Anti-hyperuricemic effect of Polydatin in model mice: promoting uric acid excretion and inhibiting XOD

Objective The aim of this study was to understand the role of anti-hyperuricemic mechanism and nephro-protective effects of polydatin. Methods The oxonate-induced hyperuricemia mice model was established and uric acids in serum were observed. Kidney tissues were used to detect gene contents of URAT1, OAT1 and OAT3 by real-time-PCR. and to detect pathological features .The activity of XOD in liver tissues of mice was detected . Results Polydatin signicantly reduced serum urate levels in hyperuricemic mice. Polydatin signicantly inhibited increasing tendency of the mRNA and the protein levels of OAT1 and OAT3 ,and decreasing tendency of the mRNA and the protein level of URAT1 in hyperuricemic mice. Polydatin signicantly inhibited the level of XOD in liver tissues of mice in a concentration-dependent manner in hyperuricemic mice. Polydatin showed a protective effect on the pathological injury of kidney in hyperuricemic mice. The renal URAT1, upregulated renal OAT1 and OAT3 and inhibition XOD in the hyperuricemic mice. renal mOAT1 and mOAT3 expression to elevate urate secretion. These results suggested that polydatin exhibited anti-hyperuricemic effects through the regulation of different urate transport-related proteins to enhance renal urate excretion in the hyperuricemic mice. In comparison withthe positive control drug, anti-hyperuricemic ecacies of polydatin seemed to be similar to those ofbenzbromarone, or even more potent at higher doses in this study.


Introduction
Hyperuricemia is de ned by a plasma concentration of uric acid levels above 7 mg/dL, is the predisposing factor for gout. In the last 20 years, epidemiological studies showed that hyperuricemia is an independent risk factor for the cardiovascular diseases, particularly in hypertensive and diabetic individuals [1][2][3][4][5]. There is strong epidemiological evidence that the prevalence of gout and hyperuricemia is on the increase worldwide [6,7]. Previous studies have shown that approximately 90% of hyperuricemia is caused by urate underexcretion from the kidneys [8,9]. The synergistic effect of a number of urate transporters in kidney is responsible for uric acid homeostasis in the body. The urate transporters regulate the excretion and reabsorption of endogenous and exogenous organic anions [10]. The urate reabsorption transporters include URAT1, OAT4, OAT10, GLUT9, and the urate excretion transporters include OAT1, OAT3, MRP2, MRP4, NPT1, UAT [11,12]. Therefore, these renal urate transport-related proteins constitute important targets for the effective agents to prevent and treat hyperuricemia and gout. Various uricosuric substances known to reduce hyperuricemia, such as probenecid, fenylbutazone, sul npyrazone, NSAIDs, and diuretic drugs, effectively inhibit URAT1.Benzbromarone (a uricosuricagent used clinically) is the most potent, completely inhibiting the urate uptake via URAT1 [13,14].
Polygonum cuspidatum Sieb.and Zucc. (Polygonaceae), also known as Huzhang in Chinese, is a traditional Chinese medicine, used for the treatment of various in ammatory diseases, hepatitis, skin burns, tumors, and diarrhea. It is o cially listed in the Chinese Pharmacopoeia. It has been traditionally used in folk medicine as a crude drug for joint pain induced by gout. As one of its main ingredients, polydatin (also named resveratroside) is a kind of glycoside, which exists widely in the plants like grape, earthnut, giant knotweed, black falsehellebore, sickle senna, etc. In human body, polydatin is hydrolyzed in intestine by glycosidase to resveratrol to display its pharmacological action, such as liver protection, anti-in ammation, anti-oxidative, antitumor, and anti-pathogenic microbe, cardio-myocyte protection, vascular smooth muscle dilation, anti-platelet aggregation, antithrombotic effect, and atherosclerosis prevention [15,16] Our previous studies have demonstrated that polydatin has signi cant uricosuric effects in the hyperuricemic mice [17]. The aim of this study was to examine the xanthine oxidase inhibition and to detect protein levels of URAT1, OAT1 and OAT3 in the hyperuricemic mice in order to understand the role of anti-hyperuricemic mechanism of polydatin. Before the experiment, all mice were adaptively raised for one week, free drinking and standard feeding during the experiment.
Potassium oxonate (approximately 2 mg) was weighted and dissolved with 200 ml 0.8% CMC-Na solution to prepare 10 mg/mL potassium oxonate injection. Trizol was provided by Invitrogen Company. Fluorescent quantitative detection kit, DNA Marker and M-MLV reverse transcription kit were purchased from Dalian Takara Biotechnology Co. Ltd. Tubes for quantitative detection PCR and sterile centrifuge tubes were provided by Axygen Company. Primer synthesis and DNA sequencing was conducted in Shanghai Invitrogen Company. All other chemicals used in this study were of analytical grade, made in china.

