Dioscin alleviates myocardial infarction injury via regulating BMP4/NOX1-mediated oxidative stress and inflammation

Abstract

Background

Dioscin, a steroidal saponin natural product, has various pharmacological activities, such as anti-inflammatory, antioxidant, lipid-lowering. However, little is known about its effects on myocardial infarction (MI) injury. Thus, the study aimed to investigate the protective effects and possible mechanisms of dioscin.

Methods

We evaluated protective effects of Dioscin on HL-1 cells after hypoxia based on MTT and ROS in vitro. In vivo, we ligated left anterior descending (LAD) of C57BL/6 mice to establish MI model and assess serum levels of LDH, CK-MB, cTnI, SOD, MDA and CAT treated by dioscin. In addition, myocardial damages were reflected by H&E, masson and ultrastructural examination and Electrocardiograph (ECG) was detected in MI mice. And the BMP4/NOX1 pathway was measured by western blotting, immunofluorescence assay and Real-time PCR. Furthermore, to investigate cardio-protective effects of dioscin via targeting BMP4, we transfected siBMP4 into HL-1 cells in vitro and injected BMP4 siRNA though tail veins in vivo.

Results

In vitro, dioscin significantly increased the viability of HL-1 cells and inhibited ROS level under hypoxia. In vivo, dioscin markedly reduced the elevation of ST segment and alleviated myocardial infarct area in mice. In terms of serology, dioscin evidently decreased LDH, CK-MB, cTnI, MDA levels, and increased SOD level. In addition, dioscin improved the pathological status of myocardial tissue and restrained the production of collagen fibers. Mechanism study proved that dioscin notablely regulated the levels of Nrf2, Keap1, HO-1, p-NF-κB, nNF-κB, TNF-α, IL-1β and IL-6 by down-regulating the protein levels of BMP4 and NOX1 against oxidative stress and inflammation. Further investigation showed that siBMP4 transfection diminished hypoxia and MI-induced oxidative and inflammation injury. The transfection decreased LDH, CK-MB and cTnI levels, improved ischemia T-wave inversion and reduced striated muscle necrosis, nucleus dissolution, collagen fibrosis and mitochondrial swelling in mice. In addition, siBMP4 decreased ROS and MDA levels, increased SOD and CAT levels and down-regulated mRNA levels of TNF-α, IL-1β and IL-6. Moreover, BMP4, NOX1 and nNF-κB protein levels were decreased and Nrf2 levels were increased by siBMP4.

Conclusion

Our study confirmed that dioscin showed an outstanding anti-myocardial infarction effect via regulating BMP4/NOX1-mediated oxidative stress and inflammation, which has a promising application value and development prospect against MI injury in the future.

Introduction

In recent 20 years, ischemic heart disease has taken its place as the first global burden of diseases and the prevalence continues to rise (Khan et al., 2020). It is expected to reach at least 1845 cases per 100,000 population by 2030 (Buja et al., 2019) . As the major one of heart ischemic diseases, myocardial infarction (MI) is mostly on the base of coronary atherosclerosis, coronary artery thrombosis and coronary artery spasm (Shiomi et al., 2013). After severely and persistently hypoxic-ischemic appears in coronary artery, MI occurs accompanied by arrhythmias, cardiogenic shock, heart failure and other serious complications. In clinical treatment, although the invasive approaches such as percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) are effective, not every patient suffering from MI can use these interventional treatments for the limited indications. In addition, drug treatments including nitrate drugs, antithrombotic drugs and angiotensin converting enzyme inhibitors (ACEI) are widely applied in clinical to alleviate cardiac load and inhibit ventricular remodeling (Barbato et al., 2020). However, we have to recognize that these drugs are generally inefficient due to short half-life, side effects and drug distribution. Drug development with minimal side effects from natural products against cardiovascular disease is attracting a growing interest recently. It was reported that Panax Notoginseng saponins induced cardiomyocyte autophagy and enhance angiogenesis against MI (Wang D et al., 2021). Ginkgo biloba extract 80 effectively inhibited AMI-induced myocardial damage via AKT/GSK3β/β-catenin signaling (Zheng et al., 2021). On the other hand, faced with difficult situation of MI treatments, demands for safe and effective preventing drugs of MI are soaring. Study suggested curcumin and curcumin nanoparticles pre-treatment significantly reduced MI induced myocardial damage and electrocardiogram alterations (Boaresu et al., 2019). Resveratrol pretreatment prevented MI induced unfavorable changes in infarct size, oxidative stress and cell apoptosis (Feng et al., 2019). There seems to be feasible to discovery effective preventative drugs of MI from natural products.

A greater understanding of MI mechanism is useful to drug development. In MI injury, reactive oxygen species induced oxidative stress injury exists throughout the whole pathogenetic process. Excessive free radicals or oxidation products cause a series of pathological changes such as mitochondrial permeability transition and Ca²⁺ overload, which can accelerate myocardial inflammation (Dogaru et al., 2019). Oxidative stress and inflammation can promote each other and jointly aggravate MI injury (Neri et al., 2015). Early inflammatory signal can enhance the dissolution of extracellular matrix, activate NF-κB nuclear translocation and trigger strong inflammatory response (Su et al., 2018). Meanwhile, the release of inflammatory factors stimulates the generation of ROS, which causes a vicious circle between oxidative stress and inflammation (Bai et al., 2020). In this case, antioxidants and from natural products are likely to protect myocardium from ischemia damages.

We have known that cell morphology, differentiation, proliferation and apoptosis are significantly affected by bone morphogenetic protein (BMP) (Chen et al., 2012; Shimizu et al., 2012). Among BMP protein family, BMP4 is one of the most characteristic members. Some studies have shown that BMP4 can activate NADPH oxidase 1 (NOX1), stimulate ROS production, up-regulate the level of cyclooxygenase-2 (COX-2), enhance oxidative stress and damage aortic endothelial function (Jo et al., 2006). In addition, as a new pro-inflammatory factor, BMP4 can induce monocyte adhesion and promote the inflammatory response of endothelial cells, which has an important impact on heart development and myocardial remodeling (McCulley et al., 2008). Multiple studies have shown that BMP4/NOX1 signaling is essential for the regulation of oxidative stress and inflammation (Koga et al., 2013; Drummond et al., 2014; Jiang et al., 2014; Youn et al., 2015). Therefore, inhibiting BMP4/NOX1 signal may have a protective effect against MI and deserves further investigation.

Dioscin, a natural product (Lin et al., 2010), widely exists in some medicinal plants such as Dioscorea and Liliaceae. Amount of researches have shown that dioscin has positive impacts on diabetes, atherosclerosis and multiple organ injury (Wu et al., 2015; Xu et al., 2017; Zhang et al., 2017; Zhao et al., 2018; Xu et al., 2020). Our previous studies also indicated that dioscin pretreatments were able to against lung and intestinal ischemia/reperfusion with the anti-inflammation and antioxidant activities (Zheng et al., 2019; Dong et al., 2021). Most notably, dioscin can inhibit platelet activation, improve myocardial energy metabolism, and alleviate oxidative stress and inflammation during myocardial ischemia-reperfusion in rats (Yang et al., 2018; Cheng et al., 2019). The effects and mechanisms of dioscin against MI have not been reported yet. Thus, this paper investigated the possible protective effects and mechanisms of dioscin against MI via BMP4/NOX1 mediated oxidative stress and inflammation. Flow chart for in vivo and in vitro experiment was showed in Fig. 1A.