Construction of Hyperuricemia Model Rats with Yeast Extract Combined with Oteracil Potassium

Background: Hyperuricemia is the most important risk factor for gout, hypertension, coronary artery disease and other cardiovascular diseases. The incidence of hyperuricemia gradually increased in recent years and it is very necessary to explore the medications of the prevention and treatment of hyperuricemia using hyperuricemia animal models. Objective: The objective of present study is to explore the optimal dose of yeast extract and oteracil potassium in the establishment of hyperuricemia rat model. Method: Sixty-four male rats were randomly divided into 8 experimental groups. Rats were treated with yeast extract by intraperitoneal injection or yeast extract by intraperitoneal injection combined with various doses of oteracil potassium by intragastric feeding or intraperitoneal injection for 28 days. The serum uric acid, urea nitrogen and creatinine levels of different groups were measured at 0th day, 7th day, 14th day, 21th day and 28th day. Results: The serum levels of uric acid in the groups of intraperitoneal injection with yeast extract alone, yeast extract by intraperitoneal injection combined with 50-200 mg/kg oteracil potassium by intragastric feeding and yeast extract by intraperitoneal injection combined with 50-100 mg/kg oteracil potassium by intraperitoneal injection were higher than that in the control group. But we found no signicant effect on rat kidney, heart or artery in the above groups. In the group of yeast extract by intraperitoneal injection combined with 200 mg/kg oteracil potassium by intraperitoneal injection, we observed the signicantly high level of serum uric acid and morphological and pathological changes in rat kidney, heart and artery. Conclusion: In the present study, we found that continuously treated with yeast extract combined with oteracil potassium is an effective method to establish rat hyperuricemia model. Intraperitoneal injection of yeast extract combined with 200 mg/kg oteracil potassium is an optimal dosage for the construction of a persistent and stable hyperuricemia animal model.


Construction of Hyperuricemia Model Rats with Yeast
Hyperuricemia caused by purine metabolic abnormalities or reduced excretion of uric acid (UA) in the body is the most important risk factor for gout [1][2][3]. With the improvement of living standards, the incidence of hyperuricemia and gout also gradually increased in recent years. Previous study has shown that the prevalence of hyperuricemia is up to 13.7% in healthy adults from northern and northeastern Chinese provinces [4]. In recent years, several studies on hyperuricemia prevalence in China have been performed and a latest meta-analysis indicated that the pooled prevalence of hyperuricemia was 13.3% in China from 2000 to 2014 [5][6][7][8]. In addition, it has been demonstrated that hyperuricemia is also signi cantly associated with hypertension, coronary artery disease, hyperlipidemia and other cardiovascular diseases [9][10][11][12][13][14][15][16]. Therefore, it is very necessary to explore the medications of the prevention and treatment of hyperuricemia.
Experimental animal models are the basis of disease pathogenesis and related drug research. However, there is currently no standard method of the establishment of hyperuricemia model rats [17]. We performed the present study to establish hyperuricemia rat model using yeast extract (YE) combined with different doses of oteracil potassium (OP) and to explore the optimal dose for model establishment. Oteracil potassium was con gured as suspension (20g/L) with sterilized distilled water and were administered to the rats once daily according to the above group. The NC group was fed with normal feed. All the treatments were continued for 28 days.

Sample collection
On the 7th day, 14th day, 21th day, 28th day and before the drug administration, blood samples were collected from the tail vein. The blood samples were centrifuged at 3000×g for 10 min and serum were collected and stored in -80 ℃ refrigerator. At the end of 28 day, all the rats were anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection) and kidney, heart and thoracic aorta were quickly dissected and stored. The removed femoral artery were xed with 15% paraformaldehyde and embedded in para n for histological analysis. The 4 μmthick para n sections were stained with hematoxylin and eosin (H&E).

Determination of SUA
The serum uric acid, urea nitrogen and creatinine levels of different groups at 0th day, 7th day, 14th day, 21th day and 28th day after drug administration were measured to determine the successful establishment of the hyperuricemia model.

Statistical analysis
The data were expressed as mean ± SD and analyzed by SPSS17.0. Signi cant differences between means were evaluated by one-way analysis of variance (ANOVA). LSD test were used for post hoc evaluations, and P < 0.05 was considered to represent a statistically signi cant difference.

Result
Changes of serum uric acid, urea nitrogen and creatinine levels of different groups The rat serum uric acid levels of different groups were shown in table 1. The serum uric acid levels of YE group and YE combined with OP group were signi cantly higher than that in NC group (P all 0.05). We found that not only the YE combined with OP group, but also the YE group may successfully establish animal models of hyperuricemia. In addition, the serum uric acid levels of YE i.p.+200 mg/kg OP i.p. group were signi cantly higher than that in other groups (P all 0.05).
The rat serum urea nitrogen and creatinine levels of different groups were shown in table 2 and table 3. The serum urea nitrogen levels of YE i.p.+200 mg/kg OP i.p. group were signi cantly higher than that in NC group at the 28th day (P 0.05). However, there exists no signi cantly difference of serum creatinine levels between different groups.
Pathological changes of kidney in rats The kidney tissue morphology of YE i.p.+200 mg/kg OP i.g. group, YE i.p.+100 mg/kg OP i.p. group and YE i.p.+200 mg/kg OP i.p. group showed slight pathological changes, including the thickening of the glomerular wall, proliferation of some endothelial cells in glomerular capillary loop and edema of renal tubular epithelial cells. There also exists little chronic in ammatory cell in ltration in the renal interstitial and urate crystal deposition in some rats of the above group. Furthermore, we also observed the endothelial cell proliferation of renal interstitial artery and lumen stenosis in the YE i.p.+200 mg/kg OP i.p. group (Fig.1).
Pathological changes of heart tissue in rats As shown in Fig. 2, morphological and pathological analysis demonstrated there exists no pathological change of the rat heart tissue in all groups except the YE i.p.+200 mg/kg OP i.p. group. There exist myocardial interstitial vasodilation with congestion and bleeding, endothelial cell proliferation, neovascularization and in ltration of plasma cells and lymphocytes in myocardial interstitial in YE i.p.+200 mg/kg OP i.p. group.

