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Original Article Hypouricemic and Nephroprotective Effects of Jianpi Huashi Decoction in a Rat Model of Hyperuricemia

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Original Article Hypouricemic and Nephroprotective Effects of Jianpi Huashi Decoction in a Rat Model of Hyperuricemia Int J Clin Exp Med 2016;9(1):455-465 www.ijcem.com /ISSN:1940-5901/IJCEM0020946 Original Article Hypouricemic and nephroprotective effects of Jianpi Huashi decoction in a rat model of hyperuricemia Xiaoying Wang1,4, Jiangbing Zhou1, Bin Shi2, Xiaolei Guo2, Qunchao Yan3 1Department of Neurosurgery, Yale University, New Haven, CT, USA 06520; 2Infinitus (China) Company Ltd, Guangzhou 510630, Guangdong, China; 3School of Medicine, Jinan University, Guangzhou, Guangdong 510632, China; 4Department of Pathogenic Biology and Immunology, School of Medicine, Southeast University, Nanjing 210009, China Received December 1, 2015; Accepted December 14, 2015; Epub January 15, 2016; Published January 30, 2016 Abstract: Hyperuricemia, indicated by elevated serum level of uric acid, is a risk factor for developing gout, hypertension, renal disease and cardiovascular disease. In the present study, we evaluated the therapeutic efficacy of a novel hypouricemic agent, Jianpi Huashi Decoction (JPHSD), derived from Chinese herbal medicines Rhizoma Smilacis Glabrae, Plantago asiatica L., kudzu root, Cichorium intybus L., seeds of Coix lacryma-jobi L., and Alismatis Rhizoma, and tuna extract in a rat model of hyperuricemia induced by potassium oxonate. The results showed that JPHSD effectively reduced the serum level of uric acid in hyperuricemic rats. The hypouricemic effect of JPHSD was achieved in two aspects: reducing the production of uric acid by inhibition of hepatic xanthine oxidase activity, and promoting renal excretion of uric acid through regulation of major urate transporters. Consequently, the oxidative stress induced by hyperuricemia was attenuated, and the renal damage was ameliorated by JPHSD treat- ment. Our study demonstrated that the novel agent JPHSD had both hypouricemic and nephroprotective effects in hyperuricemic rats. Keywords: JPHSD, hyperuricemia, uric acid, urate transporter, ion transporter, oxidative stress Introduction xanthine into xanthine and subsequently uric acid by the enzyme called xanthine dehydroge- Hyperuricemia is the underlying cause of gout, nase (XDH) and its converted form xanthine oxi- a metabolic disorder characterized by increased dase (XOD) [7, 8]. When the uric acid metabolic level of uric acid in the blood. The excessive pathway is disrupted, either by over-production serum uric acid crystallizes and deposits in the of uric acid due to increased purine intake or joints and leads to recurrent inflammatory decreased excretion of uric acid from the kid- arthritis [1]. The incidence rates of hyperurice- ney due to impaired renal function, hyperurice- mia and gout have increased rapidly over the mia develops and the risk of having gout past decade, which is believed to be associat- increases substantially [9, 10]. Inhibition of ed with the change in life styles and diet struc- XOD activity has been proved to effectively ture, and influence from environmental factors lower serum level of uric acid and prevent recur- [2-4]. Gout is also frequently accompanied by rent gout attacks. The XOD inhibitor allopurinol other medical conditions, including diabetes, is currently in clinical use to treat hyperuricemia hypertension, hyperlipemia, renal disease, ath- and chronic gout [11, 12]. Allopurinolhas long erosclerosis, and cardiovascular disease [1, 5, been the first-line therapy for gout, but the use 6]. Therefore, it will be of great significance to of suboptimal dose in patients with impaired design effective therapeutics to treat and pre- renal function is ineffective. In addition, severe vent hyperuricemia, and to improve the quality allergic reaction and hypersensitivity syn- of patients with the disease. dromes are the most common side effects of allopurinol which limit its use [13]. Febuxostat Uric acid is the metabolic product of purine is a new XOD inhibitor currently tested in clinical metabolism, which involves breakdown of hypo- trials. Available data from comparative studies Hypouricemic effect of Jianpi Huashi decoction showed that febuxostat had a similar hypouri- of uricase activity is essential to establish a rat cemic effect as allopurinol but higher risk of model of hyperuricemia. In this study, we used causing liver damage [14, 15]. When XOD inhib- potassium oxonate, a well documented effec- itors are ineffective or the patients are unre- tive uricase inhibitor, to induce a hyperuricemic sponsive to the treatment, other therapeutic condition in rats and evaluated the efficacy of agents are introduced to those patients, such JPHSD in reducing level of serum uric acid. as benzbromarone and probenecid. Benzbro- marone is a uricosuric drug with strong inhibi- Materials and methods tory activity on the uric acid transporter 1 (URAT1) mediated urate reabsorption, and thus Animals highly effective in treating hyperuricemia [16]. However, its safety has been doubted due to Male