文章信息
- 李野, 李斌超, 张雅琳, 刘宁, 刘祖望, 孔娟
- LI Ye, LI Binchao, ZHANG Yalin, LIU Ning, LIU Zuwang, KONG Juan
- 胆维丁乳对急性肺损伤小鼠肺表面活性物质的影响
- Effect of cholecalciterol cholesterol emulsion on pulmonary surfactant in a mouse model of acute lung injury
- 中国医科大学学报, 2020, 49(8): 685-689, 693
- Journal of China Medical University, 2020, 49(8): 685-689, 693
-
文章历史
- 收稿日期:2019-06-25
- 网络出版时间:2020-07-28 15:55
急性肺损伤(acute lung injury,ALI)是肺泡上皮细胞和肺毛细血管内皮细胞受损的急性肺部炎症综合征(acute respiratory distress syndrome,ARDS)[1],是一种高死亡率的急危重症。通过气管滴注脂多糖(lipopolysaccharides,LPS)可建立ALI小鼠模型[2]。
肺表面活性物质由Ⅱ型肺泡上皮细胞分泌,主要成分为二棕榈酰卵磷脂和肺表面活性物质结合蛋白(surfactant protein,SP),分布于肺泡液体分子层表面,能降低肺泡表面张力,维持大小肺泡容量的相对稳定[3],阻止肺泡毛细血管中液体向肺泡内滤出,是治疗ALI的有效手段[4]。目前已知的SP有SP-A、SP-B、SP-C、SP-D 4种。维生素D是一种脂溶性类固醇激素,在钙磷平衡和骨代谢中起重要作用[5-7],并可通过其受体(vitamin D receptor,VDR)调控多种生理反应[2, 8-16]。研究[17-19]发现,维生素D3能调节Ⅱ型肺泡上皮细胞增殖。胆维丁乳(cholecalciterol cholesterol emulsion,CCE)是一种新型的活性维生素D前体,临床上常用于治疗婴幼儿维生素D缺乏性佝偻病。SP在维生素D缺乏小鼠的肺中表达减少[20]。本研究拟探讨CCE对LPS所致ALI肺表面活性物质的影响。
1 材料与方法 1.1 制作动物模型与分组将36只健康雄性BALB/c小鼠随机分为正常对照组、CCE组、LPS组、LPS+CCE组,每组9只。正常对照组和LPS组给予饮用水,CCE组和LPS+CCE组小鼠给予10%CCE水溶液(避光,购自中国医科大学附属盛京医院)。饲喂14 d后,LPS组和LPS+CCE组小鼠行气管切开,滴注LPS(10 mg/kg),24 h后取血清、肺泡灌洗液及肺组织备用。
1.2 检测血清钙、磷的含量从小鼠心脏取血,分离血清,采用钙测试盒(带标准)微板法(C004-2,南京建成生物工程研究所)、无机磷测试盒(C006-1,南京建成生物工程研究所)检测各组小鼠血清钙、磷含量。
1.3 测定小鼠肺组织湿/干质量比取4组小鼠的右下肺,用吸水纸吸干肺组织表面血迹,立即称质量,记为“湿质量”。置于80 ℃恒温干燥箱内烘干,48 h后称质量,记为“干质量”。计算肺湿/干质量比。
1.4 肺组织HE染色4%多聚甲醛固定肺组织,石蜡包埋并切片,脱蜡,HE染色,封片,光镜下观察并拍照。
1.5 实时PCR用TRIzol试剂盒(美国Invitrogen公司)提取肺组织总RNA,按照产品说明书操作。用生物分光光度计测定RNA浓度和纯度。取2 μg RNA样本,用TaKaRa逆转录试剂盒(A2302-1,日本TaKaRa公司)进行逆转录合成cDNA,并以此为模板进行PCR扩增反应。逆转录和PCR反应条件按照试剂盒说明书设置。引物由上海生工生物工程公司合成,见表 1。
Gene name | Primer sequence |
VDR | Forward primer 5’- TGACCCTGGAGACTTTGACC -3’ |
Reverse primer 5’- GTTGAAGGGGCAGGTGAATA -3’ | |
GAPDH | Forward primer 5’- ACCACAGTCCATGCCATCAC -3’ |
