25-(OH)D3:1,25—(OH)2—D3对单侧输尿管梗阻大鼠肾小管间质巨噬细胞趋化按捺因子及核转录因子κB/P65表达的影响

来源:中国当代医药 ·2018年12月01日 13:05 浏览量:0

龙艳君+杨小翠+杨霞+达静静+袁静+田茂露+查艳

[摘要] 意图 调查巨噬细胞趋化按捺因子(MIF)及核转录因子κB/P65(NF-κB/P65)在单侧输尿管梗阻大鼠肾小管间质中的表达及1,25-(OH)2-D3对其的影响。 办法 选用单侧输尿管结扎手术树立单侧输尿管梗阻模型。将健康成年的雄性SD大鼠按随机数字表法分为3组:假手术组(n=10)、模型组(n=10)和VD组(n=10)。VD组选用6 ng/(100 g·d)的1,25-(OH)2-D3干涉医治,假手术组和模型组仅给予等量生理盐水灌胃8周。调查3组大鼠第8周肾功能、肾安排病理学改动,免疫组化法检测单核/巨噬细胞外表特异性标志抗原(ED-1)、NF-κB/P65、MIF在肾安排中的表达。 成果 与假手术组比较,模型组血肌酐增高(P<0.01),肾间质面积/核算场面积添加(P<0.01),肾安排ED-1阳性细胞显着添加(P<0.01),肾安排MIF的表达显着升高(P<0.01),NF-κB/P65核阳性细胞显着添加(P<0.01)。与模型组比较,VD组大鼠肾安排病理学改变得到必定程度的改进,肾安排ED-1、MIF的表达及NF-κB/P65核阳性细胞显着削减(P<0.01)。 定论 单侧输尿管梗阻大鼠肾间质MIF的表达和NF-κB/P65的活性显着添加,1,25-(OH)2-D3可能经过下调MIF的表达,按捺NF-κB/P65的活性来减轻肾小管间质的炎症反响,改进肾脏纤维化。

[要害词] 1,25-(OH)2-D3;巨噬细胞趋化按捺因子;核转录因子κB/P65;肾小管间质炎症反响

[中图分类号] R692.3 [文献标识码] A [文章编号] 1674-4721(2014)09(b)-0008-05

Effects of 1,25-dihydroxyvitamin D3 on the expression of macrophage chemotaxis inhibitory factor and nuclear factor-κB/P65 of tubulointerstitial cells in unilateral ureteral ligation operation model rats

LONG Yan-jun1 YANG Xiao-cui2★ YANG Xia1 DA Jing-jing1 YUAN Jing1 TIAN Mao-lu1 ZHA Yan1▲

1.Department of Nephrology,People′s Hospital of Guizhou Province,Guiyang 550002,China;2.The Fourth People′s Hospital of Zunyi City in Guizhou Province,Zunyi 563000,China

[Abstract] Objective To investigate the expression of macrophage migration inhibitory factor(MIF)and nuclear factor-κB/P65(NF-κB/P65)in the kidneys of unilateral ureteral obstruction(UUO)model rats and the effect of 1,25-dihydroxyvitamin D3 on the xpression. Methods Thirty healthy adult male SD rats were randomly divided into 3 groups:sham operation group(n=10),model group(n=10)and VD group[n=10,UUO rats treated with 6 ng/(100 g·d) 1,25-dihydroxyvitamin D3].The rats in sham group and model group were treated with equal normal saline by lavage for 8 weeks.Serum creatinine,histopatho1ogical change,renal tubulointerstitial macrophage marker antigen(ED-1),NF-κB/P65 and MIF in rats at 8 weeks was measured by immunohistochemistry respectively. Results Serum creatinine,renal interstitial area/statistical field area,the expression of MIF,the amount of NF-κB/P65 nuclear positive cell and ED-1 positive cell in model group was significantly increased compared with that in sham group respectively(P<0.01). Compared with model group,rat renal histopathological change in VD group had a certain degree of improvement,the expression of MIF,the amount of NF-κB/P65 nuclear positive cell and ED-1 positive cell in VD group was significantly decreased respectively(P<0.01). Conclusion The expression of MIF and the activition of NF-κB/P65 in UUO rats increase significantly.1,25-dihydroxyvitamin D3 may ameliorate the progression of renal tubulointerstitial inflammation and renal fibrosis by intervene the expression of MIF and decrease the activition of NF-κB/P65.

