MiR-143-3p directly targets GLUT9 to reduce uric acid reabsorption and inflammatory response of renal tubular epithelial cells.

GLUT9 is generally considered to be associated with the uric acid transport, which plays an important role in the regulation of serum uric acid level. In this study, the expression level of miR-143-3p was significantly decreased in hyperuricemia mice model group compared with the normal control by miRNA microarray, the same results were confirmed in the hyperuricemia patients and the healthy control group. It is predicted that GLUT9 may be the target gene of miR-143-3p by target scan and other net-software. GLUT9 as the downstream target gene of miR-143-3p was determinated by fluorescence enzyme activity assay. Western blotting and qRT-PCR indicated that the expression of GLUT9 in human renal tubular epithelial cells transfected with miR-143-3p mimics was significantly reduced. Meanwhile inflammatory factors IL-1ß and MCP-1 significantly decreased. In conclusion, miR-143-3p can reduce uric acid reabsorption by inhibiting its downstream target gene GLUT9.

Anti-Hyperuricemic Effect of 2-Hydroxy-4-methoxy-benzophenone-5-sulfonic Acid in Hyperuricemic Mice through XOD.

Conventionally, benzophenone-type molecules are beneficial for alleviating the UV exposure of humans. More importantly, various compounds with this skeleton have demonstrated various biological activities. In this paper, we report the anti-hyperuricemic effect of the benzophenone compound 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid (HMS). Preliminarily, its molecular docking score and xanthine oxidase (XOD) inhibition suggested a good anti-hyperuricemic effect. Then, its anti-hyperuricemic effect, primary mechanisms and general toxicity were examined on a hyperuricemic mouse model which was established using potassium oxonate and hypoxanthine together. HMS demonstrated a remarkable anti- hyperuricemic effect which was near to that of the control drugs, showing promising perspective. General toxicity was assessed and it showed no negative effects on body weight growth and kidney function. Moreover, anti-inflammatory action was observed for HMS via spleen and thymus changes. Its anti-hyperuricemic mechanisms may be ascribed to its inhibition of XOD and its up-regulation of organic anion transporter 1 (OAT1) and down-regulation of glucose transporter 9 (GLUT9).

Intrastriatal injection of hypoxanthine impairs memory formation of step-down inhibitory avoidance task in rats.

The aim of this study was to investigate the effects of intrastriatal injection of hypoxanthine, the major compound accumulated in Lesch-Nyhan disease, on performance step-down inhibitory avoidance task in the rat. Male adult Wistar rats were divided in two groups: (1) saline-injected and (2) hypoxanthine-injected group. Treated-group received intrastriatal hypoxanthine solution 30 min before training session (memory acquisition) or immediately after training session (memory consolidation) or 30 before test session (memory retrieval) on step-down inhibitory avoidance task. Results show that hypoxanthine administration caused significant memory impairment in all periods tested. These results show that intrastriatal hypoxanthine administration provoked memory process impairment of step-down inhibitory avoidance task, an effect that might be related to the cognitive memory alterations in Lesch-Nyhan patients.

Intracerebroventricular injection of oxidant and antioxidant molecules affects long-term potentiation in urethane anaesthetized rats.

Production of superoxide anions in the incubation medium of hippocampal slices can induce long-term potentiation (LTP). Other reactive oxygen species (ROS) such as hydrogen peroxide are able to modulate LTP and are likely to be involved in aging mechanisms. The present study explored whether intracerebro-ventricular (ICV) injection of oxidant or antioxidant molecules could affect LTP in vivo. With this aim in mind, field excitatory post-synaptic potentials (fEPSPs) elicited by stimulation of the perforant pathway were recorded in the dentate gyrus of the hippocampal formation in urethane-anesthetized rats. N-acetyl-L-cysteine, hydrogen peroxide (H2O2) or hypoxanthine/xanthine-oxidase solution (a superoxide producing system) were administrated by ICV injection. The control was represented by a group injected with saline ICV. Ten minutes after the injection, LTP was induced in the granule cells of the dentate gyrus by high frequency stimulation of the perforant pathway. Neither the H(2)O(2) injection or the N-acetyl-L-cysteine injection caused any variation in the fEPSP at the 10-min post-injection time point, whereas the superoxide generating system caused a significant increase in the fEPSP. Moreover, at 60 min after tetanic stimulation, all treatments attenuated LTP compared with the control group. These results show that ICV administration of oxidant or antioxidant molecules can modulate LTP in vivo in the dentate gyrus. Particularly, a superoxide producing system can induce potentiation of the synaptic response. Interestingly, ICV injection of oxidants or antioxidants prevented a full expression of LTP compared to the saline injection.

