Protection of CAPE-pNO2 Against Chronic Myocardial Ischemia by the TGF-Β1/Galectin-3 Pathway In Vivo and In Vitro

Although it is known that caffeic acid phenethyl ester (CAPE) and its derivatives could ameliorate acute myocardial injury, their effects on chronic myocardial ischemia (CMI) were not reported. This study aimed to investigate the potential effect of caffeic acid p-nitro phenethyl ester (CAPE-pNO2, a derivative of CAPE) on CMI and underlying mechanisms. SD rats were subjected to high-fat-cholesterol-diet (HFCD) and vitamin D3, and the H9c2 cells were treated with LPS to establish CMI model, followed by the respective treatment with saline, CAPE, or CAPE-pNO2. In vivo, CAPE-pNO2 could reduce serum lipid levels and improve impaired cardiac function and morphological changes. Data of related assays indicated that CAPE-pNO2 downregulated the expression of transforming growth factor-β1 (TGF-β1) and galectin-3 (Gal-3). Besides, CAPE-pNO2 decreased collagen deposition, the number of apoptotic cardiomyocytes, and some related downstream proteins of Gal-3 in the CMI rats. Interestingly, the effects of CAPE-pNO2 on TGF-β1, Gal-3, and other proteins expressed in the lung were consistent with that in the heart. In vitro, CAPE-pNO2 could attenuate the fibrosis, apoptosis, and inflammation by activating TGF-β1/Gal-3 pathway in LPS-induced H9c2 cell. However, CAPE-pNO2-mediated cardioprotection can be eliminated when treated with modified citrus pectin (MCP, an inhibitor of Gal-3). And in comparison, CAPE-pNO2 presented stronger effects than CAPE. This study indicates that CAPE-pNO2 may ameliorate CMI by suppressing fibrosis, inflammation, and apoptosis via the TGF-β1/Gal-3 pathway in vivo and in vitro.


INTRODUCTION
Myocardial ischemia (MI) refers to a pathological state of decreased blood perfusion of the heart, which results in aberrant vascular regeneration in ischemic areas, fibrosis, oxidative stress, inflammation, and even cardiovascular dysfunction [1,2], and its common cause is coronary atherosclerosis [3]. Among all MI cases, chronic myocardial ischemia (CMI) is more general in clinic, and its prevalence is increasing even in young people in recent years [4].
Galectin-3 (Gal-3) is related to cancer, inflammation, and fibrosis of various organs, which also mediates cell apoptosis and influences angiogenesis of ischemic area [10,11]. Gal-3 is often associated with fibrosis and inflammation in cardiovascular diseases [12], and it is valuable for predicting acute myocardial infarction [13]. It was reported that elevated Gal-3 level was associated with mortality risk in people with acute decompensated heart failure [14]. Knockout of galectin-3 could significantly inhibit the left ventricular ischemic area in the IR rat and reduce H/R induced cardiomyocyte apoptosis [15]. It is also a novel biomarker for right ventricle remodeling in pulmonary arterial hypertension [16]. For example, Gal-3 upregulates idiopathic pulmonary fibrosis in patients with pulmonary disease and also participates in inflammation, proliferation, and migration of lung cancer cells [17,18].
Caffeic acid phenethyl ester (CAPE), a bioactive component from propolis, has been reported to have protective effect on the injured H9c2 cell and could reduce the death of cardiomyocyte [19,20]. Our group has designed and synthesized caffeic acid p-nitro phenethyl ester (CAPE-pNO 2 ) and caffeic acid o-nitro phenethyl ester (CAPE-oNO 2 ) as two derivatives of CAPE. These two derivatives have better protective effects on mice with diabetic cardiomyopathy, and they are also effective on the heart of rats with MIRI [21][22][23].
As described above, TGF-β1 and Gal-3 are wellknown markers of vascular fibrosis and atherosclerosis [24], but studies between Gal-3 and CMI are relatively rare. This study aims to investigate whether Gal-3 is connected with pathogenesis of CMI, whether CAPE-pNO 2 has cardioprotection effect on CMI in vivo and in vitro, and whether the effect is corresponding with Gal-3, TGF-β1, and its related proteins.
CMI rat model was established by high-fat-cholesterol diet [25] (HFCD) and vitamin D3. And the H9c2 cells were treated with LPS to establish CMI model for mechanistic studies [26][27][28], because there is no mature method to establish CMI cell model, and the commonly hypoxia/reoxygenation cell method is mostly used in the study of acute myocardial injury. Studies showed that LPS could induce H9c2 cell damage, reducing cell viability, increasing apoptosis and inflammatory responses [26].

