SDS PAGE gels, all membranes were blocked using 5% non-fat dry milk in Tris-buffered saline (TBS) at 4 °C for 30 min and incubated with the following primary antibodies: anti-Bcl-2 antibody (1:600), anti-Bax antibody (1:600), anti-iNOS antibody (1:600), anti-Arg1 antibody (1:600), anti-JAK2 antibody (1:600), anti-p-JAK2 antibody (1:600), anti-STAT3 antibody (1:600), and anti-p-JAK2 antibody (1:600). Next, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody, respectively. Finally, the bands were exposed with ECL reagent (Thermo Fisher, Waltham, MA, USA). GAPDH was used as the loading control.

Statistical analysis

All the experiments were conducted at least three times, and the data were expressed as mean ± SD. Statistical analysis was carried out using IBM SPSS Statistics 25.0 (IBM, Armonk, USA). Student’s t-test or one-way ANOVA was used to evaluate the significance. p < 0.05 showed statistical significance.

Results

Baicalin preserved cardiac function after I/R

Compared with control group, the HR, LVSP, FS% and EF% were significantly decreased in I/R (p < 0.05), the situation in Table 1 confirmed that reperfusion model was successfully constructed. On the contrary, these parameters were remarkably increased in 60 or 120 mg/kg baicalin groups compared with I/R group (p < 0.05), and there was no significant difference in the low dose (20 mg/kg) group. Interestingly, LVWT had the similar trends compared with other parameters, but no statistical difference.

Table 1.

Effect of baicalin on cardiac functional parameters.

Group	Dose (mg/kg)	HR (bpm)	LVSP (mmHg)	LVFS (%)	LVEF (%)	LVWT (mm)
Control		404.17 ± 32.36	138.37 ± 21.29	28.12 ± 6.93	58.05 ± 11.28	0.75 ± 0.06
I/R		217.21 ± 27.52*	55.18 ± 12.24*	8.05 ± 1.89*	23.37 ± 8.45*	0.59 ± 0.04
Baicalin	20	223.12 ± 28.69	59.13 ± 14.73	9.49 ± 2.01	25.92 ± 7.36	0.61 ± 0.05
	60	318.25 ± 32.54#	87.36 ± 11.42#	15.05 ± 3.73#	39.16 ± 6.42#	0.64 ± 0.07
	120	369.16 ± 35.49#	123.07 ± 15.27#	24.46 ± 5.92#	49.93 ± 8.39#	0.72 ± 0.05

HR: heart rate; LVSP: left ventricular systolic pressure; LVEF: left ventricular ejection fraction; LVFS: left ventricular fractional shortening; LVWT: left ventricular wall thickness. Data values are depicted as mean ± SD, n = 10. *p < 0.05 vs control group, #p < 0.05 vs I/R group.

Baicalin repaired pathological morphology of myocardium and alleviated cardiac injury

As shown in Figure 1(A), there was no apparent difference in the morphological structures of the control group, but I/R group showed obvious myocardialfibre fracture, cellular oedema, necrosis, and neutrophil infiltration. Gratifying, compared with I/R group, myocardial tissue damage was gradually repaired with the increased dose of baicalin (60 or 120 mg/kg). As showed in Figure 1(B–D), post-reperfusion, the protein expression of CK-MB, Mb and cTnI were remarkably increased (p < 0.05). In contrast, the expression of these markers was significantly decreased with different doses of baicalin treatment (p < 0.05).

Figure 1.

Effect of baicalin on pathological changes of heart tissue. (A) HE staining of myocardial tissue sections, representative micrographs were magnified at 400×. (B, C and D) Myocardial injury markers of CK-MB, Mb and cTnI were examined by ELLSA assay. Data values are depicted as mean ± SD, n = 10. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. I/R group.

Baicalin inhibited cardiac apoptosis following I/R

As shown in Figure 2(A–C), the expression of Ki67 was increased, while the expression of cleaved-caspase-3 was decreased, compared with control (p < 0.05). However, the levels of Ki67 and cleaved-caspase-3 were reversed after diverse doses of baicalin treatment (20, 60 or 120 mg/kg) (p < 0.05). In addition, the result from Figure 2(D,E) showed that the value of Bcl-2/Bax was increased with different doses of baicalin, which was statistically different (p < 0.05).

Figure 2.

Effect of baicalin on cardiac apoptosis. (A, B and C) The protein levels of Ki67 and cleaved-caspase-3 in myocardial tissue were examined by IHC, representative micrographs were magnified at 200×. (D and E) The protein levels of Bcl-2 and Bax were examined by WB. Data values are depicted as mean ± SD, n = 10. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. I/R group.