Establishment of Hyperuricemia Mice Model and Routine Detection
Sixty male ICR mice were randomly divided into six groups with 10 mice in each group, i.e. polydatin experimental groups (5, 10, and 20 mg/kg), a positive control group (benzbromarone, 16.7 mg/kg), a model group, and a negative control group. Polydatin and benzbromarone were prepared with 0.8% CMC-Na solution to get the corresponding concentration.
The mice in the normal control group and model group were administered by gastric infusion with 0.8% CMC-Na solution, once daily for continuous 7 days, and the nal administration was given one hour after the molding. Blood samples were drawn at 2h when all the experimental groups and model group were given intraperitoneal injections of 250 mg/kg potassium oxonate while the normal control group was given with 0.8% CMC-Na solution. One hour after administration, blood samples were drawn from the ophthalmic venous plexus in mice, then 100 µL serum was collected by centrifugation (4000×g, 5min), and the blood uric acid levels were detected by TOSHIBA7060 model automatic biochemistry analyzer. Mice livers were extracted and stored at -80°C until XOD detection. Kidney tissues of mice were collected, one part was stored at -80°C and used for total RNA and protein extraction to detect the mRNA as well as protein contents, and another part was xed in 4% paraformaldehyde for pathological detection.
2.5. Content Detection of URAT1, OAT1 and OAT3 in Kidney Tissues of Mice by Western Blotting Kidney (100 mg) tissues stored at -80°C were removed with a scalpel quickly, transferred to a 1.5 mL aseptic centrifugetube, washed in 1 ml of 4°C ice-cold dPBS twice, removed to a new 1.5 mL centrifugetube, added 500 µL lysis buffer T-TER, and homogenized adequately, the lysate was transferred to a new 1.5 mL centrifugetube, centrifuged at 12000×g, 4°C for 10 min, and the supernatant was collected. The total protein level was detected by the method of BCA. Total protein (10 µg) was analyzed by SDS-PAG electrophoresis using 11% separation gel at a voltage 120 V for 100min, and transferred to PVDF membranes using the wet transfer at 400mA electric current for 90 min. The membranes were blocked in TBST containing 5% nonfat milk at room temperature for 2hours,incubated overnight at 4°C with the primary antibody (diluted with TBST, the diluent rates of the antibodies against URAT1,OAT1, OAT3 and beta actin C were 1:500, 1:300, 1:400 and 1:800 respectively), washed in 4°C ice-cold TBST three times and incubated with 1:3000 dilution of the goat anti-mouse secondary for 2h. The membrane was washed three times again and ECL solution was added, and the optical signal of HRP was determined by a scanner. The software of densitometry Totallab10.1 was used to determine the gray value of the objective band, to analyze the relative content of URAT1, OAT1 and OAT3.

Activity Detection of XOD in Liver Tissues of Mice
The activity of XOD in liver tissues of mice was detected by Xanthine Oxidase Assay Kit(ab102522). Liver tissues (200mg)stored at -80°C were obtained, added 0.2mL lysis buffer, homogenized adequately, and centrifuged at 16000×g for 10 min, and thesupernatant was collected, 50µL of which was taken to determine the activity of XOD according to the instructions of the kit. The calculation formula was as following: An enzyme-labelling measuring instrument was used to measure the absorbance value in 570 nm quickly: the rst reaction time and reading were recorded as T1 and A1 respectively; the sample was retested after being placed in 25°C for 2 min, and the second reaction time and reading were recorded as T2 and A2. The variation value of absorbance was ΔA = A2-A1, It is essential to read A1 and A2 in the reaction linear range and we will choose A1 and A2 in the reaction linear range. Where B is the amount of H2O2 (nmol) generated by XO from standard curve,T1 is the time of the rst reading (A1) (in min),T2 is the time of the second reading (A2) (in min),and V is the pre-treated sample volume (ml) added into the reaction well.

