Astragaloside IV inhibits cardiac fibrosis via miR-135a-TRPM7-TGF-β/Smads pathway

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Abstract

Ethnopharmacological relevance

Cardiac fibrosis is a common characteristic of many cardiac diseases. Our previous results showed that TRPM7 channel played an important role in the fibrosis process. MicroRNA-135a was reported to get involved in the fibrotic process. Astragalus membranaceus (Fisch.) Bunge was widely used in Chinese traditional medicine and showed cardiac protective effects in previous researches. Astragaloside IV(ASG), which is regarded as the most important ingredient of Astragalus, has been showed the effect of cardiac protection via various mechanisms, while no data suggested its action related to miRNAs regulation.

Aim of the study

The objective of this article is to investigate the inhibition effect of ASG on cardiac fibrosis through the miR-135a-TRPM7-TGF-β/Smads pathway.

Materials and methods

We extracted the active components from herb according to the paper and measured the content of ASG from the mixture via HPLC. The inhibition potency of cardiac hypertrophy between total extract of Astragalus and ASG was compared. SD rats were treated with ISO (5 mg/kg/day) subcutaneously (s.c.) for 14 days, ASG (10 mg/kg/d) and Astragalus extract (AE) (4.35 g/kg/d, which contained about ASG 10 mg) were given p.o. from the 6th day of the modeling. Cardiac fibroblasts (CFs) of neonatal rats were incubated with ISO (10 μM) and treated with ASG (10 μM) simultaneously for 24 h.

Results

The results showed that both AE and ASG treatment reduced the TRPM7 expression from the gene level and inhibited cardiac fibrosis. ASG group showed similar potency as the AE mixture. ASG treatment significantly decreased the current, mRNA and protein expression of TRPM7 which was one of targets of miR-135a. The activation of TGF-β/Smads pathway was suppressed and the expression of α-SMA and Collagen I were also decreased obviously. In addition, our results showed that there was a positive feedback between the activation of TGF-β/Smads pathway and the elevation of TRPM7, both of which could promote the development of myocardial fibrosis.

Conclusions

AE had the effect of cardiac fibrosis inhibition and decreased the mRNA expression of TRPM7. ASG, as one of the effective ingredients of AE, showed the same potency when given the same dose. ASG inhibited cardiac fibrosis by targeting the miR-135a-TRPM7-TGF-β/Smads pathway.

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.

References (31)

  • Z. Dong et al.

    Astragaloside IV alleviates heart failure via activating PPARalpha to switch glycolysis to fatty acid beta-oxidation

    Sci. Rep.

    (2017)
  • S.L. Friedman et al.

    Therapy for fibrotic diseases: nearing the starting line

    Sci. Transl. Med.

    (2013)
  • D. Huang et al.

    Effect of Astragalus membranaceus alcohol extracts on morphine-induced conditioned place preference in mice

    J. Fujian Med. Univ.

    (2006)
  • R. Khan et al.

    Fibrosis in heart disease: understanding the role of transforming growth factor-beta in cardiomyopathy, valvular disease and arrhythmia

    Immunology

    (2006)
  • P. Kong et al.

    The pathogenesis of cardiac fibrosis

    Cell. Mol. Life Sci.: CMLS

    (2014)
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    1

    These authors contributed equally to this work, and are the co-first authors.

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