Preparation of CMI Rat Model
Animal experiments were approved and carried out in accordance with the Animal Ethics Community of China. Male Sprague-Dawley rats, which weighed 200 ± 20 g, were purchased from the Experimental Animal Center of Chongqing Medical University (SCXK (Yu) 2017-002). The animals were housed in a 12-h light/ dark cycle and 22 °C conditions, which were free access to food and water.
Rats were randomly divided into two groups: the control group (normal diet) and the CMI model group. The CMI model was established by high-fat-cholesterol diet [25] (HFCD, containing 17.04 kJ/g total energy, consisting of 16.3% protein, 16.1% fat, 46.1% carbohydrate, and 2.75% cholesterol, was purchased from Botai Hongda Biotechnology Ltd., Beijing) as well as induced by intraperitoneal injection of vitamin D 3 (2000 U/kg/day, dissolved in corn oil) for 3 consecutive days. The control group was injected with the same volume of corn oil.
After 2 months of feeding, all rats' serum samples were collected. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) level in rats were measured by commercially available kits as the respective manufacturer's protocols. Besides, three rats in the model group and two rats in the control group were randomly selected to measure their two-lead electrocardiogram (ECG). After measurement, the selected rats were euthanized, and the aortas were removed and used for oil red O staining. Compared with the control, the elevation of ST segment in ECG increase of serum TC, TG, and LDL; decrease of HDL; and obvious lipid deposition in aorta were regarded as successful models.
The model rats were randomly divided into four groups (6 rats in each group): (1) the CMI (model) group treated with saline, (2) the CAPE group treated with CAPE (1 mg/kg/day), (3) the High-pNO 2 group treated with CAPE-pNO 2 (1 mg/kg/day), and (4) the Low-pNO 2 group treated with CAPE-pNO 2 (0.7 mg/kg/day), and the normal diet group remained as control group (treated with normal saline). CAPE and CAPE-pNO 2 were dissolved in 0.01% DMSO and intraperitoneally injected for 4 weeks. ECG was measured, and the blood samples were collected at the end of the experiment; then, the rats were euthanized. The entire heart and lung of all rats were removed, washed with normal saline, and weighted after being dried by filter paper. Three heart samples for tissue staining were selected from each group and fixed with 4% paraformaldehyde at 4 °C, and the other organ samples were stored at − 80 °C for the following experiments.

Detection of Lipid, Cardiac Marker Enzymes, and Gal-3 in Serum
Serum samples were obtained from blood samples by centrifugation (3000 rpm, 15 min, 4 °C). Levels of serum lipid (TC, TG, HDL, and LDL) and cardiac marker enzyme (including HBDH, LDH, CK, and CK-MB) were measured by commercially available kits as the respective manufacturer's protocols. Gal-3 level in serum was measured by rat galectin-3 ELISA assay kit according to the instructions.

