Study On The Function And Inflammation Mechanism Of Primary Gout Susceptibility Gene KCNQ1 In Mouse Monocytes

Objective We establish a mouse model of monocyte-specific KCNQ1 gene conditional knockout,and construct synovium-like subcutaneous air pouches to explore the specific inflammation mechanism of KCNQ1 involved in the pathogenesis of gout.Methods Firstly,we constructed KCNQ1^flop/floplop/flop mice by using the conditional gene knockout technique flop^-/--loxp system,and hybridized with Lyz2-cre⁺tool mouse which specifically expresses cre enzyme in monocytes to construct KCNQ1 gene knockout in monocytes.In addition to mice（cKO mice）,the progeny mouse genotypes were propagated and identified.We detect the expression of KCNQ1 protein in monocytes by western blot to see that the target gene KCNQ1 in monocytes was absent.The cKO homozygous mice were used as the experimental group,and the wild type（WT）littermates were used as the control group,and 6 mice in each group were healthy male mice of 8 weeks old.We detected the blood uric acid Liver function,kidney function,blood sugar,blood lipids and other biochemical indicators of the two groups.We constructed synovium-like subcutaneous air pouches,and observed the inflammatory reaction at the whole level,and then collected the synovial fluid,synovial membrane,serum and other samples.The synovial fluid was centrifuged to collect the precipitate,and then cells was suspended by the PBS for inflammatory cell counting and Giemsa staining.Detected the levels of inflammatory cytokines IL-1βin synovial fluid and serum.The pathological changes of synovial membrane were observed by HE staining.Detected the expressions of IL-1β,IL-18 and MCP-1 in synovium by immunohistochemistry.DetectedResults（1）The expression of KCNQ1 protein in the KCNQ1 gene conditional knockout mice was significantly decreased.The KCNQ1 gene was successfully knocked out in monocytes.The blood uric acid,blood sugar,blood lipids,transaminase,creatinine,etc.of the two groups of mice.There was no significant difference between biochemical parameters and control group（P>0.05）.（2）Synovial fluid and serum ELISA results and synovial immunohistochemistry showed that the expression of inflammatory factor IL-1βwas significantly lower than that of WT mice（4.28±0.68 VS 6.73±0.89 pg/ml,P<0.01;146.47 VS 573.09±110.16 pg/ml,t=-5.058,P<0.01;3.4±0.55 VS 5.25±0.5,P<0.05）.The results of synovial immunohistochemistry showed that the expression levels of IL-18 and MCP-1 were not significantly different from those of the control group（P=0.092,P=0.33）;HE staining results showed that MSU stimulated with wild type after 6 hours.Compared with mice,the inflammatory pathological damage of synovial membrane in KCNQ1 cKO mice was lighter（6.14±1.07 VS 3.75±0.5,t=4.153,P<0.01）.The mouse bone marrow mononuclear cells were successfully cultured and induced to differentiate in vitro.For monocytes/macrophages,urate crystals were stimulated for 2 hours,and found that compared with the WT+MSU group,cKO+MSU level of intracellular potassium ion concentration within the MSU significantly reduced（2.83±0.57 VS 1.99±0.53 mmol/L,t=-2.612,P=0.026,P<0.05）.Conclusion The KCNQ1 gene is involved in the pathogenesis of gout.It is an inflammatory susceptibility gene of gout.It may affecting the concentration of potassium ions in the cells,causing the release of inflammatory factor IL-1β and gout.