[Key words] 1,25-dihydroxyvitamin D3;Macrophage chemotaxis inhibitory factor;Nuclear factor-κB/P65;Renal tubulointerstitial inflammation

缓慢肾脏病(chronic kidney disease,CKD)正成为一个全球范围内的公共卫生问题[1]。肾小管间质炎症可促进CKD患者肾脏纤维化的进程,导致终晚期肾衰竭。巨噬细胞移动按捺因子(MIF)是一种强有力的炎症前细胞因子[2],可引起核转录因子-κB(NF-κB)的活化。在鼠的抗肾小球基底膜肾炎、系膜增生性肾炎、MRL/lpr狼疮性肾炎等均发现MIF表达添加[3],与肾脏安排学危害(间质纤维化等)、蛋白尿程度及肾功能阑珊密切相关[4-5]。本研讨在单侧输尿管梗阻(unilateral ureteral obstruction,UUO)大鼠模型中调查肾小管间质炎症反响和肾小管间质细胞MIF、NF-κB的表达状况及给予1,25-(OH)2-D3干涉后MIF、NF-κB在肾小管间质细胞中表达的改变,评论1,25-(OH)2-D3是否经过MIF影响NF-κB活性来改进肾小管间质的炎症反响,为肾纤维化药物医治供给更广泛的循证医学根据。

1 材料与办法

1.1 动物及分组

6周左右雄性SD大鼠30只,体重170~220 g,答应合格证号scxk(渝)2007-0005,由重庆试验动物中心供给。按随机数字表法分为假手术组(n=10)、模型组(n=10,行单侧输尿管结扎手术)和1,25-(OH)2-D3干涉组(VD组)(n=10,行单侧输尿管结扎手术加1,25-(OH)2-D3 6 ng/(100 g·d)入饮水中灌胃8周)。假手术组和模型组予等量生理盐水灌胃。

1.2 动物处理

在UUO术后8周麻醉大鼠从颈总动脉采血,4℃、3000 r/min离心15 min别离血清,用于测定血肌酐。脱髓处死一切大鼠,剖取左边肾脏,剥离包膜,部分肾安排10%中性甲醛溶液固定,行安排病理学、免疫安排化学检测,部分肾安排存放于-80℃,行Real-time PCR和Western blot检测。

1.3 安排病理学检测

白腊切片行惯例HE、PAS和Masson染色,光镜下双盲法调查肾小管间质的改变状况。每只大鼠选取3张切片,每张切片随机选取10个肾小管间质视界(避开肾小球和大血管),肾间质面积丈量在200倍光镜下,每个视界别离丈量肾间质面积与核算场面积的比值。选用显微图画剖析体系(Olympus C3040.ADU)对各组染色成果进行积分吸光度(A)测定,每张切片随机选取10个高倍视界(200倍),每个视界代表0.13 mm2的区域面积,核算其A值,取平均值。

1.4 免疫安排化学检测

切片脱蜡复水;抗原修正选用微波热修正,室温冷却;经3%双氧水关闭内源性酶;正常羊血清关闭15~20 min;参加一抗ED-1(Santa Cruz)1∶50、NF-κB/P65(Cell Signaling Technology)1∶400、MIF(Santa Cruz)1∶50,4℃孵育过夜;滴加HRP Polymer(酶标二抗),在室温下孵育30 min;DAB显色,苏木素复染,脱水,通明。试验一起以磷酸盐缓冲液替代一抗作阴性对照。

1.5 核算学处理

选用SPSS 18.0核算软件对数据进行剖析和处理,计量材料以x±s表明,选用单因素方差剖析或q查验,以P<0.05为差异有核算学含义。

2 成果

2.1 3组大鼠血肌酐及肾间质面积/核算场面积的比较

术后8周,模型组的血肌酐及肾间质面积/核算场面积显着高于假手术组及VD组(P<0.01);VD组的血肌酐及肾间质面积/核算场面积显着高于假手术组(P<0.01)(表1)。

表1 3组大鼠血肌酐及肾间质面积/核算场面积的比较(x±s,n=10)

与假手术组比较,*P<0.01;与模型组比较,#P<0.01

2.2 3组大鼠肾安排病理查看成果

假手术组肾小管间质形状正常;模型组可见肾小管管腔增大,小管上皮细胞空泡状变性、萎缩、掉落,肾间质结构紊乱、炎症细胞滋润及纤维化;VD组肾小管间质病变较模型组显着改进(图1)。

