The effects of a hirudin/liposome complex on a diabetic nephropathy rat model

Wang et al. BMC Complementary and Alternative Medicine https://doi.org/10.1186/s12906-019-2531-7 (2019) 19:118 RESEARCH ARTICLE Open Access The effects of a hirudin/liposome complex on a diabetic nephropathy rat model Hongwu Wang1*†, Huantian Cui1†, Lan Lin2, Yue Ji1, Qing Ni2, Junchen Li1, Jianli Pang3, Gongyan Bing4 and Yuhong Bian1 Abstract Background: Hirudin, an extract from Hirudo spp., is an anticoagulant used to treat a variety of renal diseases, including diabetic nephropathy (DN). Currently, hirudin has to be used at high dosages to treat DN because it poorly targets the kidneys, although at high dosages it can have severe side effects. Developing a targeted drug delivery system for hirudin, then, could boost its positive therapeutic effects while lowering the risk of side effects. Liposomes have been demonstrated to have significant renal targeting potential, but here we show that a hirudin-loaded liposome is an effective delivery method for patients with DN. Method: In this study, we prepared a hirudin/liposome complex and tested its efficacy by injecting it into a rat model. We then compared the renal accumulation of hirudin between complex-injected rat models and rat models that received injections of hirudin alone. We also investigated the mechanisms behind the complex’s effects. Result: The hirudin/liposome complex increased the accumulation of hirudin in kidney tissues and relieved the renal injury in DN rat models. Moreover, the hirudin/liposome complex down-regulated the expression of TGF-β1 and VEGF in the kidneys. Conclusion: We demonstrated that a hirudin/liposome complex can have a significant positive effect on DN. The mechanism may be that the complex inhibits the expression of VEGF and TGF-β1. Keywords: Diabetic nephropathy, Hirudin/liposome complex, Target therapy Background Diabetic nephropathy (DN) is a common complication from diabetes. Approximately 25–50% of diabetes patients suffer from the condition [1], which causes a progressive loss of renal function. Poor renal function can trigger end-stage renal disease (ESRD), and ultimately lead to renal failure [1]. Currently, the pathogenesis of DN is unclear. Recent studies have shown the progression of DN is related to the dysfunction of vascular endothelial growth factor (VEGF) and transforming growth factor β-1(TGF-β1), which may contribute to the pathological changes of glomeruli and renal tubules [2, 3]. Traditional Chinese medicine (TCM) has shown promise in the treatment of DN. Shuxuetong injection * Correspondence: † Hongwu Wang and Huantian Cui contributed equally to this work. 1 Tianjin University of Traditional Chinese Medicine, No.10, Poyang Road, Jinghai District, Tianjin 301617, China Full list of author information is available at the end of the article can significantly improve the renal function in DN patients [4], while Huangkui Capsule, an extract from Abelmoschus manihot (L.), improves their microinflammatory status [5]. Bailing capsules relieve microinflammation and oxidative stress in patients with DN-induced chronic renal failure [6]. Finally, the Tang-Wei-Kang pills, made of extract from Astragalus, hirudo, and Schisandra chinensis, are known to have a positive effect on patients with DN [7, 8], up-regulating the expression of Matrix metallopeptidase 9 (MMP-9), while downregulating the expression of TIMP metallopeptidase inhibitor 1 (TIMP-1) in the kidneys of DN rat models [8, 9]. However, nonspecific targeting is a disadvantage in the clinical use of TCM. Patients normally need long periods of treatment with TCM, and this can be expensive. Hirudin, an extract from Hirudo spp., has been used as an anticoagulant in many renal diseases, including chronic renal failure, glomerulonephritis, and DN [10, 11]. Hirudin © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. BMC Complementary and Alternative Medicine (2019) 19:118 Page 2 of 10 Table 1 Primer sequences of target genes in the rat model Methods Genes Primer sequence (5′-3′) Animals and reagents β-actin Forward: ACC CGC GAG TAC AAC CTT CT The following were used in the course of our experiments: Male Sprague Dawley rats (Weitonglihua CO., LTD., Beijing, China); Hirudin (Kekang medical science CO., LTD., Nanning, China); distearoyl phosphatidylcholine (DSPC) (TC1, Shanghai, China); streptozotocin (STZ) (Sigma-Aldrich Inc., St. Louis, USA); Cr, BUN, and total urine protein test kit (Beihuakangtai CO., LTD., Beijing, China); Rabbit-anti-rat VEGF and TGF-β antibody (Biosynthesis Biotechnology Co., Beijing, China); PV-6001 immunostaining test kit (Zhongshanjinqiao biology science CO., LTD., Beijing, China); Hirudin Elisa test kit (American Diagnostica Inc., USA); extract total RNA kit, first-stand cDNA reverse transcription kit, polymerase chain reaction kit and primers (TianGen Biotechnology Co., Ltd., Beijing, China). Reverse: TCA GGG TCA GGA TGC CTC T TGF-β1 Forward: CTT TGG ATG CCG CCT ATT GC Reverse: CCC CAG CAC AGA AGT TAG CA VEGF Forward: CCT GGC TTT ACT GCT GTA CCT Reverse: GCT GGT AGA CGT CCA TGA ACT can be used clinically as an alternative anticogulant for heparin in hemodialysis [12]. It decreases urine albumin and improves hypercoagulable states in the kidneys of DN patients [13]. However, the targeting efficiency of hirudin is low, obligating that it be used in high doses. This is both wasteful and can result in significant side effects, such as hemorrhaging [14]. Enhancing the drug’s ability to accumulate in the kidneys, therefore, could increase hirudin’s therapeutic effects while decreasing the potential for harmful side effects at the same time. Targeted therapy uses small (10-200 nm) pharmaceutical carriers, such as low molecular weight proteins, microspheres, micro-capsules, and liposomes to encapsulate drugs and deliver them to specific organs [15, 16]. Liposomes consist of phospholipid and quaternized cholesterol. They have high permeability, are widely used to deliver drugs to a target position [17, 18], and have high renal targeting potential. A recent study showed that the accumulation of doxorubicin in the kidneys of rats that received an injection of doxorubicin-loaded liposomes was significantly higher compared to rats that received an injection of doxorubicin alone [19]. Given the effects of hirudin on DN, and the renal targeting potential of liposomes, we hypothesized that a hirudin-loaded liposome could incre (...truncated)