| Hyperuricemia refers to a body state where uric acid concentration exceeds normal value. In recent years, prevalence rate of hyperuricemia has an obvious elevation with the improvement of people's living standards and the change of diet. Hyperuricemia, as a metabolic disease, is closely correlated with cardiovascular disease, chronic kidney disease and metabolic syndrome (diabetes, insulin resistance and obesity). The phenomenon of uric acid concentration higher than normal value in serum is mainly attributed to two aspects:one reason is more production of uric acid, the other one is reduction of uric acid excretion. Various transport proteins fixed in tubular epithelial cells are responsible for transporting uric acid, of which urate-anion transporter 1 (URAT1) on renal epithelial cells play a main role in uric acid reabsorption and is a main target for hyperuricemia treatment. Mouse renal-specific transporter (RST) as murine homologue provides an available experimental material for URAT1 targeting drug research.Currently, in the light of side effects of western medicine, Chinese traditional medicine becomes a new trend for hyperuricemia treatment due to its safety and long-term applicability. Xiezhuochubi decoction is a prescription of Chinese traditional medicine and has achieved some certain clinical results. Anthoxanthin formerly separated by our research group is applied to investigate its therapeutic effects for hyperuricemia. Specific experiments are as follows:Firstly, conditions of RTECs orimary culture were optimized and cultures underwent functional tests. The adherent rate of segments of renal proximal tubules reached 54.5% after the digestion using 400U collagenase type I for 20 minutes; the most suitable time for the first change of the culture medium was 72 hours; Log phase began from the fifth day and the cell's growth conditions became unsuitable in the tenth day. In the transport medium contained 1500μmol/L urate acid, the 30-min uptake of uric acid was the highest and was taken as a measure of an initial rate. Different urate uptake inhibitory effects appeared due to different concentrations of the indicate drugs probenecid and benzbromarone. The inhibition influence of benzbromarone was higher than probenecid. The mouse renal proximal tubular epithelial cells cultured by the modified method will yield rich and homogeneous harvest, and present well function of urate acid uptake.Next, effects of anthoxanthin on cell proliferation and uric uptake were investigated. The RTECs were isolated from the renal of mouse by the utilized method and were cultured with anthoxanthin from xiezhuochubi decoction at different concentrations. The proliferation of RTECs was assenssed by MTT assay. The uric uptake medium was prepared and the uric uptake values were determined by the kit. The interest cells were isolated and it has the function of uric uptake. The proliferation of the RTECs was inhibited by high concentrations (10,7.5,5g·L-1) of the drug, while no significant effects on the proliferation of the RTECs were observed at the low concentrations (2.5,0.5g·L-1). Moreover, uric uptake tests were undertaken at this range of concentrations. And different inhibitory degrees by the drug were also determined.Effects of anthoxanthin on gene RST expression were detected to reveal its molecular machnism. The uric acid uptake essay was carried out on the mouse renal tubular epithelia cells and the different effects of different levels of anthoxanthin in Xiezhuochubi decoction on the uric acid uptake were studied. The expression of mouse renal-specific transporter (RST) in the cells was detected by RT-PCR. Results indicated that anthoxanthin in Xiezhuochubi decoction at the concentration range from 0.5g·L-1 to 2.5g·L-1 was proved to inhibit the uric acid uptake function of RTECs and the expression of RST was lower under treatment at these concentrations. The uric acid uptake can be reduced by anthoxanthin in Xiezhuochubi decoction by inhibiting the expression of RST.Ultimately, inhibition of total anthoxanthin in Xiezhuochubi decoction on xathine oxidase was investigated in vitro, proving itself a well inhibitor. Its half inhibitory effect concentration (IC50) is 44.1μg/ml.This research proved the inhibitory influences of anthoxanthin in Xiezhuochubi decoction on uric uptake and revealed molecular mechanism of inhibition with RST as targets. The positive result indicated its certain therapeutic effects on hyperuricemia. |
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