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

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

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