2.3 3组大鼠肾小管间质ED-1的表达状况

免疫组化显现假手术组ED-1表达很少;与假手术组比较,模型组ED-1表达显着升高(P<0.01),首要表达于肾小管上皮细胞及肾间质细胞的细胞质中;与模型组比较,VD组ED-1表达显着削减(P<0.01)(图2)。

图2 3组大鼠肾小管间质ED-1的表达状况(×200)

A.假手术组;B.模型组;C.VD组;与假手术组比较,*P<0.01;与模型组比较,#P<0.01

2.4 3组大鼠肾小管间质MIF的表达状况

免疫组化显现假手术组MIF表达很少;与假手术组比较,模型组MIF显着升高(P<0.01),首要表达于肾小管上皮细胞及肾间质细胞的细胞质中;与模型组比较,VD组MIF表达显着削减(P<0.01)(图3)。

2.5 3组大鼠肾小管间质NF-κB/P65的表达状况

免疫组化显现假手术组NF-κB/P65在肾小管上皮细胞中首要表达于细胞质,未见显着核阳性细胞;模型组肾小管NF-κB/P65核阳性细胞较假手术组显着升高(P<0.01);VD组肾小管NF-κB/P65核阳性细胞显着削减(P<0.01)(图4)。

3 评论

肾脏纤维化,尤其是肾小管间质纤维化,几乎是一切CKD的终究成果[6]。肾小管间质炎症是肾小管间质纤维化的重用病理生理机制之一。MIF是免疫和炎症反响中的要害成分[2],是一种含有115个氨基酸的蛋白质,相对分子质量约为12.5 kD[7]。MIF的活化状况是由含2个反向平行α螺旋和6个β片层的三个单体组成的同源三聚体,构成一结尾敞开的中空结构。MIF首要由巨噬细胞发生,其他细胞如淋巴细胞、树突状细胞、中性粒细胞、内皮细胞、滑润肌细胞、上皮细胞、成纤维细胞、心肌细胞、神经细胞、生殖安排、脂肪细胞和激素排泄细胞也可表达MIF[8]。MIF首要经过按捺巨噬细胞游走移动,促进巨噬细胞在炎症部分的集合、增生及排泄多种细胞因子如白介素-1(IL-1)、肿瘤坏死因子-α(TNF-α)等发挥其增强炎症反响程度的生物学效应。在正常人、大鼠的肾安排中,MIF首要在肾小球脏层和壁层上皮细胞表达,在肾小管上皮细胞弱表达[9]。在人类各种原发和继发肾小球疾病中,肾脏MIF表达均添加[3]。有研讨发现,在体系性红斑狼疮小鼠模型中给予MIF拮抗剂处理可改进肾脏的功能及安排学目标[10]。本研讨发现,UUO大鼠肾小管间质ED-1阳性细胞显着升高,MIF的表达显着添加,成果提示MIF可能参加UUO大鼠肾小管间质的炎症反响,与既往在发展性肾炎模型大鼠上的相关研讨共同[11]。

NF-κB归于NF-κB/Rel宗族,由NF-κB1(p50/p105)、NF-κB2(p52/p100)、RelA(p65)、RelB及c-Rel五个成员组成,是调控许多基因的重要转录因子,参加炎症反响、免疫应对、细胞增生及转化和凋亡等重要的生理病理进程[12-13]。许多与肾脏损害相关的影响(如高糖、蛋白尿、糖基化终产物等)均可引起NF-κB的活化[14-15],其间起首要效果的是NF-κB1/P65异源二聚体。有研讨发现,MIF能够经过效果于一种转录因子ETS宗族成员PU1来上调TCL-R4的表达,引起NF-κB过度激活,导致炎症反响的级联扩大效应[16-17]。还有研讨发现,MIF在IgA大鼠肾损害中的效果机制可能与活化NF-κB/P65有关[18]。本研讨发现,在正常大鼠肾小管间质中NF-κB/P65核阳性细胞很少,而在UUO大鼠肾小管间质的NF-κB/P65核阳性细胞显着添加,成果提示在UUO大鼠中,MIF可能经过诱导NF-κB/P65活化,参加肾小管间质炎症反响。

研讨发现,1,25-(OH)2-D3经过减轻梗阻性肾病模型中肾小球硬化的损害而发挥抗肾脏纤维化的效果[19],已被证明能够推迟肾小球硬化及肾间质纤维化的发展[20],但详细机制尚不清晰。本研讨发现,在UUO大鼠肾脏,MIF表达显着升高(P<0.01),且ED-1、NF-κB/P65核阳性细胞添加;使用1,25-(OH)2-D3处理降低了MIF在UUO模型大鼠肾脏中的表达和集合,削减了NF-κB/P65、ED-1核阳性细胞数量,显着减轻了肾小管间质病变,改进了肾功能。