Pathological Detection in Kidney Tissues of Mice
The tissues were xed, cut into sections and stained with HE staining according to the routine procedure.

Data Analysis
The statistical analysis in the experimental data was performed using statistic software SPSS 10.0. The analysis of variance test was applied in comparing the intergroup difference of measurement data, while Ridit analysis was used for ranked data. A statistical signi cance was considered when P < 0.05.

Effects of Polydatin on Serum Uric Acid Level in Hyperuricemic Mice
According to the results in Fig. 1, the polydatin experimental groups (5, 10, and 20 mg/kg) and the positive control group (benzbromarone, 26 mg/kg) showed signi cantly inhibited levels of serum uric acid when compared with the model group. Variance analysis indicated that the level of serum uric acid was remarkably elevated in the model group when compared with the normal control group, and the statisticallysigni cant differences between groups (P < 0.01) suggested that the model was successfully established. The level of serum uric acid was lower in the benzbromarone control group compared with the model group, and there were no statistical differences between benzbromarone control group and normal control group (P < 0.05), suggesting that benzbromarone decreased the level of serum uric acid to the normal one. The levels of serum uric acid were higher in the polydatin experimental groups compared with the normal control group, but lower than the model group, and there were no statistical differences among three dosage groups (P > 0.05) suggesting that polydatin signi cantly decreased the level of serum uric acid, however, the e cacy of polydatin had no obvious relation with the doses within a range of 5-20 mg/kg.

3.2.Assay Analysis of the mRNA Levels
The relative contents of URAT1, OAT1 and OAT3 were calculated according to the value of Ct with beta actin as the reference gene. The data in Fig. 2 showed the relative level of URAT1 was obviously increased in the model group compared with normal control group (P < 0.01), and polydatin signi cantly inhibited the increasing tendency of URAT1 levels. There were statistically signi cant differences between the high dosage group and the model group (P < 0.01), suggesting that the inhibitory action of polydatin on URAT1 was obviously concentration-dependent. The data in Fig. 2 also showed the relative levels of OAT1 and OAT3 were signi cantly decreased in the model group compared with the normal control group (P < 0.01), and polydatin signi cantly inhibited the decreasing tendency of OAT1 and OAT3 levels. There were statistical differences between the high, middle dosage group, and the model group (P < 0.05), suggesting that the inhibitory action of polydatin on OAT1 and OAT3 was obviously concentrationdependent. Similar results were observed in the benzbromarone group.

3.3.Assay Detection of Proteins
The data of protein detection in Fig. 3 showed the protein levels of OAT1 and OAT3 were signi cantly increased in the hyperuricemic mice while the protein level of URAT1 was decreased remarkably, and there were statistical differences among groups (P < 0.05). The data also showed the inhibitory action of polydatin on the protein level of OAT1, URAT1 and OAT3 was obviously concentration-dependent, and there were statistical differences between the high, middle dosage groups and the model group (P < 0.05).
Similar results were observed in the benzbromarone group, indicating the high reliability of the experimental system.

3.4.Content Detection of XOD in Liver Tissues of Mice
According to the results in Fig. 4, the levels of XOD in the normal group and model group were 2.41 ± 0.40 mU/mL and 11.15 ± 2.09 mU/mL, respectively, and there was a statistic signi cance between the two groups (P < 0.05). The levels of XOD in the high, middle and low dose groups were 7.89 ± 1.20, 7.89 ± 0.79 and 10.41 ± 0.79 mU/mL, respectively, of which the level in the high dosage group was signi cantly lower than that of the model group(P < 0.05). The level of XOD in positive control group (benzbromarone) was 5.18 ± 1.04 mU/mL, signi cantly lower than that of the model group (P < 0.05). The results indicated the model had high reliability, and polydatin signi cantly inhibited the level of XOD in liver tissues of mice in a concentration-dependent manner.

3.5.Pathological Detection
The results of HE staining in Fig. 5 suggested that there were kidney tissue pathological changes in hyperuricemic mice, and polydatin and benzbromarone showed a protective effect on the pathological injury of kidney. The gure showed in comparison with normal structure of kidney, no cloudy swelling and normal size of glomeruli in the normal control group, there were glomerular disturbance, severe hemorrhage and in ammatory cells in ltration of glomerular periphery in the model group. The gure also indicated that glomeruli were arranged nearly uniformlywith a few of in ammatory cells around them in the low dosage group. In the middle dosage group, the glomeruli were arranged relatively uniformly,without obvious hemorrhage but a few of in ammatory cells around them. The glomeruliwere arranged highly uniformly with a relatively complete structure where there was a little hemorrhage in the high dosage group. The gure also indicated that glomeruli were arranged relatively uniformlywith a little hemorrhage in the benzbromarone group.

