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

doi:10.16098/j.issn.0529-1356.2017.01.014

摘要/Abstract

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。

关键词: 活性维生素D3, miR-29a, 肺纤维化, 实时定量聚合酶链反应, Masson染色, 免疫组织化学, 大鼠

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.

Key words: Active vitamin D3, miR-29a, Pulmonary fibrosis, Real-time PCR, Masson staining, Immunohistochemistry, Rat

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.

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.

参考文献

［1］King TJ, Pardo A, Selman M. Idiopathic pulmonary fibrosis［J］. Lancet，2011, 378(9807):1949-1961.

［2］Wang YX, Zhang L, Zhang X, et al. Anti-fibrotic effect of kudancao tablets on bleomycin-induced pulmonary fibrosis in rats［J］. Journal of Kunming Medical University, 2016, 37(5):38-41. (in Chinese)

王应霞, 张丽, 张旋, 等. 苦胆草对博来霉素致大鼠肺纤维化的防治作用［J］. 昆明医科大学学报, 2016, 37(5):38-41.

［3］Lin JR, Xiong F, Zhang XY, et al. The effect of minoxidil on transforming growth gactor β1-induced proliferation in rat lung fibroblasts［J］. Guangdong Medical Journal, 2015, 36(19):2933-2936. (in Chinese)

林洁如, 熊方, 张湘燕, 等. 米诺地尔对转化生长因子β1诱导的大鼠肺成纤维细胞增殖的影响［J］. 广东医学, 2015, 36(19):2933-2936.

［4］Xiao J, Meng XM., Huang XR, et al. miR-29 inhibits bleomycin-induced pulmonary fibrosis in mice［J］. Mol Ther, 2012, 20(6):1251-1260.

［5］Montgomery RL, Yu G, Latimer PA, et al. MicroRNA- mimicry blocks pulmonary fibrosis［J］. EMBO Mol Med, 2014, 6(10):1347-1356.

［6］Chen L, Wu BQ, Chen WQ, et al. Dynamic expression changes of microR-146a and microR-29a in neonatal rats with acute lung injury［J］. Chinese Journal of Applied Clinical Pediatrics, 2014, 29(22):1747-1750. (in Chinese)

陈丽, 吴本清, 陈文清, 等. microR-146a、microR-29在新生大鼠急性肺损伤中的动态表达［J］. 中华实用儿科临床杂志, 2014, 29(22):1747-1750.

［7］Holick MF. Resurrection of vitamin D deficiency and rickets［J］. J Clin Invest, 2006, 116(8):2062-2072.

［8］Chen YH, Yu Z, Fu L, et al. Vitamin D3 inhibits lipopolysaccharideinduced placental inflammation through reinforcing interaction between vitamin D receptor and nuclear factor kappa B p65 subunit［J］. Sci Rep, 2015, 5:10871.

［9］Xu S, Chen YH, Tan ZX, et al. Vitamin D3 pretreatment alleviates renal oxidative stress in lipopolysaccharide-induced acute kidney injury［J］. J Steroid Biochem Mol Biol, 2015, 152:133-141.

［10］Mert H, Yoruk I, Ertekin A, et al. Vitamin levels in lung tissue of rats with bleomycin induced pulmonary fibrosis［J］. J Nutr Sci Vitaminol (Tokyo), 2009, 55(2):186-190.

［11］Ramirez AM, Wongtrakool C, Welch T, et al. Vitamin D inhibition of pro-fibrotic effects of transforming growth factorβ1 in lung fibroblasts and epithelial cells［J］. J Steroid Biochem Mol Biol, 2010, 118(3):142-150.

［12］Matheu V, Back O, Mondoc E, et al. Dual effects of vitamin D-induced alteration of TH1/TH2 cytokine expression: Enhancing IgE production and decreasing airway eosinophilia in murine allergic airway disease［J］. J Allergy Clin Immunol, 2003, 112(3):585-592.

［13］Liu NG, Zhang WQ, Hu FA, et al. Expression and effect of let-7a, miR-21 and miR-27a in oral squamous cell carcinomas［J］. Acta Anatomica Sinica, 2015, 46(2):221-226. (in Chinese)

刘乃国, 张卫群, 胡凤爱, 等. 口腔鳞癌中let-7a、miR-21和miR-27a的表达及其作用［J］. 解剖学报, 2015, 46(2):221-226.

［14］Jin XG, Dai HP, Pang BS, et al. Kinetics of pathologic changes in bleomycin-induced murine pulmonary fibrosis model［J］. Chinese Journal of Pathophysiology, 2009, 25(4):708-714. (in Chinese)

金晓光, 代华平, 庞宝森, 等. 博来霉素致大鼠肺纤维化模型肺组织的动态病理变化及其发生机制［J］. 中国病理生理杂志, 2009, 25(4):708-714.

［15］Dimitrov V, Salehi-Tabar R, An BS, et al. Non-classical mechanisms of transcriptional regulation by the vitamin D receptor: insights into calcium homeostasis, immune system regulation and cancer chemoprevention［J］. J Steroid Biochem Mol Biol, 2014, 144 Pt A: 74-80.

［16］Tan ZX, Chen YH, Xu S, et al. Calcitriol inhibits bleomycin-induced early pulmonary inflammatory response and epithelial-mesenchymal transition in mice［J］. Toxicol Lett, 2016, 240(1):161-171.

