Tanshinone II A Relieves Adriamycin-induced Myocardial Injury in Rat Model

As an effective antineoplastic agent, adriamycin (ADR) remains a use for the treatment of cancer. However, it is limited by the serous cardiotoxicity. Tanshinone II A is the main effective component of Salvia miltiorrhiza Bunge which has been used for treatment of cardiovascular diseases. The purpose of this study is to evaluate the protective effect of Tanshinone II A on adriamycin-induced myocardial injury in rat and explore the mechanism of this effect. Male Wistar rats (200 ± 20 g) were divided into three groups, control (CON) group, adriamycin (ADR) group and ADR + Tanshinone II A (TRA) group. At the end of the 4 week treatment period, cardiac function was evaluated by transthoracic echocardiography. Molecular and cellular measurements were performed in atrial muscle to examine histopathological changes, the formation of fibrosis, Inflammation and apoptosis. Cardiac dysfunction was induced by adriamycin, as indicated by significant decreases in ventricular fractional shortening and ejection fraction. This adriamycin-induced cardiac dysfunction was prevented by the treatment of Tanshinone II A. Adriamycin induced pathological changes and fibrosis, activated apoptosis (increased TUNEL index, apoptotic DNA fragmentation,and caspase-3 activity and decreased Bcl-2/Bax ratio), inflammation and suppressed phosphorylation status (eIF2α and PERK) in atrial. All these molecular and cellular alterations induced by ADR were not found in the rats treated with Tanshinone II A. These findings demonstrate clearly that Tanshinone II A protects the cardiomyocytes against the ADR-induced cardiomyopathy by preventing the activation of cardiac fibrosis and apoptosis, and the effects are probably mediated through ERS pathway.


Introduction
As an anthracycline antibiotic, adriamycin (ADR) is widely used for treatment of cancer (Blum et al., 1974).After the crossing of the cellular membrane, adriamycin inhibits the synthesis of RNA and DNA, killing various growth cycle of tumor cells (Shi et al., 1993).As same as other anticancer drugs, adriamycin has a series of side effects, such as nausea and vomiting which disappear after stopping medication (Huang et al., 2004).However, high concentrations of ADR tend to cause severe toxicity to normal tissues, including cardiotoxicity, limiting the application of adriamycin (Lefrak et al., 1973).
As traditional Chines medicine, the root of Salvia miltiorrhiza Bunge has been used for treatment of cardiovascular diseases for a long time (Bristow et al., 1978).Tanshinone II A is one of the major active components of Salvia miltiorrhiza Bunge.It has been reported that Tanshinone II A inhibit atherosclerotic calcification (Futian et al., 2007).It also relieves the apoptosis of cardiomyocytes (Lin et al., 2015).Additionally, Tanshinone II A has been found to anti-inflammation (Gao et al., 2008).Some researchers have reported Tanshinone II A protect against cardiotoxicity induced by adriamycin.Jiang et al provided evidence on the potential of Tanshinone II A as a proteictive agent against adriamycin induced cardiotoxicity ( Zhi-yuan et al., 2008).Tanshinone II A significantly inhibits adriamycin-induced cardiomyocyte apoptosis in a dose-dependent manner also has been reported (Baohong et al., 2009), and Akt-signaling pathways related with this phenomenon (Hong-Jye et al., 2012).Some studies have implicated the role of ER stress in myocardial pathology (Xin et al., 2007;Toth et al., 2007).ER stress is related with the development of ischemic heart disease in mice (Azfer et al., 2006).Whether Tanshinone II A protect against cardiotoxicity induced by adriamycin through ERS or other activity.Therefore, this study determined to examine the effect of Tanshinone II A on adriamycin-induced cardiomyocythy.We test Tanshinone II A whether protect against myocardial fibrosis and apoptosis induced by adriamycin.To further examine whether the effects of Tanshinone II A are mediated by ERS pathway.Last, we test the effects of Tanshinone II A on adriamycin-induced inflammation related factors.

Materials
Adriamycin was purchased from the medicine of WanDong (ShenZhen, China).Terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay kit was purchased from Beyotime Biotechnology (ShangHai, China).Tanshinone II A was provided from SHANGHAI NO.1 Biochemical and Pharmaceutical.Co., Ltd.The structure of Tanshinone II A is shown in figure 1.
Figure 1. the chemical structure of Tanshinone II A

Experimental Animals and Rat Model of Adriamycin Induced
Male Wistar rats (200 ± 20 g) were obtained from YiSi Laboratory Animal Technology Co., Ltd (ChangChun).All of rats were housed under standard conditions.All experiments in this research were performed in accordance with the Guidelines of Animal Experiments form the Committee of Medical Ethics, National Health Department of China (1998).These rats were divided into three groups, control (CON) group, adriamycin (ADR) group and ADR + Tanshinone II A (TRA) group.CON group intraperitoneal injected saline at the indicated time; ADR group were intraperitoneal injected 2mg/kg/week adriamycin for 3 weeks and ADR + TRA group intraperitoneal injected 2mg/kg/week adriamycin and 2mg/kg/week Tanshinone II A for 3 weeks.All the rats were maintained on a 12 : 12 hour light-dark cycle, at the temperature of 20 ± 2 ℃ and were allowed free access to food and tap water.After 4 weeks, the rats were sacrificed.

