活性维生素D3在大鼠肺纤维化发生发展中的作用及对miR-29a表达的影响

许梦婷 刘乃国* 董洪亮 郑静 倪娜 王楠

doi:10.16098/j.issn.0529-1356.2017.01.014

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解剖学报 ›› 2017, Vol. 48 ›› Issue (1) : 78-86. DOI: 10.16098/j.issn.0529-1356.2017.01.014

组织学胚胎学发育生物学

活性维生素D3在大鼠肺纤维化发生发展中的作用及对miR-29a表达的影响

许梦婷刘乃国* 董洪亮郑静倪娜王楠

作者信息 +

Impact of active vitamin D3 on the expression of miR-29a and on the production and development of pulmonary fibrosis in rats

XU Meng-ting LIU Nai-guo* DONG Hong-liang ZHENG Jing NI Na WANG Nan

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文章历史 +

摘要

目的探讨活性维生素D3［1,25(OH)₂D₃］在大鼠肺纤维化发生发展中的作用及对miR-29a的影响。方法 150只SD雄性大鼠随机分为纤维化发生干预组（90只）和纤维化后干预组（60只），纤维化发生干预组分为模型组Ⅰ、给药组Ⅰ和对照组Ⅰ（n=30），纤维化后干预组分为模型组Ⅱ、给药组Ⅱ和对照组Ⅱ（n=20）。模型组Ⅰ/Ⅱ和给药组Ⅰ/Ⅱ经气管注入博来霉素（5mg/kg）建立肺纤维化模型，对照组Ⅰ/Ⅱ经气管注入等体积生理盐水。给药组Ⅰ/Ⅱ分别于手术后第2天和第14天腹腔注射活性维生素D3，模型组Ⅰ/Ⅱ分别于手术后第2天和第14天腹腔注射等量的活性维生素D3溶剂（0.1%乙醇和99.9%的丙二醇），对照组Ⅰ/Ⅱ分别在术后第2天和第14天腹腔注射等量的生理盐水。各种处理均为两天1次。纤维化发生干预组分别于手术后第14天、第21天和第28天处死大鼠取材，纤维化后干预组分别于手术后第21天和第28天处死大鼠取材，各小组每个时间点10只大鼠。用Masson染色法观察各组实验大鼠肺中胶原纤维的差异，用碱水解法检测羟脯氨酸含量的变化，用实时定量PCR检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白 (Col)ⅠmRNA 及miR-29a的相对表达量，用免疫组织化学方法检测α-SMA和ColⅠ蛋白质表达水平,并经图像分析进行量化。结果博来霉素处理后，第14天大鼠肺部已经出现纤维化，随着时间推移，纤维化进一步加重。模型组Ⅰ/Ⅱ和给药组Ⅰ/Ⅱ中的羟脯氨酸含量、α-SMA和Col Ⅰ的mRNA和蛋白质表达都明显高于对照组Ⅰ/Ⅱ，而其miR-29a表达与对照组Ⅰ/Ⅱ相比则明显减少。给药组Ⅰ/Ⅱ中miR-29a的表达与模型组Ⅰ/Ⅱ相比有所增加（P<0.05）。在纤维化发生干预组中，与模型组Ⅰ相比，给药组Ⅰ在3个时间点羟脯氨酸含量、α-SMA、ColⅠ的mRNA和蛋白质表达都显著降低（P<0.05），但在纤维化后干预组中的给药组Ⅱ中羟脯氨酸含量、α-SMA、ColⅠ的mRNA和蛋白质表达比模型组Ⅱ虽有所降低，但差异无显著性（P>0.05）。结论活性维生素D3对大鼠肺纤维化的发生发展具有一定的抑制作用，其预防效果更好一些，并且能促进miR-29a的表达，活性维生素D3可能是通过促进miR-29a的表达来抑制肺纤维化的发生发展。

