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Effects of Xie-Zhuo-Chu-Bi-Fang on Mir-34A and URAT1 and Their

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Effects of Xie-Zhuo-Chu-Bi-Fang on Mir-34A and URAT1 and Their Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 1 Contents lists available at ScienceDirect 2 3 4 Journal of Ethnopharmacology 5 6 journal homepage: www.elsevier.com/locate/jep 7 8 9 Research Paper 10 11 12 Effects of Xie-Zhuo-Chu-Bi-Fang on miR-34a and URAT1 and their 13 relationship in hyperuricemic mice 14 15 Wei-Feng Sun a,n,1, Ming-Min Zhu b, Jing Li a,b, Xian-Xian Zhang a, Ying-Wan Liu a, 16 a,nn,1 a Q1 17 Xin-Rong Wu , Zhi-Gang Liu 18 a Department of Traditional Chinese Medicine, General Hospital of Guangzhou Military Command of PLA, Guangzhou 510010, China 19 b Guangzhou University of Chinese Medicine, Guangzhou 510006, China 20 21 22 article info abstract 23 24 Article history: Ethnopharmacological relevance: Xie-Zhuo-Chu-Bi-Fang (XZCBF) is an empirical formula that was 25 Received 13 October 2013 developed based on the principles of traditional Chinese medicine, for the therapeutic purpose of Received in revised form 26 treating hyperuricemia. XZCBF has been clinically utilized in the Department of Traditional Chinese 14 November 2014 Medicine at General Hospital of Guangzhou Military Command of PLA for many years and has exhibited 27 Accepted 1 December 2014 favorable efficacy. The aim of the study is to evaluate the effects of XZCBF on the expression of uric acid Q3 28 transporter 1 (URAT1) and miR-34a in hyperuricemic mice and to determine, the correlation between the 29 Chemical compounds studied in this article: two expression levels. 30 Astilbin (Pubchem CID: 119258) Materials and methods: A hyperuricemic animal model was created by administering adenine and Colchicine (Pubchem CID: 6167) 31 allantoxanic acid potassium salt to mice. The blood uric acid levels were measured in these model mice Ecdysterone (Pubchem CID: 5459840) 32 after treatment with XZCBF for 15 days. The potential targets of miR-34a were screened. The expression 33 Keywords: levels of miR-34a and URAT1 in the renal tissues collected from the model mice were determined by 34 Hyperuricemia quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and their correlation was further 35 Kidney established by immunohistochemistry and in situ hybridization. miR-34a 36 Results: The uric acid levels in the model mice were significantly higher than those in the blank controls URAT1 o fi 37 (P 0.05). These levels were signi cantly lower in the three groups receiving different doses of XZCBF (Po0.05), which was, in agreement with the downregulation of URAT1 and the upregulation of miR-34a 38 in each group. The mRNA expression level of URAT1 was positively correlated with the concentration of 39 uric acid but, negatively correlated with the expression level of miR-34a. 40 Conclusions: The ability of XZCBF to facilitate the excretion of uric acid and to lower its level in the model 41 group was mediated by the upregulation of miR-34a and the inhibition of URAT1 mRNA expression, 42 which suggests that XZCBF could be an option for the treatment of hyperuricemia in mice. 43 & 2014 Published by Elsevier Ireland Ltd. 44 45 67 46 68 fi 47 1. Introduction with conditions such as renal insuf ciency, cardiovascular disease 69 48 and hyperlipidemia. 70 49 Epidemiological surveys from various countries have shown Hyperuricemia is characterized by a state of over-saturation of 71 fi 50 that the prevalence of hyperuricemia and gout has risen signi - extracellular urate, which is caused by a malfunction in purine 72 51 cantly, while the age of incidence has decreased (Roddy et al., metabolism or a decrease in uric acid production. Uric acid is a 73 52 2007; Wu et al., 2008). The asymptomatic period of hyperuricemia product of purine metabolism, and a large majority is excreted in 74 fi 53 is relatively long; however, deposits of uric acid salt cause injury to the urine through four steps, described as ultra ltration, reabsorp- 75 54 tissues and internal organs, which have been closely associated tion, secretion, and post-secretion reabsorption. Ninety-eight 76 fi 55 percent of glomerular- ltered uric acid is reabsorbed by the renal 77 56 tubules, a process that depends on human urate acid transporter 1 78 57 (hURAT1, SLC22A12). hURAT1 is a transporter protein that has 79 n 58 Correspondence to: Department of Traditional Chinese Medicine, General been shown to be closely related to the reabsorption of uric acid. 80 59 Hospital of Guangzhou Military Command of PLA, No.111 Liuhua Road, Yuexiu This transporter is selectively localized on the luminal surface of 81 District, Guangzhou (510010), China. Tel.: þ86 20 36653520. the epithelial lining along the proximal renal tubules (Wang, 60 nn Corresponding author. Tel.: þ86 20 88653476. 82 2004), where it maintains the electrical balance across the cell 61 E-mail addresses: [email protected] (W.-F. Sun), 83 62 [email protected] (X.