Hypoxanthine Induces Neuroenergetic Impairment and Cell Death in Striatum of Young Adult Wistar Rats.

Hypoxanthine is the major purine involved in the salvage pathway of purines in the brain. High levels of hypoxanthine are characteristic of Lesch-Nyhan Disease. Since hypoxanthine is a purine closely related to ATP formation, the aim of this study was to investigate the effect of intrastriatal hypoxanthine administration on neuroenergetic parameters (pyruvate kinase, succinate dehydrogenase, complex II, cytochrome c oxidase, and ATP levels) and mitochondrial function (mitochondrial mass and membrane potential) in striatum of rats. We also evaluated the effect of cell death parameters (necrosis and apoptosis). Wistar rats of 60 days of life underwent stereotactic surgery and were divided into two groups: control (infusion of saline 0.9%) and hypoxanthine (10 µM). Intrastriatal hypoxanthine administration did not alter pyruvate kinase activity, but increased succinate dehydrogenase and complex II activities and diminished cytochrome c oxidase activity and immunocontent. Hypoxanthine injection decreased the percentage of cells with mitochondrial membrane label and increased mitochondrial membrane potential labeling. There was a decrease in the number of live cells and an increase in the number of apoptotic cells by caused hypoxanthine. Our findings show that intrastriatal hypoxanthine administration altered neuroenergetic parameters, and caused mitochondrial dysfunction and cell death by apoptosis, suggesting that these processes may be associated, at least in part, with neurological symptoms found in patients with Lesch-Nyhan Disease.

Hypoxanthine Intrastriatal Administration Alters Neuroinflammatory Profile and Redox Status in Striatum of Infant and Young Adult Rats.

Hypoxanthine, the major oxypurine metabolite involved in purine's salvage pathway in the brain, is accumulated in Lesch-Nyhan disease, an inborn error of metabolism of purine. The purpose of this study was to investigate the effects of hypoxanthine intrastriatal administration on infant and young adult rats submitted to stereotactic surgery. We analyzed the effect of hypoxanthine on neuroinflammatory parameters, such as cytokine levels, immunocontent of NF-κB/p65 subunit, iNOS immunocontent, nitrite levels, as well as IBA1 and GFAP immunocontent in striatum of infant and young adult rats. We also evaluate some oxidative parameters, including reactive species production, superoxide dismutase, catalase, glutathione peroxidase activities, as well as DNA damage. Wistar rats of 21 and 60 days of life underwent stereotactic surgery and were divided into two groups: control (infusion of saline 0.9 %) and hypoxanthine (10 µM). Intrastriatal administration of hypoxanthine increased IL-6 levels in striatum of both ages of rats tested, while TNF-α increased only in 21-day-old rats. Hypoxanthine also increased nuclear immunocontent of NF-κB/p65 subunit in striatum of both ages of rats. Nitrite levels were decreased in striatum of 21-day-old rats; however, the immunocontent of iNOS was increased in striatum of hypoxanthine groups. Microglial and astrocyte activation was seen by the increase in IBA1 and GFAP immunocontent, respectively, in striatum of infant rats. All oxidative parameters were altered, suggesting a strong neurotoxic hypoxanthine role on oxidative stress. According to our results, hypoxanthine intrastriatal administration increases neuroinflammatory parameters perhaps through oxidative misbalance, suggesting that this process may be involved, at least in part, to neurological disorders found in patients with Lesch-Nyhan disease.

Intrastriatal hypoxanthine administration affects Na+,K+-ATPase, acetylcholinesterase and catalase activities in striatum, hippocampus and cerebral cortex of rats.

The aim of this study was to investigate the effects of a single intrastriatal injection of hypoxanthine, the major metabolite accumulating in Lesch-Nyhan disease, on Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities in striatum, cerebral cortex and hippocampus of rats at different post-infusion periods. Adult Wistar rats were divided in two groups: (1) vehicle-injected group (control) and (2) hypoxanthine-injected group. For Na(+),K(+)-ATPase activity determination, the animals were sacrificed 3h, 24h and 7 days after drug infusion. For the evaluation of acetylcholinesterase and catalase activities, the animals were sacrificed 30min, 3h, 24h and 7 days after hypoxanthine infusion. Results show regional and time dependent effects of hypoxanthine on Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities. The in vitro effect of hypoxanthine on the same enzymes in striatum was also investigated. Results showed that hypoxanthine inhibited Na(+),K(+)-ATPase, but not the activities of acetylcholinesterase and catalase in rat striatum. We suggest that these modification on cerebral biochemical parameters (Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities) induced by intrastriatal administration of hypoxanthine in all cerebral structures studied, striatum, hippocampus and cerebral cortex, could be involved in the pathophysiology of Lesch-Nyhan disease.