Discussion
In this study, we examined the anti-hyperuricemic effect of polydatin in the oxonate-induced hyperuricemic mice. The effect was related to the regulation of renal urate transporter 1 (mURAT1), organic anion transporter 1 (mOAT1) and organic anion transporter 3 (mOAT3) in the hyperuricemic mice. Polydatin was found to down-regulate mRNA and protein levels of mURAT1, as well as up-regulate mOAT1 and mOAT3 in the kidney of the hyperuricemic mice. Moreover, the inhibition of XOD in liver tissues of mice as well as protective effects of renal function also contributed to the anti-hyperuricemic effect of polydatin. These ndings suggested that polydatin might inhibit uric acid formation, enhance uric acid excretion and decrease of uric acid reabsorption, thus in turn reduce serum uric acid level in the hyperuricemic mice.
Uric acid exit from blood is mainly controlled by the kidney [18]. In the kidney, uric acid and urate are initially ltered and additionally secreted. However, the largest part (90%) is usually reabsorbed and returns to blood [1]. The kidney plays a dominant role in maintaining plasma urate levels through the excretion process; it eliminates ~ 70% of the daily urate production. Therefore, it is important to understand the mechanism of renal urate handling because underexcretion of urate has been demonstrated in the majority of hyperuricemic patients [18]. URAT1 mediates urate reabsorption from the kidney tubule lumen to blood, transports urate in exchange for Cl-or other organic anions and maintains blood urate homeostasis. URAT1 is inhibited by the classical uricosuric agent benzbromarone [19]. URAT1 is the sole transporter whose physiological role in renal urate reabsorption is established, based on the fact that loss-of-function mutations in URAT1 cause renal hypouricemia [20]. The present study revealed that polydatin reduced serum urate levels by downregulating mURAT1 expression to inhibit urate reabsorption in the kidney of the hyperuricemic mice. The polydatin and benzbromarone groups showed the same kind ofeffects. The organic anion transporters OAT1 and OAT3 operate as organic ion/dicarboxylate exchangers, responsible for the uptake of organic anions from the blood across the basolateral membrane into the proximal tubule cells, which are recognized as the rst step of renal organic anion secretion, and may mediate renal urate secretion. Our data suggest that polydatin signi cantly inhibited the decreasing tendency of OAT1 and OAT3 levels in the hyperuricemic mice. Down-regulation of renal rOAT1 and rOAT3 was previously observed in the hyperuricemicrats induced by oxonic acid, indicating that OAT1 and OAT3 may play an important role in the pathogenesis of hyperuricemia. Our data suggested that polydatin exerted anti-hyperuricemic actions by simultaneously upregulating renal mOAT1 and mOAT3 expression to elevate urate secretion. These results suggested that polydatin exhibited anti-hyperuricemic effects through the regulation of different renal urate transport-related proteins to enhance renal urate excretion in the hyperuricemic mice. In comparison withthe positive control drug, anti-hyperuricemic e cacies of polydatin seemed to be similar to those ofbenzbromarone, or even more potent at higher doses in this study.
XOD has been implicated as a key oxidative enzyme that catalyzes purine catabolism. The highest activity of XOD is detected in liver, intestine, and endothelium. In speci c, uric acid derives from the conversion of hypoxanthine to xanthine and of xanthine to uric acid, both reactions being catalyzed by the enzyme XOD. XOD plays a pivotal role in regulating the production of uric acid that is important to the occurrence of hyperuricemia. The results from experimental studies imply that polydatin at the low, middle and high doses signi cantly inhibited the activity of XOD in liver tissues of mice in a concentration-dependent way. Beside controlling purine catabolic pathway as a key oxidative enzyme, XOD has also been implicated to induce the expression of cyclooxygenase-2, translocation of nuclear factor-κB(NF-κB), synthesis of TNF-γ, activation of neutrophils, and phagocytic killing, being therefore a potent modulator of innate immune response. In ammatory cytokines may be responsible for upregulated synthesis of XOD, and in turn XOD has a putative role in in ammatory signal transduction. An alternative hypothesis to explain the association between hyperuricemia and increased vascular risk has been proposed: hyperuricemia may simply represent a surrogate marker for high levels of damaging oxidative stress associated with increased xanthine oxidase activity, rather than being directly responsible for vascular injury and the subsequent increase in risk. Dijana et al. have demonstrated that an upregulated XOD may be implicated in hemodialysis-induced oxidative injury [21]. In this study, we observed that polydatin has a protective effect on the pathological injury of kidney as benzbromarone in the hyperuricemic mice. A series of studies have recently demonstrated that polydatin has been suggested to have the properties of anti-oxidative, anti-in ammatory and nephroprotective effects. Chen et al. have reported that polydatin has inhibitory activities on the xanthine oxidase to repress the level of serum uric acid in vivo and in vitro. This demonstrated that the nephroprotective activities of polydatin were not only due to the effects on remarkably attenuating the oxidative stress induced by uric acid, but also on markedly suppressing the oxidative stress-related in ammatory cascade. Hyperuricemia is one of well-described risk factors for kidney function disorders. Our results showed that there were kidney tissue pathological changes in the hyperuricemicmice,consistent with a previous study that polydatin ameliorates renal injury [16]. Our results should give strong pathophysiological support to the positive correlation between XOD activity and renal damage. Although uric acid is a low potency antioxidant outside the cell, inside the cell it enhances oxidative stress by activating NADPH oxidase while activating the renin-angiotensin system [21]. In fact, uric acid re ects upregulated XOD activity, and is associated with a pro-in ammatory state in human subjects and particularly with an increase in in ammatory markers. The previous studies showed that hyperuricemia was frequently noted in patients either with cardiovascular disease or at a high risk of cardiovascular disease such as hypertension, coronary heart disease, peripheral vascular disease, heart failure, metabolic syndrome, and stroke. Findings from this study support the potential use of serum urate as an independent biomarker for poor all-cause and coronary heart disease mortality in patients with recent acute myocardial infarction through increased oxidative stress and in ammation. Therefore, we propose that polydatin has nephroprotectiveeffects through inhibiting XOD activity for uric acid reduction and suppressing in ammatory response.
The current pathological classi cation of hyperuricemia is based on the understanding of its mechanism by which hyperuricemia results from either overproduction of urate due to a metabolism disorder, or underexcretion by abnormal renal urate transport activity, or the combination of the two. Our study provides two facts: polydatin not only increases renal urate excretion of uric acid through the regulation of different renal urate transport-related proteins; but also reduces uric acid production by inhibiting the levels of XOD enzymes in liver. Existing urate-lowering agents in clinical practice usually have single mechanism-uricosuricagents such as benzbromarone and probenecidenhance the excretion of uric acid, and xanthine oxidase inhibitors such as allopurinolattenuate the production of urate. Because polydatin has the anti-hyperuricemic effect with a potentially dual mechanism-combination of the underproduction of urate and increasing renal urate excretion, polydatin is expected to become a more effective and safer uric acid lowering agent.

