Effects of Alismatis Rhizoma and Rhizoma Smilacis Glabrae Decoction on Hyperuricemia in Rats

The combination of Alismatis Rhizoma (AR) and Rhizoma Smilacis Glabrae (RSG), as Chinese herb medicine, has been used for their uric acid-lowering effect. However, the effects and mechanism of the combination of the two medicines have not been fully reported. Therefore, to explore the effects of AR-RSG combination decoction on the treatment of chronic hyperuricemia (HUA) in rats as well as the underlying mechanisms, in this study, at the first stage, a long-term HUA rats model was established by gavage of oteracil potassium plus adenine; allopurinol was used as the positive control, and the uric acid-lowering effects of AR or RSG decoction alone with low and high dose were evaluated, respectively. Serum uric acid (UA) and xanthine oxidase (XOD) were determined mainly, and pathological analysis of the kidney and liver was carried out after sacrifice of the animals. And then, at the second stage, four dose groups of AR-RSG combination treatment were investigated in HUA rats. In addition to the indicators measured at the first stage, the expression of urate anion exchanger 1 (URAT1) in rat kidney was determined by immunohistochemistry. We discovered that the UA levels of the model group in both stages were significantly and steadily higher than those of control groups. AR and RSG alone or in combination possess ability to decrease serum UA level of HUA rats, with effects more marked in the combination groups. The uric acid-lowering mechanism of AR-RSG combination may be related to its inhibiting activity of XOD, improving kidney damage and downregulating the expression of URAT1 in kidney.


Introduction
Hyperuricemia (HUA) refers to an abnormal state in which the fasting serum uric acid value is higher (7.0 mg/dL) under normal diet [1][2][3]. HUA is the biochemical basis of gout and a risk factor of other chronic diseases as well (e.g., acute and chronic kidney diseases, diabetes mellitus, coronary heart diseases, and metabolic syndrome) [4][5][6][7][8]. Currently, the medications used for lowering uric acid are mainly western pharmaceuticals, which if used in long term may cause adverse outcomes. For example, allopurinol has been reported to induce hypersensitivity reaction [9], benzbromarone may cause cells apoptosis and has certain hepatotoxicity [10], and uricase may lead to severe immunogenic and hemolytic reactions [11]. On the contrary, traditional Chinese medicine (TCM) has been used to treat gout for almost a thousand years and shows promising curative effect, and some of them are even as effective as western medicines [12]. A recent meta-analysis of 11 randomized controlled clinical trials showed that the efficacy of TCM is similar to that of western pharmaceuticals in treating HUA [12]. TCM, with natural sources and mild effects, may be more promising on long-term maintenance of serum uric acid (UA) level. Lu et al. has analyzed the role of TCM used for HUA and gout and found that Alismatis Rhizoma (AR) and Rhizoma Smilacis Glabrae (RSG) were frequently used in the treatment of HUA and gout [13].
However, due to the unclear material basis and mechanism of action of TCM, there is a certain gap in the application between TCM and western pharmaceuticals such as allopurinol currently used in clinical practice. In addition, problems such as nonuniform criteria of diagnosis and efficacy evaluation, lack of mechanism studies, and unclear long-term efficacy studies also exist in the medicine development for HUA.
Animal models play an important role in the screening of drugs for HUA and the preliminary exploration of the mechanism of action. At present, there are a number of methods to establish animal models of HUA. Rats and mice are used as model animals commonly, and the common model drugs are adenine, oteracil potassium, and so on. Studies have shown that modeling with a single drug often fails to achieve good results; currently, the combination of modeling agents has been mostly adopted, which could maintain longer and higher serum UA [14]. It should be noted that most animal models established before were not suitable for screening slow-acting agents such as TCM because of short-time treatment for both modeling and intervention [15][16][17]. Huo et al. from our research group have published the prophase study on establishment of rat models for screening slow-acting drugs of hyperuricemia before [18]; our previous reports indicate that the combination of adenine and oteracil potassium can establish a stable rat model of chronic HUA and allopurinol can be used as an effective positive control [18]. erefore, based on the rat model we have established already, the uric acidlowering effects of AR and RSG decoction alone were investigated firstly in this study and then based on the results of the first stage, the combination of AR-RSG decoction was studied in order to provide a basis for the subsequent research and the development of TCM prescriptions for UA lowering.

Preparation of AR and RSG Decoction.
e decoction administered to rats was prepared as comparable to real-life scenario. At the first stage, the proper amount of AR pieces was weighed, added with 10 times of water, soaked for 30 min, and decocted for 45 min in a gallipot. After collection of the liquid, an equal amount of water was added again and decocted for 45 min and then the two collections were combined together and concentrated to the required doses. RSG pieces were also prepared as stated above. At the second stage, AR and RSG pieces were weighed proportionally and mixed together and then the corresponding combination decoction concentrates were prepared in the same way.

