Astragaloside IV inhibits cardiac fibrosis via miR-135a-TRPM7-TGF-β/Smads pathway
Graphical abstract
Introduction
Cardiac fibrosis is one of the major processes which leads to various cardiac diseases (Khan and Sheppard, 2006; Yue et al., 2011). In response to pathological stimuli such as oxidative stress and inflammation, cardiac fibroblasts (CFs) can differentiate into myofibroblasts, which initiates fibrosis (Swynghedauw, 1999). The characteristics of fibrosis include excess accumulation of extracellular matrix (ECM) components as well as myofibroblasts cells within the insulted tissues (Kania et al., 2009; Kong et al., 2014). Transient receptor potential melastatin 7 (TRPM7), as an important profibrotic mediator (Huang et al., 2006), can promote the proliferation and differentiation of fibroblasts and increase the synthesis of ECM such as Collagen I (Zhou et al., 2015). Our previous data showed that hypoxia would cause an up-regulation of TRPM7 function. TRPM7 channel played an important role in regulating fibrosis including cell proliferation and differentiation (Lu et al., 2017). Wu et al. illustrated that TRPM7 channel played an essential role in regulating fibrosis, and that miR-135a protected against ISO-induced cardiac fibrosis by targeting TRPM7 channel (Wu et al., 2018).
Several studies have showed that TGF-β/Smads signaling was considered to be the main pathway leading to fibrosis in various tissues (Friedman et al., 2013). TGF-β1 can promote cardiac fibrosis by regulating its downstream molecules like Smad2/3 and JNK, while activated Smad7 can ameliorate fibrosis by triggering the degradation of TGF-β receptor I and Smad proteins, which brings about a negative feedback to the TGF-β/Smads signaling (Overstreet et al., 2014). Although the importance of both TRPM7 and TGF-β/Smads for fibrogenesis has been recognized, there seems to be no work to discuss their relationship in fibrosis process.
Astragalus membranaceus (Fisch.) Bunge, which was first described in the Chinese book Shen Nong Ben Cao Jing, has been widely used in traditional Chinese medicine and is also used in modern science to treat cardiac diseases (Cui et al., 2005; Yang et al., 2010). Astragaloside IV (ASG) is one of the active ingredients of Astragalus membranaceus (Li et al., 2014; Ren et al., 2013) and reported to have cardiac protective effects (Dong et al., 2017; Li et al., 2017). It is used as the quality control of Astragalus membranaceus in Chinese Pharmacopoeia (2015). To our best knowledge, there is little work to compare the action between Astragalus membranaceus total extract (AE) and ASG.
Our previous studies found that ASG could inhibit fibrosis by inhibiting TRPM7 channel (Lu et al., 2017). As previous results showed that miR-135a was the upstream regulator of TRPM7 (Wu et al., 2018), we supposed that miR-135a might be a target of ASG in cardiac fibrosis treatment. In this article, we demonstrated that ASG could promote miR-135a expression in ISO-treated rats and attenuate TRPM7 up-regulation. In addition, our work confirmed that there is a positive feedback between enhanced TRPM7 function and TGF-β/Smads pathway activation, which promotes fibrosis progression.
Section snippets
Materials and reagents
ASG was obtained from Zelang Medical Technology CO., Ltd. (Nanjing, China). The HPLC profile of ASG was shown in Supplementary Fig. 1. Isoproterenol Hydrochloride (CAS: 51-30-9) was purchased from Sigma Aldrich (USA). Dry root of Astragalus membranaceus was purchased from Hangzhou Huadong Chinese Herbal Pieces Co., Ltd.
Preparation of AE and high-performance liquid chromatography (HPLC) analysis
The dry root of Astragalus membranaceus was authenticated by Professor Jianqin Jiang (Chemistry of Natural Medicine, China Pharmaceutical University), and the AE was prepared by
ASG showed the same potency as AE on cardiac protection in ISO induced hypertrophy model
We used 70% EtOH as solvent to extract active components from the dry root of Astragalus membranaceus and the extraction rate is 27.63%. The content of ASG in mixture was measured via HPLC-ELS method (Wang, 2017). In our experimental condition, the content of ASG in AE is 0.23%. The data suggested that 4.35 g AE contained about 10 mg ASG. We used 4.35 g/kg/d AE and 10 mg/kg/d ASG to compare the cardiac protection. As our previous paper reported, subcutaneous injection of ISO can establish the
Discussion
Astragalus membranaceus (Fisch.) Bunge was reported to have the effect of invigorating Qi and Yang, promoting the production of body fluid, nourishing blood, activating stagnancy and freeing Bi according to Pharmacopoeia of People's Republic of China (2015 edition). Recently, some well-controlled clinical trials have showed that the cardiovascular protection of Astragalus membranaceus and its extract (Li et al., 2018; Piao and Liang, 2014). ASG is one of the active ingredients with high content
Conclusion
AE had the effect of cardiac inhibition and decreased the mRNA expression of TRPM7. ASG, as one of the effective ingredients of AE, showed the same potency when given with same dose. ASG inhibited cardiac fibrosis by targeting the miR-135a-TRPM7-TGF-β/Smads pathway.
Declaration of competing interest
The authors hereby declare that there are no conflicts of interest.
Acknowledgement
This work was supported by the National Key Program for New Drug Research Development (grant number 2011ZX09401-021) and the Graduate Research and Innovation Program of Jiangsu Province (grant number KYCX17_0725). We thank Professor Jianqin Jiang for crude drug authentication and Professor Jianbo Sun for crude drug extraction and AE analysis. We also thank Minhui Sun for technical support from Cellular and Molecular Biology Center of China Pharmaceutical University.
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These authors contributed equally to this work, and are the co-first authors.