Original ArticleCaffeic acid phenethyl ester alleviated hypouricemia in hyperuricemic mice through inhibiting XOD and up-regulating OAT3
Graphical abstract
Caffeic acid phenethyl ester alleviated hypouricemia in hyperuricemic mice through inhibiting XOD and up-regulating OAT3.
Introduction
Hyperuricemia is a progressively chronic disease defined by high serum uric acids (SUAs). Generally, hyperuricemia is attributed to the imbalanced metabolism of purines from food, the impaired uric acid synthesis, and/or the dysfunctional uric acid excretions into the urine or gastrointestinal tract (Zhu et al., 2011), which directly causes gout and then is related to diabetes (Lou et al., 2020), hypertension (Zhang et al., 2020), cardiovascular disease (Fu et al., 2015), and kidney disease (Isaka et al., 2016). The risks of hyperuricemia are multi-factorial, including aging, obesity (Zhang et al., 2018), diets of high purines (Yokose et al., 2021), high alcohol consumptions, insulin resistances, hypertension, and some medications. Generally, SUAs are modulated genetically and several genes correlate with SUA fluctuations closely, especially including organic anion transporter 3 (OAT3) (Dehghan et al., 2008), glucose transporter type 9 (GLUT9, also known as SLC2A9), and urate transporter 1 (URAT1, also known as SLC22A12) (Kottgen, 2013), which were identified with genome-wide association studies (GWAS). It is known that Western diet is rich in purine and adenine, which may impair the uric acid metabolism and then lead to hyperuricemia. In addition, some purine compounds may increase the burdens of purine metabolism (Klerk et al., 2013), such as disodium 59-guanylate and disodium 59-inosinate, which are the main components of many flavor enhancers for diets and are widely used in modern food industry. Presently, several clinically available drugs are used to manage gout, such as allopurinol and benzbromarone. However, most patients with hyperuricemia are unable to achieve long-term managements over hyperuricemia due to the limits of the allopurinol and benzbromarone. In detail, allopurinol is effective for only 40% patients of hyperuricemia and also it may induce severe Steven-Johnson syndrome (Gan et al., 2020), which may lead to deaths of patients. Also, alopurrinol was limited by its renal toxicity (Horiuchi et al., 2000). Meanwhile, benzbromarone may raise serious hepatic toxicity (Takemura et al., 2019). Thus, it has been forbidden by many countries or areas, such as USA and European. Nowadays, bio-enzyme drugs, such as pegloticase (Lipsky et al., 2014) and rasburicase (Xu et al., 2020), have attracted important attentions, but they have been limited by their immune-arising problems, which made their usages even less than one month. Therefore, searching for agents of high efficacy and safety is highly demanded.
For thousands of years, natural medicines, taking herbs, higher fungi, and animals as examples, have been used widely to prevent and treat diseases (Tu, 2011). From these natural medicines, bioactive compounds were isolated and characterized, and then the medicine industry was pushed into modern times (Hu et al., 2021). Of which, propolis of honey hives (Ramezani et al., 2021) has been used as a nutrition food and also as a medicine (Chiu et al., 2020) for thousands of years, according to the Compendium of Materia Medica. Caffeic acid phenethyl ester (CAPE, Fig. 1a), a natural flavonoid-like compound, is the characteristic active component of the propolis of honey hives, which was reported to show antiviral, anti-inflammatory (Karaboğa, 2019), immunomodulatory, antioxidant (Wan et al., 2019) and cancer inhibitory effects. Structurally, it presents a phenolic and two-cyclic feature, which may render it exerting properties resembling redox shuttle and then suppressing xanthine oxidase (XOD), a key target for hyperuricemia. Also, its specific inhibition against NF-κB (Liu et al., 2018) may alleviate the renal dysfunction caused by hyperuricemia. However, its hypouricemic effect has not been reported .
