Continuing Treatment with Salvia miltiorrhiza Injection Attenuates Myocardial Fibrosis in Chronic Iron-Overloaded Mice

April Continuing Treatment with Salvia miltiorrhiza Injection Attenuates Myocardial Fibrosis in Chronic Iron-Overloaded Mice Data Availability Statement: All relevant data are within the paper. 0 1 2 Ying Zhang 0 1 2 Hao Wang 0 1 2 Lijing Cui 0 1 2 Yuanyuan Zhang 0 1 2 Yang Liu 0 1 2 Xi Chu 0 1 2 Zhenyi Liu 0 1 2 Jianping Zhang 0 1 2 Li Chu 0 1 2 0 1 Department of Pharmacology, Hebei Medical University , Shijiazhuang , China , 2 Department of Chinese Materia Medica, Hebei Medical University , Shijiazhuang , China , 3 Department of Pharmaceutics, Hebei University of Chinese Medicine , Shijiazhuang , China , 4 The Fourth Hospital of Hebei Medical University , Shijiazhuang , China 1 Funding: This study was supported by the Nature Fund of Hebei Province, China http://hensf.hebstd. gov.cn/ (No. C2011206025 and H2014206244) and the Fund of Hebei Science and Technology Bureau http://www.hebstd.gov.cn/ (No. 10276105D-2). In this study, the funder conceived and designed the experiments, and wrote the paper 2 Academic Editor: Michael Bader, Max-Delbruck Center for Molecular Medicine (MDC) , GERMANY Iron overload cardiomyopathy results from iron accumulation in the myocardium that is closely linked to iron-mediated myocardial fibrosis. Salvia miltiorrhiza (SM, also known as Danshen), a traditional Chinese medicinal herb, has been widely used for hundreds of years to treat cardiovascular diseases. Here, we investigated the effect and potential mechanism of SM on myocardial fibrosis induced by chronic iron overload (CIO) in mice. Kunming male mice (8 weeks old) were randomized to six groups of 10 animals each: control (CONT), CIO, low-dose SM (L-SM), high-dose SM (H-SM), verapamil (VRP) and deferoxamine (DFO) groups. Normal saline was injected in the CONT group. Mice in the other five groups were treated with iron dextran at 50 mg/kg per day intraperitoneally for 7 weeks, and those in the latter four groups also received corresponding daily treatments, including 3 g/kg or 6 g/kg of SM, 100 mg/kg of VRP, or 100 mg/kg of DFO. The iron deposition was estimated histologically using Prussian blue staining. Myocardial fibrosis was determined by Masson's trichrome staining and hydroxyproline (Hyp) quantitative assay. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) content and protein expression levels of type I collagen (COL I), type I collagen (COL III), transforming growth factor-1 (TGF-1) and matrix metalloproteinase-9 (MMP-9) were analyzed to investigate the mechanisms underlying the effects of SM against iron-overloaded fibrosis. Treatment of chronic iron-overloaded mice with SM dose-dependently reduced iron deposition levels, fibrotic area percentage, Hyp content, expression levels of COL I and COL III, as well as upregulated the expression of TGF- 1 and MMP-9 proteins in the heart. Moreover, SM treatment decreased MDA content and increased SOD activity. In conclusion, SM exerted activities against cardiac fibrosis induced by CIO, which may be attributed to its inhibition of iron deposition, as well as collagen metabolism and oxidative stress. - Competing Interests: The authors have declared that no competing interests exist. Iron is an essential element for cell metabolism and the function of various cellular enzymes, and its level is tightly regulated physiologically [1]. However, the body has no mechanism to excrete excess iron, which is highly toxic when present in high quantities and unbound from proteins [2]. Iron overload is a common clinical problem, arising from disorders of increased iron absorption, such as hereditary hemochromatosis or thalassaemia intermedia syndromes, or as a consequence of chronic blood transfusions for various blood disorders [35]. In these conditions, iron homeostasis is perturbed, and the excessive iron deposits in the liver, spleen, heart, bone marrow, pituitary, pancreas and central nervous system. Iron overload cardiomyopathy results from the accumulation of iron in the myocardium and is a leading cause of morbidity and mortality in patients with iron overload [6,7]. Iron overload cardiomyopathy, regardless of its origin, is characterized by a restrictive cardiomyopathy with early diastolic dysfunction which invariably progresses to a dilated cardiomyopathy [6]. Diastolic dysfunction occurs when the ventricle cannot fill properly due to an adverse accumulation and structural remodeling of the heart extracellular matrix (ECM) components defined as cardiac fibrosis [8 10]. The available body of evidence implicates iron itself in the initiation of fibrosis [11]. Additionally, in patients with iron overload the significant presence of myocardial fibrosis is a timedependent process correlating with cardiovascular risk factors and cardiac complications [12]. For iron overload diseases, the current mainstays of therapy for excessive iron deposition in patients are phlebotomy and iron chelation, which are designed to remove whole-body iron [5,13]. Iron chelator was designed to bind with iron ions to remove the metal from the body. In accordance with its relatively high molecular weight and highly hydrophilic properties, chelators do not readily enter most types of cells (indirect action), including cardiomyocytes, but they may not prevent uptake of iron in organs, especially in those in which iron enters cells through specific ion channels [14,15]. Unfortunately, chelation therapy is cumbersome and associated with toxic adverse effects, including ophthalmological, auditory and bone toxicity and growth retardation [15]. For example, cardiac morbidity and mortality continue to occur in patients with thalassemia major treated with deferoxamine (DFO), a traditional iron chelator, presumably related to difficulties with adherence to chelation therapy [15,16]. However, phlebotomy and iron chelation are less effective for the treatment of myocardial fibrosis complicated with iron overload. Statin, baicalin and green tea were reported to inhibit or delay iron deposition in vitro, but their effects on iron-overloaded fibrosis have not been determined [1719]. Encouragingly, recent studies have shown that calcium channels provide a major portal for iron uptake into cardiomyocytes in iron overload cardiomyopathy, and calcium channel blockers (CCBs) used in routine cardiovascular treatment can inhibit iron entry into cardiomyocytes and reduce the collagen volume in heart tissue [5,20 22]. Furthermore, a pilot trial investigating the effect of amlodipine (a CCB) on iron overload in patients with thalassemia major reported that it can serve as a complementary treatment to standard chelation regimens and may improve the efficacy of iron removal in the heart without the burden of significant side effects [23]. The above information suggests us that an ideal drug treatment should prevent iron accumulation and iron-related cardiac fibrosis, as well as have few side effects. In China and other Asian countries, herbal medicines have been widely applied in (...truncated)