Experimental Animals.
Adult male SD rats (200 ± 20 g, SPF-grade) were procured from the Dashuo Laboratory Animal Reproduction Center (Chengdu, China) with animal license: SCXK (Chuan) 2013-24. Two batches of rats were used for two stages in this study. e rats were kept at the Animal Laboratory Center of West China School of Public Health (Chengdu, China) with license: SYXK (Chuan) 2013-011 and given free access to standard commercial rodent feed and drinking water. Animal treatment protocol followed the guidance of the Ethical Committee for Research on Laboratory Animals of Sichuan University and was in accordance with the international guidelines for laboratory animal use and care found in the European Community guidelines.
All treatments were via oral gavage in a volume of 10 mL/ kg·bw for 45 days. e modeling agent was dissolved with 1% CMC-Na. In the positive control group, allopurinol was dissolved with pure water. AR, RSG, and their combination treatment were the corresponding decoction concentrates. e details about the treatment are summarized in Table 1.

Samples Collection and Parameters Measurement.
For two stages, body weight was measured and recorded before treatment on the first day of dosing and twice per week thereafter and prior to necropsy.

Blood Collection and Measurement.
Blood collection and indicators detection in two stages were the same. e rats were fasted eight hours prior to blood collection with free access to water. Peripheral blood from posterior venous 2.6.2. Histopathology Analysis. After sacrificing, the livers and kidneys of the rats were excised and weighed. Livers and kidneys were fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Light microscopy studies were done under a photomicroscope (Olympus BX50F4, Japan). e extent of the tissue damage was evaluated by the Image-Pro Plus System 5.0 (Media Cybernetics Inc., Silver Spring, MD, USA). e images of whole tissue sections from all animals were captured under ×200 optical magnification. All histological determinations were done by a blinded observer.

UART1 Expression in Kidney with
Immunohistochemistry Analysis. Only in the second stage, immunohistochemistry was conducted to determine the expression of kidney urate anion transporter 1 (URAT1). Kidney tissues were dehydrated in a graded series of alcohols, embedded in paraffin, and sectioned at 3 μm. After deparaffinization and rehydration, endogenous peroxidase activity was blocked by using 3% H 2 O 2 solution for 25 min. Sections were then incubated for 30 min at room temperature in blocking solution containing 3% bovine serum albumin, followed by incubation with primary antibodies diluted in PBS at 4°C overnight (anti-URAT1 antibody, 1 : 50). Secondary antibody incubation was performed at room temperature for 50 min, followed by developing with DAB, restaining with hematoxylin, water washing, staining with blue dye, air drying, and film sealing. e tissues were observed under a microscope, and high-quality images were collected by CCD software (1600 × 1200 pixels); then, the integrated optical density (IOD) of the selected areas was measured by IPP program software.

Statistical Analyses.
Results were expressed as mean values with standard deviations (mean ± SD). Statistical analysis was performed using Statistical Product and Service Solutions (SPSS) Version 20.0, and the level of significance was set at α � 0.05. Multiple means were compared using one-way analysis of variance (ANOVA) after checking for homogeneity of variance, or the Kruskal-Wallis rank sum test was performed. Comparison between groups was undertaken with Dunnett's test while P ≤ 0.05 in ANOVA.

Results
No rat died during the experiment both in the first and second stage. Later in the 45 consecutive days, yellow and clutter in furs, apathy, and burnout in spirit were observed in model groups, positive control groups, and all treatment groups except the control groups.

Body Weights and Organ Weights.
At the first stage, body weights at sacrifice and relative liver/kidney-to-body weights of rats in each group are summarized in Table 2.
Significant decrease (P < 0.05 or P < 0.01) of body weights was observed in rats of model and the treatment groups compared with those of control. Except for the AR-H group, Evidence-Based Complementary and Alternative Medicine the relative kidney weights of the other groups were significantly higher than those of control group (P < 0.05). Compared with those in group M, the body weight of rats in group RSG-H was significantly lower (P < 0.01) and the relative liver weight was significantly higher (P < 0.05).

Necropsy and Histopathologic Findings.
At the first stage, the kidney tissue structure of rats in the control group was normal. In group M, the kidney volume was increased, with yellow vesicles or particles on the surface, moderate glomerular atrophy, moderate tubular dilation, epithelial necrosis, and exfoliation with severe inflammatory cell infiltration and more brown crystalline precipitation in the renal tubes. In the TCM groups (except group AR-L) and group A, there was mild inflammatory cell infiltration in the kidney tubules and only a small amount of crystallization was observed in the tubules, indicating their effect on alleviating kidney damage, as shown in Figure 1. e pathological microscopic examination of the livers showed intercellular pigment crystallization in some areas with flaky necrosis in group RSG-H, as shown in Figure 2.

