The flow of our study was represented in Fig. 1. All experimental animals were procured from the Experimental Animal Center of Guangxi Medical University. All experimental protocols and steps according to the NIH (National Institutes of Health) Guidelines for the Use of Laboratory Animals, and had the approval of the Animal Ethics Committee. Firstly, 40 male adult Sprague-Dawley (SD) rats of 250–300 g were randomly divided into CME, CME + HUK treatment, CME + HUK + LY, and sham operation groups, with ten rats in each group. We then built a CME model based on the previously reported process [9]. Briefly, an intraperitoneal injection of pentobarbital sodium (30–40 mg/kg) was administered to anesthetize the SD rats. We used a small-animal ventilator to assist with breathing. We conducted a left-sided thoracotomy in the incision position between the third and fourth ribs of the rat. The ascending aorta was separated and gripped for 10 s. Simultaneously, we mixed 3000 plastic microspheres of 42 $\mu$m diameter (BioSphere Medical Inc., USA) in 0.1 mL sodium lauryl sulfate, which were injected into the left ventricle via the apical part of the heart. An equal volume of saline was injected into the Sham group in the same way. The CME + HUK and CME + HUK + LY groups were administered HUK 0.016 PNA/kg via tail vein for seven consecutive days prior to surgery [13, 14]. And the CME + HUK + LY group was given 10 mg/kg LY294002 (a specific inhibitor of PI3K) (APExBIO, Houston, USA) preoperatively via the peritoneal cavity for 30 min [15]. Penicillin was applied to all animals to resist infection.

Fig. 1.

The flow chart of our study.

Based on Chen et al. [4] study, 12 hours after modeling is the best time to measure cardiac function in rats while at the lowest level. At that stage, we evaluated the cardiac function of the rat using the Hewlett-Packard Sonos (Philips Sonos7500 system, Philips Technologies, USA) to measure the left ventricular fraction shortening (LVFS), left ventricular ejection fraction (LVEF), left ventricular end-systolic diameter (LVESd), and left ventricular end-diastolic diameter (LVEDd), respectively. The measurement was repeated thrice for each rat, and the average value was calculated. All the above operations were performed using an S12 probe with a 12 MHz probe. Experienced cardiac sonographers were used to perform all ultrasound examinations.

Blood samples were drawn from the abdominal aorta before sacrifice. The levels of serum cardiac troponin I (cTnI), creatine kinase myocardial band isoenzyme (CK-MB), lactate dehydrogenase (LDH), IL-1$\beta$, and TNF-$\alpha$ were estimated using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. All the above tests were performed using standardized ELISA kits (Bio-Swamp Biological Technology Co., Ltd., USA).

Twelve hours after the operation, the rats were sacrificed for sampling after completing the ultrasound examination. We administered an intraperitoneal injection of pentobarbital sodium (60 mg/kg) to anesthetize rats. Blood was collected from the abdominal aorta and the heart was isolated shortly after opening the chest. The auricle and atrium were then excised, and the cardiac tissue was separated into upper, middle, and bottom sections. For the comparability of each group, the same inspection index was performed using the same part of cardiac tissue. The upper and middle sections were kept at –80 °C to prepare for real-time quantitative PCR (RT-qPCR) and western blotting. The bottom sections were immobilized in 4% paraformaldehyde for 12 h. It was then embedded in paraffin and used for slice preparation. After hematoxylin-eosin (H&E) staining, cardiomyocytes were observed after CME using an optical microscope. Hematoxylin-basic fuchsin-picric acid (HBFP) staining was used to identify myocardial ischemia, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to identify cardiomyocyte apoptosis.

Myocardial ischemia was detected via HBFP staining, which enabled the staining of ischemic cardiomyocytes and cytoplasm (red), and the nuclei of infarcted (yellow) and healthy (blue) cardiomyocytes. Each stained section was observed with a pathology image analyzer (DMR + Q550, Leica, Wetzlar, Germany). Five fields of view were obtained from each slice. Leica Qwin software (2.7, Leica Microsystems Inc, Buffalo Grove, IL, USA) and the planar method were used to calculate the infarct area. The infarct area was then divided by the entire observation area to measure the infarct percentage.

