| Objective:The role of LncRNA RP11-413M3.4,Notch1 in the pathogenesis of idiopathic pulmonary fibrosis was explored through in vivo and in vitro studies.Methods:1.The study in vitro was designed to induce myofibroblast model induced by TGF-?1 intervention in fibroblasts.The cells were divided into two groups: N group was normal control group,and TGF-? group was model group.All cells were starved for 12 h to synchronize them before modeling.Cells were harvested after cells were treated with 5 ng/ml TGF-?1 for 24 h,and cells in N group were treated with equal volume of PBS for 24 h.Masson staining was used to detect the content of collagen fibers in the two groups of cells.The expression of Notch1 protein was detected by Western Blot,and the expression of LncRNA RP11-413M3.4 and Notch1 mRNA was detected by RT-PCR in both groups.2.The study in vivo was designed to induce IPF model by intratracheal injection of bleomycin(BLM)in C57BL/6 mice.Twenty-four mice were randomly divided into three groups according to a random number table: N group was normal control group,I group was IPF model group,I+D group was DAPT intervention group,and each group had 8 mice.In I group and I+D group,mice were injected intratracheally with 5 mg/kg of BLM,and mice in N group were injected with an equal volume of saline in the trachea.In I+D group,mice were injected with 5 mg/kg DAPT intraperitoneally for 10 days after modeling for 7d.Mice were sacrificed by cervical dislocation after modeling 28 days,left lungs were frozen at-80°C for RT-PCR and Western Blot,right lungs were fixed with formaldehyde and embedded in paraffin for HE staining,Masson staining,immunohistochemistry and in situ hybridization.HE and Masson staining were used to show the histopathological changes of the three groups of mice and scored according to histopathological changes using the Ashcroft criteria.Western Blot and immunohistochemistry were used to detect the expression of Notch1 protein in the lungs of the three groups of mice.In situ hybridization was used to detect the expression of LncRNA RP11-413M3.4 in the three groups of mice.RT-PCR was used to detect the expression of LncRNA RP11-413M3.4 and Notch1 mRNA in the three groups of mice.Results:1.In vitro studies:(1)After intervention with TGF-?1,fibroblasts proliferated in large numbers,and the number of fibroblasts increased significantly,and their morphogenesis changed.They became thin and flat and differentiated into myofibroblasts.Collagen fibers produced by TGF-?1 intervention were significantly higher than those in N group,and the difference was statistically significant(P<0.05).This demonstrated that fibroblasts successfully induced to myofibroblast model with 5 ng/ml TGF-?1.(2)Western Blot results showed that compared with N group,the expression of Notch1 in TGF-? group increased,and the difference was statistically significant(P<0.05).RT-PCR results showed that LncRNA RP11-413M3.4,Notch1 mRNA expression increased,the difference was statistically significant(P <0.05).Linear correlation analysis showed that the expression of LncRNA RP11-413M3.4 and Notch1 mRNA in the cells was linearly correlated,and the correlation coefficient r was 0.8902,which was statistically significant(P<0.05).2.In vivo study:(1)HE staining showed that a large number of alveolar structures were destroyed,some alveolar collapsed and the alveolar septum thickened in the lung tissue of I group.Masson staining of alveolar space and interstitial muscle fibroblasts proliferated and accumulated,showing a large number of broadband and flaky collagen fibers,showing diffuse fibrosis changes.The destruction of alveolar structure and fibrosis in mice of I+D group were lighter than those in I group.Compared with group N,the Ashcroft score of I group was significantly increased,and there was a significant fibrosis change,suggesting that I group mice using BLM induction of IPF model is feasible.The Ashcroft score was significantly lower in the I+D group compared with the I group,suggesting that DAPT can reduce the degree of pulmonary fibrosis.(2)Results of Western Blot showed that compared with N group,the expression of Notch1 protein in I group increased,and the difference between groups was statistically significant(P<0.05).Compared with I group,the expression of Notch1 protein in I+D group was decreased,and the difference between groups was statistically significant(P<0.05).(3)Results of RT-PCR showed that compared with N group,LncRNA RP11-413M3.4,Notch1 mRNA expression in I group increased,the difference between the groups was statistically significant(P<0.05).Compared with I group,the expression of LncRNA RP11-413M3.4 and Notch1 mRNA in I+D group was decreased,and the difference between groups was statistically significant(P<0.05).Correlation analysis showed that LncRNA RP11-413M3.4 and Notch1 mRNA were linearly correlated,and the correlation coefficient r was0.6259,which was statistically significant(P<0.05).(4)The results of immunohistochemistry indicated that Notch1 was mainly expressed in the cytoplasm of bronchial epithelial cells and alveolar cells,and the results of in situ hybridization suggested that LncRNA RP11-413M3.4 was also expressed in the cytoplasm.The results of in situ hybridization and immunohistochemistry showed that compared with group N,the expression of LncRNA RP11-413M3.4 and Notch1 protein in I group increased,and the difference between groups was statistically significant(P<0.05).Compared with I group,the expression of LncRNA RP11-413M3.4 and Notch1 protein in the I+D group decreased,and the difference between the two groups was statistically significant(P<0.05).Correlation analysis showed that LncRNA RP11-413M3.4 was linearly related to the expression of Notch1 protein,and the correlation coefficient r was 0.7647,which was statistically significant(P<0.05).Conclusion:1.Under the action of cytokine TGF-?1,upregulation of LncRNA RP11-413M3.4 in fibroblasts may promote the up-regulation of the adjacent gene Notch1 expression,thereby promoting its proliferation and differentiation into myofibroblasts and producing a large number of collagen fibers.2.In the bleomycin-induced IPF mouse model,the up-regulated LncRNA RP11-413M3.4 in the cytoplasm may regulates the expression of the adjacent gene Notch1 in cis,thus promoting the development of pulmonary fibrosis,while DAPT can inhibit the expression of both and reduce the pulmonary fibrosis. |
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