阿托伐他汀对草酸钙晶体诱导的细胞炎症反应的影响及其机制探讨  

Effect and mechanism of atorvastatin on cellular inflammatory response induced by calcium oxalate crystals

作　　者：孙焱 陶芝伟[1] 康珏宁 刘权[1] 王翔[1] 龙诗彬 李德荣 邓耀良 Sun Yan;Tao Zhiwei;Kang Juening;Liu Quan;Wang Xiang;Long Shibin;Li Derong;Deng Yaoliang(Department of Urology,the First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China;Department of Urology,Affiliated Langdong Hospital of Guangxi Medical University,Nanning 530000,China)

机构地区：[1]广西医科大学第一附属医院泌尿外科,南宁530021 [2]广西医科大学附属埌东医院泌尿外科,南宁530000

出　　处：《中华泌尿外科杂志》2019年第10期780-785,共6页Chinese Journal of Urology

基　　金：广西青年科学基金(2018GXNSFBA138011);国家自然科学基金(81960138);广西医科大学青年科学基金(GXMUYSF201815).

摘　　要：目的探讨阿托伐他汀(Atorvastatin,ATV)对草酸钙晶体诱导的人肾小管上皮细胞(HK-2细胞)炎症反应的影响及其作用机制。方法取HK-2细胞分为对照组(正常培养基培养)、ATV组(40μmol/LATV预处理3h后换成正常培养基)、草酸钙晶体刺激组(含4mmol/L草酸钙晶体的培养基)和ATV干预组(40μmol/LATV预处理3h后,换成含4mmol/L草酸钙晶体的培养基)。培养12h后收集细胞,采用免疫组化染色检查和蛋白质印迹法检测Nod样受体蛋白3(NLRP3)和Cleavedcaspase-1的表达水平;采用免疫荧光染色法和蛋白质印迹法检测NF-κB的表达水平;收集细胞培养上清液,采用酶联免疫吸附测定法(ELISA)检测白细胞介素(IL)-1β和IL-18的浓度。结果蛋白质印迹法检测结果显示,ATV组较对照组NLRP3(0.125±0.013与0.135±0.007)和Cleaved caspase-1(0.090±0.014与0.095±0.006)的相对表达量有所减少,但差异均无统计学意义(P>0.05);草酸钙晶体刺激组较对照组NLRP3(0.315±0.021与0.135±0.007,P<0.001)和Cleavedcaspase-1(0.235±0.008与0.095±0.006,P<0.001)的相对表达量明显增多;ATV干预组较草酸钙晶体刺激组NLRP3(0.245±0.007与0.315±0.021,P<0.05)和Cleavedcaspase-1(0.170±0.017与0.235±0.008,P<0.05)的相对表达量明显下降。免疫组化染色检查结果显示各组的NLRP3和Cleavedcaspase-1表达趋势与蛋白质印迹法检测结果一致。ELISA结果显示,ATV组较对照组细胞培养液中炎症因子IL-1β[(162.00±21.21)pg/ml与(183.50±7.78)pg/ml,P>0.05]和IL-18[(176.50±24.12)pg/ml与(182.50±20.51)pg/ml,P>0.05]浓度有所减低,但差异无统计学意义;草酸钙晶体刺激组较对照组细胞培养液中炎症因子IL-1β[(850.50±48.79)pg/ml与(183.50±7.78)pg/ml,P<0.001]和IL-18[(526.00±39.61)pg/ml与(182.50±20.51)pg/ml,P<0.001]浓度显著增高;ATV干预组较草酸钙晶体刺激组细胞培养液中炎症因子IL-1β[(452.50±36.06)pg/ml与(850.50±48.79)pg/ml,P<0.01]和IL-18[(403.50±23.33)pg/ml与(526.00±39.61)pg/ml,P<0.05]Objective To investigate the effect and mechanism of atorvastatin (ATV) on the inflammatory response of human renal tubular epithelial cells (HK-2 cells) induced by calcium oxalate crystals. Methods HK-2 cells were divided into control group (normal medium), ATV group (after 3 h pretreatment with 40 μmol/L ATV, replaced with normal medium), calcium oxalate crystal stimulation group (4 mmol/L calcium oxalate crystal) and ATV treatment group (after 3 h pretreatment with 40 μmol/L ATV, replaced with 4 mmol/L calcium oxalate crystals). After 12 h, the cells were collected, and the expression levels of NLRP3 and Cleaved caspase-1 were detected by immunohistochemical staining and Western blotting. The expression level of NF-κB was detected by immunofluorescence and Western blotting. The cell culture supernatant was collected to detecte the concentrations of interleukin-1β(IL-1β) and interleukin-18 (IL-18) by enzyme linked immunosorbent assay (ELISA). Results Western blot analysis showed that the relative expression of NLRP3 (0.125±0.013 vs. 0.135±0.007) and Cleaved caspase-1 (0.090±0.014 vs. 0.095±0.006) was decreased in the ATV group compared with the control group, but the difference was not statistically significant (P>0.05). The relative expression of NLRP3 (0.315±0.021 vs. 0.135±0.007, P<0.001) and Cleaved caspase-1 (0.235±0.008 vs. 0.095±0.006, P<0.001) was significantly increased in the calcium oxalate crystal stimulation group compared with the control group. While the relative expression of NLRP3 (0.245±0.007 vs. 0.315±0.021, P<0.05) and Cleaved caspase-1 (0.170±0.017 vs. 0.235±0.008, P<0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group. The results of immunohistochemical staining showed that the expression trends of NLRP3 and Cleaved caspase-1 in each group were consistent with those obtained by Western blotting. The ELISA results showed that the concentration of inflammatory factors IL-1β[(162.00±21.21)pg/ml vs.(183.50±7.78)

关 键 词：炎症 细胞因子 阿托伐他汀 草酸钙晶体 人肾小管上皮细胞 

分 类 号：R9[医药卫生—药学]