Establishment Of Animal Model Of Hyperuricemia And Studies On Hyperuricemia Related Complications

OBJECTIVE: The objectives of our study were to analyze the incidence and related factors of bone erosion in gout patients;to investigate the drug treatment of gout and its pathophysiological mechanisms;and to establish an animal model of hyperuricemia,observe its serum uric acid level and hyperuricemia-related renal damage and glycolipid metabolism.METHODS:(1)Determination of general characters and biochemical indicators of gout patients,which recruited in the clinic of endocrinology of our hospital.The lower limb joints of the patient were scanned by ultrasound,including the knee joint,the ankle joint,the interphalangeal joint and the metatarsophalangeal joint.The diagnosis of gout was based on the 2015 ACR / EULAR gout diagnostic criteria,and bone erosion was diagnosed through ultrasound by experienced radiological doctors.The prevalence of bone erosion in gout patients was calculated,and independent correlation factors of bone erosion in gout patients were screened by binary logistic regression analysis.(2)Isolation of mouse bone marrow-derived macrophages to detect the effect of chaetocin on the expression of inflammatory cytokine interleukin-1?(IL-1?),further explore its mechanism,and detect the effect of chaetocin on glycolytic pathway and NLRP3 inflammation activation.Furthermore,the effect of chaetocin on sodium monosodium citrate(MSU)induced mouse peritonitis and arthritis were detected.(3)The urate oxidase(UOX)gene of Wistar rats was knocked out by CRISPR-Cas9 system,and fasting blood was collected every 4 weeks to detect serum uric acid,serum lipid profile,creatinine and urea nitrogen levels.24 h urine was collected to test the urine volume and urine protein levels of UOX-KO rats and WT rats.Renal pathology includes HE,?-SAM,PAS,Masson,type 1 collagen,F4/80 and IL-1? staining to detect renal involvement.Rats were tested by glucose tolerance test and insulin tolerance to test glucose metabolism.Uric acid-lowering drugs such as allopurinol,febuxostat and benzbromarone were given for 4 weeks of uric acid-lowering treatment to observe changes in serum uric acid levels in rats.RESULTS:(1)Among 980 gout patients,44% of patients found bone erosion under ultrasound.The first metatarsophalangeal joint was the most common site of bone erosion,and 78.4% of bone erosion occurred in this joint.Bone erosion was independently associated with age,gout course,tophi,synovial thickening,and joint effusion.The presence of tophi was the most important risk factor for bone erosion in gout patients,and the number of tophi was related to bone erosion.(2)Western blot and ELISA showed that chaetocin could significantly inhibit the secretion of IL-1? induced by MSU.Further studies found that chaetocin inhibits the expression of hypoxia-inducible factor-1?(HIF-1?)and inhibits NLRP3 inflammasome Activation;in vitro,chaetocin could ameliorate MSU-induced joint swelling,inflammatory cell infiltration and hyperalgesia,and inhibit MSU-induced increase of HIF-1?,NLRP3-related genes and Il1 b m RNA levels in peritoneal macrophages in MSU-induced peritonitis.(3)The UOX gene of Wistar rat was successfully knocked out by CRISPR-Cas9 technology,and the expression of UOX protein in liver was significantly decreased.Serum uric acid level of UOX-KO rats was significantly higher than that of wild type rats;UOX-KO rats showed severe kidney damage characterized by elevated serum creatinine and urea nitrogen levels,elevated urine volume and urinary protein,kidney stones,renal fibrosis,renal inflammatory cell infiltration and apoptosis.The total blood cholesterol level and liver lipid droplets increased.UOX-KO female rats showed elevated fasting blood glucose.Allopurinol and febuxostat significantly reduced blood uric acid levels in UOX-KO rats,while benzbromarone was no significant effect of serum uric acid levels in UOX-KO rats.CONCLUSIONS:(1)The prevalence of bone erosion in gout patients was high,and tophi was the most important risk factor for bone erosion.(2)Chaetocin could significantly reduce the expression of inflammatory factors induced by MSU,and provided a new drug choice for the treatment of gout.(3)UOX-KO rats spontaneously have elevated seum uric acid levels,accompanied by renal damage and glycolipid metabolism disorder;UOX-KO rats were sensitive to uric acid-lowering drug such as allopurinol and febuxostat.UOX-KO rat could be used as good animal models of pathophysiological studies of hyperuricemia and related complications and drug screening.