DOX is currently one of the most commonly used anthracycline anti-tumor drugs and is widely used to treat various malignant tumors, including hematological malignancies and solid tumors. However, the use of DOX was often restricted due to left ventricular dysfunction, and even cardiotoxic reactions such as dilated cardiomyopathy and CHF. At present, the pathogenesis of cardiotoxicity caused by the DOX is not fully understood. Traditional studies believed that the DOX produces semiquinone free radicals due to metabolism in the body, which in turn leads to oxidative damage to cardiomyocytes. However, recent studies have found that apoptosis is still an important cause of myocardial damage caused by the DOX[5, 6]. Therefore, it is urgent to find drugs that can alleviate the apoptotic damage of cardiomyocytes caused by the DOX without affecting the use of anthracyclines. It is also a new strategy to improve the survival rate of cancer patients and improve the long-term quality of life of patients.
SMI is a form of modern Chinese medicine, derived from the classic prescription Shengmai San. In recent years, it has been proven that SMI can improve the efficacy of anti-tumor drugs and reduce their toxicity. Our previous studies have also shown that SMI has the effect of reducing the myocardial damage caused by the DOX. It mainly promotes the expression of miR-30a, which leads to the decrease of Beclin 1 expression, inhibits the excessive autophagy of the myocardium, and protects the myocardium from the damage of the DOX. Beclin 1 is an important target downstream of miR-30a. As we all know, Beclin 1 is a key node in the crosstalk between autophagy and apoptosis. On the one hand, Beclin 1 is a component of the class III PI3K/Vps34 complex and is essential for the formation of autophagic vesicles. On the other hand, Beclin 1 binds to the anti-apoptotic protein Bcl-2, leading to the dissociation of the pro-apoptotic protein Bax, which initiates the apoptosis cascade[26, 27]. Therefore, the down-regulation of Beclin 1 expression is consistent with previous related research results and helps protect cardiomyocytes from apoptosis. And a related study has also shown that SMI can alleviate myocardial endoplasmic reticulum stress and caspase 12 dependent apoptosis, and play a role in protecting the myocardium. However, whether SMI also exerts an anti-cardiomyocyte apoptosis effect through miR-30a/Beclin 1 in cardiotoxicity induced by the DOX remains to be further studied.
To verify the protective mechanism of SMI on the myocardial injury induced by the DOX, we used rat tail vein injection of the DOX to replicate the DOX cardiotoxicity model. Since anthracycline anti-tumor drugs can damage myocardial cell membranes and cause myocardial cell damage, the markers of myocardial injury are of great significance for diagnosing the cardiotoxicity of anthracyclines. Cardiac enzymes, troponin, and brain natriuretic peptides were traditional biomarkers for detecting myocardial injury. Creatine kinase (CK) and lactate dehydrogenase (LDH) were also widely used in the detection of cardiotoxicity of anticancer drugs[29, 30]. Troponin can be used as an independent predictor of cardiotoxicity after chemotherapy, troponin T (cTnT),and N-terminal pro-brain natriuretic peptide (NT-proBNP) can be used as an important marker for the evaluation of early myocardial dysfunction in cardiotoxicity induced by the anthracyclines. However, studies have shown that myocardial enzymes, cTnT, and NT-proBNP still have certain differences in predicting and diagnosing cardiotoxicity[32, 33], and the combined application can further clarify the existence of cardiotoxicity. In this study, we found that the serum CK, LDH, cTnT, and NT-proBNP levels of rats in the model group increased, while miR-30a agomir and SMI treatment groups could reduce the rat serum CK, LDH, cTnT and NT-proBNP levels. Moreover, the SMI-L effect of the Chinese medicine treatment group was significant. The above results suggest that SMI can reduce the myocardial damage caused by DOX and improve the heart function of rats.
The changes in the rat myocardial tissue were further detected and observed under an optical microscope that the red collagen fibers in the myocardium of the model group increased significantly compared with the control group, indicating that the DOX caused the proliferation of the myocardial collagen knot tissue and interstitial fibrosis in the rat lesions. While miR-30a agomir group and SMI treatment group significantly alleviated myocardial fibrosis induced by the DOX in rats. The soluble ST2 (sST2), a member of the family of interleukin-1 receptors, was produced by cardiomyocytes and cardiac fibroblasts and was associated with structural and functional changes of left ventricular caused by myocardial damage, fibrosis, and poor remodeling, and can be used as an early sensitive predictor of left ventricular dysfunction caused by anthracycline drugs[34, 35]. Growth differentiation factor-15 (GDF-15) was a member of the transforming growth factor-β superfamily and was closely related to collagen renewal, deposition, and the degree of cardiac fibrosis. Under normal circumstances, GDF-15 was weakly expressed by cardiomyocytes, but it was induced to be expressed and secreted under conditions of myocardial ischemia, tissue damage, oxidative stress, and inflammation. It was a predictor of a variety of cardiovascular adverse events and mortality risk assessment and has diagnostic value in oncology drug cardiotoxicity[37–39]. The main characteristics of myocardial pathological response are myocardial fibrosis, collagen deposition, and the increase in the synthesis of type I collagen and type III collagen, leading to cardiac dysfunction. In the cardiotoxicity induced by the DOX, we found that SMI can reduce the levels of serum sST2 and GDF-15 in rats, and reduce the expression of Collagen I and Collagen III, indicating that SMI can reduce the cardiac fibrosis induced by the DOX and reduce collagen deposition.
Cardiomyocyte apoptosis was considered to be an important mechanism of DOX cardiotoxicity[5, 6]. Apoptosis can remove damaged aging or harmful cells to maintain cell homeostasis, while cardiomyocytes are non-renewable cells, and cardiomyocyte apoptosis was the main feature of myocardial injury. The gold standard of the transmission electron microscope was used to detect apoptosis, and the phenomenon of the disappearance of microvilli, condensation of chromatin, expansion of endoplasmic reticulum, the disappearance of nuclear fragmentation, and formation of crescent apoptotic bodies was observed under the electron microscope. This study found that miR-30a agomir and SMI can alleviate the apoptosis of the rat cardiomyocytes and reduce the apoptosis index of cardiomyocytes. Apoptosis was the result of multi-molecule co-regulation, among which the Bcl-2 family and the Caspase family are two important parts of regulating apoptosis. The Bcl-2 protein family includes both Bax pro-apoptotic members and Bcl-2 anti-apoptotic members, which were closely related to the Caspase family. Bcl-2 can regulate cell apoptosis through Caspase-9 and Caspase-3 dependent pathways, which means that by increasing Bax and reducing Bcl-2 expression, it can stimulate mitochondria to release cytochrome C, and cytochrome C further recruits Caspase-9 to promote the formation of apoptotic bodies,and then induces the activation of Caspase-3 and triggers the Caspase cascade, which ultimately leads to apoptotic cell death[45, 46]. Bcl-2 can be used as the upstream regulatory mechanism of Caspase. Overexpression of Bcl-2 can effectively inhibit Caspase activation and apoptosis induced by various factors. In this study, we found that SMI can increase the expression of anti-apoptotic protein Bcl-2 and decrease the expression of pro-apoptotic protein Bax, thereby inhibiting the expression of Caspase-9 and Caspase-3, suggesting that SMI can inhibit rat cardiomyocyte apoptosis. In summary, combined with our previous research results, it is suggested that SMI can increase the expression of miR-30a and decrease the expression of Beclin 1. It can not only inhibit the excessive autophagy of the myocardium but also reduce the myocardial damage caused by DOX by inhibiting the apoptosis of myocardial cells.