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

doi:10.16098/j.issn.0529-1356.2017.01.014

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Acta Anatomica Sinica ›› 2017, Vol. 48 ›› Issue (1) : 78-86. DOI: 10.16098/j.issn.0529-1356.2017.01.014

Histology,Embryology and Developmental Biology

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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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

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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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