-R. Wu). membrane by transporting uric acid in exchange for anions (Hong 84 1 63 Contributed equally to this work. et al., 2005). Nonsynonymous mutations in the coding region of Q2 85 64 http://dx.doi.org/10.1016/j.jep.2014.12.001 86 65 0378-8741/& 2014 Published by Elsevier Ireland Ltd. 87 66 Please cite this article as: Sun, W.-F., et al., Effects of Xie-Zhuo-Chu-Bi-Fang on miR-34a and URAT1 and their relationship in hyperuricemic mice. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.001i 2 W.-F. Sun et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 1 SLC22A12 leading to loss of function were reportedly found in miR-146a) were closely associated with XZCBF. In addition, miR- 67 2 patients diagnosed with familial hypouricemia (Yan and Zhou, 34a was found to be capable of binding to the 30UTR of URAT1, 68 3 Q4 2007), and downregulation of URAT1 could result in elevated which is consistent with the inhibitory role of miR-34a that was 69 4 excretion of uric acid (Cheong et al., 2008). identified in a sensor reporter assay. In this study, we located the 70 5 The promoter of uric acid excretion, benzbromarone, is an binding site of miR-34a in URAT1 by predicting target sites and 71 6 effective inhibitor of URAT1, which facilitates the excretion of uric exploring the relationships between XZCBF, miR-34a, and URAT1 72 7 acid by directly inhibiting URAT1 on the top surface of the renal using biomedical approaches, including vector construction, 73 8 tubules. However, benzbromarone has adverse effects including immunohistochemistry, and in situ hybridization. 74 9 functional impairment of the liver, gastrointestinal reactions, and 75 10 rash. Traditional Chinese medicine (TCM) has definite curative 76 11 effects in the treatment of hyperuricemia without serious adverse 2. Materials and methods 77 12 effects, and an understanding of the underlying mechanism of 78 fi 13 TCM has important practical signi cance. 2.1. Reagents 79 14 Xie-Zhuo-Chu-Bi-Fang (XZCBF) is an empirical formula that was 80 15 developedbasedontheprinciplesofTCMandisusedtotreat The following reagents were acquired: 97% allantoxanic acid 81 16 hyperuricemia. This formula has obtained a national invention patent potassium salt (Sigma-Aldrich, Shanghai), adenine (Sigma-Aldrich, 82 17 (Patent no.: ZL 2007 1 0032363.3). XZCBF consists of the following Shanghai), a uric acid assay kit (liquid, enzymatic method; Eastern 83 18 herbs: Smilax glabra Roxb., rhizome (tu fu ling, Smilacis Glabrae China Diagnostic [Shanghai] Co. Ltd), SYBR Green PCR Master Mix 84 19 Rhizoma), Heterosmilax japonica Kunth, rhizome (bi xie, Heterosmila- (TOYOBO Company, Japan), TRIzols Reagent (Invitrogen, USA), 85 20 cis Rhizoma), Cremastra appendiculata (D.Don) Makino, rhizome (shan anti-digoxigenin-AP (Roche, Switzerland), hsa-miR-34a (Exiqon, 86 21 ci gu, Cremastrae Pseudobulbus), Vaccaria hispanica (Mill.) Rauschert, USA); anti URAT1 (rabbit IgG; Abbiotec, USA), secondary antibody 87 22 seed (wang bu liu xing, Vaccariae Semen), and Achyranthes bidentata (DAKO ChemMate™, Denmark), and a detection kit (DAKO Envi- 88 23 Blume, root (niu xi, Achyranthis Bidentatae Radix). XZCBF has been sion™, Denmark). 89 24 applied clinically in the Department of TCM at General Hospital of 90 25 Guangzhou Military Command for many years, and its overall efficacy 91 26 rate was statistically estimated to reach approximately 90% (Sun et al., 2.2. Chemicals 92 27 2009). For more convenient use, we developed a granular form of 93 28 XZCBF: Compound Tufuling Granules (approved by Guangdong Food The herbs we used in this study were all commercially available 94 29 and Drug Administration, license no. Guang Zhi Zi 2011-F01009). as dry matter and were purchased from Nanhai Pengyang Phar- 95 30 According to the theory of Chinese Materia Medica (Chinese maceutical Co., Ltd. (Table 1). These herbs were identified by 96 31 Pharmacopoeia Commission, 2010), Smilax glabra Roxb., Hetero- Professor Xin-Rong Wu (Pharmacy Department of General Hospi- 97 32 smilax japonica Kunth, Vaccaria hispanica (Mill.) Rauschert and tal of Guangzhou Military Command, Guangzhou, China) and 98 33 Achyranthes bidentata Blume can induce diuresis to dispel “Damp- Director Hui-Chan Hou (Chinese Medicine Department of Guangz- 99 34 ness” and “Turbidity”; Smilax glabra Roxb. and Heterosmilax hou Institute for Drug Control, Guangzhou, China). Voucher speci- 100 35 japonica Kunth can smoothen joint movement to eliminate Impe- mens were deposited in the Herbarium of Guangzhou Institute for 101 36 diment; Cremastra appendiculata (D.Don) Makino, Vaccaria hispa- Drug Control. The dosages of the herbs in XZCBF are based on the 102 37 nica (Mill.) Rauschert, and Achyranthes bidentata Blume can theory of Chinese Materia Medica. 103 38 activate “Blood” and resolve “Stasis”; Achyranthes bidentata Blume These herbs were processed into Compound Tufuling Granules 104 39 can tonify the “Liver” and “Kidney”, and conduct “Blood” down- by the Pharmacy Department of General Hospital of Guangzhou 105 40 ward.
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