The TUNEL assay (Roche, USA) was performed using a TUNEL apoptosis detection kit according to the manufacturer’s instructions to detect cardiomyocyte apoptosis. The nuclei of apoptotic cells appeared yellowish-brown under light microscopy. Forty nonoverlapping regions were randomly selected from each slice ($\times$400 magnification). The total number of apoptotic and normal cardiomyocytes was counted. The number of apoptotic cardiomyocytes was obtained using the formula of the count of apoptotic cardiomyocytes/gross count of cardiomyocytes.

We prepared slices of 4 $\mu$m thickness and performed immunofluorescence (IF) staining using the manufacturer’s instructions, following which the slices were incubated with the three antibodies (cleaved caspase-3 [#9661, Cell Signaling Technologies, Danvers, MA, USA], Bax [ab53154, Abcam, Cambridge, UK], and Bcl-2 [ab194583, Abcam]) at 4 °C overnight. PBS was used to rinse the slices five times after incubation with primary antibodies. The slices were then incubated with fluorescent secondary antibodies for 50 min at room temperature. Subsequently, images of 4’,6-diamidino-2-phenylindole (DAPI) counterstained cell nuclei for 7 min were acquired with a fluorescence microscope (Olympus, Tokyo, Japan).

Samples of heart tissue (200 mg) were processed by RT-qPCR. Following the manufacturer’s instructions, we extracted apoptosis-associated RNAs such as Bax, Bcl-2, and caspase-3, which were determined by RT-qPCR for relative levels in CME. The ABI PRISM-7500 machine (CA, USA) was used to perform RT-qPCR using the TB Green® Premix Ex Taq™ II (TaKaRa, Japan). The specific primer sequences used in this study were listed in Table 1.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (stacking gel: 5% SDS-PAGE, separating gel: 10–15% SDS-PAGE) was utilized to separate the total protein obtained from cardiac tissues and then transferred it to a polyvinylidene fluoride membrane (PVDF, Millipore, Atlanta, Georgia, United States). The above experimental operations were all completed at a constant voltage of 100 V. The membrane was then sealed with BSA at 25 °C for 1 hour. Next, the membrane was hatched with corresponding primary antibodies at 4 °C for 24 hours. After 24 hours, the membrane was washed five times. We then incubated the secondary antibody (ab6721, 1:10000, Abcam) coupled to the enzyme for 2 hours.

All data were analyzed in SPSS software (version 23.0, IBM, Chicago, IL, USA). The results of our experiments are presented as mean standard deviation (SD). ANOVA was conducted to test the significance of differences in sample means, and a p-value 0.05 was set as the threshold. Graphpad Prism software (version 8.0, San Diego, CA, USA) for data visualization.

To assess circulatory changes in rats, we measured the LVEF, LVFS, LVEDd, and LVESd after 12 h in the CME model. The echocardiographic results of each group demonstrated that the CME group had lower LVEF and LVFS values than the Sham group, while LVEDd and LVESd were reversed (Fig. 2A,B). Additionally, cardiac function was improved in the CME + HUK group compared with the CME group, showing higher LVEF/LVFS and lower LVEDd/LVESd.

Fig. 2.

Measurement of cardiac function. (A) Cardiac function evaluated by echocardiography in each group (n = 6, per group). (B) The cardiac function parameters of left ventricle ejection fraction (LVEF), left ventricle fractional shortening (LVFS), left ventricular end-diastolic diameter (LVEDd), and left ventricular end-systolic diameter (LVESd) were measured quantitatively. Data were presented as the mean $\pm$ standard deviation (SD). *p 0.05, **p 0.01, ***p 0.001. Abbreviations: CME, coronary microembolization; HUK, Human Urinary Kallidinogenase; LY, LY294002.