Conclusions
This study has clari ed that polydatin has an anti-hyperuricemic effect in oxonate-induced hyperuricemic mice. The effect was related to the down-regulation of renal URAT1, up-regulated renal OAT1 and OAT3 and inhibition XOD in the hyperuricemic mice. Effects of polydatin on serum uric acid levels. Mice were administrated with 0.8% CMC-Na(normal or model groups) or polydatin (5, 10, or 20 mg/kg) orbenzbromarone (Benz, 16.7 mg/kg) once daily for continuous 7 days, and received 0.8% CMC-Na (normal) or 250 mg/kg potassium oxonate (model, poly-H, poly-M, ploy-L and benz) by intraperitoneal injections one hour before the last administration of the drugs. Blood samples were collected 2 hours later and the blood uric acid levels were detected. Results are expressed as mean ± SEM. * P<0.05 and **P < 0.01 vs. the model group. Effects of polydatin on liver XOD activity. The XOD activities in theliver tissues from the treated mice were detected withXanthine Oxidase Assay Kit. Results are expressed as mean ± SEM. * P<0.05 and **P < 0.01 vs. the model group. Effects of polydatin on pathological changes in kidney tissues. The kidney tissues from the treated mice were xed, cut into sections and stained with HE according to the routine procedure and the representative images are shown.