Pathological changes of thoracic aorta in rats
There exists no pathological change of the rat thoracic aorta in all groups except the YE i.p.+200 mg/kg OP i.p. group. There exist exfoliation of arterial endothelial cells, proliferation of vascular smooth muscle cells and light proliferation of adventitial nutrient vessels with congestion in YE i.p.+200 mg/kg OP i.p. group (Fig.3).

Pathological changes of femoral artery in rats
There exists no pathological change of the rat thoracic aorta in all groups except the YE i.p.+200 mg/kg OP i.p. group. There exist endothelial cell proliferation, lumen stenosis and neovascularization in peripheral adipose tissue in YE i.p.+200 mg/kg OP i.p. group (Fig.4).

Discussion
The establishment of hyperuricemia animal model is an effective way to develop and verify therapeutic drugs of hyperuricemia. However, there is currently no standard method of the establishment of hyperuricemia model rats. In the present study, we designed to establish hyperuricemia rat model using yeast extract combined with different doses of oteracil potassium and to explore the optimal dose for model establishment. Our results showed that intraperitoneal injection of yeast extract combined with 200 mg/kg oteracil potassium is an optimal dosage for the establishment of a persistent and stable hyperuricemia animal model.
Hyperuricemia is one of the increasingly common diseases, which is reported to have a icted more than 2 million men and women in the United States alone, and is growing rapidly in China due probably to changes in dietary habits [18][19]. Hyperuricemia is associated with abnormal uric acid concentrations in the body, resulting in the deposition of urate crystals in the joints and kidneys that lead to in ammation, as well as gouty arthritis and uric acid nephrolithiasis. In addition to an increased risk of hyperuricemia and gout, excess uric acid is also related to cardiovascular disorders, nephrolithiasis and diabetes [20][21][22][23][24][25][26].
Two major mechanisms have been proposed for hyperucicemia in man, excess production and insu cient metabolisation of uric acid. Yeast extract paste and oteracil potassium were used to mimic both mechanisms: yeast represents excess production of UA, probably the main mechanism in man, and oteracil potassium impairs metabolisation [21,27]. Potassium oxonate is most frequently employed to develop an animal model of hyperuricemia by inhibiting uricase that converts uric acid to allantoin. However, there exist great differences among different researchers in the type of drug compatibility, the way of drug use, the dosage and the method of animal selection and there is currently no standard method of the establishment of hyperuricemia model rats.
In the present study, serum uric acid levels were signi cantly increased on 7th, 14th, 21th and 28th day in the groups of intraperitoneal injection with yeast extract alone, yeast extract by intraperitoneal injection combined with 50-200 mg/kg oteracil potassium by intraperitoneal injection and yeast extract by intraperitoneal injection combined with 50-200 mg/kg oteracil potassium by intragastric feeding.
Hyperuricemia has been closely associated with renal dysfunction. High level of uric acid could increase the burden of the kidney and cause renal damage [28]. In the present study, YE i.p. +200 mg/kg OP i.p. group developed the elevated levels of serum urea nitrogen and creatinine levels compared with NC group, and showed pathological changes of kidney, including the thickening of the glomerular wall, proliferation of some endothelial cells in glomerular capillary loop and edema of renal tubular epithelial cells, chronic in ammatory cell in ltration in the renal interstitial and urate crystal deposition and endothelial cell proliferation of renal interstitial artery and lumen stenosis ( Fig. 1), which is consistent with the results of various studies, but the mechanism involved is not clear.
Purine metabolism in the circulatory system yields uric acid as its nal oxidation product, which is believed to belinked to the development of gout and kidney stones. In addition, hyperuricemia is closely correlated with cardiovascular disease too. A number of epidemiologic studies have confrmed an association between hyperuricemia and CVD [16]. A study in Japan has reported that hyperuricemia is positively associated with obesity, hypertension and dyslipidemia, and hyperuricemic subjects tend to have a clustering of these cardiovascular risk factors. In the present study, we found morphological and pathological changes in heart, thoracic aorta and femoral artery in the YE i.p.+200 mg/kg OP i.p. group, indicating that intraperitoneal injection of yeast extract combined with 200 mg/kg oteracil potassium is an optimal dosage for the construction of a hyperuricemia animal model with pathological damages related to secondary cardiovascular diseases.

Conclusion
In the present study, we found that continuously treated with yeast extract combined with oteracil potassium is an effective method to establish rat hyperuricemia model.   Figure 1 Histopathological changes of rat renal tissues (Hematoxylin-eosin staining, ×400). a: Blank control group; b: YE i.p.+200 mg/kg OP i.p. group.