Actions of water extract from Cordyceps militaris in hyperuricemic mice induced by potassium oxonate combined with hypoxanthine.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps militaris was recorded in the classic traditional Chinese medicine book with the main functions of "protecting liver and enhancing kidney functions", influencing serum uric acid levels. AIM OF STUDY: The aim is to investigate the hypouricemic effects and possible mechanism of C. militaris in hyperuricemic mice. MATERIALS AND METHODS: A water extract (WECM) was prepared by decocting C. militaris directly at 80 °C in water bath, followed by lyophilization. WECM at 50, 100 and 200mg/kg was orally administered to hyperuricemic mice induced by potassium oxonate and hypoxanthine combinedly and allopurinol (5mg/kg) was served as a positive control. RESULTS: WECM exhibited excellent hypouricemic activity, which could decrease the serum uric acid levels of the hyperuricemic mice (306µmol/L) to 189, 184 and 162µmol/L at different doses respectively (P<0.01), approaching the levels of normal mice (184µmol/L). The urate transporter 1 (URAT1) protein levels of kidney at different doses of WECM were 28.15, 17.43, 9.03pg/mL respectively, much lower than that in the hyperuricemia group (93.45pg/mL, P<0.01); and suggested WECM may interact with URAT1. Docking simulations using modeled structure of URAT1 suggested that LYS145, ARG325, ARG477 and ASP168 of URAT1 are key functional residues of URAT1. Four active compounds in C. militaris were identified and their interaction energies with target were estimated between -200 and -400kcal/mol. CONCLUSIONS: These findings suggested that C. militaris produced significant hypouricemic actions and the hypouricemic effects of WECM may be attributed to the inhibitive effect of WECM on URAT1 protein levels. The results of blood urine nitrogen and serum creatinine levels and liver, kidney and spleen coefficients showed that WECM have no negative impacts on liver, renal and spleen functions. The screened four active compounds using molecular docking method deserve further investigation in other work.

Intrastriatal injection of hypoxanthine alters striatal ectonucleotidase activities: a time-dependent effect.

The aim of this study was to investigate the effects of intrastriatal injection of hypoxanthine on ectonucleotidase (E-NTPDases and ecto-5'-nucleotidase) activities and expressions in the striatum of rats. The effect of pre-treatment with vitamins E and C on the effects elicited by this oxypurine on enzymatic activities and on thiobarbituric reactive substances (TBARS) was also investigated. The effect of pre-incubation with hypoxanthine on nucleotide hydrolysis in striatum homogenate was also determined. Adult Wistar rats were divided into (1) control and (2) hypoxanthine-injected groups. For ectonucleotidase activity determination, the animals were sacrificed at 30 min, 24 h and 7 days after drug infusion. For the evaluation of the expression of NTPDase 1-3 and also ecto-5'-nucleotidase, TBARS assay and the influence of the pre-treatment with vitamins on ectonucleotidase activities, the animals were sacrificed 24 h after hypoxanthine infusion. Results show that hypoxanthine infusion significantly inhibited ectonucleotidase activities and increased TBARS only 24 h after administration. Pre-treatment with vitamins was able to prevent these effects. Moreover, ecto-5'-nucleotidase expression was increased (80%) at 24 h after hypoxanthine infusion. We suggest that these hypoxanthine-induced biochemical modifications could, at least in part, participate in the pathophysiology of Lesch Nyhan disease.

Addition of hypoxanthine to culture media allows in vitro cultivation of Babesia bovis and B. bigemina at reduced serum concentrations.

The microaerophilous stationary phase system (MASP) was introduced in 1980 and successfully used as a standard technique for Babesia bovis and B. bigemina in vitro culture. The percentage of serum in the medium and the dependence on specific serum donors have been recognized as important constraints both for immunochemical studies and for the logistics of culture routine. In the present study the supplementation of RPMI 1640 with hypoxanthine at a concentration between 50 and 200 microM has enabled patterns of growth of B. bovis and B. bigemina to be achieved comparable to the standard technique with a simultaneous reduction of serum concentration from 40% to 5%. With hypoxanthine-supplemented medium it was possible to either replace the bovine serum from a specific donor with horse serum or use commercial adult bovine serum or foetal calf serum at 10%. When the serum replacement media Albumax II and GF21 were used, the growth of both B. bovis and B. bigemina markedly decreased after 3 x 72 h cycles. However, when these species were cultivated in culture flasks previously coated with cells from a murine peritoneal lavage, continuous parasite growth was achieved.