Serum Liver Biological Indexes (ALT, AST, and T-BIL).
Levels of ALT, AST, and T-BIL in group RSG-H were significantly higher than those in group M, suggesting that there was liver damage of rats in the RSG-H group, as shown in Table 3.

Serum UA, Cr, and Ur Levels.
e level of UA of rats in group M was significantly higher than that in the control group, suggesting that the rats model of HUA was successfully developed and maintained till the 45 th day. e UA level in group A and RSG-H was significantly lower than that in group M on the 15 th , 30 th , and 45 th day (P < 0.05 or P < 0.01). e UA levels in groups AR-L, AR-H, and RSG-L were significantly lower than those in group M at the 45 th day (P < 0.05). e descent rate of UA in TCM groups was lower than that in group A, as shown in Figure 3. e percentage of decrease in UA is shown in Table 4.
e Cr and Ur levels in group M were significantly higher than those of the control group (P < 0.01). e Ur level in group A was significantly lower than that of group M (P < 0.01). In group RSG-H, the Cr levels, consistent with Ur levels, were all significantly lower than those in group M on the 30 th and 45 th day (P < 0.01), suggesting that RSG decoction may have protective effect on the kidney function of HUA rats, as shown in Table 5.

Serum UA Metabolism-Related Enzyme (XOD and ADA) Levels.
Serum XOD and ADA levels in group M were significantly higher than those in the control group. e levels of XOD and ADA in group A were significantly lower than those in group M. Only in group RSG-H, the XOD level was significantly lower than that in group M (P < 0.01), as shown in Figure 4.

Body Weights and Organ
Weights. Body weights at sacrifice and relative liver/kidney-to-body weights of rats in each group are summarized in Table 6. Compared with those of the control group, the relative kidney weights of the other groups increased significantly (P < 0.01) and there was no significant difference of body weights and relative liver weights among all groups (P > 0.05).

Necropsy and Histopathologic Findings.
Kidney tissue structure of the control group was normal. In group M, the kidney volume was increased with yellow vesicles or graywhite particles on the surface, moderate atrophy of glomeruli, moderate dilatation of renal tubules, more necrosis and shedding of epithelium with severe inflammatory cell infiltration, and more brown crystalline precipitation in the tubes. Mild inflammatory cell infiltration was observed in kidney tubules of rats in PF-1, -2, -3, and -4 groups and group A, and only a small amount of crystallization was observed in the tubules, indicating their effects of alleviating kidney injury, but there was no significant difference among the treatment groups, as shown in Figure 5.
A small amount of inflammatory cell infiltration accompanied by mild cell atrophy and crystallization was observed in the liver of rats in group M, group A, and all TCM treatment groups (PF-1, -2, -3, and -4), without significant difference among these groups. e observation might be attributed to increased intake of adenine (picture not shown).

Serum Liver Biological Indexes (ALT, AST, and T-BIL).
ere was no statistical difference of serum ALT, AST, and T-BIL among all groups, as shown in Table 7.

Serum UA, Cr, and Ur Levels. Levels of UA in group
A was significantly lower than those in group M (P < 0.01) at any timepoint. e UA levels of group PF-4 on the 30 th and 45 th day were significantly lower than those in group M (for both, P < 0.05), while the UA levels of group PF-1, -2, and -3 were significantly lower than those of group M at the 45 th day (for all, P < 0.05). e UA level of group PF-4 decreased by more than 50% on the 45 th day, as shown in Figure 6. e percentage of decrease in UA is shown in Table 8.
e Cr and Ur levels in group M were significantly higher than those of the control group (P < 0.01), and the Cr and Ur levels in group A were significantly lower than those of group M (P < 0.01). e Cr levels of group PF-2, -3, and -4 were significantly lower than those of group M on the 30 th and 45 th day (P < 0.05 or P < 0.01).
e Ur levels in all combination groups were significantly lower than those of group M on the 45 th day (P < 0.01), as shown in Table 9.

Serum UA Metabolism-Related Enzyme (XOD and ADA) Levels.
Compared with those in the control group, the XOD and ADA levels in group M were significantly increased. e levels of XOD and ADA in group A were significantly lower than those of group M (P < 0.01). e XOD levels of group PF-2, -3, and -4 were significantly lower than those of group M (P < 0.05 or P < 0.01). e ADA level in group PF-2 was significantly lower than that in group M, as shown in Figure 7.

Expression of URAT1 in Kidney.
Expression of URAT1 was significantly increased in group M compared with the control group (P < 0.01). Compared with that of group M, the expression of URAT1 in group A and all TCM groups were decreased to different extent. Only in group PF-3 and PF-4, the expression of URAT1 was significantly decreased, as shown in Figures 8 and 9.

