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).

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.

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.

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.

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.

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.

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).

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).

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.

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′,

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

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.

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.

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.

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).

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).

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).

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)).

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).

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.