胰岛素样生长因子-1可促进油酸诱导急性肺损伤小鼠纤维化及可能机制

doi:10.3877/cma.j.issn.1674-6902.2017.03.006

目的

探讨胰岛素样生长因子(IGF)-1在急性肺损伤小鼠肺纤维化发病机制中的作用。

方法

健康BALB/c小鼠60只，随机分为油酸组(n＝35)和对照组(n＝25)。油酸组从尾静脉注入0.3 ml/kg的油酸，对照组从尾静脉注入同等体积的生理盐水；两组分别于8 h、1、3、7、14、21、28 d各处死3只小鼠，取右肺用于qRT-PCR法检测IGF-1和IGF-1R mRNA表达水平，Western Blotting检测IGF-1、IGF-1R蛋白表达水平，免疫组织化学法检测IGF-1、IGF-1R和胶原Ⅰ/Ⅲ，α-平滑肌肌动蛋白，CD68和增殖细胞核抗原阳性细胞数；取左肺用于HE和Masson染色观察肺组织的病理改变。

结果

油酸组小鼠第1～28天肺组织中IGF-1、IGF-1R mRNA、蛋白表达水平和阳性细胞数逐步升高，第21天达高峰，第28天缓慢下降；胶原Ⅰ、胶原Ⅲ、α-平滑肌肌动蛋白、CD68和增殖细胞核抗原阳性细胞数随时间持续升高，第21天后升高势头趋向平缓；与对照组相比，差异有统计学意义(P<0.05)。油酸组小鼠第1～3天病理特征为明显肺泡炎性改变，第7～21天肺泡炎性改变减轻，纤维增生明显，支气管周围、肺泡间隔及肺泡腔内出现纤维化，第28天纤维增生趋向于平缓。

结论

胰岛素样生长因子-1可能通过促进胶原Ⅰ和胶原Ⅲ以及α-平滑肌肌动蛋白、CD68和增殖细胞核抗原的生成、增加肺间质胶原的沉积、加重肌成纤维细胞的增殖，促进急性肺损伤小鼠肺纤维化。

关键词: 油酸, 急性肺损伤, 肺纤维化, 胰岛素样生长因子-1, 胰岛素样生长因子-1

Objective

To investigate the role of insulin-like growth factor-1 in the development of pulmonary fibrosis in acute lung injury induced by oleic acid in a rat model.

Methods

The mice were injected with oleic acid (0.3 ml/kg) intravenously to induce ALI. Sixty BALB/c mice were randomly divided into an oleic acid group (n=35) and a control group (n=25). The oleic acid group was treated with oleic acid (0.3 ml/kg) intravenously and the control group was treated with same amount of normal saline. 3 rats in each group respectively were sacrificed on 1st, 3rd, 7 th, 14 th, 21 th and 28 th day after the treatment. The expression levels of IGF-1 and IGF-1R mRNA were detected by quantitative real-time polymerase chain reaction ( qRT-PCR), the protein levels of IGF-1 and IGF-1R were measure by western blotting assay and the number of positive cells of IGF-1, IGF-1R, collagen Ⅰ, collagenⅢ, α-smooth muscle actin, CD68 and proliferating cell nuclear antigen were measure by immunohistochemical assay, meanwhile, pathological changes were observed in sections of left lung stained with Hematoxylin Eosin and Masson.

Results

Compared with control group, the expression levels of IGF-1 and IGF-1R mRNA, the protein levels and the number of positive cells of IGF-1 and IGF-1R in lung homogenate of the rats in the oleic acid group were significantly increased with time(P<0. 05). And the number of positive cells of IGF-1, IGF-1R collagen Ⅰ, collagenⅢ, α-smooth muscle actin, CD68 and proliferating cell nuclear antigen synchronously increased with time in the oleic acid group. The phase in early (1-3 d) was characterized by acute alveolitis. The phase in later stage (7-21 d) was characterized by marked proliferation of fibrous tissue. It would become slower after 28 th day.

Conclusions

The expression of IGF-1, IGF-1R, collagen Ⅰ, collagenⅢ, α-smooth muscle actin, CD68 and proliferating cell nuclear antigen are increased strong with time in lung homogenate in acute lung injury induced by oleic acid in a rat model which are consistent with the progression of pulmonary fibrosis in the comparison of the lung histopathological examination. The study indicated that insulin-like growth factor-1 probably promoted pulmonary fibrosis by up-regulating the expression of collagen Ⅰ, collagenⅢ, α-smooth muscle actin, CD68 and proliferating cell nuclear antigen which could increase pulmonary interstitial collagen deposition and proliferation of myofibroblasts in acute lung injury induced by oleic acid in a rat model.