Baicalin changed the macrophage phenotype and reduced inflammatory response

Treatment with baicalin (Figure 3(A,B)) significantly reduced CD86+ cells and increased CD206+ cells. Besides, as shown in Figure 3(C,D), compared with control, we found a significant increase in the ratio of pro-inflammatory macrophages (F4-80+/CD86+) (p < 0.05), and a decrease in anti-inflammatory macrophages (F4-80+/CD206+) (p < 0.05) post-reperfusion. Treated with diverse doses of baicalin (20, 60 or 120 mg/kg), the macrophage was switched from pro-inflammatory subset (M1) to anti-inflammatory subset (M2).

Figure 3.

Effect of baicalin on phenotype polarisation of macrophages M1 and M2. (A) Representative pseudocolour flow cytometry showed the distribution of CD86 and CD206 macrophages. (B) Proportion of CD86+ and CD206+ macrophages in myocardium. Data values are depicted as mean ± SD, n = 10. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs I/R group.

Baicalin transformed expression of macrophage M1 and M2 biomarkers

As shown in Figure 4(A), compared with control, the mRNA levels of iNOS, IL-1β and IL-6 (M1 biomarkers) were markedly increased in I/R group, and baicalin treatment (20, 60 or 120 mg/kg) effectively decreased the mRNA levels of these biomarkers (p < 0.05). Besides, the expression of M2 biomarkers (Arg-1, IL-10 and TGF-β) were down-regulated in I/R group, while baicalin reversed the down-regulation effect of I/R on biomarkers (Figure 4(B)). Furthermore, the results of iNOS and Arg-1 in proteins were consistent with that in mRNA (Figure 4(C)).

Figure 4.

Effect of baicalin on inflammatory cytokines. (A) The mRNA levels of macrophage M1 biomarkers (iNOS, IL-1β and IL-6) were examined by qRT-PCR. (B) The mRNA levels of macrophage M2 biomarkers (Arg-1, IL-10 and TGF-β) were examined by qRT-PCR. (C) The expression of iNOS and Arg-1 were examined by WB. Data values are depicted as mean ± SD, n = 10. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. I/R group.

Baicalin regulated the activition of JAK-STAT

To elucidate the underlying mechanism of baicalin, we thus detected the activation of JAK2 and STAT3 proteins. Compared with thevcontrol group, our results revealed that the phosphorylation levels of JAK2 and STAT3 were remarkably increased in I/R group (p < 0.05). Conversely, the phosphorylated expression of JAK2 and STAT3 proteins were decreased with baicalin treatment (Figure 5).

Figure 5.

Effect of baicalin on the protein levels of JAK2 and STAT3. (A) The expression of p-JAK2, JAK2, p-STAT3 and STAT3 were examined by WB. (B) The value of p-JAK/JAK in different doses of baicalin. (C) The value of p-STAT3/STAT3 in different doses of baicalin. Data values are depicted as mean ± SD, n = 10. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. I/R group.

Baicalin attenuated MI/RI through suppressing JAK–STAT pathway

We injected 2.5 μM JAK inhibitor (AG-490) into the jugular vein of rats and randomly separated the rats into groups: control group, I/R group, I/R + AG-490 (2.5 μM) group, and I/R + baicalin (60 mg/kg) group, AG-490 + baicalin group. As shown in Figure 6(A,B), the expression of cleaved-csapase3, iNOS and IL-1β were significantly increased in the I/R group, while the expression of Arg-1 and IL-10 were significantly decreased, compared with the control (p < 0.05). In addition, AG-490 or baicalin alone antagonised I/R-induced content changes of these proteins (p < 0.05). Moreover, AG-490 and baicalin co-treatment further reduced the levels of cleaved-capase3, iNOS and IL-1β and increased Arg-1 and IL-10.

Figure 6.

Effect of baicalin on MI/RI in the absence or presence of AG490. (A and B) After adding JAK inhibitor AG-490, the protein levels of cleaved-csapase3, iNOS, IL-1β, Arg-1and IL-10 were examined by WB. Data values are depicted as mean ± SD, n = 10. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05 vs. I/R group; &p < 0.05 vs. AG-490 or baicalin group.

Discussion

It is universally acknowledged that blood flow recovery in ischaemic myocardial tissue has become one of the most feasible treatments for ischaemia-induced heart disease. In this experiment, we constructed a model of myocardial I/R in rats to investigate the protective role of baicalin in myocardium post-reperfusion. This study demonstrated baicalin could improve cardiac function confirmed by echocardiography and histopathological examination. Baicalin inhibited myocardial tissue apoptosis and reduced inflammatory response through macrophage-mediated release of inflammatory factors. Moreover, baicalin down-regulated the protein expression of JAK2 and STAT3 in myocardial tissues.