HE, Sirius Red, TUNEL, and Immunohistochemistry Staining of Heart Tissues
Heart tissues were fixed in 4% paraformaldehyde solution for 48 h and embedded in paraffin to prepare 5-μm slices. After dewaxing and rehydration, slices were stained with hematoxylin-eosin (HE) and Sirius Red (SR), respectively. Optical microscopy and digital camera were used to observe and photograph the images of the dyed parts.
As for TUNEL staining, after dewaxing and rehydration, the sections were treated with protease K solution fixed and sealed in turn and washed with PBS three times after each step. Subsequently, TdT enzyme reaction solution was added for labeling, 0.3% H 2 O 2 /PBS was added for blocking endogenous peroxidase activity, and DAB solution was used for color rendering. TUNEL-positive cells were observed by optical microscopy and photographed by digital camera.
For immunohistochemistry experiments, the effect of endogenous peroxidase was eliminated by adding 0.3% hydrogen peroxide methanol solution after dewaxing and rehydration of paraffin slices. Then, the slices were incubated overnight with primary antibodies against TGF-β1, IL-6, and Gal-3 (1:100) at 4 °C, respectively, and incubated with goat anti-rabbit IgG antibody conjugated with HRP for 2 h. Then, the sections were re-dyed with DAB and hematoxylin. The slices were observed under an optical microscope and photographed with a digital camera. All the stained areas were measured and quantified by IPP.

Cell Culture and Preparation of CMI Cell Model
H9c2 cells were cultured in DMEM high-glucose medium with 10% FBS and 1% penicillin-streptomycin solution in a humidified incubator with 5% CO 2 at 37 °C.

Immunofluorescence
H9c2 cells were seeded into 96-well plates at a density of 2 × 10 3 -2 × 10 4 and treated as previously described. Then, H9c2 cells were fixed with 4% paraformaldehyde for 15 min, washed with PBST 3 times, and permeated with 0.25% Triton-100 in PBST. Following the H9c2 cells were blocked with 1% bovine serum albumin and then incubated with primary antibodies against TGF-β1, Gal-3, Col-I, Col-III, TNF-α, and IL-6 (1:100) overnight at 4 °C. Washed again, H9c2 cells were incubated with Cy3-conjugated secondary antibodies (1:50) for 1.5 h in dark and then stained by DAPI for 5 min. Images were captured under a fluorescence microscope and analyzed by IPP.

AO Staining
H9c2 cells were planted into 6-well plates at a density of 1 × 10 6 and treated as previously described. H9c2 cells were incubated with AO dye solution (100 μg/ mL) for 15 min in dark; then, the apoptosis cells were observed using a fluorescence microscope.

Western Blot Assay
Heart tissues were lysed on ice with RIPA lysis buffer for 5 min to obtain homogenates. The obtained homogenates and H9c2 cells were decomposed in RIPA lysis solution on ice for 30 min and then centrifuged at 4 °C for 10 min at 12,000 rpm to obtain supernatant for subsequent experiments. The total protein concentration in the supernatant was determined by BCA protein analysis kit and operated strictly according to the instructions. The protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes, and then, the membranes were blocked in 5% non-fat dry milk resolved in PBST for 1.5 h. Subsequently, the blots were incubated with primary antibodies against Gal-3, MMP-9, Col-I, Col-III, TGF-β1, Bcl-2, Bax, TNF-α, IL-6, caspase 3, smad 2, NF-κB, IL-1β, PI3K, and β-actin (1:1000) overnight at 4 °C. After washing with PBST, the blots were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) for 1.5 h. Washed again, the bound antibodies on the blots were visualized with enhanced chemiluminescence (ECL) reagent and quantified by Quantity One.

Statistical Analysis
All experiments were performed at least in triplicate. Data were analyzed by SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA) and are presented as the mean value ± SD. Statistical analysis of data was carried out by single-factor analysis of variance (ANOVA) and postevent multiple comparisons. p < 0.05 was considered of statistical significance.

Effects on Body Weight and Organ Index in the CMI Rats
During the experimental period, the bodyweight of the control was always heavier than the other groups (p < 0.05). After CAPE or CAPE-pNO 2 treatment, the bodyweight of CMI model rats increased significantly (p < 0.05), and the increment of body weight in the high-pNO 2 group was higher than that of CAPE group (p < 0.01) (Fig. 1a).
The heart index of the model group was lower than the control, which increased after CAPE or CAPE-pNO 2 treatment, but there was no statistical significance (p = 0.320). The lung index of the model group increased significantly compared to the control and decreased after CAPE or CAPE-pNO 2 treatment (p < 0.01) (Fig. 1b, c).

