Study On The Regulation Of MiR-126 On VEGF In Rats With Pulmonary Fibrosis And The Intervention Of Jiawei Buyang Huanwu Decoction Based On Angiogenesis

Objective:Pulmonary fibrosis is a common outcome of many different causes of interstitial lung disease.It is typically characterized by excessive deposition of extracellular matrix proteins,angiogenesis and remodeling,and irreversible scarring of the lung parenchyma,leading to reduced gas exchange and impaired lung function.The purpose of this study was to observe the angiogenesis of bleomycin-induced pulmonary fibrosis in rats,and to explore the protective effects of Jiawei Buyang Huanwu Decoction on pulmonary fibrosis in vivo and in vitro through miR-126 and VEGF.The role and mechanism are intended to provide new ideas and methods for the treatment of pulmonary fibrosis.Methods:1.In vivo experiments: Healthy male clean-grade SD rats,age 7 weeks,weight 180 ±20g.Randomly divided into control group,model group,prednisone group,Buyang Huanwu Decoction group and Jiawei Buyang Huanwu Decoction group.A pulmonary fibrosis model was established using the bleomycin tracheal instillation method.24 hours after modeling,the control group was administered with saline,the prednisone group was administered with prednisone suspension(4.2mg/kg?d),and the Buyang Huanwu Decoction group was treated with Buyang Huanwu Decoction(4.9ml/kg?d)was administered to the stomach,and the Jiawei Buyang Huanwu Decoction group was administered with Jiawei Buyang Huanwu Decoction(7.3ml/kg?d).Animals in each group were sacrificed in random batches on days14 and 28.The lung tissues of the rats were taken.HE staining was used to observe the pathological changes in the lung tissue.Masson staining was used to observe the collagen content,distribution,and morphological changes in the lung tissue.Pulmonary blood vessels were labeled with VIII factor staining,the number of microvessels was counted under a microscope,the microvessel density(MVD)was measured,the levels of miR-126 and VEGF m RNA in lung tissue were detected by RT-PCR,and the expression of VEGF protein in lung tissue was detected by Western blot and immunohistochemistry.Immunohistochemical method was also used to compare the expression of HIF-lα protein in lung tissue of each group.2.In vitro experiment One: miR-126 mimics or inhibitors and their negative controls were added to MRC-5 stimulated by TGF-β1 to investigate relationship of miR-126 and VEGF.MRC-5 was randomly divided into 6 groups: control group,model group,miR-126 mimics group,miR-126 mimics NC group,miR-126 inhibitor group,miR-126 inhibitor NC group.Cells were collected 48 hours later,and cell proliferation was detected by MTT assay.The control group and model group were tested for the expression of α-SMA and fibronectin gene by RT-PCR to verify the effect of modeling.The levels of miR-126 and VEGF m RNA were detected by RT-PCR in all 6 groups.The levels of VEGF,collagen I,and collagen III in each group were compared by Western blot,and the expressions of collagen I and collagen III were observed by immunofluorescence.Two: To explore the effect of Jiawei Buyang Huanwu Decoction serum on MRC-5 miR-126,HIF-1α m RNA and VEGF induced by TGF-β1.MRC-5 was randomly divided into 4 groups: the control group,the model group,the Normal serum group,and the Jiawei Buyang Huanwu Decoction serum group.RT-PCR was used to detect the levels of miR-126,HIF-1α m RNA and VEGF m RNA in MRC-5.Western blot was used to compare the expression of VEGF and collagen I in each group.Results:In vivo experiments:(1)HE and Masson staining results show that Jiawei Buyang Huanwu Decoction can significantly reduce inflammation and fibrosis in lung tissue of fibrotic rats,and the effect is better than Buyang Huanwu Decoction;(2)Ⅷ factor staining results showed that bleomycin induced pathological angiogenesis in rats with pulmonary fibrosis and increased microvessel density;Jiawei Buyang Huanwu Decoction can significantly inhibit pathological angiogenesis in lung tissue of fibrotic rats;(3)Jiawei Buyang Huanwu Decoction can reduce the expression of miR-126,VEGF m RNA and protein,and HIF-1α protein in the lung tissue of fibrotic rats.In vitro experiments:(1)miR-126,VEGF m RNA and protein expression were significantly increased in fibrotic cell models;(2)miR-126 mimics can increase VEGF m RNA and protein levels,and promote collagen synthesis;miR-126 inhibitor can inhibit the expression of miR-126,VEGF m RNA and protein,and reduce collagen synthesis;(3)Jiawei Buyang Huanwu Decoction serum can reduce collagen synthesis of MRC-5 caused by TGF-β1;(4)Jiawei Buyang Huanwu Decoction serum can reduce miR-126,HIF-1α,VEGF m RNA and protein expression level in MRC-5 caused by TGF-β1.Conclusion:Jiawei Buyang Huanwu Decoction has a significant protective effect on rats with bleomycin-induced pulmonary fibrosis,and the effect is better than Buyang Huanwu Decoction.The mechanism of action may be related to the inhibition of miR-126/VEGF expression and thus inhibit fibrosis pathological angiogenesis associated with lung tissue.