Manipulation of plasma uric acid in broiler chicks and its effect on leukocyte oxidative activity.

Birds have high metabolic rates, body temperatures, and plasma glucose concentrations yet physiologically age at a rate slower than comparably sized mammals. These studies were designed to test the hypothesis that the antioxidant uric acid protects birds against oxidative stress. Mixed sex broiler chicks (3 wk old) were fed diets supplemented or not with purines (0.6 mol hypoxanthine or inosine). Study 1 consisted of 18 female Cobb x Cobb broilers that were fed purines for 7 days, whereas study 2 consisted of 12 males in a 21-day trial. Study 3 involved 30 mixed sex broilers that were fed 40 or 50 mg allopurinol/kg body mass (BM) for 21 days, a drug that lowers plasma uric acid (PUA). PUA and leukocyte oxidative activity (LOA) were determined weekly for all studies. For study 2, pectoralis major shear force, relative kidney and liver sizes (RKS and RLS), and plasma glucose concentrations were also determined. In study 1, PUA concentration was increased three- and twofold (P < 0.001) in birds fed inosine or hypoxanthine, respectively, compared with control birds. LOA of birds supplemented with inosine was lower (P < 0.05) than that of control or hypoxanthine birds. In study 2, PUA concentrations were increased fivefold (P < 0.001) in birds fed inosine and twofold (P < 0.001) in birds fed hypoxanthine compared with control birds at day 21. RKS (g/kg BM) was greater (P < 0.001) for chicks fed purine diets compared with control chicks. Muscle shear value was lower (P < 0.05) in chicks fed purine diets. PUA concentration was decreased (P < 0.001) in birds consuming allopurinol diets, whereas LOA was increased (P < 0.01) in study 3. These studies show that PUA concentrations can be related to oxidative stress in birds, which can be linked to tissue aging.

Oxidative stress induced cytokine production in isolated rat pancreatic acinar cells: effects of small-molecule antioxidants.

Reactive oxygen species are considered important regulators in the pathogenesis and in the development of pancreatitis. The transcription factor nuclear factor kappaB (NF-kappaB) is activated by reactive oxygen species and regulates the gene expressions of inflammatory cytokines. The present study investigates (1) the susceptibility of isolated rat pancreatic acinar cells to oxidant attacks produced by adenosine diphosphate/ferrous iron, hypoxanthine/xanthine oxidase, and neutrophils primed with 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA) and (2) the potential of small-molecule antioxidants (N-acetylcysteine, beta-carotene, rebamipide, allopurinol) and superoxide dismutase (SOD) to prevent such injury and oxidant-mediated NF-kappaB activation and inflammatory cytokine production in the cells. As a result, oxidative stress resulted in a time-dependent increase in lipid peroxide production in pancreatic acinar cells which was inhibited by small-molecule antioxidants and SOD. PMA-primed neutrophils induced NF-kappaB activation and increased the production of cytokines (IL-6, TNF-alpha) in the cells. This was in parallel with lipid peroxide production. Small-molecule antioxidants and SOD inhibited NF-kappaB activation and cytokine production in acinar cells caused by PMA-primed neutrophils. In conclusion, oxidative stress activates NF-kappaB, resulting in upregulation of inflammatory cytokines in pancreatic acinar cells. Small-molecule antioxidants might be clinically useful anti-inflammatory agents by inhibiting oxidant-induced cytokine production.

Sindrome de Lesch-Nyhan a propodito de 3 casos / Syndrome of Lesch-Nyhan report of three cases

El sindrome de Lesch-Nyhan es un desorden metabòlico causado por la ausencia cingènita de una enzima, la hipoxantina fosforibosil-transferasa (HPRT). Este sindrome se caracteriza clìnicamente por coreoatetosis, automutilaciòn, hiperuricemia y retardo mental. Presentamos a continuaciòn tres pacientes diagnosticados en nuestro medio por las caracteristicas clinicas, ayuda laboratorial de una relaciòn del àcido ùrico y creatinina en orina, ademàs de la confirmaciòn realizada en el exterior midiendo la actividad de la enzima de bulbo piloso y glòbulos rojos de dos pacientes