Discussion
Hyperuricemia is mainly determined by serum uric acid levels, the clinical symptoms of which are not obvious, but it

Evidence-Based Complementary and Alternative Medicine
is the biochemical basis and key risk factor of gout attack [19]. Some researchers have used TCM for treating gout and found that herbs such as Alismatis Rhizoma (AR), Rhizoma Smilacis Glabrae (RSG), and their extracts have certain effects on reducing UA [20][21][22]. As we all know, TCM treatment takes a longer time and shows slower progress than western medicine does, and the effect of TCM is less immediate but perhaps more substantial. Meanwhile, the modeling time for most HUA animal models at present is relatively short. erefore, we established a long-term effective hyperuricemia rat model firstly [18] and then used the rat model to evaluate the efficacy of TCM in treating HUA. Considering that the way of taking TCM for most patients is decoction instead of single purification or several extracts, our study used the decoction of AR and RSG as interventional treatment, which has important practical significance to the research of TCM prescription of uric acidlowering effects. In this study, the UA levels of the model group in both stages were significantly and steadily higher than those of control groups at any timepoint, which were consistent with the results of model establishment we have published before [18], indicating that the model of HUA in rats was established successfully.
It is recognized that it would be better if the doses effective in animal experiments could be deduced to actual human intake. According to the relevant requirements of dose selection in the Chinese national standard of "functional evaluation procedures and testing methods for health food (2003)," in general, 5 to 20 times of the minimum recommended dose for human are suggested as the effective dose range for test substances of function evaluation in animal studies. erefore, in the first stage, the low-and high-dose settings of AR or RSG were equivalent to 5 and 20 times of the minimum recommended dose for human (AR: 0.1 g/kg·bw; RSG: 0.25 g/kg·bw). e results showed that AR and RSG  Results of the first stage showed that 0.50∼2.00 g/kg·bw of AR decoction and 1.25∼5.00 g/kg·bw of RSG decoction could reduce UA but the high dose of RSG caused liver damage. erefore, combined with UA-lowering effect and its possible mechanism, in the second stage, 1.00 g/kg·bw of AR was used as the reference and the dose rate of AR: RSG � 1 : 1, 2 : 1, 1 : 2, and 2 : 2 was used for dose design.
Results showed that the four AR-RSG combination groups all had the effect of decreasing UA and, according to the declining trend of UA levels, it was speculated that           Evidence-Based Complementary and Alternative Medicine 9 the effect of reducing UA was time dependent. When the content of AR was the same (2.00 g/kg·bw), the drop of UA in group PF-2 and group PF-4 was greater than that in group AR-H (shown in Tables 4 and 8.), indicating that the effect of reducing UA of the combination group was better than that of the single drug. e four formulations can reduce the serum Cr and Ur levels to different degrees, and improve the kidney injury in rats. In the second stage, the XOD levels of groups PF-2, -3, and -4 were significantly lower than those in group M, suggesting that the UA-lowering effect of the combination may be related to decreasing XOD activity. e kidney damage of rats with * * * * * * *   HUA was all alleviated in combination groups, and the protective effect on the kidney may also be one of the mechanisms of the combination treatment to reduce UA.
Furthermore, URAT1 is a membrane transporter encoded by SLC22A12 gene of the organic anion-encoding family SLC22A, which is mainly located in the lumen side of the brush border epithelial cells of the proximal convoluted tubule of the kidney cortex and involved in the resorption of uric acid, thereby regulating the concentration of serum uric acid [23]. Studies have shown that the loss of URAT1 function is strongly associated with kidney hypouricemia and the UA excretion rate of these people can even reach 100% [24,25]. Inhibiting the expression of URAT1 can effectively reduce the serum uric acid level in vivo, which is an important approach to treat hyperuricemia. Studies have reported that TCMs such as Rhizoma Smilacis Glabrae and Semen Plantaginis can reduce the expression of URAT1 in HUA rats [26]. In our study, immunohistochemical results of kidney URAT1 in rats showed that PF-3 and PF-4 could significantly downregulate the expression of URAT1 in the kidney of HUA rats, suggesting that combination of AR and RSG could promote UA excretion by inhibiting the expression of URAT1 in the kidney of HUA rats. And according to the dose setting of combination groups, it can be speculated that the effect of RSG on inhibiting the expression of URAT1 may be stronger than that of AR.

Conclusion
Combination of Alismatis Rhizoma and Rhizoma Smilacis Glabrae decoction could reduce UA in HUA rats, which is more efficient together than treated alone. e combination decoction may play its role in reducing UA by inhibiting activity of XOD, improving kidney damage and downregulating the expression of URAT1 in kidney.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Shupin Cheng and Hanxiao Sun contributed equally to this research work.