Effects on Serum Lipid Levels in the CMI Rats
As shown in Fig. 2, the levels of TC, TG, and LDL in serum of CMI rats were significantly higher (p < 0.01) then decreased after CAPE or CAPE-pNO 2 treatment (p < 0.05). The effect of high-pNO 2 was flat or better than that of CAPE, and the treatment effect of Low-pNO 2 was equal to or weaker ( Fig. 2a, b, d). As for HDL, the level in the model group was significantly decreased compared to the control (p < 0.01), treating with CAPE-pNO 2 decreased HDL level much stronger than CAPE did (p < 0.05) (Fig. 2c).

Effects on Changes of ECG and Cardiac Structure in the CMI Rats
The model rats had significantly higher heart rate, and the ST segment of the lead II ECG was elevated. The positive charge of QRS wave group increased, the negative charge decreased, and the U wave charge increased. After treatment of CAPE or CAPE-pNO 2 , the heart rate The changes of heart index. c The changes of lung index. Data are expressed as mean ± SD and repeated three times (n = 6). ** p < 0.01 vs. control group; ## p < 0.01 vs. model group; && p < 0.01 vs. CAPE group; $$ p < 0.01 vs. high-pNO 2 group. became slower, the positive and negative charges of QRS wave group tended to be balanced, and the charge of U wave decreased. The above effects of High-pNO 2 were better than CAPE (Fig. 3a).
As illustrated in Fig. 3, the levels of HBDH, LDH, and CK-MB were all promoted in the model rats (p < 0.01) and were also decreased with the treatment of CAPE or CAPE-pNO 2 (p < 0.05). The effects of CAPE-pNO 2 were more remarkable than CAPE even in the lower dose (Fig. 3b, c, e). The level of serum CK had the same trend as the others, though there was no significant statistical difference (Fig. 3d).
The HE staining of the heart was shown in Fig. 3f. The cardiomyocytes of the model were not long spindle with regular arrangement but appeared irregular interstitial spaces (as indicated by the arrows in Fig. 3f). The shape of cardiomyocytes had been improved to long spindle in good order with no meaningless intercellular cavities when treated with CAPE or CAPE-pNO 2 .

Effects on Expression of TGF-Β1 and Gal-3 in the CMI Rats
The results of immunohistochemistry showed that the expression of TGF-β1 in CMI rats enhanced significantly (p < 0.01), and the level of TGF-β1 decreased after CAPE or CAPE-pNO 2 treatment (p < 0.01) (Fig. 4a). From Western blot results, it was visible that the level of TGF-β1 in the heart was upregulated in the model rats (p < 0.01) and could be downregulated after treatment of CAPE or CAPE-pNO 2 (p < 0.01) (Fig. 4d). The decreasing effect of CAPE was better than that of high-pNO 2 in both immunohistochemistry and western blot results (p < 0.05).
As in Fig. 4b, the level of Gal-3 in CMI rat heart increased significantly (p < 0.01) and decreased after CAPE or CAPE-pNO 2 treatment (p < 0.01). The effect of high-pNO 2 was better than CAPE (p < 0.01). The Gal-3 levels of serum and heart exhibited a similar trend to immunohistochemistry result both before and after CAPE or CAPE-pNO 2 treatment, while the content of Gal-3 in low-pNO 2 group was approaching or lower than that in CAPE group in the results of serum ELISA assay and western blot test (Fig. 4c, d).

