Myocardial ischemia is one of the main causes of cardiac remodeling and heart failure , 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 . 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 .
CAPE-pNO2 was synthesized based on CAPE in our lab, and it showed better protective effects on heart of rats with MIRI [21, 23]. Therefore, in this study, CAPE was considered as a positive control, and the doses of CAPE-pNO2 were set to be consistent with or lower than that of CAPE.
Our results demonstrated that CAPE-pNO2 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-pNO2 also reduced the serum levels of cardiac enzyme including HBDB, LDH, CK and CK-MB in CMI rats, which were positively correlated with cardiac damage. CAPE-pNO2 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 , and lowering the heart rate can improve regional blood flow and systolic function of ischemic myocardium . ECG is usually used in clinical diagnosis and monitoring of cardiovascular diseases such as myocardial ischemia and myocardial infarction , of which elevated ST segment of ECG is a sensitive and specific marker . QRS complex waves also produce electrical changes during depolarization of ischemic ventricular tissue . After treatment with CAPE-pNO2, 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-pNO2 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 . TGF-β1 is up-regulated in remodeling after myocardial infarction and heart failure, and it can intensify MIRI . In this study, the level of TGF-β1 increased in CMI heart and LPS-induced H9c2, and CAPE-pNO2 could significantly decrease the expression of TGF-β1. Therefore, it is probable that the inhibition effect of CAPE-pNO2 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 LPS-induced H9c2, and CAPE-pNO2 significantly decreased Gal-3 levels.
To further explore the mechanism of cardio-protective effect of CAPE-pNO2, we co-treated with Gal-3 inhibitor MCP and CAPE-pNO2. Not as anticipation, the CAPE-pNO2-mediated Gal-3 decrease was eliminated. We speculate that co-treated with MCP and CAPE-pNO2 may have competitive binding with Gal-3, or inhibit the effect of CAPE-pNO2 in other ways, which needs further studied.
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 . 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-pNO2 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 TGF-β1 regulates the expression of Gal-3 in nuclear pulposus cells through a typical Smad3 signaling pathway . Moreover, in mice deficient in galectin-3, TGF-β1 and bleomycin-induced lung fibrosis was dramatically reduced , 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-pNO2 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 . 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 . In the present study, CAPE-pNO2 treatment significantly reduced the apoptosis rate of cardiomyocytes, up-regulated the expression of Bcl-2, and down-regulated 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-pNO2 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 . Furthermore, Cardiac inflammation increases the level of Gal-3 in the heart and releases it into the systemic circulation . Gal-3 regulates typical inflammatory factors like TNF-α and IL-6 . In this study, CAPE-pNO2 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-pNO2 ameliorated 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-pNO2 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 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-pNO2 could up-regulate the expression of Bcl-2 and down-regulate 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-pNO2 could down-regulate TGF-β1 and Gal-3 in the lung of CMI rat, too. In addition, CAPE-pNO2 could alleviate pulmonary fibrosis, apoptosis and inflammation through TGF-β1/Gal-3 pathway.
In conclusion (Fig. 13), CAPE-pNO2 could significantly reduce the expression of Gal-3 and TGF-β1in the heart, lung of CMI rats and LPS-induced H9c2, and had regulatory effect on related downstream proteins of Gal-3. These results showed that the protective effect of CAPE-pNO2 on CMI may be via TGF-β1/Gal-3 pathway.