Key words: Oleic acid, Acute lung injury, Pulmonary fibrosis, Insulin-like growth factor-1, Insulin-like growth factor-1 receptor

表1 油酸组各时段小鼠肺泡炎和肺纤维化程度评分(n＝3，±s)

状态时段	d0	d1	d3	d7	d14	d21	d28
肺泡炎(分)	0.00±0.00	1.00±0.00	2.33±0.58	2.00±0.00	1.67±0.58	1.33±0.57	1.00±0.00
肺纤维化(%)	0.00±0.00	0.00±0.00	3.33±5.77	20.00±0.00	23.33±5.77	36.67±5.77	41.67±2.87

表1 油酸组各时段小鼠肺泡炎和肺纤维化程度评分(n＝3，±s)

状态时段	d0	d1	d3	d7	d14	d21	d28
肺泡炎(分)	0.00±0.00	1.00±0.00	2.33±0.58	2.00±0.00	1.67±0.58	1.33±0.57	1.00±0.00
肺纤维化(%)	0.00±0.00	0.00±0.00	3.33±5.77	20.00±0.00	23.33±5.77	36.67±5.77	41.67±2.87

表2 两组小鼠肺组织中IGF-1mRNA和IGF-1R mRNA表达水平(n＝3，±s)

时相点		d0	d1	d3	d7	d14	d21	d28
IGF-1mRNA		?	?	?	?	?	?	?
?	油酸组	2.15±0.13	2.20±0.04	2.44±0.10^a	2.89±0.08^b	2.94±0.10^b	3.68±0.16^b	3.32±0.05^b
?	对照组	2.18±0.19	2.17±0.12	2.12±0.10	2.19±0.05	2.21±0.04	2.15±0.06	2.11±0.16
IGF-1R mRNA		?	?	?	?	?	?	?
?	油酸组	2.28±0.13	2.33±0.40	2.52±0.17^a	3.02±0.09^b	3.08±0.09^b	3.81±0.18^b	3.46±0.04^b
?	对照组	2.26±0.18	2.25±0.11	2.19±0.12	2.25±0.05	2.28±0.05	2.21±0.05	2.19±0.15

表2 两组小鼠肺组织中IGF-1mRNA和IGF-1R mRNA表达水平(n＝3，±s)

时相点		d0	d1	d3	d7	d14	d21	d28
IGF-1mRNA		?	?	?	?	?	?	?
?	油酸组	2.15±0.13	2.20±0.04	2.44±0.10^a	2.89±0.08^b	2.94±0.10^b	3.68±0.16^b	3.32±0.05^b
?	对照组	2.18±0.19	2.17±0.12	2.12±0.10	2.19±0.05	2.21±0.04	2.15±0.06	2.11±0.16
IGF-1R mRNA		?	?	?	?	?	?	?
?	油酸组	2.28±0.13	2.33±0.40	2.52±0.17^a	3.02±0.09^b	3.08±0.09^b	3.81±0.18^b	3.46±0.04^b
?	对照组	2.26±0.18	2.25±0.11	2.19±0.12	2.25±0.05	2.28±0.05	2.21±0.05	2.19±0.15

图1 油酸组小鼠肺组织中IGF-1和IGF-1R蛋白表达水平

表3 两组小鼠肺组织中IGF-1和IGF-1R蛋白表达水平(n＝3，±s)

时相点		d0	d1	d3	d7	d14	d21	d28
IGF-1蛋白		?	?	?	?	?	?	?
?	油酸组	134.33±9.50	142.33±8.50	164.33±8.02^a	202.00±10.82^b	214.33±6.03^b	276.33±10.21^b	259.33±10.60^b
?	对照组	132.67±7.02	131.33±9.07	134.00±8.72	133.33±4.51	136.00±7.00	134.33±7.51	134.00±9.85
IGF-1R蛋白		?	?	?	?	?	?	?
?	油酸组	153.33±7.57	163.00±8.54	183.67±10.50^a	223.00±9.54^b	235.00±6.56^b	297.67±10.02^b	279.67±11.68^b
?	对照组	157.00±10.54	150.67±9.07	154.67±5.69	156.00±10.54	154.00±8.00	154.00±5.57	155.00±9.54

表3 两组小鼠肺组织中IGF-1和IGF-1R蛋白表达水平(n＝3，±s)