CAPE-pNO 2 Attenuated Cardiac Fibrosis in the CMI Rats
The results of Sirius red staining indicated that the level of collagen deposition in the heart of CMI rats was higher than that of the control (p < 0.01). After treatment of CAPE or CAPE-pNO 2 , the collagen deposition was significantly reduced (p < 0.01). The high-pNO 2 displayed better effect than CAPE (p < 0.05) (Fig. 5a). The paraffin section of heart tissue stained with hematoxylin and eosin. The cells in the control group were arranged regularly and in a long spindle shape, but the cells in the model group were disordered, with irregular interstitial spaces appearing (as indicated by the arrows in Fig. 3f). Scare bar = 100 µm. Data are expressed as mean ± SD and repeated three times (n = 6). ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. model group; & p < 0.05 vs. CAPE group.
The expression of MMP-9, Smad2, Col-I, and Col-III was increased in the heart of model rats (p < 0.01). After treatment with CAPE or CAPE-pNO 2 , the expression of these proteins decreased (p < 0.05), and the effect of High-pNO 2 was more potent than CAPE (p < 0.05). The effects of low-pNO 2 on MMP-9 and Smad2 were stronger compared with CAPE (p < 0.05), but weaker on Col-I and Col-III (p < 0.01) (Fig. 5b).

CAPE-pNO 2 Decreased Cardiomyocytes Apoptosis in the CMI Rats
Results of TUNEL staining manifested that apoptosis of cardiomyocytes in CMI rat increased (p < 0.01); CAPE and CAPE-pNO 2 could effectively reduce the apoptotic rate of cardiomyocytes (p < 0.05), and the effect of high-pNO 2 was similar to that of CAPE (Fig. 6a).
There was a rising trend of expressions of Bax and caspase 3, and a declining trend of Bcl-2 expression in the heart of CMI rat (p < 0.01). After treatment with CAPE or CAPE-pNO 2 , the expression of Bax and caspase 3 decreased (p < 0.01), and Bcl-2 expression increased. High-pNO 2 showed a better effect on these proteins than CAPE. The effects on cardiomyocytes apoptosis were in accordance with the TUNEL results (p < 0.05) (Fig. 6b).

CAPE-pNO 2 Weakened Myocardial Inflammation in the CMI Rats
It was revealed in the immunohistochemistry results in the heart that the expression of IL-6 was higher in the CMI rats than the control (p < 0.01), and it was reduced with the treatment of CAPE or CAPE-pNO 2 (p < 0.01). High-pNO 2 showed better effects than CAPE did (p < 0.05) (Fig. 7a).
As shown in Fig. 7b, CMI rats had higher expressions of TNF-α, NF-κB, IL-1β, IL-6, and PI3K in the heart (p < 0.01). The expressions of these proteins can The paraffin section of heart tissue detected by immunohistochemistry to measure TGF-β1 and Gal-3 expression. The sections were incubated with primary antibody (anti-TGF-β1 and anti-Gal-3) and goat anti-rabbit IgG antibody conjugated with HRP, then re-dyed with DAB and hematoxylin. The density value of brown part was used to measure the expression level of TGF-β1 and Gal-3. Scare bar = 100 µm. c Serum Gal-3 level. d The expressions of TGF-β1 and Gal-3 in the heart of CMI rats detected by western blot. Data are expressed as mean ± SD and repeated three times (n = 6). ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. model group; & p < 0.01, && p < 0.01 vs. CAPE group; $$ p < 0.01 vs. High-pNO 2 group. be downregulated by CAPE and CAPE-pNO 2 , and the regulating effect of High-pNO 2 was similar to or stronger than that of CAPE.

CAPE-pNO 2 Decreased TGF-Β1 and Gal-3 Expression in LPS-Induced H9c2
As shown in Fig. 9, the results of immunofluorescence and western blot experiment indicated that the content of TGF-β1 and Gal-3 in model cells stimulated by LPS was increased significantly (p < 0.01), and the level of TGF-β1 and Gal-3 could be downregulated after CAPE or CAPE-pNO 2 treatment. CAPE-pNO 2 showed better effects on Gal-3 than CAPE (p < 0.01). After cotreatment with CAPE-pNO 2 and MCP, the decreasing effect on Gal-3 was not as good as treatment of CAPE-pNO 2 alone (p < 0.01). While the effect of CAPE-pNO 2 on TGF-β1 was approaching to CAPE or co-treatment with CAPE-pNO 2 and MCP in immunofluorescence and western blot experiment.