In this paper, we investigated the hypouricemic effect of CAPE in hyperuricemic mice. SUAs and urine uric acids (UUAs) were assayed to examine its uric acid-lowering efficacy. Serum creatinine and urea nitrogen (BUN) were determined to assess its effects on kidney filtration function. Also, serum AST/ALT and ALP was detected to examine its influence on hepatic function. Moreover, body weights and inner organ index were recorded to estimate its general toxicity in hyperuricemic mice. Furthermore, sections of liver and kidney were observed by hematoxylin and eosin (H&E) staining to characterize its effects on the histomorphologic changes of liver and kidney. In order to investigate the mechanisms of its hypouricemic effects, XOD activities were assayed and mRNAs and proteins of ATP binding cassette subfamily G member 2 (ABCG2), GLUT9, organic anion transporter 1 (OAT1), OAT3, organic cation transporter member 2 (OCT2) and URAT1 were assayed. Moreover, molecular simulation was performed to unveil the interaction mode of CAPE to its target protein. This study may provide evidence and novel insights into the CAPE as a hypouricemic agent.
Section snippets
Drugs and reagents
Potassium oxonate (PO, 98%) and hypoxanthine (HX, 99%) for model establishment were purchased from Aladdin Reagent Co. (Shanghai, China). CAPE (> 97%), xanthine (99%), allopurinol (98%) and benzbromarone (98%) were obtained from Sigma-Aldrich LLC. (St. Louis, USA). Kits of uric acid, BUN, creatinine, ALT, AST and ALP were offered by Mindray Medical Corp. (Shenzhen, China). TRIZOL reagent was supplied by Invitrogen Corp. (Carlsbad, USA). Primers for RT-PCR were offered by Sangon Biotech Co. (
CAPE exhibited a remarkable hypouricemic effect and some protections for renal and liver
The structure of CAPE is shown in Fig. 1a, which is featured with two aromatic moieties linked by an alkyl ester. Also, the aromatic rings, double bond of the alkene and the carbonyl group formed a large conjugated structure, wherein electrons could be distributed over this large structure. These result in the ionization of the proton of hydroxyl group and the forming of a quinone-like structure, which may shuttle electrons and then inhibit biochemical redox reaction, especially in terms of
Discussion
Uric acid, which is an important metabolite, is distributed into body unanimously and plays an important role in metabolism and cell fate (Cantor et al., 2017). In addition, it presents striking effects on de novo pyrimidine biosynthesis as a direct inhibitor of UMP synthase. Also, it antagonizes the cytotoxicity of 5-fluorouracil in cancer cells. Most importantly, uric acid was focused because it primes hyperuricemia and gout directly (Crisan et al., 2017). Since it is unsolvable in blood, it
Conclusion
In conclusion, CAPE demonstrated an excellent hypouricemic effect through inhibiting XOD and up-regulating OAT3, which reduced the SUAs of hyperuricemic mice (401 ± 111 µmol/l) to 209 ± 56, 204 ± 65 and 154 ± 40 µmol/l (p < 0.01) at the doses of 15, 30 and 60 mg/kg correspondingly, depicting efficacies of 48–62% and approaching to the efficacy of allopurinol (52%). Serum parameters, body weights, inner organ coefficients and H&E staining suggested that CAPE displayed no general toxicity and
Author contributions
Tianqiao Yong, Danling Liang and Qingping Wu conceived the conception and prepared the manuscript; Shaodan Chen, Chun Xiao and Xiong Gao and Yizhen Xie conducted the experiments and the analysis; Longhua Huang, Huiping Hu, Xiangmin Li, Yuancao Liu and Manjun Cai helped perform the analysis with constructive discussions. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy
Declaration of Competing Interest
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Acknowledgments
This work was supported by the Key-Area Research and Development Program of Guangdong Province (2018B020206001, 2018B020205001), the National Natural Science Foundation of China (31901696, 81803393), the Science and Technology Program of Guangzhou (202002030225, 201707020022), the Natural Science Foundation of Guangdong Province of China (2022A1515011066, 2021A1515010960) and the Guangdong Province Sail Plan (2017YT05S115) and GDAS’ Project of Science and Technology Development (
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