脂多糖对小鼠肾脏炎症因子、过氧化物酶体增殖物活化受体γ及其辅调节因子表达的影响

Effects of intraperitoneal injection of LPS on expression of MCP-1,iNOS,PPAR-γ and related coregulators in mice kidneys

目的:观察腹腔注射脂多糖(LPS)对小鼠肾脏单核细胞趋化蛋白1(MCP-1)、诱导型一氧化氮合酶(iNOS)、过氧化物酶体增殖物活化受体γ(PPAR-γ)及其辅调节因子表达的影响。方法:雄性云南昆明小白鼠[(20±2)g]随机分成两组:LPS组,腹腔注射LPS(5mg/kg);对照组,腹腔注射等体积磷酸盐缓冲液。分别于0～72h后处死小鼠,检测血清尿素氮和肌酐变化;留取肾脏,EMSA法检测NF-κB及PPAR-γ的DNA结合活性;Real-time PCR法检测肾组织MCP-1、iNOS、PPAR-γ及其辅激活因子1(PGC-1)、类固醇受体辅激活因子1(SRC-1)、SRC-2、SRC-3及核辅抑制因子(NCoR)mRNA的表达;ELISA检测肾组织MCP-1蛋白表达,Western Blot检测肾组织核蛋白PPAR-γ表达;HE染色观察病理改变,免疫组织化学法观察肾组织巨噬细胞的浸润情况。结果:小鼠腹腔注射LPS后血清尿素氮升高明显(P<0.05),但血肌酐无明显变化。肾组织NF-κB的DNA结合活性在0.5h后即明显增高,而PPAR-γ的DNA结合活性早期增强,8h后开始减弱。与同时间点对照组及基础值相比,小鼠腹腔注射LPS后6h,12h及24h,肾组织MCP-1 mRNA及蛋白表达均显著增加(P<0.01);而iNOS mRNA表达在6h及12h后显著增加(P<0.01)。PPAR-γ及PGC-1 mRNA表达则显著下调(P<0.01),核内PPAR-γ蛋白含量早期升高,24h时显著下降(P<0.01)。SRC-1、SRC-2、SRC-3及NCoR mRNA的表达与对照组相比无显著差异。LPS作用后肾组织HE染色未见显著改变,免疫组化可见肾组织巨噬细胞浸润,最初主要分布在髓质肾小管周围,在48h及72h浸润更为明显,皮质肾小球周围也可见巨噬细胞浸润。结论:小鼠腹腔注射LPS后肾组织中NF-κB活性及相关炎症因子MCP-1和iNOS表达上调,肾组织内有明显的巨噬细胞浸润,表明处于炎症状态,而PPAR-γ及其辅激活因子PGC-1的表达下调可能与炎症发展相关。

Objective：To observe the effects of hpopolysaccharide （LPS） by intraperitoneal injection on expression of MCP-1, iNOS, PPAR-γ and related coregulators in mice kidneys. Methodology：Male KM mice （weight：20±2g） were randomized to two groups. They were LPS group injected with LPS（5mg/kg） by intraperitoneal route and control group injected with equal volume of PBS. The mice were sacrificed after 0-72 hours, then blood urea nitrogen （BUN） and serum creatinine （SCr） levels were measured. The DNA binding activity of NF-KB and PPAR-γ, in kidneys was measured with electrophoretic mobility shift assay （EMSA）, and the mRNA expression of MCP-1, iNOS, PPAR-γ, PGC-1, SRC-1, SRC-2, SRC-3 and NCoR in kidneys were detected by real-time PCR. ELISA and Western blot were applied to analyze the protein expression of MCP-1 and PPAR-γ, respectively. The pathologic change and macrophage recruitment to the kidney were detected by HE and immunohistochemistry （IHC） staining. Results：The level of BUN was increased significantly after LPS injection（P 〈 0. 05 ）while SCr was no significant change between the two groups. The DNA binding activity of NF-KB was significantly enhanced at 0. 5h after intraperitoneal injection of LIPS, while the DNA binding activity of PPAR-γ was elevated at early time, then gradually down-regulated after 8 hours. Compared with control group and baseline, the mRNA and protein expression of MCP-1 in kidneys of mice was significantly up-regulated after 6, 12, and 24 hours （P 〈 0. 01 ）. The mRNA expression of iNOS was significantly up-regulated after 6 and 12 hours （ P 〈 0.01 ）. The mRNA expression of PPAR-γ and PGC-1 was down-regulated after 6 and 12 hours （P 〈 0.01 ）and the nuclear protein level of PPAR-3, was up-regulated at early time bat down-regulated at 24 hours （P 〈 0. 01 ）. The mRNA expression of SRC-1, SRC-2, SRC- 3 and NCoR was no significant changes. The morphological changes in the mice kidneys intraperitoneal injected with LPS were not observed by HE staining, but IHC staining showed obvious infiltration of macrophages which located around renal tubules at early time, then became much more at 48 and 72 hours with the distribution around cortex glomeruli. Conclusion：The up-regulation of NF-KB activity and related inflammation cytokines such as MCP-1 and iNOS as well as the evident infiltration of macrophages in mice kidneys after intraperitoneal injecton of LPS suggest the inflammation status of kid- neys. The down-regulation of PPAR-γ and PGC-1 may be related to inflammation development.