Reverse primer 5’- TCCACCACCCTGTTGCTGTA -3’ | |
SP-A | Forward primer 5’- AATGGGAGTCCTCAGCTTGC -3’ |
Reverse primer 5’- CCGGCTCTGGTACACATCTC -3’ | |
SP-B | Forward primer 5’- TGCTTGATGTCTACCTGCCC -3’ |
Reverse primer 5’- AGCAGGAGAACTGTGTAGCG -3’ | |
SP-C | Forward primer 5’- TGATGGAGAGTCCACCGGAT -3’ |
Reverse primer 5’- CCACCACAACCACGATGAGA -3’ | |
SP-D | Forward primer 5’- CGAGCCTGACAAACAGAGGT -3’ |
Reverse primer 5’- GGAGAGAAAGGGCAGCATGT -3’ |
1.6 Western blotting
取肺组织,研磨匀浆后,蛋白裂解,BCA法检测总蛋白浓度。取50 μg总蛋白,行SDS-PAGE电泳,电转至PVDF膜,用5%脱脂奶粉封闭1 h。加入一抗(VDR抗体1:1 000稀释,购自美国SANTA CRUZ BIOTECHNOLOGY公司;GAPDH抗体1:5 000稀释,购自美国Proteintech公司),4 ℃孵育过夜。TBST洗3次,室温孵育二抗1 h,ECL显色。Image J图像分析软件对条带进行定量分析比较。
1.7 绘制生存曲线另取健康雄性BALB/c小鼠20只,分为LPS组、LPS+CCE组,每组10只,处置同1.1,绘制并分析2组小鼠生存时间曲线。
1.8 统计学分析采用Graph Pad Prism 5.0软件进行统计学分析,计量结果均采用x±s表示,采用方差分析或配对t检验进行组间比较。P < 0.05为差异有统计学意义。
2 结果 2.1 CCE对ALI模型小鼠血清钙、磷含量的影响如表 2所示,喂饲CCE 2周后,4组小鼠血清钙、磷水平无统计学差异。说明CCE对LPS所致ALI模型小鼠血清钙、磷的影响较小,故可排除血清钙、磷水平的高低对实验结果的干扰。
Item | Control group | CCE group | LPS group | LPS+CCE group | P |
Serum calcium(mmol/L) | 2.216±0.421 | 2.051±0.219 | 2.256±0.466 | 2.033±0.348 | 0.491 |
Serum phosphorus(mmol/L) | 2.618±0.490 | 2.854±0.217 | 2.799±0.487 | 2.678±0.210 | 0.535 |
CCE,cholecalciterol cholesterol emulsion;LPS,lipopolysaccharides. |
2.2 CCE对ALI模型小鼠生存时间的影响
如图 1所示,LPS+CCE组小鼠平均生存时间长于LPS组,小鼠死亡的首发时间晚于LPS组,给予LPS处理后25 h,LPS+CCE组小鼠仍有80%存活,LPS组则有40%小鼠死亡;53 h以内LPS组小鼠全部死亡,2组小鼠生存时间有显著的统计学差异(P < 0.05)。表明CCE能显著延长ALI小鼠的生存时间,对ALI起保护作用。
2.3 CCE对ALI模型小鼠肺组织及肺水肿的影响
肉眼观察可见,与正常对照组相比,LPS组小鼠双肺肿胀,体积变大,肺表面可见出血区。光镜下(图 2A)可见,正常对照组与CCE组小鼠肺泡结构完整,肺泡腔和间隙内无炎症细胞浸润;LPS组小鼠肺泡和肺间质广泛充血、水肿、大量炎性渗出液,肺泡腔可见破裂的红细胞和渗出物,肺泡结构紊乱,肺泡腔狭窄,肺泡间隔增厚;与LPS组相比,LPS+CCE组小鼠肺泡和肺间质微血管充血扩张程度及炎症细胞浸润减轻,肺泡腔内红细胞减少。
为了观察ALI时肺泡和肺间质充血、水肿的程度,本研究进一步检测了肺湿/干质量比。结果如图 2B所示,LPS组小鼠肺湿/干质量高于正常对照组,差异有统计学意义(P < 0.01);LPS+CCE组较LPS组显著降低,差异有统计学意义(P = 0.023)。提示CCE能改善ALI肺组织水肿程度。
