多巴胺受体5活化对内毒素介导的急性肾脏炎症的影响

摘要

目的 探讨多巴胺受体5(D5R)激动剂非诺多泮对内毒素介导的急性肾脏炎症的影响.方法 提取C57BL/6J小鼠骨髓单核巨噬细胞,蛋白质印迹法检测细胞中D1R ～ D5R蛋白水平,免疫荧光方法观察D5R在骨髓单核巨噬细胞中的表达.21只雄性C57BL/6J小鼠完全随机分为盐水组、盐水联合脂多糖组、非诺多泮联合脂多糖组,每组7只,采用腹腔注射的方法相应组别分别给予0.9％氯化钠注射液和非诺多泮;30 min后分别给予脂多糖和0.9％氯化钠注射液腹腔注射.于注射后6h采用实时荧光定量-聚合酶链反应(qRT-PCR)的方法检测肾脏D5R,肿瘤坏死因子(TNF)-α,干扰素-γ mRNA的水平;免疫组织化学染色观察肾脏单核巨噬细胞的浸润;检测血清中TNF-α,干扰素-y的水平.分离骨髓细胞,以巨噬细胞集落刺激因子诱导制备骨髓单核巨噬细胞.分别给予磷酸盐缓冲液和非诺多泮预处理巨噬细胞,5 min后给予脂多糖刺激12 h,酶联免疫吸附试验的方法检测巨噬细胞内环磷酸腺苷水平,qRT-PCR方法检测单核巨噬细胞中TNF-α,干扰素-γ mRNA水平.结果 核细胞中以D5R表达为主.与盐水组比较,盐水联合脂多糖组小鼠肾脏D5R mRNA水平明显高[(110.5±3.7)比(1.2±1.0),P＜0.001].与盐水联合脂多糖组比较,非诺多泮联合脂多糖组可以明显抑制脂多糖介导的肾脏炎症因子TNF-α、干扰素-γmRNA水平的升高[(0.089 ±0.007)比(0.231 ±0.019)、(0.363 ±0.102)比(0.920±0.007),P＜0.01],骨髓单核细胞在肾脏的浸润以及血清炎症因子TNF-α及干扰素-γ的水平[(22±5)比(236 ±20)、(18±6)比(76±10),P＜0.01或P＜0.05].体外培养骨髓单核巨噬细胞给予非诺多泮后环磷酸腺苷的产生明显增多[(8.726±0.909)nmol/L比(0.348 ±0.024) nmol/L,P＜0.01]、同时明显抑制TNF-α、干扰素-γ的产生[(0.669 ±0.010)比(1.362 ±0.199)、(0.896±0.071)比(2.854±0.270),P＜0.05].结论 D5R在骨髓单核巨噬细胞上有表达,D5R活化可以明显抑制内毒素介导的急性肾脏炎性反应.%Objective To investigate the effect of dopamine receptor 5 (D5R) agonist fenoldopam on endotoxin induced acute renal inflammation.Methods The protein levels of D1R-D5R in bone marrow mononuclear macrophages in C57BL/6J mouse were detected by Western blotting,the expression level of D5R in bone marrow mononuclear macrophages then was detected by immunofluorescence.Totally 21 C57BL/6J mice were randomly divided into saline group,saline + LPS group,fenoldopam + LPS group (n =7 in each group).The mice were intraperitoneally injected with 0.9％ saline or fenoldopam for 30 minutes,then were intraperitoneally injected with lipopolysaccharide or 0.9％ saline for 6 hours in corresponding group.The mRNA level of D5R,tumor necrosis factor (TNF)-α and interferon (INF)-γ in renal were measured by using real-time fluorescent quantitative polymease chain reaction (qRT-PCR);the mononuclear macrophages infiltration in renal was observed by immunohistochemical staining;the levels of TNF-α,INF-γ in serum were detected.After obtaining bone marrow cells of mice,the bone marrow mononuclear macrophages were induced by macrophage colony-stimulating factors (M-CSF);then the macrophages were pretreated with PBS or fenoldopam for 5 minutes,followed by lipopolysaccharide for 12 hours.The level of cyclic adenosine monophosphate in macrophages was measured by using enzyme linked immunosorbent assay,the mRNA levels of TNF-α and INF-γin mononuclear macrophages were detected by using qRT-PCR.Results In myelomonocyte,D5R was mainly expressed.The mRNA level of D5R in renal in saline + LPS group was significantly higher than that in saline group [(110.5 ± 3.7) vs (1.2 ± 1.0)] (P ＜ 0.001);the mRNA levels of TNF-α and INF-γin renal in fenoldopam + LPS group were significantly lower than those in saline + LPS group [(0.089 ±0.007) vs (0.231 ±0.019),(0.363 ±0.102) vs (0.920 ±0.007)] (P ＜0.01);the infiltration of monocyte in renal in fenoldopam + LPS group was significantly improved compared with saline + LPS group;the serum levels of TNF-α and INF-γin fenoldopam + LPS group were significantly lower than those in saline+LPSgroup [(22±5) vs (236±20),(18±6) vs (76±10)] (P＜0.01 orP＜0.05).In vitro culture,fenoldopam promoted the production of cyclic adenosine monophosphate [(8.726 ± 0.909) nmol/L vs (0.348 ±0.024) nmol/L] (P ＜0.01),and also supressed the production of TNF-α and INF-γinduced by LPS in myelomonocyte [(0.669 ± 0.010) vs (1.362 ± 0.199),(0.896 ± 0.071) vs (2.854 ± 0.270)] (P ＜ 0.05).Conclusions D5R is expressed in bone marrow mononuclear macrophages;D5R agonist fenoldopam can inhibite endotoxin induced acute renal inflammation.