Study On The Relationship Between MUTYH Deficiency Or Aging And The Pathogenesis Of Pulmonary Fibrosis

Idiopathic Pulmonary Fibrosis(IPF)is the most common clinical interstitial lung disease.It is a typical aging-related disease with unknown etiology,and the age of onset is mostly over 60 years old.Clinical manifestations of IPF are progressive dyspnea accompanied by lung interstitial infiltration and restrictive ventilation impairment.The pathological characteristics show excessive extracellular matrix deposition and destruction of lung tissue structure,lung tissue appears fibrosis,honeycomb lungs,and fibroblast foci.Chest radiograph clearly suggests fibrotic lines,nets,honeycombs,etc.,and usually accompanied by traction bronchiectasis.In general,oxidative damage is one of the causes of IPF during aging.A large number of clinical observations found that the airway and bronchoalveolar lavage fluid of IPF patients with significantly elevated oxidative stress markers.Accumulating evidence shows that the oxidative damage/apoptosis of alveolar epithelial cells(AECs)plays an impaortant role in the development of IPF.Oxidative stress induce excessive production of reactive oxygen species(ROS),which directly damage a variety of biological macromolecules such as DNA.To ensure the integrity of genomic DNA,a variety of biochemical repair pathways has been developed to defense the DNA oxidative damage in cell.Base excision repair system(BER)is one of the most important DNA damage repair systems.MUTYH(human Mut Y homolog,MUTYH),as the main BER repair gene,is responsible for removing the A base that is mismatched with 8-oxoguanine(8-oxo-G),the product of oxidative damage during DNA replication.Our laboratory previous found that there is an Alu Yb8 insertion variant in MUTYH gene in the Chinese population,which can decrease the stability of mitochondrial DNA(mt DNA)with IPF patients and affect the clinical development and prognosis of IPF.In view of the key role of base excision repair genes and aging in the oxidative stress,we focused on the role of MUTYH gene or aging in the progress of IPF.Part 1:The role of MUTYH gene deficiency on the pathogenesis of pulmonary fibrosisThe incidence of IPF significantly increase with age,which is considered to the accumulation of age-related DNA oxidative damage.8-oxo-G is the most important product of DNA oxidative damage,which can cause mitochondrial dysfunction and multiple organ damage.8-oxo-G is a major cause of G:C to T:A transversions in genomic DNA.Under physiological condition,the oxidized lesion can be corrected by the BER system in cells.In the system,the OGG1 gene(oxoguanine glycosylase 1,OGG1)can remove 8-oxo-G produced by guanine oxidation in DNA,MUTYH removes adenine that is misincorporated opposite to 8-oxo-G in template DNA,which prevents the base substitutions of G:C to T:A in DNA replication.Notably,the BER system is essential for maintaining the fidelity of DNA replication.The Alu Yb8(Alu Yb8MUTYH)insertion mutation in MUTYH damage the stability of mt DNA in patients with IPF,and is related to the age of onset of IPF.In this study,we used Mutyh knockout mice and bleomycin(BLM)-induced pulmonary fibrosis model to test the effect of Mutyh deficiency on the progression of pulmonary fibrosis.Unexpectedly,a much less severe lesion of pulmonary fibrosis was observed in Mutyh knockout than in wide type mice,which was supported by assay on protein levels of tissue fibrosis and epithelial markers such as TGF-?1,?-SMA,Vimentin and E-cadherin in pulmonary tissues of the model animals.At the same time,we observed that Mutyh deficiency prevented the genomic DNA of pulmonary tissue cells from the buildup of single-strand breaks(SSBs)of DNA and maintained the integrity of mt DNA.In addition,we found that Mutyh deficiency could maintain mitochondrial homeostasis in the lung tissue of BLM induced mice and reduce alveolar epithelial cell apoptosis.Based on the results,we considered that MUTYH gene deficiency reduce the damage of external stimuli to lung tissue under severe oxidative stress,which can help us to understand the complexity of the phenotypic effects of MUTYH gene abnormality under different conditions.Part 2:Preliminary study on molecular basis of susceptibility to aging-related pulmonary fibrosisThe pathogenesis of IPF has not been fully elucidated.Accumulating evidence shows that repair function of oxidative damage is less efficient with age,resulting in the accumulation of DNA damage,which lead to aging-related pulmonary fibrosis under environmental stimulation.D-galactose(D-Gal)was used to successfully construct an aging mice model in our laboratory.In the present study,we firstly used relatively low doses of bleomycin to construct a pulmonary fibrosis in aging mice,and observed the characteristics of pulmonary fibrosis in aging mice.We also explored the mitochondrial stress-related regulatory mechanism in the progression of aging-related pulmonary fibrosis.The results suggested that aging or low dose of BLM induced a slight collagen deposition in the alveolar compartment,respectively;aging significantly aggravated the phenotype of BLM induced pulmonary fibrosis in mice.QPCR and Western blot results suggested that aging and BLM interactively promoted the activation of TGF-?1 signaling pathway in lung tissue of mice.At the same time,we also found that D-Gal dose dependently upregulated the expression levels of mitochondrial unfolded protein response(UPR^mt)genes mt Hsp70,Hsp60,Hsp10,revealing that aging can activate UPR^mt to compensate for normal mitochondrial function.However,we also observed that UPR^mt associated proteins were significantly reduced when D-Gal plus with low-concentration BLM,accompanied with the increased expression levels of mitochondrial fusion protein MFN2,and decreased fission expression levels of Fis1.Additionally,our results showed the imbalance of MTCO1/SDHA,which revealed the abnormality of mitochondrial steady in aging-related pulmonary fibrosis.Our study suggested that exogenous oxidative stress in aging mice could cause decompensation of UPR^mt,impaired mitochondrial proteostasis and dynamics,and eventually exacerbated the phenotype of pulmonary fibrosis.In summary,we demonstrated that aging and exogenous oxidative stress interactively disturb the expression levels of molecular chaperone mt Hsp70,and facilitate abnormality of UPR^mt,which lead to TGF-?1/Smad signal activation,aggravate the pulmonary fibrosis in mice.