High Frequency Echocardiography Analysis
The Color Ultragraphy was used for the echocardiography of rats.Echocardiography was performed according to a previously described protocol (Xiao et al., 2014).Animals in each group were anaesthetised with sodium pentobarbital (100 mg/kg).Hair from chests was removed with an electrical clipper and hair removal gel prior to the examination.Two-dimensional grayscale ultrasound scanning was performed to assess the cardiac structures in the parasternal short-axis view at the midpapillary level.The grayscale echocardiographic view was used to position the M-mode echocardiographic line.Left ventricle (LV) internal dimensions and anterior and posterior wall thickness were then measured.LV end-diastolic (LVEDD) and end-systolic dimensions (LVESD) were assessed from the M-mode tracing.Fractional shortening (FS), the percent change in LV cavity dimension, was calculated using the equation FS (%)=[(LVEDD-LVESD)/LVEDD]×100.Ejection fraction (EF) represents stroke volume as a percentage of end-diastolic LV volume and was derived as EF (%)=Y+[(100-Y)×0.15],where Y=[(LVEDD2-LVESD2)/LVEDD2]×100.All measurements were averaged over three consecutive cardiac cycles.

Enzyme-linked Immunosorbent Assay
Heart tissue levels of TNF-α and TGF-β1 were determined using the rat TNF-α and TGF-β1 ELISA kit (NEOBIOSCIENCE, China), according to the manufacturer's instructions.

HE staining, Masson Staining and VG Staining
Harvested hearts were fixed in 4 % formalin and embedded in paraffin for hematoxylin and HE, masson or VG staining.For observation of fibrosis, HE staining, VG staining and masson staining were used to stain collagen fibers.The results were detected by microscopy.ologicalObser showed that no the formation sions (figure 3 The effects of T brosis signific nshinone II A tr

Discussion
In the present which is used The underlyin mitochondrial In our researc adriamycin in intraperitonea killed them 7 rats but not in HE staining, t than CON an adriamycincar prevented by T injury.basis of ffects on mitochondria.By electron microscopy, we observed marker mitochondrial damage and vacuolization in adriamycin-treated rats' hearts but minimal ultrastructural changes in adriamycin/Tanshinone II A-treated rats.Thus, Tanshinone II A inhibited adriamycin-induced ultratructural changes in cardiomyocyte mitochondria.

Abnormality i the cardiopro
To gain further insight into the treatment of Tanshinone II A, we performed TUNEL assay to explore the mechanism of Tanshinone II A inhibition adriamycin-inducted myocardial injury.From the result, it indicated that Tanshinone II A inhibited cardiomyocyte apoptosis inducted by adriamycin which was similar to previous reports (Hong-Jye et al., 2012).In addition, ER stress was suggested to contribute to cardiomyopathy (Xu et al., 2012).To investigate the reasons of Tanshinone II A prevent adriamycin-induced myocardial injury.We examined apoptosis related protein Bax, Bcl-2 and Caspase-3, the ratio of Bax/Bcl-2 and Caspase-3 increased in ADR+TRA group compared with CON and ADR group.In the present study, the observed inhibition of PERK (figure 9A), eIF2α (figure 9B), and the activation of ATF-4 (figure 9C) by Tanshinone II A suggests that the Tanshinone II A relieves adriamycin-induced myocardial injury in rat model by down-regulation ERS related apoptosis.It was evidenced that Tanshinone II A had anti-inflammatory response (Ren et al., 2010).Finally, we tested heart tissue levels of TNF-α and TGF-β, and found tanshinone II A reduce adriamycin-induced inflammatory response.

Conclusion
In summary, the present studies demonstrated Tanshinone II A inhibited adriamycin-induced myocardial injury in rat model.Then we explored the underlying mechanisms of this effect.We found the cardioprotective effects of Tanshinone II A involved with cardiac function, myocardial fibrosis, mitochondrial damage, inflammatory response and ER stress associated apoptosis.
Figure 4. e photomicrog sue Levels of T evels of TNF-α A treatment si