本研讨成果证明,使用1,25-(OH)2-D3干涉可减轻UUO大鼠肾小管间质的炎症反响,缓解肾小管间质纤维化。1,25-(OH)2-D3可能经过按捺MIF在肾小管间质的表达,削减NF-κB/P65的活化,改进炎症反响,减轻肾脏损害。

[参考文献]

[1] McClellan AC,Plantinga L,McClellan WM.Epidemiology,geography and chronic kidney disease[J].Curr Opin Nephrol Hypertens,2012,21(3):323-328.

[2] Matsumoto K,Maruyama T.Elevated macrophage migration inhibitiory factor(MIF)levels in the urine of patients with focal glomerular sclerosis[J].Clin Exp Immunol,2005,139(2):338-347.

[3] Sanchez-Ni?觡o MD,Sanz AB,Ruiz-Andres O,et al.MIF,CD74 and other partners in kidney disease:tales of a promiscuous couple[J].Cytokine Growth Factor Rev,2013, 24(1):23-40.

[4] Nakima K,Tanaka Y,Nomiyama T,et al.RANTES promoter genotype is associated with diabetic nephropathy in type 2 diabetic subjects[J].Diabetes Care,2003,26(3):892.

[5] 孔耀中,黄英伟.巨噬细胞移动按捺因子在原发性肾小球肾炎安排中的表达和含义[J].中华肾脏病杂志,2000, 12(16):383.

[6] Lan A,Du J.Potential role of Akt signaling in chronic kidney disease[J].Nephrol Dial Transplant,2014.[Epub ahead of print]

[7] Mitchell R,Bacher M,Bernhagen J,et al.Cloning and characterization of the gene for mouse macrophage migration inhibitory factor(MIF)[J].J Immunol,1995,154(8):3863-3870.

[8] Asare Y,Schmitt M,Bernhagen J.The vascular biology of macrophage migration inhibitory factor(MIF).Expression and effects in inflammation,atherogenesis and angiogenesis[J].Thromb Haemost,2013,109(3):391-398.

[9] Bruchfeld A,Carrero JJ,Qureshi AR,et al.Elevated serum macrophage migration inhibitoryfactor(MIF)concentrations in chronic kidney disease(CKD)are associated with markers of oxidative stress and endothelial activation[J].Mol Med,2009,15(3-4):70-75.

[10] Leng L,Chen L,Fan J,et al.A small-molecule macrophage migration inhibitory factor antagonist protects against glomerulonephritis in lupus-prone NZB/NZW F1 and MRL/lpr mice[J].J Immunol,2011,186(1):527-538.

[11] 査艳,赵盈亭,杨霞,等.氯沙坦对发展性肾炎模型大鼠肾小管-间质细胞巨噬细胞移动按捺因子的影响研讨[J].我国药房,2011,22(33):3098-3100.

[12] Wan F,Lenardo MJ.Specification of DNA binding activity of NF-kappaB proteins[J].Cold Spring Harb Perspect Biol,2009,1(4):a000067.

[13] Hayden MS,Ghosh S.Shared principles in NF-kappaB signaling[J].Cell,2008,132(3):344-362.

[14] Sanz AB,Sanchez-Ni?觡o MD,Ramos AM,et al.NF-kappaB in renal inflammation[J].J Am Soc Nephrol,2010,21(8):1254-1262.

[15] Okabe C,Borges RL,de Almeida DC,et al.NF-κB activation mediates crystal translocation and interstitial inflammation in adenine overload nephropathy[J].Am J Physiol Renal Physiol,2013,305(2):F155-F163.

[16] Daun JM,Cannon JG.Macroghage migration inhibitory factor antagonizes hydrocortisone- induced increase in cytosolic IκB[J].Am J Physiol,2000,279(2):1045-1049.

[17] Salminen A,Kaarniranta K.Control of p53 and NF-κB signaling by WIP1 and MIF:role in cellular senescence and organismal aging[J].Cell Signal,2011,23(5):747-752.

[18] 刘延霞.巨噬细胞移动按捺因子在IgA肾病大鼠模型肾损害中的效果[J].温州:温州医学院,2008.

[19] Li Y,Spataro BC,Yang J,et al.1,25-dihydroxyvitamin D inhibits renal interstitial myofibroblast activation by inducing hepatocyte growth factor expression[J].Kidney Int,2005,68(4):1500-1510.