［17］Stefani G, Slack FJ. Small noncoding RNA-s in animal development［J］. Nat Rev Mol Cell Biol, 2008, 9(3):219-230.

［18］Chung AC, Dong Y, Yang W, et al. Smad7 suppresses renal fibrosis via altering expression of TGF-beta/Smad3-regulated microRNA-s［J］. Mol Ther, 2013, 21(2):388-398.

［19］Zhang Y, Huang XR, Wei LH, et al. miR-29b as a therapeutic agent for angiotensin II-induced cardiac fibrosis by targeting TGF-beta/Smad3 signaling［J］. Mol Ther, 2014, 22(5):974-985.

［20］Maurer B, Stanczyk J, Jungel A, et al. MicroRNA-29, a key regulator of collagen expression in systemic sclerosis［J］. Arthritis Rheum, 2010, 62(6):1733-1743.

［21］Sengupta S, den Boon JA, Chen IH, et al. MicroRNA-29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNA-s encoding extracellular matrix proteins［J］. Proc Natl Acad Sci USA, 2008, 105(15):5874-5878.

［22］Cushing L, Kuang PP, Qian J, et al. miR-29 is a major regulator of genes associated with pulmonary fibrosis［J］. Am J Respir Cell Mol Biol, 2011, 45(2):287-294.

［23］Cushing L, Kuang PP, Lü J. The role of miR-29 in pulmonary fibrosis［J］. Biochem Cell Biol, 2015, 93(2):109-118.

[1]	谢惠兰方芳林毅. Chir99021调控Wnt/β-catenin 信号通路抑制大鼠牙髓干细胞成骨诱导分化的机制[J]. 解剖学报, 2024, 55(1): 67-72.
[2]	魏茜茜李超红赵晨露唐家萍刘玉珍. 大鼠颈上神经节中Ⅰ组代谢型谷氨酸受体表达及慢性间歇性低氧对其的影响 [J]. 解剖学报, 2024, 55(1): 3-9.
[3]	袁蕾杨智伟杨惠刘政海何洁万炜. 三七总皂苷抑制小鼠星形胶质细胞活化缓解完全弗氏佐剂所致炎性痛 [J]. 解剖学报, 2024, 55(1): 25-31.
[4]	郑丽平刘霞杜晓华吴亚娟刘珊珊 . 脑源性神经营养因子在牦牛与黄牛端脑不同区域的表达与分布 [J]. 解剖学报, 2024, 55(1): 10-16.
[5]	苏芃王兴仪梁景岩熊天庆. 一种低密度及高纯度胎鼠原代海马神经元培养方法的优化及鉴定[J]. 解剖学报, 2024, 55(1): 113-119.
[6]	郝永明郭磊周程辉裴汉军李莉赵哲 . 改良腹主动脉缩窄法建立心肌肥厚模型[J]. 解剖学报, 2024, 55(1): 120-124.
[7]	许亚平余悦欣陈金福王柯柯郭志坤 . 高龄大鼠心室肌间质弹性纤维和胶原纤维的结构分布特征及其机制 [J]. 解剖学报, 2023, 54(6): 716-721.
[8]	刘丽平朱梦妮徐慧 . 白细胞介素6对脂多糖诱导所致类子痫前期大鼠有核红细胞的影响 [J]. 解剖学报, 2023, 54(6): 722-729.
[9]	赵卫明李晓余国营王改平常翠芳 . 丝氨酸肽酶抑制因子Kazal型1对肝癌细胞增殖的影响及其机制 [J]. 解剖学报, 2023, 54(6): 695-702.
[10]	王文娟丁雨桐苏昊任捷孙艳荣王涵菲陆嘉莉张林倩白钰秦丽华. 低雌激素状态下大鼠海马及纹状体Nogo-A的表达变化[J]. 解剖学报, 2023, 54(6): 620-627.
[11]	张琴杨俊霞蒋青松. 福辛普利对糖尿病视网膜病变小鼠血管紧张素转化酶2和血管内皮生长因子的影响[J]. 解剖学报, 2023, 54(6): 628-634.
[12]	梁盼盼禹爱梅杜静寇文辉王欢欢宋爱霞. 基于c-Jun氨基末端激酶/p38丝裂原活化蛋白激酶信号通路探讨阿魏酸钠对偏头痛大鼠炎症反应的抑制作用[J]. 解剖学报, 2023, 54(6): 652-659.
[13]	姚素华鄢骏谢芳林春华黄争春 . 奥氮平介导环磷酸腺苷反应元件结合蛋白/脑源性神经营养因子/酪氨酸蛋白激酶受体B通路对精神分裂模型大鼠的神经修复作用 [J]. 解剖学报, 2023, 54(6): 660-667.
[14]	徐颖刘斌郑兴何宗钊. 补体C3a受体在盲肠结扎穿孔致脓毒症大鼠认知障碍中的作用及机制 [J]. 解剖学报, 2023, 54(6): 668-675.
[15]	李霞朱世杰唐中生罗亚非范瑞娟谢高宇寇云芳陆莹 . 电针智三针调节酪氨酸激酶受体的配体通路改善血管性痴呆的作用机制[J]. 解剖学报, 2023, 54(6): 689-694.