时相点		d0	d1	d3	d7	d14	d21	d28
IGF-1蛋白		?	?	?	?	?	?	?
?	油酸组	134.33±9.50	142.33±8.50	164.33±8.02^a	202.00±10.82^b	214.33±6.03^b	276.33±10.21^b	259.33±10.60^b
?	对照组	132.67±7.02	131.33±9.07	134.00±8.72	133.33±4.51	136.00±7.00	134.33±7.51	134.00±9.85
IGF-1R蛋白		?	?	?	?	?	?	?
?	油酸组	153.33±7.57	163.00±8.54	183.67±10.50^a	223.00±9.54^b	235.00±6.56^b	297.67±10.02^b	279.67±11.68^b
?	对照组	157.00±10.54	150.67±9.07	154.67±5.69	156.00±10.54	154.00±8.00	154.00±5.57	155.00±9.54

表4 两组小鼠肺组织中IGF-1和IGF-1R阳性细胞数水平(n＝3，±s)

时相点		d0	d1	d3	d7	d14	d21	d28
IGF-1阳性细胞数		?	?	?	?	?	?	?
?	油酸组	4.33±0.58^a	4.33±0.58^a	9.00±1.00^a	14.00±1.00^a	17.00±1.00^a	26.33±1.53^a	24.00±2.00^a
?	对照组	1.33±0.58	1.33±0.58	1.00±0.00	1.67±0.58	1.33±0.58	1.67±0.58	2.00±1.00
IGF-1R阳性细胞数		?	?	?	?	?	?	?
?	油酸组	5.33±0.58^a	5.00±1.00^a	7.67±0.58^a	16.00±1.00^a	20.67±0.58^a	30.33±1.53^a	27.00±1.00^a
?	对照组	2.33±0.58	2.00±0.00	3.00±1.00	3.00±1.00	2.67±1.15	2.33±0.58	2.67±1.15

表4 两组小鼠肺组织中IGF-1和IGF-1R阳性细胞数水平(n＝3，±s)

时相点		d0	d1	d3	d7	d14	d21	d28
IGF-1阳性细胞数		?	?	?	?	?	?	?
?	油酸组	4.33±0.58^a	4.33±0.58^a	9.00±1.00^a	14.00±1.00^a	17.00±1.00^a	26.33±1.53^a	24.00±2.00^a
?	对照组	1.33±0.58	1.33±0.58	1.00±0.00	1.67±0.58	1.33±0.58	1.67±0.58	2.00±1.00
IGF-1R阳性细胞数		?	?	?	?	?	?	?
?	油酸组	5.33±0.58^a	5.00±1.00^a	7.67±0.58^a	16.00±1.00^a	20.67±0.58^a	30.33±1.53^a	27.00±1.00^a
?	对照组	2.33±0.58	2.00±0.00	3.00±1.00	3.00±1.00	2.67±1.15	2.33±0.58	2.67±1.15

表5 油酸组小鼠肺组织中IGF-1、IGF-1R和胶原Ⅰ/Ⅲ、α-平滑肌肌动蛋白、CD68和增殖细胞核抗原阳性细胞数(n＝3，±s)

项目时段	d0	d1	d3	d7	d14	d21	d28
IGF-1	4.33±0.58	4.33±0.58	9.00±1.00	14.00±1.00	17.00±1.00	26.33±1.53	24.00±2.00
IGF-1R	5.33±0.58	5.00±1.00	7.67±0.58	16.00±1.00	20.67±0.58	30.33±1.53	27.00±1.00
胶原Ⅰ	3.33±0.58	3.33±0.58	4.00±1.00	7.00±1.00	15.67±1.53	21.33±1.53	23.00±1.00
胶原Ⅲ	5.33±0.58	5.33±3.21	7.33±3.21	9.67±3.06	18.33±1.53	24.67±1.53	26.33±1.53
actin	4.33±0.58	4.67±0.58	11.00±1.00	13.67±1.53	19.00±1.00	23.00±1.00	24.00±1.00
CD68	2.33±0.58	3.00±0.00	8.00±1.00	12.00±1.00	17.33±2.08	21.67±1.53	22.33±1.53
PCNA	2.67±0.58	3.33±0.58	5.33±0.58	8.67±0.58	17.33±1.53	20.00±1.00	21.00±1.00

表5 油酸组小鼠肺组织中IGF-1、IGF-1R和胶原Ⅰ/Ⅲ、α-平滑肌肌动蛋白、CD68和增殖细胞核抗原阳性细胞数(n＝3，±s)