2.4 CCE对ALI模型小鼠肺中VDR表达的影响Western blotting和实时PCR结果如图 3所示,CCE组小鼠肺组织中VDR蛋白表达水平高于正常对照组,LPS+CCE组VDR蛋白表达水平高于LPS组,差异有统计学意义(P = 0.012,P = 0.005)。提示饲喂CCE能增加小鼠肺组织内VDR表达。
2.5 CCE对ALI模型小鼠肺组织SP-A、SP-B、SP-C、SP-D mRNA表达的影响
如表 3所示,CCE小鼠肺组织SP-A、SP-C和SP-D的表达水平高于正常对照组,差异有统计学意义(P = 0.007,P = 0.011,P = 0.006);与正常对照组相比,LPS组SP-A、SP-B、SP-C、SP-D的表达水平均显著下降(P = 0.006,P = 0.016,P = 0.001,P = 0.001),而LPS+CCE组SP-A、SP-B、SP-C、SP-D的表达水平均较LPS组显著上升(P = 0.001,P = 0.049,P = 0.001,P = 0.042),差异有统计学意义。表明CCE能促进小鼠肺泡中肺表面活性物质的产生。
Group | SP-A | SP-B | SP-C | SP-D |
Control | 2.403±1.551 | 0.003±0.001 | 7.522±1.998 | 3.758±0.511 |
CCE | 3.175±0.6111) | 0.005±0.002 | 11.233±3.3322) | 5.189±1.2781) |
LPS | 0.750±0.0011) | 0.001±0.0002) | 1.985±0.8011) | 0.861±0.1271) |
LPS+CCE | 0.998±0.0311) | 0.002±0.0012) | 3.452±0.0531) | 1.233±0.1302) |
1)P < 0.01,2)P < 0.05 vs control group. |
3 讨论
尽管临床研究[21-22]发现,活性维生素D可影响血清钙磷水平,但本研究结果显示CCE并未影响小鼠的血清钙磷水平,因此排除了血清钙磷对后续实验结果的影响。
本研究发现,LPS+CCE组小鼠死亡首发时间晚于LPS组,且平均生存时间长于LPS组,提示维生素D前体CCE有显著延长ALI小鼠生存时间的作用,原因可能与维生素D的抗炎特性有关[23-24]。本研究还发现,CCE具有减轻ALI所致肺组织损伤的作用。HE染色显示,LPS组小鼠肺组织可见肺泡上皮细胞及间质水肿、肺泡腔内渗出、充血、炎症细胞浸润等典型ALI病理改变[25],而LPS+CCE组ALI病理改变较LPS组减轻。小鼠肺湿/干质量比能反映ALI所致肺水肿的程度,ALI时肺水肿严重,肺湿/干质量比升高[26]。本研究结果显示,LPS+CCE组肺湿/干质量比较LPS组降低,提示CCE在一定程度上缓解了LPS对小鼠造成的ALI肺水肿。
本研究发现,与正常对照组和LPS组相比,CCE组和LPS+CCE组小鼠肺组织内VDR表达水平显著升高,表明饲喂CCE能使小鼠体内VDR表达增多。研究[27-29]表明,ALI会造成肺泡不稳定、肺泡去复张及相应的组织重塑,肺表面活性物质可降低肺泡表面张力,从而防止低肺容量时发生肺泡塌陷及水肿[30-31],因此增加肺表面活性物质可治疗ALI [4]。维生素D3可加速胎儿肺成熟和Ⅱ型肺泡上皮细胞分化,并增加大鼠肺组织和Ⅱ型肺泡上皮细胞中肺表面活性物质的表达和分泌[32-34]。有研究[35]表明,维生素D3可直接作用于原代培养的Ⅱ型肺泡上皮细胞,诱导SP-B mRNA的产生,故推测维生素D前体CCE对ALI的保护作用有可能是因为增加了肺表面活性物质,因此本研究检测了各组小鼠肺组织中SP-A、SP-B、SP-C、SP-D的表达情况,结果证实CCE确能增加SP的表达。
综上所述,本研究证实了维生素D前体物质CCE能缓解LPS造成的ALI,改善并延长ALI小鼠的生存时间,改善肺组织病理学变化及肺水肿,增加SP的表达。因此,维生素D前体CCE增加肺表面活性物质可能是缓解ALI的机制之一,为探索ALI治疗方法提供了新的思路。
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