Effects Of CY-09 On NLRP3 Inflammatory Pathway In Human Renal Tubular Epithelial Cells Co-culturing With Different Concentrations Of Glucose And Uric

BackgroundDiabetic kidney disease(DKD)is one of the common and serious complications of chronic microangiopathy of diabetes(DM).Renal tubular injury is one of the important pathological types of DKD,and it may even be earlier than glomerular disease.A number of studies have confirmed that the NLRP3 inflammasome pathway plays an important role in renal tubular injury.High concentration of glucose and high uric acid activate the NLRP3 inflammasome pathway of human renal tubular epithelial cells(HK-2),and increase the production of effector molecules IL-1β and IL-18.The long-term chronic inflammation mediated by it may be the main pathophysiological mechanism of renal tubular injury.We then tried to make the pathogenic mechanism more clear by cultivating tubular epithelia in different concentrations of glucose and uric acid by adding CY-09,a specific blocker for NLRP3 inflammasome.ObjectiveTo explore the effect of CY-09 on the gene expression levels and protein expression levels of five factors in the NLRP3 inflammasome pathway of HK-2 cells cultured in different concentrations of glucose and uric acid,and to explore its protection effect after blocking the NLRP3 inflammasome pathway.Methods1.Cultivating the human renal tubular epithelial cells.2.Using a 2×3×2 three-factor factorial design method,the experiment was divided into 12 groups according to different concentrations of glucose(5.5 mmol/L,25 mmol/L),uric acid(0 mmol/L,0.2mmol/L,0.4 mmol/L)and the presence or absence of CY-09.Each group was cultured for 72 h.3.q PCR and Western-Blot methods were used to detect m RNA expression levels and protein expression levels of NLRP3,ASC,caspase-1,IL-1β,IL-18 in the inflammatory pathway of NLRP3 in each group.Results m RNA expression of the five factors:Main effect: 1)The gene expression of caspase-1 and IL-1β increased at high glucose concentration(P<0.05).Different concentrations of glucose did not affect the gene expression levels of ASC,NLRP3 and IL-18.2)With the increase of uric acid concentration,the gene expression of the five factors showed an increasing trend(P<0.05).3)CY-09 intervention did not affect the gene expression of ASC and IL-18(P>0.05),but increased the gene expression of the other three factors.Interaction effect: 1)The increase of glucose and uric acid concentration has a synergistic effect on the gene expression of the five factors(P<0.05).2)CY-09 can improve the gene expression of the other four factors differently between normal glucose and high glucose concentrations except for Caspase-1.3)With the increase of uric acid concentration,CY-09 increased the gene expression of the five factors and basically showed a trend of first increasing and then decreasing(P<0.05).Protein expression of the five factors:Main effect: 1)The protein expression of the other four factors increased at high glucose concentration except for Caspase-1(P<0.05).2)With the increase of uric acid concentration,the protein expression of the other four factors showed an increasing trend except for NLRP3(P<0.05).3)CY-09 intervention has inconsistent effects on the protein expression of the five factors in the inflammatory pathway of NLRP3.Interaction effect: 1)The increase of glucose and uric acid concentration has a synergistic effect on the protein expression of the five factors(P<0.05).2)CY-09 intervention increases the tendency of the protein expression of the five factors in normal glucose concentration;however,the trend of protein expression of the five factors in high glucose concentration was significantly reduced(P<0.05).3)With the increase of uric acid concentration,CY-09 increased the protein expression of the four factors and basically showed a trend of first increasing and then decreasing except for IL-18(P<0.05).Conclusions1.With the increase of glucose and uric acid concentration,the gene expression of five components in NLRP3 inflammasome pathway of HK-2 cells increased to different degrees,and they had synergistic effect.CY-09 intervention increased gene expression of some factors,but the trend was different in different glucose and uric acid environments.2.With the increase of glucose and uric acid concentration,the protein expression of five components in NLRP3 inflammasome pathway of HK-2 cells increased to different degrees,and they had synergistic effect.CY-09 intervention increased the protein expression of the five components in the normal glucose environment,but decreased their expression in the high glucose environment;the trend of five components is different in different uric acid concentrations.