项目时段	d0	d1	d3	d7	d14	d21	d28
IGF-1	4.33±0.58	4.33±0.58	9.00±1.00	14.00±1.00	17.00±1.00	26.33±1.53	24.00±2.00
IGF-1R	5.33±0.58	5.00±1.00	7.67±0.58	16.00±1.00	20.67±0.58	30.33±1.53	27.00±1.00
胶原Ⅰ	3.33±0.58	3.33±0.58	4.00±1.00	7.00±1.00	15.67±1.53	21.33±1.53	23.00±1.00
胶原Ⅲ	5.33±0.58	5.33±3.21	7.33±3.21	9.67±3.06	18.33±1.53	24.67±1.53	26.33±1.53
actin	4.33±0.58	4.67±0.58	11.00±1.00	13.67±1.53	19.00±1.00	23.00±1.00	24.00±1.00
CD68	2.33±0.58	3.00±0.00	8.00±1.00	12.00±1.00	17.33±2.08	21.67±1.53	22.33±1.53
PCNA	2.67±0.58	3.33±0.58	5.33±0.58	8.67±0.58	17.33±1.53	20.00±1.00	21.00±1.00

1	金发光. 急性肺损伤的诊治研究现状及进展[J/CD]. 中华肺部疾病杂志(电子版), 2013, 6(1): 1-3.
2	马李杰，李王平，金发光. 急性肺损伤/急性呼吸窘迫综合征发病机制的研究进展[J/CD]. 中华肺部疾病杂志(电子版), 2013, 6(1): 65-68.
3	施卉，任成山. 急性肺损伤/急性呼吸窘迫综合征基础及临床研究进展[J/CD]. 2013, 6(4): 350-355.
4	吴秀琳，陈复辉，钱斓兰，等. 活化转录因子3与急性肺损伤[J/CD]. 中华肺部疾病杂志(电子版), 2017, 10(2): 215-217.
5	Bellani G, Laffey JG, Pham T, et al. Epidemiology, patterns of care，and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries[J]. JAMA, 2016, 315(8): 788-800.
6	Barnes PJ. The cytokine network in chronic obstructive pulmonary disease[J]. Am J Respir Cell Mol Biol, 2009, 41(6): 631-638.
7	刘峰，黄华萍，李羲，等. 胰岛素样生长因子-1在油酸致急性肺损伤小鼠中的作用[J/CD]. 中华肺部疾病杂志(电子版), 2016, 9(5): 503-506.
8	Szapiel SV, Elson NA, Fulmer JD, et al. Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse[J]. Am Rev Respir Dis, 1979, 120(4): 893-899.
9	张云，王维群. 胰岛素样生长因子-1(IGF-1)在运动性脊髓损伤中的作用[J]. 当代体育科技，2013, 3(6): 22-23.
10	王杰华，李国前，杨小霞，等. 尤瑞克林对脑缺血再灌注大鼠脑组织中IGF-1和IGF-1R表达水平的影响及其机制[J]. 吉林大学学报(医学版), 2015, 41(2): 338-342.
11	许春花，薛东生，金正勇. 胰岛素样生长因子1对新生大鼠高氧肺损伤的影响及其机制探讨[J]. 山东医药，2016, 56(42): 37-39.
12	Kim TH, Chow YH, Gill SE, et al. Effect of insulin-like growth factor blockade on hyperoxia-induced lung injury[J]. Am J Respir Cell Mol Biol, 2012, 47(3): 372-378.
13	Ahasic AM, Tejera P, Wei Y, et al. Predictors of circulating insulin-like growth factor-1 and insulin-like growth factor-binding protein-3 in critical illness[J]. Crit Care Med, 2015, 43(12): 2651-2659.
14	Jian XD, Li M, Zhang YJ, et al. Role of growth factors in acute lung injury induced by paraquat in a rat model[J]. Hum Exp Toxicol, 2011, 30(6): 460-469.
15	许春花. 内质网应激反应在高氧性肺损伤中的作用及IGF-1的干预效应[D]. 延边大学，2013.
16	Krein PM, Sabatini PJ, Tinmouth W, et al. Localization of insulin-like growth factor-I in lung tissues of patients with fibroproliferative acute respiratory distress syndrome[J]. Am J Respir Crit Care Med, 2003, 167(1): 83-90.
17	李蓓，陈力，马劲夫，等. 胰岛素样生长因子1对全氟异丁烯吸入性肺损伤小鼠防护作用的研究[J]. 中华危重病急救医学，2009, 21(4): 219-221
18	Daian T, Ohtsuru A, Rogounovitch T, et al. Insulin-like growth factor-I enhances transforming growth factor-beta-induced extracellular matrix protein production through the P38/activating transcription factor-2 signaling pathway in keloid fibroblasts[J]. J Invest Dermatol, 2003, 120(6): 956-962.
19	Andonegui G, Krein PM, Mowat C, et al. Enhanced production of IGF-I in the lungs of fibroproliferative ARDS patients[J]. Physiol Rep, 2014, 2(11):e12197.
20	Krein PM, Sabatini PJ, Tinmouth W, et al. Localization of insulin-like growth factor-I in lung tissues of patients with fibroproliferative acute respiratory distress syndrome[J]. Am J Respir Crit Care Med, 2003, 167(1): 83-90.