CAPE-pNO 2 Attenuated Fibrosis in LPS-Induced H9c2
The results of immunofluorescence showed that Col-I and Col-III levels were markedly increased in LPSinduced H9c2 (p < 0.01). CAPE and CAPE-pNO 2 treatment decreased Col-I and Col-III expression, and CAPE-pNO 2 showed the similar effect on Col-III (p > 0.05) and showed the better effect on Col-I (p < 0.01) than CAPE. The downregulation effect of CAPE-pNO 2 decreased after MCP treatment (Fig. 10a, b). Western blot results Fig. 6 CAPE-pNO 2 decreased cardiomyocytes apoptosis in CMI rats. a The paraffin section of heart tissue detected by TUNEL staining. TUNELpositive cell rate was used to measure cardiomyocyte apoptosis. It was calculated by the ratio of apoptotic cell number to total cell number (the apoptotic cell nucleus was reddish brown, the normal cell nucleus was blue). Scare bar = 100 µm. b The expressions of Bcl-2, Bax, and caspase 3 in the heart of CMI rats detected by western blot. Data are expressed as mean ± SD and repeated three times (n = 6). ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. model group; && p < 0.01 vs. CAPE group; $$ p < 0.01 vs. high-pNO 2 group.
of Col-I and Col-III showed the similar regulation trend as immunofluorescence (Fig. 10c).

CAPE-pNO 2 Attenuated Apoptosis in LPS-Induced H9c2
The results of AO staining exhibited that the number of surviving cells decreased and apoptosis appeared in LPS-induced H9c2. CAPE-pNO 2 could increase the viable cell quantity and reduce the LPS-induced apoptosis. However, after treatment with MCP suppressed the protection effect of CAPE-pNO 2 (Fig. 11a).
Western blot assay analysis showed the Bax and caspase 3 upregulations, and Bcl-2 downregulations in LPS-induced H9c2 (p < 0.01). CAPE and CAPE-pNO 2 treatment decreased the Bax and caspase 3 expressions and increased Bcl-2 expression. Co-treatment with MCP and CAPE-pNO 2 eliminated the effect of CAPE-pNO 2 (p < 0.01; Fig. 11b). Fig. 7 CAPE-pNO 2 weakened cardiac inflammation in CMI rats. a The paraffin section of heart tissue detected by immunohistochemistry to measure IL-6 expression. The sections were incubated with primary antibody (anti-IL-6) and goat anti-rabbit IgG antibody conjugated with HRP, then re-dyed with DAB and hematoxylin. The density value of brown part was used to measure the expression level of IL-6. Scare bar = 100 µm. (b) The expressions of TNF-α, NF-κB, IL-1β, IL-6, and PI3K in the heart of CMI rats detected by western blot. Data are expressed as mean ± SD and repeated three times (n = 6). ** p < 0.01 vs. control group; ## p < 0.01 vs. model group; & p < 0.05, && p < 0.01 vs. CAPE group; $ p < 0.05, $$ p < 0.01 vs. High-pNO 2 group.

