ABSTRACT
Xanthine oxidoreductase (XOR), the key enzyme catalyzing purine to produce uric acid, including two subtypes, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), respectively, in vivo. Usually, XDH and XO can transform to each other. In this study, based on the principle that the subtype XO or XDH uses different electron acceptors, the methods for the measuring the activities of bovine milk XOR (pure enzyme) and its subtypes were established. The optimal concentrations of substrate xanthine (50 μmol·L-1) and electron acceptor NAD+ (50 μmol·L-1), pH value (7.80) were investigated. The ranges of the XOR, XO, XDH activity which could be determined were 0.97-17.5 U·L-1, 1-9 U·L-1, and 66-1 191 mU·L-1, respectively. Furthermore, the methods for determining the activities of XOR and its subtypes in mouse liver were established. The preparation of liver samples, the optimal concentrations of xanthine (100 μmol·L-1) and NAD+ (100 μmol·L-1) were researched. And the activity ranges of XOR, XO and XDH in mouse liver which could be determined were 0.67-3.98, 0.19-1.08, and 0.52-3.55 U·gprot-1, respectively. With the methods above, the effects of classic XOR inhibitor allopurinal (Allo) on XOR, XO and XDH from both milk and mouse liver were determined. All animal experiments have been approved by the Animal Experimental Center, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College (00003346). This study established new methods for the determination of XOR and its subtypes activity in pure enzyme system and in mouse liver, respectively, which were accurate and convenient. It laid the experimental foundation for exploring the different pathophysiological effects of XOR in the body and developing new XOR inhibitors.
ABSTRACT
Hyperuricemia is not only the biochemical basis of gout, but also closely related to the development of metabolic syndrome, cardiovascular diseases, chronic kidney disease, etc. Xanthine oxidase (XOD) is the key catalytic enzyme for uric acid biosynthesis, therefore the vital target for anti-hyperuricemic drugs. In this study, compound CC18022 was designed and synthesized specifically targeting to XOD. Molecular docking analysis indicated a fairly tight binding between CC18022 and XOD. In the in vitro study, CC18022 significantly inhibited XOD activity with a half maximal inhibitory concentration (IC50) value in the order of nmol·L-1, which is relative to the XOD inhibitor febuxostat. By using both acute and chronic hyperuricemic mice model, compound CC18022 was found to have serum uric acid-lowering effect in a dose-dependent manner in vivo. The animal welfare and experimental processes were in accordance with the provisions of the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences. In the acute hyperuricemic mice, CC18022 significantly inhibited serum XOD activity, and also the XOD activity in intestine and liver, which were related to purine absorption and metabolism. Therefore, the novel compound CC18022 exhibited significant inhibition on XOD activity and anti-hyperuricemic effects, making it a favorable candidate for further research.
ABSTRACT
To investigate the effects of small molecule compound bicyclol on type 2 diabetes mellitus (T2DM) and its mechanism of action, KKAy mice were treated with various doses of bicyclol (100, 200, and 400 mg·kg-1·d-1) with metformin (200 mg·kg-1·d-1) as a positive control, respectively. Age-matched C57BL/6J mice were used as the non-diabetic control (Con). The effect on hyperglycemia was evaluated by the levels of no-fasting blood glucose, fasting blood glucose (FPG), and glucose tolerance. Whole body insulin sensitivity was evaluated by fasting plasma insulin (FPI) and homeostasis model assessment-insulin resistance (HOMA-IR). The hepatic response to insulin was evaluated by insulin-induced activation of insulin signaling pathway. Western blot was performed to detect hepatic protein expressions. All animal experimental procedures were approved by the Animal Ethics Committee of Chinese Academy of Medical Sciences. KKAy mice showed T2DM characteristics such as hyperglycemia and insulin resistance, including attenuated response to insulin in the liver. A 28-day treatment of bicyclol suppressed both FPG and no-fasting blood glucose, in a dose- and time-dependent manner. Moreover, FPI and HOMA-IR values were both significantly decreased, and hepatic insulin-induced-phosphorylation of IRβ and Akt were up-regulated in KKAy mice after bicyclol treatment. Phosphorylation of FoxO1, the key transcription factor for regulating gluconeogenesis, was also significantly elevated by bicyclol treatment. These results suggested that bicyclol has some therapeutic effects on hyperglycemia in a time- and dose-dependent manner in KKAy mice. Its mechanism might be attributed to improving insulin resistance, enhancing hepatic insulin signaling pathway, and inhibiting gluconeogenesis. No significant interference on the hypoglycemic effect of metformin by bicyclol was observed in this study.