Abstract

Objective To investigate the preventive and therapeutic effects of active vitamin D3［1,25(OH)₂D₃］ on the production and development of pulmonary fibrosis in rats, and the effect on miR-29a. Methods A total of 150 male SD rats were randomly divided into fibrogenic intervention group (n=90) and fibrotic-intervention group (n=60), then subdivided into model group Ⅰ, treatment group Ⅰ and control group Ⅰ（n=30）for fibrogenic intervention group, and model group Ⅱ, treatment group Ⅱ and control group Ⅱ（n=20）for fibrotic-intervention group. Bleomycin（5mg/kg）was injected into the rat trachea to establish the model of pulmonary fibrosis in the model groups Ⅰ/Ⅱ and treatment groups Ⅰ/Ⅱ, while the control groupsⅠ/Ⅱ were injected with isopyknic sterile saline. The treatment groups Ⅰ/Ⅱ were intraperitoneal injected with active vitamin D3 on the 2nd day and 14th day after surgery respectively. The model groupsⅠ/Ⅱ were intraperitoneally injected with solvent of vitamin D3 (0.1% ethanol in 99.9% propylene glycol ), the control groupsⅠ/Ⅱ were injected intraperitoneally with sterile saline, on the 2nd day and 14th day after surgery respectively. All injections were carried out once every other day. In the fibrogenic intervention group, 10 rats were euthanized at 14th, 21st and 28th day in each group, and in the fibrotic-intervention group, 10 rats were euthanized at 21st and 28th day in each group respectively. After obtaining lung tissues from experimental rats, the differences of collagen fiber and hydroxyproline content were compared by the Masson staining and basic-hydrolysis method Respectively. The expression of α-smooth muscle actin (α-SMA), type Ⅰ collagen (ColⅠ) mRNA and miR-29a were detected by Real-time PCR. The protein expressions of α-SMA and Col Ⅰwere measured by immunohistochemical technology, and quantized with image analysis. Results Fibrosis appeared in lungs of experimental rats treated with bleomycin after 14 days, and gradually aggravated with time. At 14th, 21st and 28th day, the hydroxyproline content, the mRNA and protein expression levels of α-SMA and ColⅠin model groupⅠ/Ⅱ and treatment group Ⅰ/Ⅱ were significantly higher than that of control group Ⅰ/Ⅱ, but the expressions of miR-29a in model groupⅠ/Ⅱ and treatment group Ⅰ/Ⅱ were significantly lower than that of control group Ⅰ/Ⅱ. Compared with model groupsⅠ/Ⅱ, the expressions of miR-29a was increased in treatment groups Ⅰ/Ⅱ (P<0.05). In the fibrogenic intervention group, the hydroxyproline content, the mRNA and protein expression of α-SMA and Col Ⅰin treatment group Ⅰwere all obviously reduced at three time points compared with model groupⅠ (P<0.05). In the fibrotic-intervention group, the hydroxyproline content, the mRNA and protein expression of α-SMA and Col Ⅰwere slightly decreased in treatment group Ⅱ compared with model groupⅡ, but no significant difference was found (P>0.05). Conclusion Active vitamin D3 may have an inhibiting effect on genesis and progression of pulmonary fibrosis in rats, with better preventive effect than therapeutic effect, and may promote the expression of miR-29a, suggesting that active vitamin D3 inhibits the genesis and progression of pulmonary fibrosis probably by promoting the expression of miR-29a.

导出引用

许梦婷刘乃国董洪亮郑静倪娜王楠. 活性维生素D3在大鼠肺纤维化发生发展中的作用及对miR-29a表达的影响[J]. 解剖学报. 2017, 48(1): 78-86 https://doi.org/10.16098/j.issn.0529-1356.2017.01.014

XU Meng-ting LIU Nai-guo DONG Hong-liang ZHENG Jing NI Na WANG Nan. Impact of active vitamin D3 on the expression of miR-29a and on the production and development of pulmonary fibrosis in rats[J]. Acta Anatomica Sinica. 2017, 48(1): 78-86 https://doi.org/10.16098/j.issn.0529-1356.2017.01.014

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