In the present study, we demonstrated for the first time that muscone inhibits Ang II-induced myocardial hypertrophy and myocardial injury in vivo. The present results showed that muscones significantly attenuated Ang II-induced myocardial fibrosis, remodeling, and inflammation. Mechanistically, the MAPK, STAT3, and TGF-β/SMAD pathways are closely associated with muscone cardiac protection, and muscone has no significant toxicity or side effects in vivo or in vitro (Fig. 6). In conclusion, the present study suggested that muscone may be a promising candidate for the treatment of cardiac hypertrophy and heart failure.
In pathological conditions, myocardial hypertrophy is harmful to the organism and is a key risk factor for the progression of heart failure, often leading to cardiac injury, irregular heartbeat rhythm, angina, and heart attack, and the risk of death is also severely increased with myocardial hypertrophy (Reiter, Manchester et al. 2010, Jiang, Lan et al. 2018). Despite many reports describing the pathophysiological mechanisms of myocardial hypertrophy as well as cutting-edge studies, the clinical outcome of myocardial hypertrophy remains poor. Therefore, the development of new drugs to prevent and treat myocardial hypertrophy is urgent. In recent years, many researchers have extensively studied compound Chinese medicines and natural products derived from Chinese medicines, and their cardioprotective effects have been explored and applied in the clinic (Wu, Lin et al. 2017, Bian, Li et al. 2020, Cheng, Shen et al. 2021). As a monomeric compound, muscone is the main component of Heart-protecting Musk Pill (HMP) (Wang, Xing et al. 2020). There is growing evidence that muscone exerts significant beneficial effects in the cardiovascular setting. For example, muscone attenuates myocardial ischemia-reperfusion injury by inhibiting oxidative stress and enhancing SIRT3 (Wei, Hua et al. 2021). Hong et al. found that muscone inhibits the excessive inflammatory response in myocardial infarction by targeting TREM-1. Yingqiang et al. found that inhibition of NF-κB and NLRP3 inflammasomes attenuates chronic inflammation mediated by cardiac macrophages and improves cardiac function in mice after myocardial infarction (Du, Gu et al. 2018, Zhang, Ye et al. 2022). However, the studies addressing the mechanism of action of muscone in relation to myocardial hypertrophy have not investigated whether muscone has a therapeutic effect on cardiovascular disease. In the present study, we investigated the relationship between muscone and myocardial hypertrophy on the basis of cellular and animal models. For a positive control, we used valsartan, an angiotensin II receptor blocker (ARBs), which has been clinically used to treat hypertension. Nordén and Burke et al. found that valsartan reduces cardiac fibrosis during left ventricular pressure overload by restoring protein kinase G (PKG) signaling in cardiac fibroblasts, and they reported that valsartan improves cardiac hypertrophy and preserves diastolic during cardiac pressure overload (Burke, Lighthouse et al. 2019, Nordén, Bendiksen et al. 2021). Therefore, we evaluated the therapeutic effect of muscone in this model of cardiac hypertrophy, using valsartan as a positive control. We found that muscone inhibited Ang II-induced myocardial hypertrophy in vivo, and the therapeutic value of high-dose muscone was comparable to that of valsartan.
Myocardial fibrosis is a phenomenon of collagen accumulation within the myocardium, and it is usually characterized by elevated collagen levels (Nakamura and Sadoshima 2018). In the presence of excessive left ventricular pressure, myocardial fibrosis occurs, initially as an adaptive response that maintains the firing capacity, or active (contractile) stiffness, of the hypertrophied myocardium. In the later stages of hypertrophy, myocardial interstitial fibrosis has a detrimental effect on diastolic and systolic stiffness of the myocardium, which may lead to pathological hypertrophy and heart failure (Turto 1977, Weber, Jalil et al. 1989). In the present study, we found that muscone significantly inhibited Ang II-induced myocardial fibrosis. Moreover, muscone effectively reduced the expression of COL1A1 and COL3A1 in vivo, and we found that the SMAD pathway may play a key role in this process. These results suggested that muscone significantly ameliorates Ang II-induced myocardial fibrosis in mice.