H&E and HBFP staining suggested that there were sporadic ischemic areas in the Sham group and most areas were normal filling tissues (Fig. 3A,B). In the CME, CME + HUK, and CME + HUK + LY groups, micro-infarct areas were observed; most of the infarct areas were located in the left ventricle, and embolized microspheres could be seen in the field of vision. The results of H&E staining indicated that cardiomyocytes in the micro-infarct area had no nuclei or ruptured nuclei; the cytoplasm was also red. In addition, the area around the infarct showed swelling and degeneration of cardiomyocytes, red blood cell effusion, and infiltration of inflammatory cells. The HBFP staining results showed that the infarct area of CME rats was significantly wider than that of the Sham group (p 0.05). The infarct area in the CME group was also significantly wider than that of the CME + HUK group (p 0.05).

Fig. 3.

H&E and HBFP staining of myocardial tissues ($\mathrm{\times }$200 magnification; bar = 50 $\mu$m) (n = 6, per group). (A) H&E staining. Microspheres were observed in the CME, CME + HUK, and CME + HUK + LY groups but not in the Sham group. The arrows indicated the microspheres. (B) HBFP staining. An ischemic myocardium was highlighted in red. Data were presented as the mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001. Abbreviations: CME, coronary microembolization; HUK, Human Urinary Kallidinogenase; LY, LY294002.

TUNEL staining results (Fig. 4) have distinguished infarcted and normal myocardial nuclei; we found that apoptotic cardiomyocytes were mainly distributed in the peri-infarct region of myocardial microinfarction. The apoptosis rate was dramatically higher in the CME group than in the Sham and CME + HUK groups (both p 0.05), and LY294002 and HUK treatment balanced the anti-apoptotic function of HUK. Meanwhile, we assessed mRNA translation and protein levels using RT-qPCR and western blotting. RT-qPCR analysis (Fig. 5A) showed that the expression levels of caspase-3 and Bax in the myocardial tissue were significantly higher in the CME group than in the Sham and CME + HUK groups, and Bcl-2 was lower (both p 0.05). Additionally, the expression levels of Bax and caspase-3 in cardiomyocytes in the CME + HUK + LY group were significantly higher than those in the CME + HUK group (p 0.05). In contrast, the expression of Bcl-2 in the CME group was significantly downregulated (p 0.05). Western blotting (Fig. 5B) results were concordant with RT-qPCR.

Fig. 4.

TUNEL staining results of the infarcted and normal cardiomyocyte nuclei and the apoptosis rate of cardiomyocytes in each group (n = 6, per group). The arrows indicated the microspheres. The nuclei of normal cardiomyocytes were stained light blue, while apoptotic cardiomyocytes’ nuclei were stained yellow-brown. Data were presented as the mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001. Abbreviations: CME, coronary microembolization; HUK, Human Urinary Kallidinogenase; LY, LY294002.

Fig. 5.

Pretreatment with HUK attenuated CME-induced myocardial apoptosis. (A) The mRNA expression levels of Bcl-2, Bax, and caspase-3 in cardiac tissues (n = 6, per group). (B) The protein expression levels of Bcl-2, Bax, and cleaved caspase-3 in cardiac tissues (n = 3, per group). Data were presented as the mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001. Abbreviations: CME, coronary microembolization; HUK, Human Urinary Kallidinogenase; LY, LY294002.

Furthermore, we observed from the IF results that Bax and cleaved caspase-3 expression levels were significantly decreased in the Sham and CME + HUK groups compared to those in the CME and CME + HUK + LY groups, and Bcl-2 in the CME and CME + HUK + LY groups were significantly downregulated (Fig. 6).

Fig. 6.

