肾小管上皮细胞中可溶性表氧化物水解酶对小鼠巨噬细胞极化的调控作用

Role of soluble epoxide hydrolase in renal tubular epithelial cells in the regulation of macrophage polarization in mice

目的探讨肾小管上皮细胞中可溶性表氧化物水解酶(sEH)在小鼠巨噬细胞极化中的作用。方法以人近端肾小管上皮细胞系HK-2细胞及小鼠巨噬细胞系RAW264.7细胞为研究对象。将HK-2细胞分为正常对照组、sEH抑制剂组、尿蛋白组及sEH抑制剂联合尿蛋白组;正常对照组细胞不给予任何干预处理;sEH抑制剂组细胞给予1μmol·L^(-1)sEH抑制剂;尿蛋白组细胞给予10 g·L^(-1)尿蛋白;sEH抑制剂联合尿蛋白组细胞给予10 g·L^(-1)尿蛋白和1μmol·L^(-1)sEH抑制剂;各组细胞均培养24 h。将RAW264.7细胞分为A、B、C、D组及干扰素-γ(IFN-γ)阳性对照组、白细胞介素(IL)-4阳性对照组。A、B、C、D组细胞分别加入正常对照组、sEH抑制剂组、尿蛋白组、sEH抑制剂联合尿蛋白组培养24 h的HK-2细胞培养基孵育24 h;IFN-γ阳性对照组和IL-4阳性对照组细胞分别加入M1型巨噬细胞诱导剂IFN-γ及M2型巨噬细胞诱导剂IL-4。采用Western blot法检测HK-2细胞中sEH蛋白表达,实时荧光定量聚合酶链反应检测HK-2细胞中单核细胞趋化蛋白-1(MCP-1)、IL-6、集落刺激因子-1(CSF-1)、肿瘤坏死因子-α(TNF-α)mRNA表达及RAW264.7细胞中诱导型氮氧化物合酶(iNOS)、IL-6、精氨酸酶-1(Arg^(-1))及IL-10 mRNA表达。酶联免疫吸附试验检测各组HK-2细胞培养上清液中14,15-环氧二十碳三烯酸(14,15-EET)和14,15-脱氧二十碳三烯酸(14,15-DHET)水平,计算14,15-EET/14,15-DHET比值。结果正常对照组和sEH抑制剂组HK-2细胞中sEH蛋白表达及MCP-1、IL-6、CSF-1、TNF-αmRNA表达、细胞培养上清液中14,15-EET/14,15-DHET比较差异均无统计学意义(P>0.05)。与正常对照组比较,蛋白尿组HK-2细胞中sEH蛋白及MCP-1、IL-6、CSF-1、TNF-αmRNA表达均显著增加(P<0.05),细胞培养上清液中14,15-EET/14,15-DHET显著降低(P<0.05)。与尿蛋白组比较,sEH抑制剂联合尿蛋白组HK-2细胞中MCP-1、IL-6、CSF-1及TNF-αmRNA表达显著下降(P<0.05),培养上清液中14,15-EET/14,15-DHET显著升高(P<0.05)。sEH抑制剂联合尿蛋白组与尿蛋白组HK-2细胞中sEH蛋白表达比较差异无统计学意义(P>0.05)。A组与B组RAW264.7细胞中IL-6、iNOS、Arg^(-1)及IL-10 mRNA表达比较差异均无统计学意义(P>0.05)。与A组比较,C组、IFN-γ阳性对照组RAW264.7细胞中iNOS、IL-6 mRNA表达显著增加(P<0.05,P<0.01);IL-4阳性对照组RAW264.7细胞中Arg^(-1)、IL-10 mRNA表达显著增加(P<0.05)。与C组比较,D组RAW264.7细胞中iNOS、IL-6 mRNA表达显著降低(P<0.05),Arg^(-1)、IL-10 mRNA表达显著增加(P<0.05)。结论肾小管上皮细胞中的sEH可上调诱导M1型巨噬细胞极化的细胞因子表达。