[1]	梁哲浩, 方明笋, 胡弘毅, 陶涛, 徐孝平, 孙华琴. 基于生物信息学分析筛选脓毒症诱导急性肺损伤的关键基因[J]. 中华危重症医学杂志(电子版), 2022, 15(05): 360-366.
[2]	沈纵, 魏晨如, 朱邦晖, 包郁露, 伍国胜, 孙瑜. 间充质干细胞治疗吸入性损伤的动物实验研究进展[J]. 中华损伤与修复杂志(电子版), 2023, 18(02): 180-183.
[3]	蔡维霞, 曹涛, 赵明, 肖丹, 贾艳慧, 王璟, 张月, 王克甲, 韩军涛, 胡大海. Notch信号通路对烧伤大鼠血清诱导的肺血管内皮细胞细胞间黏附分子-1的影响[J]. 中华损伤与修复杂志(电子版), 2022, 17(04): 292-299.
[4]	李安琪, 徐祎琳, 向天新. 新型冠状病毒感染后肺纤维化病变诊治进展[J]. 中华实验和临床感染病杂志(电子版), 2023, 17(05): 294-298.
[5]	闫凯悦, 邓慧玲, 张玉凤, 宋鹤, 陈媛, 席淼. 胰岛素样生长因子-1与手足口病重症化的相关性研究[J]. 中华实验和临床感染病杂志(电子版), 2023, 17(03): 151-157.
[6]	中华医学会器官移植学分会肺移植学组, 国家肺移植质控中心. 新型冠状病毒感染肺移植受者选择中国专家建议[J]. 中华移植杂志(电子版), 2023, 17(01): 13-16.
[7]	熊欢庆, 李玉娟, 陈键, 刘刚, 李志超, 金发光. 丹参酮IIA及苦参碱组方对脂多糖致小鼠急性肺损伤的协同保护作用[J]. 中华肺部疾病杂志(电子版), 2023, 16(04): 455-459.
[8]	李埝, 赵建军, 张建勇, 赵睿桢. hAMSCs调控MAPK信号通路对急性肺损伤AQP1的影响[J]. 中华肺部疾病杂志(电子版), 2023, 16(02): 156-163.
[9]	李青原, 冯同, 邱雪琴, 李万成. 迷迭香提取物防治肺纤维化研究进展[J]. 中华肺部疾病杂志(电子版), 2022, 15(06): 908-911.
[10]	王赛妮, 徐旺, 李华娟, 唐英俊, 李卫霞, 李羲, 黄华萍. 影像表现为肺纤维化的肉芽肿性多血管炎一例报告并文献复习[J]. 中华肺部疾病杂志(电子版), 2022, 15(04): 603-605.
[11]	朱冠能, 汪洋, 宋海苗, 汪骏东. 血清铁蛋白及C反应蛋白水平对胸部创伤后急性肺损伤的预测意义[J]. 中华肺部疾病杂志(电子版), 2022, 15(03): 379-381.
[12]	杜文峰, 周玲, 李琪. 血清KL-6对特发性肺纤维化患者诊断及预后的意义[J]. 中华肺部疾病杂志(电子版), 2022, 15(02): 218-220.
[13]	刘雪娇, 盛伟利, 丁艳艳, 尹凤先. 特发性肺纤维化急性加重患者预后危险因素及意义[J]. 中华肺部疾病杂志(电子版), 2022, 15(02): 212-214.
[14]	王召, 田进杰, 郭朝, 王蕾, 严红燕, 冯素娟, 张毅. 血浆PGK1早期检测对创伤性颅脑损伤患者病情严重程度及预后的预测价值[J]. 中华神经创伤外科电子杂志, 2023, 09(03): 154-159.
[15]	吕昆明, 王沙沙, 万军, 令狐恩强. 胃食管反流病与特发性肺纤维化关系的研究进展[J]. 中华胃肠内镜电子杂志, 2023, 10(02): 121-124.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Insulin-like growth factor-1 promotes pulmonary fibrosis with time in acute lung injury induced by oleic acid in a rat model

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Insulin-like growth factor-1 promotes pulmonary fibrosis with time in acute lung injury induced by oleic acid in a rat model