Immunofluorescence staining of Bax, Bcl-2, and cleaved caspase-3 in cardiac tissues (magnification $\mathrm{\times }$400, scale bar = 20 $\mu$m) (n = 3, per group). (A) IF staining detected the expression of Bcl-2. (B) IF staining detected the expression of Bax. (C) IF staining detected the expression of cleaved caspase-3. Abbreviations: CME, coronary microembolization; HUK, Human Urinary Kallidinogenase; LY, LY294002; IF, Immunofluorescence.

To explore the effect of HUK on cardiomyocyte inflammation, two methods (ELISA and western blotting) were used to identify the relative expression levels of IL-1$\beta$ and TNF-$\alpha$ in the different samples (Fig. 7). Interestingly, consistent results were obtained. Briefly, the measurement results of the aforementioned methods all indicated that the expression levels of IL-1$\beta$ and TNF-$\alpha$ in the CME group were significantly higher than that of the Sham group. In addition, compared with the CME group, the expression levels of IL-1$\beta$ and TNF-$\alpha$ in the CME + HUK group were significantly lower (both p 0.05). The expression levels of IL-1$\beta$ and TNF-$\alpha$ in the CME + HUK + LY group were significantly higher than those in the CME + HUK group (p 0.05).

Fig. 7.

Human Urinary Kallidinogenase reduced the myocardial inflammation. (A) The serum levels of TNF-$\alpha$ and IL-1$\beta$ in the four groups (n = 6, per group). (B) The protein expression levels of TNF-$\alpha$ and IL-1$\beta$ in the four groups (n = 3, per group). Data were presented as the mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001. Abbreviations: CME, coronary microembolization; HUK, Human Urinary Kallidinogenase; LY, LY294002.

In this study, we quantified serum myocardial injury biomarkers (including cTnI, LDH, and CK-MB) between groups (Fig. 8A–C). Briefly, the degree of myocardial damage was assessed by calculating the serum cTnI, LDH, and CK-MB levels. The CME group had significantly higher serum levels of CK-MB, cTnI, and LDH compared to the Sham group. In addition, serum LDH, cTnI, and CK-MB levels in the CME + HUK group were significantly lower than those in the CME group. Compared with the CME + HUK group, serum CK-MB, cTnI, and LDH levels were significantly higher in the CME + HUK + LY group (all p 0.05).

Fig. 8.

Human Urinary Kallidinogenase reduced the serum levels of myocardial injury markers (n = 6 per group). (A) The blood levels of cTnI in the four groups. (B) The blood levels of LDH in the four groups. (C) The blood levels of CK-MB in the four groups. Data were presented as the mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001. Abbreviations: CME, coronary microembolization; HUK, Human Urinary Kallidinogenase; LY, LY294002.

Here, we determined the relative expression levels of PI3K/Akt/FoxO1 signaling pathway marker genes (p-PI3K, PI3K, p-Akt, Akt, p-FoxO1, and FoxO1) at the protein levels‘1 using western blotting (Fig. 9). This indicated that the expression levels of p-PI3K, p-Akt, and p-FoxO1 in the CME group were significantly lower than those in the Sham and CME + HUK groups (all p 0.05). Compared to the CME + HUK group, the expression levels of p-PI3K, p-Akt, and p-FoxO1 in the CME + HUK + LY group were significantly lower (all p 0.05). In addition, compared with the CME group, the rates of p-PI3K/PI3K, p-Akt/Akt, and p-FoxO1/FoxO1 in the Sham and CME + HUK groups were significantly higher (all p 0.05). In addition, the levels of p-PI3K/PI3K, p-Akt/Akt, and p-FoxO1/FoxO1 were significantly lower in the CME + HUK + LY group than in the CME + HUK group (p 0.05).

Fig. 9.

Effect of HUK on PI3K/Akt/FoxO1-associated protein expression in cardiac tissues (n = 3, per group). Data were presented as the mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001. Abbreviations: CME, coronary microembolization; HUK, Human Urinary Kallidinogenase; LY, LY294002.