Numerous complex signal pathways, such as PI3K/Akt, Akt/NF-κB, p38/MAPK, Nrf2-ARE and JAK/STAT pathways, are involved in the development of MI/RI. Among them, the JAK/STAT signalling pathway plays an important role in the progression of MI/RI (Bolli et al. 2001). It is well known that JAKs are rapidly recruited to the receptor and activated after receiving the signal from the upstream receptor molecule, and activated JAK then catalyses tyrosine phosphorylation of this receptor, thus supplying binding sites for the SH2 domain of STATs, ultimately leading to gene transcription. In particular, JAK1 and JAK2 were activated by MI/RI, which in turn activates STAT1 and STAT3, STAT1 promotes apoptosis, whilst STAT3 protects cardiomyocyte (Stephanou 2004). Apoptosis is a crucial pathophysiological feature in the early I/R that triggers more severe heart failure (Hilbert et al. 2018). Hattori et al. (2001) found that activated JAK/STAT up-regulated Bcl-2 and down-regulated Bax, enhancing myocardial pro-survival pathways. In this study, baicalin decreased the level of cleaved-caspase-3 and increased Bcl-2/Bax ratio post-I/R (Figure 2). In order to understand whether JAK/STAT signal is involved in baicalin-regulated myocardial apoptosis, we also detected the expression of cleaved-caspase-3 after the addition of JAK inhibitor AG-490, the result showed AG-490 or baicalin alone inhibited I/R-induced cleaved-capase3 increase, AG-490 and baicalin co-treatment further reduced the levels of cleaved-capase3 (Figure 6), indicating baicalin inhibited myocardial apoptosis induced by I/R via JAK/STAT pathway.

Inflammation plays a crucial role in the recovery of MI and MI/RI (Li et al. 2015). IL-1β is an outstanding inflammatory mediator in the pre-MI/RI period (Takahashi 2011) that can increase endothelial cell permeability and stimulate the release of chemokines, which further leads to the accumulation of inflammatory cells including neutrophils and macrophages (Swirski and Nahrendorf 2013). Baicalin has been shown to have anti-inflammatory effects in several diseases. Guo et al. (2019) reported that baicalin substantially decreased the levels of IL-1β, IL-6, and TNF-α in the depressive-like symptoms. Luan et al. (2019) found that baicalin inhibited myocardial inflammation through down-regulating the levels of TNF-α, IL-1β, IL-6, and IL-8 and up-regulating IL-10. Similar to these studies, we found that baicalin weakened neutrophils infiltration (Figure 1) and decreased the release of iNOS, IL-1β and IL-6 post-I/R (Figure 4), supporting the previous views.

Macrophages are the core mediums of cardiac inflammation (Ben-Mordechai et al. 2015; Wynn and Vannella 2016) and contribute to the onset and regression of inflammation. In fact, stem cells can modify anti-inflammatory macrophages (M2) after heart damage, thereby promoting cardiac recovery (de Couto et al. 2015). In addition, the production of pro-inflammatory cytokines (such as TNF-α, IL-1β and IL-6) and their harmful effects could be reduced by regulating macrophage phenotypes (Saini et al. 2005). we present evidence for the first time in this study that baicalin transformed macrophages from pro-inflammatory/M1 to anti-inflammatory/M2 phenotype and played an immunomodulatory role after I/R. Flow cytometry data (Figure 3) showed a significant decrease in CD86+ (anti-inflammatory macrophages) cells and a significant increase in CD206+ (anti-inflammatory macrophages) cells after baicalin treatment. Moreover, the expression of M1 biomarkers (iNOS, IL-1β and IL-6) were down-regulated, while that of M2 (Arg-1, IL-10 and TGF-β) were up-regulated (Figure 4). Importantly, the transcription of JAK1/STAT3 pathway increased iNOS and contributed to the development in the late phase of I/R preconditioning (Xuan et al. 2003), while JAK2/STAT3 signalling was critically involved in inflammation (Amani et al. 2019). Interestingly, we observed that baicalin reduced the phosphorylation of JAK2 and STAT3 (Figure 5) and decreased the levels of iNOS and IL-1β (Figure 6). AG-490 alone reduced the levels of iNOS and IL-1β and increased Arg-1 and IL-10, AG-490 and baicalin co-treatment further extended the results of AG-490 alone processing. Taken together, our work confirmed that baicalin-attenuated myocardial inflammatory response and switched macrophage polarisation from M1 to M2 by inhibiting JAK/STAT activation. These findings strongly suggested that baicalin regulated the polarisation conversion of macrophage phenotypes post-I/R but are there other cells involved in baicalin-mediated cardiac protection? It is worth our further study.

Conclusions

Our study demonstrated the protective role of baicalin on MI/RI, which is mediated via the JAK/STAT pathway. In addition, baicalin alleviated myocardial injury and inhibited apoptosis following I/R, weakened the myocardial inflammation response through switching the polarization of macrophages from M1 to M2. Hence, it could be a candidate for the treatment of MI/RI.