[20] 林建明,周洁,杨霞,等.1,25-(OH)2-D3对发展性肾炎大鼠肾小管间质胶原蛋白Ⅲ表达的影响[J].我国当代医药,2014,21(4):4-6,9.

(收稿日期:2014-08-01 本文修改:李亚聪)

[11] 査艳,赵盈亭,杨霞,等.氯沙坦对发展性肾炎模型大鼠肾小管-间质细胞巨噬细胞移动按捺因子的影响研讨[J].我国药房,2011,22(33):3098-3100.

[12] Wan F,Lenardo MJ.Specification of DNA binding activity of NF-kappaB proteins[J].Cold Spring Harb Perspect Biol,2009,1(4):a000067.

[13] Hayden MS,Ghosh S.Shared principles in NF-kappaB signaling[J].Cell,2008,132(3):344-362.

[14] Sanz AB,Sanchez-Ni?觡o MD,Ramos AM,et al.NF-kappaB in renal inflammation[J].J Am Soc Nephrol,2010,21(8):1254-1262.

[15] Okabe C,Borges RL,de Almeida DC,et al.NF-κB activation mediates crystal translocation and interstitial inflammation in adenine overload nephropathy[J].Am J Physiol Renal Physiol,2013,305(2):F155-F163.

[16] Daun JM,Cannon JG.Macroghage migration inhibitory factor antagonizes hydrocortisone- induced increase in cytosolic IκB[J].Am J Physiol,2000,279(2):1045-1049.

[17] Salminen A,Kaarniranta K.Control of p53 and NF-κB signaling by WIP1 and MIF:role in cellular senescence and organismal aging[J].Cell Signal,2011,23(5):747-752.

[18] 刘延霞.巨噬细胞移动按捺因子在IgA肾病大鼠模型肾损害中的效果[J].温州:温州医学院,2008.

[19] Li Y,Spataro BC,Yang J,et al.1,25-dihydroxyvitamin D inhibits renal interstitial myofibroblast activation by inducing hepatocyte growth factor expression[J].Kidney Int,2005,68(4):1500-1510.

[20] 林建明,周洁,杨霞,等.1,25-(OH)2-D3对发展性肾炎大鼠肾小管间质胶原蛋白Ⅲ表达的影响[J].我国当代医药,2014,21(4):4-6,9.

(收稿日期:2014-08-01 本文修改:李亚聪)

[11] 査艳,赵盈亭,杨霞,等.氯沙坦对发展性肾炎模型大鼠肾小管-间质细胞巨噬细胞移动按捺因子的影响研讨[J].我国药房,2011,22(33):3098-3100.

[12] Wan F,Lenardo MJ.Specification of DNA binding activity of NF-kappaB proteins[J].Cold Spring Harb Perspect Biol,2009,1(4):a000067.

[13] Hayden MS,Ghosh S.Shared principles in NF-kappaB signaling[J].Cell,2008,132(3):344-362.

[14] Sanz AB,Sanchez-Ni?觡o MD,Ramos AM,et al.NF-kappaB in renal inflammation[J].J Am Soc Nephrol,2010,21(8):1254-1262.

[15] Okabe C,Borges RL,de Almeida DC,et al.NF-κB activation mediates crystal translocation and interstitial inflammation in adenine overload nephropathy[J].Am J Physiol Renal Physiol,2013,305(2):F155-F163.

[16] Daun JM,Cannon JG.Macroghage migration inhibitory factor antagonizes hydrocortisone- induced increase in cytosolic IκB[J].Am J Physiol,2000,279(2):1045-1049.

[17] Salminen A,Kaarniranta K.Control of p53 and NF-κB signaling by WIP1 and MIF:role in cellular senescence and organismal aging[J].Cell Signal,2011,23(5):747-752.

[18] 刘延霞.巨噬细胞移动按捺因子在IgA肾病大鼠模型肾损害中的效果[J].温州:温州医学院,2008.

[19] Li Y,Spataro BC,Yang J,et al.1,25-dihydroxyvitamin D inhibits renal interstitial myofibroblast activation by inducing hepatocyte growth factor expression[J].Kidney Int,2005,68(4):1500-1510.

[20] 林建明,周洁,杨霞,等.1,25-(OH)2-D3对发展性肾炎大鼠肾小管间质胶原蛋白Ⅲ表达的影响[J].我国当代医药,2014,21(4):4-6,9.

(收稿日期:2014-08-01 本文修改:李亚聪)

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