The Role Of DNA Demethylase TET2 In The Development Of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis(IPF)is a chronic,progressive and diffuse interstitial lung disease with unknown etiology,which will endanger human physical and mental health,and finally lead to respiratory failure and death.At present,there are insufficient treatment methods and drugs for pulmonary fibrosis,and it is urgent to find new prevention and treatment targets.DNA methylation is a reversible epigenetic modification mechanism to maintains its dynamic balance mainly through methylation and demethylation in vivo,which is involved in the occurrence and development of pulmonary fibrosis.TET2 is a DNA demethylase whose role in pulmonary fibrosis has not been reported.Objective:This paper mainly observes the role of TET2 in the occurrence and development of pulmonary fibrosis,and explore its potential regulatory mechanism preliminarily,for the purpose of providing a new theoretical basis for the prevention and treatment of pulmonary fibrosis.Method:1 Effects of overexpression of TET2 on bleomycin-induced pulmonary fibrosis in mice.The mouse pulmonary fibrosis model was established by tracheal injection of bleomycin(BLM),and the m RNA levels and protein expression of TET2 and IDAX in lung tissue were detected at 7 days and21 days after modeling.After TET2 overexpression virus was expressed in lung tissue for 30 days,established BLM-induced pulmonary fibrosis model,and collected bronchoalveolar lavage fluid and lung tissue at 7 days.TNF-α,IL-1βand IL-10 were detected by q-PCR and ELISA.The pathological changes of lung tissue were observed by HE staining,the number of leukocytes in the bronchoalveolar lavage fluid was counted by hemocytometer,and the protein concentration in the bronchoalveolar lavage fluid was detected by BCA,and evaluated the pathological condition of lung tissue by HE and Masson staining at 21 days.The histopathological changes ofα-SMA and Vimentin were observed by immunohistochemistry,and the expressions ofα-SMA,ColⅠ,ColⅢand FN were detected by Western blot and q-PCR.2 Effects of overexpression and knockdown of TET2 on transdifferentiation of Human fetal lung fibroblasts1(HFL1).(1)Effects of TGF-β1 on TET2 in HFL1.The human fetal lung fibroblasts1(HFL1)was treated with 2 ng/ml TGF-β1,after 48h,detected the expression of TET2 and IDAX by Western blot.(2)Effects of overexpression and knockdown of TET2 on transdifferentiation of Human fetal lung fibroblasts1(HFL1).HFL1 cells were infected with TET2 overexpression and knockdown virus at 1×10~7TU/ml respectively,after 72h,they were treated with 2 ng/ml TGF-β1 for 48h,and then extracted total protein,detected the expression ofα-SMA,ColⅠ,ColⅢand FN by Western blot.(3)The effect of TGF-β1/Smad3-IDAX signaling pathway on the expression of TET2 in fibroblasts.HLF1 cells were treated with 2 ng/ml TGF-β,and after 15 min,detected the expression of Smad3 and P-Smad3 by Western blot.Then,the HFL1 cells were treated with 20μm Smad3 inhibitor(SIS3)for 18h,and treated with 2 ng/ml TGF-β1 for 48h,detected the expression of TET2 and IDAX by Western blot.Result:1 Effects of overexpression of TET2 on bleomycin-induced pulmonary fibrosis in mice.By post-BLM day 7 and day 21,the lung tissue had both decreased TET2 m RNA and protein,and both increased IDAX m RNA and protein.These results showed that during the progress of pulmonary fibrosis,the expression level of TET2 was decreased and the expression level of IDAX was increased.After overexpression of TET2,the lung tissue damage was alleviated during the inflammatory period,the expressions of TNF-αand IL-1βwas decreased,and the expression of IL-10 was increased,and the number of leukocytes and the protein concentration in the bronchoalveolar lavage fluid also decreased.These results suggested that overexpression of TET2can reduce the degree of damage and inflammation in the lung tissue of mice during the period of inflammation.After overexpression of TET2,the injury of lung tissue was alleviated,the deposition of collagen and the expression ofα-SMA,ColⅠ,ColⅢwere decreased significantly,and immunohistochemical showed the positive areas ofα-SMA and vimentin were reduced,which implied that overexpression of TET2 can reduce the degree of damage and fibrosis of lung tissue in mice during the period of pulmonary fibrosis.2 Effects of overexpression and knockdown of TET2 on transdifferentiation of human lung fibroblasts.(1)Effects of TGF-β1 on TET2 in HFL1.After treatment of HFL1 with 2 ng/ml TGF-β1 for 48 h,TET2 protein expression decreased and IDAX protein expression increased.The results suggest that TGF-β1 down-regulates the expression of TET2 in HFL1.(2)Effects of overexpression and knockdown of TET2 on transdifferentiation of human lung fibroblasts.After overexpression of TET2,HFL1 was stimulated with TGF-β1,and the expressions of Col I and Col III were significantly decreased.The results suggest that overexpression of TET2 can alleviate the transdifferentiation of HFL1.After knockdown of TET2,HFL1 was stimulated with TGF-β1,and the expressions of Col I,Col III and FN were significantly increased.The results suggest that knockdown of TET2 can promote the transdifferentiation of HFL1.(3)The effect of TGF-β1/Smad3-IDAX signaling pathway on the expression of TET2 in fibroblasts.After the addition of Smad3 inhibitor,the expression of IDAX decreased and the expression of TET2 increased.The results suggest that TGF-β1 may regulate the expression of TET2 through the TGF-β1/Smad3-IDAX signaling pathway,thereby affecting the transdifferentiation of HFL1.Conclusion:1.The expression of TET2 was decreased during BLM-inducedpulmonary fibrosis,and overexpression of TET2 can alleviateinflammatory response and fibrotic changes of BLM-inducedpulmonary fibrosis in mice.2.TGF-β1 can downregulated the expression of TET2 in fibroblastsvia regulated by TGF-β1/Smad3-IDAX signal pathway.Overexpression of TET2 can inhibited the transdifferentiation ofHFL1,while knockdown of TET2 can promote thetransdifferentiation of HFL1.