Objective To investigate the role of soluble epoxide hydrolase（ sEH） in renal tubular epithelial cells in the regulation of macrophage polarization in mice. Methods Human proximal tubular epithelial cell line（ HK-2） and mouse macrophage cell line（ RAW264. 7） were studied in the present study. HK-2 cells were divided into normal control group,sEH inhibitor group,urine protein group and sEH inhibitor combined urine protein group. The cells in the normal control group wasn’t given any intervention. The cells in the sEH inhibitor group and the urine protein group were given 1 μmol·L-1 sEH inhibitor and 10 g · L-1 urine protein,respectively; while the cells in the sEH inhibitor combined urine protein group were given 1 μmol·L-1 sEH inhibitor and 10 g·L-1 urine protein. All the cells in each group were cultured for 24 hours. RAW264. 7 cells were divided into group A,group B,group C,group D,interferon gamma（ IFN-γ） positive control group and interleukin（ IL）-4 positive control group. The cells of group A,group B,group C and group D were respectively added to the HK-2 cell cultured medium of the normal control group,sEH inhibitor group,urine protein group and sEH inhibitor combined urine protein group; then the cells were cultured for 24 h. IFN-γ and IL-4 were respectively added in IFN-γ positive control group and IL-4 positive control group. The expression of sEH protein in HK-2 cells was measured by Western blot. The expression of monocyte chemotactic protein-1（ MCP-1）,IL-6,colony stimulating factor-1（ CSF-1）,tumor necrosis factor-α（ TNF-α） mRNA in the HK-2 cells and the expression of inducible nitric oxide synthase（ iNOS）,arginase（ Arg-1）,IL-10 mRNA in the RAW264. 7 cells were measured by real-time polymerase chain reaction. The levels of 14,15-epoxyeicosatrienoic acids（ 14,15 EET） and 14,15-dihydroxyeicosatrienoic acid（ 14,15-DHET） in the supernate of HK-2 cell medium in each group were detected by enzyme linked immunosorbent assay,and the 14,15-EET/14,15-DHET was accounted. Results There was no significance in the expression of sEH protein,and MCP-1,IL-6,CSF-1,TNF-α mRNA,14,15-EET/14,15-DHET between the normal control group and the sEH inhibitor group（ P 〉 0. 05）. The expressions of sEH protein and MCP-1,IL-6,CSF-1,TNF-α mRNA in the urine protein group were significantly higher than those in the normal control group（ P 〈 0. 05）,while the 14,15-EET/14,15-DHET was significantly lower than that in the normal control group（ P 〈 0. 05）. Compared with the urine protein group,the 14,15-EET/14,15-DHET in sEH inhibitor combined urine protein group increased significantly（ P 〈 0. 05）,while the expression of MCP-1,IL-6,CSF-1 and TNF-α mRNA decreased significantly（ P 〈 0. 05）. There was no significant difference in the expression of sEH protein between the sEH inhibitor combined urine protein group and the urine protein group（ P 〉 0. 05）.There was no significant difference in the expression of IL-6,iNOS,Arg-1 and IL-10 mRNA between the group A and the group B（ P 〉 0. 05）. Compared with the group A,the expression of iNOS and IL-6 mRNA in RAW264. 7 cells in the IFN-γ positive control group increased significantly（ P 〈 0. 05,P 〈 0. 01）; the expression of Arg-1 and IL-10 mRNA in RAW264. 7 cells in the IL-4 positive control group increased significantly（ P 〈 0. 05）. Compared with the group C,the expression of iNOS and IL-6 mRNA in RAW264. 7 cells in the group D decreased significantly（ P 〈 0. 05）,while the expression of Arg-1 and IL-10 mRNA increased significantly（ P 〈 0. 05）. Conclusion The sEH in tubular epithelial cells could promote M1 macrophage polarization.