Astragaloside IV from Astragalus membranaceus ameliorates renal interstitial fibrosis by inhibiting inflammation via TLR4/NF-кB in vivo and in vitro

doi:10.1016/j.intimp.2016.11.006

International Immunopharmacology

Volume 42, January 2017, Pages 18-24

https://doi.org/10.1016/j.intimp.2016.11.006 Get rights and content

Highlights

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UUO-induce renal fibrosis and increase inflammation.
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Astragaloside IV ameliorates renal fibrosis through anti-inflammatory effects.
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TLR4/NF-кB signal pathways are involved in inhibiting inflammation.

Abstract

Renal fibrosis is characterized by infiltration of inflammatory cells, activation and proliferation of fibroblasts, and accumulation of extracellular matrix (ECM). Astragalus membranaceus (AM) is traditional Chinese medicine and has a range of pharmacological effects. Astragaloside IV (As IV) is the main compound of AM and has anti-inflammation activities. Whether As IV ameliorates renal interstitial fibrosis by inhibiting inflammation remains unknown. Accordingly, this study investigated the ameliorating effect of As IV on renal fibrosis. Renal fibrosis was induced in vivo using the unilateral ureteral obstruction (UUO) model. UUO mice were administered intragastrically with As IV (20 and 40 mg/kg/day). After a week, ECM including fibronectin and collagen I was examined by Immunohistochemistry and Western blot, inflammatory cells (CD68 and CD3) were detected by Immunohistochemistry, the release of inflammatory cytokines (tumor necrosis factor-α and interleukin-1β) was inspected by polymerase chain reaction, and signaling pathway was determined by Western blot. In vitro, 100 ng/ml lipopolysaccharide (LPS) stimulated epithelial cells to construct the inflammatory model; these cells were treated by As IV (10 and 20 μM) with or without TAK-242 (1 μM) for 48 h. The released inflammatory cytokines were assayed by enzyme-linked immunosorbent assay, and signaling pathway was evaluated by Western blot. As IV decreased accumulation of ECM and infiltration of inflammatory cells in UUO-induced renal fibrosis. Furthermore, As IV markedly attenuated pro-inflammatory cytokines in UUO mouse and LPS-induced epithelial cells. As IV also inhibited the TLR4 and nuclear factor (NF)-кB signaling pathway in vivo and vitro. These results demonstrate that As IV protects against the progression of renal fibrosis by inhibiting inflammation via the TLR4/NF-кB signaling pathway.

Introduction

Renal fibrosis is a common pathogenic pathway of chronic kidney disease (CKD) [1]. However, the treatment for CKD is very limited. Therefore, clarifying the molecular cellular mechanisms underlying renal fibrosis is important to delay the development of CKD and find effective intervention strategies for this disease [2]. When the kidney incurs an injury, almost all kidney cells, including fibroblasts, renal tubular epithelial cells, pericytes, endothelial cells, and podocytes are mobilized [3]. These mobilized cells promote the repair and production of extracellular matrix (ECM). Meanwhile, lymphocytes, monocytes/macrophages, and other inflammatory cells are involved in the repair of the different pathways [4]. Renal fibrosis is abnormally repaired and is mainly characterized by infiltration of inflammatory cells, activation and proliferation of fibroblasts, accumulation of ECM, disappearance of intrinsic renal cells [5], [6], [7]. Inflammatory response occurs with renal fibrosis. Interleukin (IL)-1, IL-4, IL-6, IL-10, and tumor necrosis factor (TNF) participate in renal fibrosis [8]. Release of local inflammatory factors also aggravates the progression of fibrosis [9].

Astragalus membranaceus (AM) is a traditional Chinese herbal medicine and has been studied extensively in therapy of a various diseases. Astragaloside IV (As IV) is one of the main active ingredients in AM and exhibits various activities, including vasodilating effect, preventing endothelial dysfunction, improving cardiac cell energy metabolism ,anti-inflammatory effect, and antioxidant effects [10]. This study examined the anti-inflammatory effect of As IV on renal fibrosis and elucidated the underlying mechanism.

Section snippets

Reagents

The purified natural product Astragaloside IV was obtained from Chengdu Conbon Bio-tech Co., LTD. (Chengdu, China). DMEM/F12 culture medium and fetal bovine serum (FBS) were purchased from American Gibco Company. PrimeScript™ RT reagent kit was from TaKaRa, Japan. PCR primers were from Sangon Biotech, Shanghai, China. Collagen I and ELISA kit were from Boshide, Wuhan, China. CD68, CD3, and fibronectin were from Abcam, Cambridge, UK. TLR4, NF-кB, and IkBa were from Cell Signaling Technology

As IV inhibited ECM accumulation in UUO mice

The results of Immunohistochemical staining and quantification showed that fibronectin and collagen I significantly increased in the UUO model group compared with the sham group. Fibronectin and collagen I significantly decreased in the treatment groups compared with the UUO group (Fig. 1A–C). The results of the Western blot were consistent with Immunohistochemical staining (Fig. 1D–F).

As IV inhibited inflammatory cell infiltration and inflammatory cytokine secretion in UUO mice

The results showed the infiltration of macrophages (CD68) and lymphocytes (CD3) in the UUO model group

Discussion

Renal interstitial fibrosis is the deposition of excessive ECM caused by infiltration of interstitial inflammatory cells, production of inflammatory mediators, and activation and proliferation of interstitial cells [11]. After kidney injury, renal interstitial lymphocytes and macrophages, as well as interstitial collagen fibers increase. Studies using the obstructive nephropathy model showed that the lack of mature T and B lymphocytes significantly reduces renal fibrosis in mice [12]. In

Conflict of interest

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Acknowledgments

This work was also supported by the Taihe Hospital of Hubei University of Medicine (2013PY02) and Renmin Hospital of Wuhan University (30972989).

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Astragaloside IV from Astragalus membranaceus ameliorates renal interstitial fibrosis by inhibiting inflammation via TLR4/NF-кB in vivo and in vitro

Highlights

Abstract

Introduction

Section snippets

Reagents

As IV inhibited ECM accumulation in UUO mice

As IV inhibited inflammatory cell infiltration and inflammatory cytokine secretion in UUO mice

Discussion

Conflict of interest

Acknowledgments

Nat. Rev. Nephrol.

Pharmacol. Ther.

Transplant. Proc.

Int. Immunopharmacol.

Int. Immunopharmacol.

Kidney Int.

Semin. Nephrol.

Semin. Nephrol.

J. Surg. Res.

J. Ethnopharmacol.

Cytokine

Understanding the mechanisms of kidney fibrosis

Nat. Rev. Nephrol.

Modulation of renal inflammation: therapeutic strategies

Saudi J. Kidney Dis. Transpl.

Mechanisms of tubulointerstitial fibrosis

J. Am. Soc. Nephrol.