Kruppel样因子15通过调控核苷酸结合寡聚化结构域样受体蛋白3表达影响胰岛素抵抗实验研究

Kruppel-like factor 15 affects insulin resistance by regulating expression of NOD-like receptor protein 3

目的:探讨Kruppel样因子15(KLF15)是否通过调控核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)表达影响胰岛素抵抗。方法:建立小鼠模型和细胞模型,细胞蛋白电泳检测细胞内目的蛋白表达,聚合酶链反应(PCR)检测KLF15表达量,Western blot检测NLRP3相对表达量,免疫组化法检测组织KLF15表达,免疫荧光染色检测细胞增殖相关蛋白。结果:在链脲佐菌素(STZ)诱导小鼠模型中,小鼠诱导后1~4个月,血清KLF15 mRNA表达量水平被抑制。用10、20、40 mmol/L葡萄糖诱导HepG2细胞24 h后,细胞内KLF15 mRNA表达量水平随葡萄糖浓度升高而减少(均P<0.05)。小鼠组织中,KLF15、葡萄糖转运蛋白4(GLUT4)、胰岛素受体底物1(IRS1)在空白组表达最高,其次分别为1、2、4个月组;NLRP3在空白组表达最低,1、2、4个月组依次升高(均P<0.05)。在体外模型中,si-NLRP3质粒可以降低NLRP3表达量;抑制NLRP3表达可以减少干扰素-γ(INF-γ)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和IL-1β表达,同时增加IRS1、GLUT4表达量;NLRP3过表达质粒可以促进NLRP3表达量,过表达NLRP3可以促进INF-γ、TNF-α、IL-6和IL-1β表达,同时减少IRS1、GLUT4表达量;上调KLF15可以抑制NLRP3蛋白表达,抑制KLF15可以促进NLRP3蛋白表达(均P<0.05)。结论:KLF15表达量与2型糖尿病(T2DM)胰岛素抵抗有关,KLF15低表达量可能会增加胰岛素抵抗。KLF15可能通过抑制NLRP3表达量减少T2DM胰岛素的炎症水平,抑制胰岛素抵抗。KLF15可能为胰岛素抵抗的T2DM的潜在治疗和诊断靶点,为以后的相关研究提供实验依据。

Objective:To investigate whether Kruppel-like factor 15(KLF15)affects insulin resistance by regulating the expression of NOD-like receptor protein 3(NLRP3).Methods:Mouse model and cell model were established.Cell protein electrophoresis was used to detect the expression of target protein in cells,and PCR was used to detect the expression of KLF15,and Western blot was used to detect the relative expression of NLRP3,and immunohistochemistry was used to detect the expression of KLF15 in tissues,and immunofluorescence staining was used to detect cell proliferation-related proteins.Results:In streptozotocin(STZ)-induced mouse model,the expression level of KLF15 mRNA in serum was inhibited 1 to 4 months after the induction.After inducing HepG2 cells with 10,20 and 40 mmol/L glucose for 24 hours,the expression level of KLF15 mRNA in cells decreased with the increase of glucose concentration(all P<0.05).In rat tissues,the expression of KLF15,GLUT4 and IRS1 was the highest in the blank group,followed by the 1-,2-and 4-month groups respectively;the expression of NLRP3 was the lowest in the blank group,and increased in turn in the 1-,2-and 4-month groups(all P<0.05).In vitro model,si-NLRP3 plasmid could reduce the expression of NLRP3;inhibition of NLRP3 expression could reduce the expression levels of INF-γ,TNF-α,IL-6 and IL-1β,and increase the expression levels of IRS1 and GLUT4;overexpression of NLRP3 plasmid could promote the expression of NLRP3,and overexpression of NLRP3 could promote the expressions of INF-γ,TNF-α,IL-6 and IL-1β,while reducing the expression levels of IRS1 and GLUT4;up-regulation of KLF15 could inhibit the expression of NLRP3 protein,while inhibition of KLF15 could promote the expression of NLRP3 protein(all P<0.05).Conclusion:The level of KLF15 expression is related to insulin resistance in T2DM.The low expression of KLF15 may increase insulin resistance.KLF15 may reduce the expression level of NLRP3,reduce the level of inflammation and inhibit insulin resistance in T2DM.KLF15 may be a potential therapeutic and diagnostic target for T2DM with insulin resistance,and provide experimental basis for future related research.