DISCUSSION
Myocardial ischemia is one of the main causes of heart failure [31], which is also closely related to atherosclerosis and myocardial infarction. The preconditioning and treatment on chronic and acute ischemia have clinical significance in the treatment of coronary artery disease [32]. Many cardiovascular pathological changes can lead to MI, including oxidative stress, inflammation, fibrosis, and apoptosis, and these changes are also related to variety of activating proteins and factors, such as NF-κB, TNF-α, and TGF-β1 [33]. c The expressions of apoptosis-related proteins (Bcl-2, Bax, and caspase 3) in the lung of CMI rats detected by western blot. d The expressions of inflammation-related proteins (TNF-α, NF-κB, and IL-6) in the lung of CMI rats detected by western blot. Data are expressed as mean ± SD and repeated three times (n = 6). ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. model group; & p < 0.05, && p < 0.01 vs. CAPE group; $ p < 0.05, $$ p < 0.01 vs. High-pNO 2 group.  CAPE-pNO 2 was synthesized based on CAPE in our lab, and it showed better protective effects on the heart of rats with MIRI [21,23]. Therefore, in this study, CAPE was considered a positive control, and the doses of CAPE-pNO 2 were set to be consistent with or lower than that of CAPE.
Our results demonstrated that CAPE-pNO 2 could decrease the lung index, the contents of TC, TG, and LDL in the serum of CMI rat, as well as increase the body weight, heart index, and the level of HDL. This compound could better promote lipid metabolism and growth and improve the lipid accumulation in the heart of the model rats. Besides, CAPE-pNO 2 also reduced the serum levels of cardiac enzyme including HBDH, LDH, CK, and CK-MB in CMI rats, which were positively correlated with cardiac damage. CAPE-pNO 2 treatment improved the disordered arrangement of cardiomyocytes and irregular cavities in HE staining figures, which means the cardiac structure has changed to recovery.
Studies have confirmed that high heart rate can aggravate myocardial ischemia, ventricular arrhythmia, vascular oxidative stress, endothelial dysfunction, and atherosclerosis [34], and lowering the heart rate can increase stroke volume and improve myocardial infarction [35]. ECG is usually used in clinical diagnosis and monitoring of cardiovascular diseases such as myocardial ischemia and myocardial infarction [36], of which elevated ST segment of ECG is a sensitive and specific marker [37]. QRS complex waves also produce electrical changes during depolarization of ischemic ventricular tissue [38]. After treatment with CAPE-pNO 2 , the heart rate of CMI rats became slower, elevated ST  segment dropped, and the positive and negative charge of the QRS wave group tended to be balanced. Thus CAPE-pNO 2 treatment exhibited repair efficacy on heart function.
TGF-β1 is closely related to heart disease, which aggravates cardiomyocyte apoptosis and cardiac hypertrophy, and it also takes part in myocardial fibrosis [39]. TGF-β1 is upregulated in remodeling after myocardial infarction and heart failure, and it can intensify MIRI [5]. In this study, the level of TGF-β1 increased in CMI heart and LPS-induced H9c2, and CAPE-pNO 2 could significantly decrease the expression of TGF-β1. Therefore, it is probable that the inhibition effect of CAPE-pNO 2 on cardiomyocyte apoptosis and myocardial fibrosis would be related to the decreased expression of TGF-β1 in heart tissue.
Gal-3 has important significance in the diagnosis, prognosis, and treatment of cardiovascular diseases in clinic. Higher serum and plasma Gal-3 levels are related to more serious MI and fibrotic cardiomyopathy [40,41]. In this study, the contents of Gal-3 increased significantly in CMI rat heart tissue, serum, and LPSinduced H9c2, and CAPE-pNO 2 significantly decreased Gal-3 levels.
To further explore the mechanism of the cardioprotective effect of CAPE-pNO 2 , we co-treated with Gal-3 inhibitor MCP and CAPE-pNO 2 . Not as anticipation, the CAPE-pNO 2 -mediated Gal-3 decrease was eliminated. We speculate that co-treated with MCP and CAPE-pNO 2 may have competitive binding with Gal-3 or inhibit the effect of CAPE-pNO 2 in other ways, which needs further study. Fig. 13 Experimental design. The experiment aimed to explore the effect of CAPE-pNO 2 on the expression of Gal-3 and on CMI rat. It's reported that TGF-β1 can regulate the Gal-3 expression, so we regarded TGF-β1/Gal-3 as an existing pathway, and it can regulate many related downstream proteins. Considering the pathway and the pathological mechanism of CMI, this article mainly discussed the protective effects of CAPE-pNO 2 on CMI rats in terms of fibrosis, apoptosis, and inflammation. The red lines represented the inhibiting effects, and the green arrows represented the promoting effects.