Sprague-Dawley (SD) rats (age: 7-8 weeks; the irreversible adverse effects and serious weight: 180-220 g) were purchased from the hepatotoxicity [17]. It has been withdrawn from Experimental Animal Center in Guangzhou the US market since 2003. Probenecid targets University of Chinese Medicine (Guangzhou, the organic anion transporters (OAT) in the kid- Guangdong, China) with the batch number of ney, thereby blocks the transport of uric acid SCXK-2013-0020. Rats were allowed to accli- across the cell membrane into plasma and matize for 1 week before the experiment. All eventually reduces the serum concentration of rats were maintained under specific pathogen uric acid [18]. However, severe and potentially free (SPF) laboratory conditions with tempera- life-threatening adverse reaction and liver dam- ture at 22 ± 1°C, relative humidity 55 ± 5%, and age have been reported in several cases [17, automatic 12-h light/12-h dark cycle. They 19]. A new therapeutic alternative with high were allowed free access to food and water. All efficacy and low risk is in urgent need. the cages, food, and water were processed by 121°C steam sterilization. All protocols for ani- In this study, we evaluated the efficacy of a mal experiments were approved by the Animal novel agent developed in our laboratory, desig- Care Committee of Guangzhou University of nated as Jianpi Huashi Decoction (JPHSD), in Chinese Medicine. reducing serum uric acid levels in a rat mo- del of hyperuricemia. JPHSD contains extra- Reagents and preparation of JPHSD cts from Chinese herbal medicines Rhizoma Smilacis Glabrae, Plantago asiatica L., kudzu JPHSD was developed and prepared in our lab- root, Cichorium intybus L., seeds of Coix lacry- oratory. Briefly, mixture of Chinese herbal medi- ma-jobi L., and Alismatis Rhizoma, all of which cines Rhizoma Smilacis Glabrae (from Guang- have long been used in traditional Chinese xi, China), Plantago asiatica L. (from Jiangxi, medicine to treat gout orhave documented China), kudzu root (from Anhui, China), Cicho- anti-gout and anti-inflammatory effects [20, rium intybus L. (from Heilongjiang, China), seed 21]. In addition, recent studies have suggested of Coix lacryma-jobi L. (from Hebei, China), and that peptides might have anti-hyperuricemic Alismatis Rhizoma (from Sichuan, China) (mixed effect. In a study using fractions of the proteo- according to 10:5:5:4:10:4 by weight) were sub- lytic digest of shark cartilage, peptides have jected to extraction by refluxing with distilled been shown to function as the bioactive com- water (1:12, w/v) for 1.5 h. The extract process ponent to modulate hyperuricemia in rats [22]. was repeated with distilled water (1:8, w/v) for Further investigation showed that the dipep- 1 h. The filtrates were combined and evaporat- tides derived from milk protein could non-com- ed, and the concentrated herbal extract was petitively inhibit the XOD activity [23, 24]. To obtained [yield 20% (w/w)]. Wild tunas were develop a novel agent with high anti-hyperurice- caught and stored at -20°C until use. After rehy- mic efficacy, we combined the Chinese herbal dration, heads and internal organs were re- medicines with tuna extract which is a rich moved, and the remaining parts were grounded source of peptides, and studied the combined followed by crude proteolytic digestion with effect in rats. Humans lack uricase, a uric acid- food-grade protease including Papain (Nanning oxidizing enzyme, so the end product of purine pangbo, China) and Neutrase (Novozymes, metabolism, uric acid, gets excreted in urine. China) at 50-60°C for 4 hours. The digested While in rats, uric acid is further oxidized to samples were heated at 95°C for 20 minutes to allantoin by uricase [25]. Therefore, inhibition stop protease activity, and then centrifuged at 456 Int J Clin Exp Med 2016;9(1):455-465 Hypouricemic effect of Jianpi Huashi decoction 8000 rpm for 20 minutes at 4°C. Next, the was used as vehicle control in the normal supernatants were collected, clarified by filtra- group. Allopurinol (27.0 mg/kg·d), benzbroma- tion, and spray dried. The resulting powder was rone (4.5 mg/kg·d), and JPHSD (1.67, 3.33, and resolved in the concentrated herbal extract to 6.67 mL/kg·d) were given to the groups Oxo + make JPHSD. After sterilization, JPHSD was ali- AP, Oxo + BB, Oxo + JPHSD_L, Oxo + JPHSD_M, quoted and ready for use. and Oxo + JPHSD_H, respectively, by oral gavage once daily at 1:00 p.m. for 30 consecu- Concentrated JPHSD was diluted to the required tive days. 0.1% CMC-Na solution was given to concentration using 0.1% carboxymethylcellu- the normal group and Oxo group as control. All lose sodium (CMC-Na, purchased from Matrix doses of drug administered were determined Laboratories Ltd, Xiamen, Fujian, China). Three based on the body weight measured immedi- different doses of JPHSD - 1.67,3.33, and 6.67 ately prior to each treatment. The volume of all mL/kg per day- were used in the experiments. the gavages was 10 mL/kg·d. The activity level, Allopurinol and potassium oxonate (Oxo) were health status, and the amount of food intake purchased from Matrix Laboratories Ltd. Ben- for all rats were monitored daily.
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