Proinflammatory cytokines are small molecule proteins involved in signaling pathways that have been shown to be involved in cardiovascular disease, and they have also been shown to be involved in the transition from adaptive to pathological cardiac hypertrophy during cardiac remodeling (Ionita, Arslan et al. 2010, Oldfield, Duhamel et al. 2020). Previous studies have shown that proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, are closely associated with myocardial fibrosis, pathological cardiac remodeling, and myocardial hypertrophy (Sun, Chen et al. 2007, Meier, Bullinger et al. 2009, Meléndez, McLarty et al. 2010, Dobaczewski, Chen et al. 2011). In the present study, we examined the expression levels of the proinflammatory and anti-inflammatory cytokines in mice. The proinflammatory cytokines were significantly lower after muscone treatment compared to the Ang II group alone, and opposite results were observed for the anti-inflammatory cytokines. The regulation of inflammatory responses and myocardial hypertrophy by Ang II via the MAPK and SMAD signaling pathways has been extensively studied (Ai, Zhang et al. 2010, Chen, Li et al. 2019, Liao, Zhang et al. 2019, Zeng, Yan et al. 2020). In addition, IL-6, which is produced by cardiomyocytes in response to hypertrophic stimuli, activates the STAT3 and MAPK signaling pathway in myocardial hypertrophy (Fahmi, Smart et al. 2013, Tham, Bernardo et al. 2015). Muscone has been shown to improve diabetic peripheral neuropathy by activating the AKT/mTOR signaling pathway (Dong, Li et al. 2019). Yu et al. found that muscone alleviates inflammatory pain by inhibiting the NOX4/JAK2-STAT3 pathway and NLRP3 inflammasomes via microglia activation-mediated inflammatory responses (Yu, Zhao et al. 2020). Qian et al. (2010) reported that muscone inhibits the expression of prostaglandin E2, 6-keto-prostaglandin F1 alpha, IL-1β, and TNF-α as well as restores the structural deformation of degenerative intervertebral discs (Liang, Zhang et al. 2010). In addition, Liu et al. (2022) reported that muscone exerts antidepressant-like effects by reducing neuroinflammation and oxidative stress in a mouse model of chronic restraint stress (Liu, Liu et al. 2022). In the present study, molecular docking predicted that muscone may function in the heart through binding molecules in the MAPK and SMAD pathway. The present experiments clearly demonstrated that muscone significantly reduced the expression of p-ERK, p-JNK, p-P38, p-SMAD2, and p-SMAD3 compared to Ang II treatment alone. These results suggested that muscone attenuates ANG II-induced myocardial hypertrophy, at least in part, by inhibiting the MAPK and TGF-β/SMAD signaling pathways. However, more detailed studies are needed in the future to determine the exact molecular pathways underlying the anti-hypertrophic effects of muscone.
As we mentioned previously, several studies have shown that muscone is protective against certain diseases. For example, muscone ameliorates lipopolysaccharide (LPS)-induced depressive-like behaviors and inhibits neuroinflammation in the prefrontal cortex of mice (He, Shi et al. 2020). In addition, muscone suppresses inflammatory responses and neuronal damage in a rat model of cervical spondylotic myelopathy by modulating Drp1-dependent mitochondrial fission (Zhou, Yao et al. 2020). These studies imply that muscone has the potential to be developed as a clinical agent to alleviate neuroinflammation. Therefore, studies on the toxicity and adverse effects of muscone are particularly important. In this regard, we conducted a study on the toxicity of muscone to the body by using a muscone treatment group as a negative control to test liver and kidney functions as well as cell viability using a CCK-8 assay. The results showed that muscone had no significant toxicity or adverse effects on normal organs, such as the heart, liver, and kidneys.