Gal-3 participates in fibrosis and it can increase the accumulation of Col-Ia in the fibrosis of pulmonary adventitia fibroblasts induced by TGF-β1 [42]. Besides, Gal-3 is the binding substrate of MMP-9. In our study, the collagen accumulation in the heart of CMI rats was significantly decreased after CAPE-pNO 2 treatment, along with a decrease of MMP-9, Smad2, Col-I, and Col-III. Similarly, this effect was inhibited by MCP in LPS-induced H9c2. It has been reported that Gal-3 induces atrial fibrosis through interacting with TGF-β1 and stimulating Smad signaling pathway [43]. Moreover, in mice deficient in Gal-3, TGF-β1-and bleomycininduced lung fibrosis was dramatically reduced [44], which also confirmed that Gal-3 mediated the actions of TGF-β1 and that Gal-3 was the downstream protein of TGF-β1. Thus, we call it TGF-β1/Gal-3 pathway in this article for short. It can be inferred from the above experimental results and literature that the effect of CAPE-pNO 2 on alleviating myocardial fibrosis in CMI was by regulating MMP-9 and TGF-β1/Gal-3/Col pathway.
Overexpression of Gal-3 has been reported to reduce myocardial cell viability and induce apoptosis [45]. The expression of proliferating cell nuclear antigen and Bcl-2 decreased in cardiomyocytes with high expression of Gal-3, while the expression of Bax and caspase 3 increased [14]. In the present study, CAPE-pNO 2 treatment significantly reduced the apoptosis rate of cardiomyocytes, upregulated the expression of Bcl-2, and downregulated the expression of Bax and caspase-3 in vivo and in vitro, while its effect was inhibited by MCP in LPS-induced H9c2. It implied that the anti-apoptosis effect of CAPE-pNO 2 on the heart of CMI rats might be through the TGF-β1/Gal-3/Bcl-2 signaling pathway.
Myocardial ischemic injury, such as MIRI, is closely related to inflammatory response [46]. Furthermore, cardiac inflammation increases the level of Gal-3 in the heart and releases it into the systemic circulation [41]. Gal-3 regulates typical inflammatory factors like TNF-α, IL-1β, and IL-18 [47]. In this study, CAPE-pNO 2 decreased the level of IL-6 expression in CMI heart and significantly decreased the contents of some typical inflammatory-related factors like TNF-α, NF-κB, IL-1β, IL-6, and PI3K. In LPS-induced H9c2, CAPE-pNO 2ameliorated inflammatory by suppressing the expression of IL-6 and TNF-α, but this effect was eliminated after treating with MCP. It can be concluded that CAPE-pNO 2 effectively weaken myocardial inflammation in CMI rats and LPS-induced H9c2, and the effect might be through TGF-β1/Gal-3/TNF-α and TGF-β1/Gal-3/IL-6 pathways.
Because of the circulation, the pathological changes of the lung and heart are always correlated. For example, after myocardial ischemia and reperfusion, pulmonary congestion may be caused by delayed production of reactive nitrogen species, and acute lung injury in diabetic rats can be caused after myocardial ischemia-reperfusion [48,49]. Moreover, in our study, increase of lung index in CMI rats could be seen, and it may be due to inflammation and other injury to enlarge lung. Further study found that the level of Gal-3, TGF-β1, MMP-9, Smad2, Col-I, Col-III, Bax, caspase 3, TNF-α, NF-κB, and IL-6 were increased, and the level of Bcl-2 was decreased in CMI rats. CAPE-pNO 2 could upregulate the expression of Bcl-2 and downregulate the other proteins. The changing trend of these proteins was consistent with that in the heart. It came to a conclusion that CMI also induced the lung injury, and CAPE-pNO 2 could downregulate TGF-β1 and Gal-3 in the lung of CMI rat, too. In addition, CAPE-pNO 2 could alleviate pulmonary fibrosis, apoptosis, and inflammation through TGF-β1/Gal-3 pathway.
In conclusion (Fig. 13), CAPE-pNO 2 could significantly reduce the expression of Gal-3 and TGF-β1 in the heart, lung of CMI rats, and LPS-induced H9c2, and had a regulatory effect on related downstream proteins of Gal-3. These results showed that the protective effect of CAPE-pNO 2 on CMI may be via TGF-β1/Gal-3 pathway.

DATA AVAILABILITY
Data supporting the results of this research are available to the corresponding author on request.

CODE AVAILABILITY
Not applicable.