Study On The Effect Of D-gal-induced Increase Of Senescent Cells On Cardiac Fibrosis In Mice

Cardiac fibrosis is characterized by a net accumulation of extracellular matrix in heart,which is the pathological change of most heart diseases.Aging is one of the causes of heart fibrosis.With the increase of age,collagen deposition in the heart gradually increases,which ultimately affects heart function.D-galactose（D-gal）can accelerate the aging of young animals and can induce stress-induced premature senescence of cells.The D-gal injection can cause cardiac fibrosis in young rats,but the effect of D-gal on cardiac fibrosis in aged rodents has not been studied.Therefore,the purpose of this study is to investigate the effect of D-gal on cardiac fibrosis in young and aged mice,with a view to increasing the understanding of the effect of D-galactose,contribute to establish a more accurate aging model,and provide theoretical basis research for clinical treatment of cardiac fibrosis,especially for the elderly patients.In this study,D-gal with a dose of 125 mg/kg bw was administered subcutaneously to young and aged C57B/6 mice for 6 weeks.Samples were collected after anesthesia.First assess the effects of D-gal on senescence markers:Western blot was used to detect the expression levels of p53,p21,p16^INK4a,p Rb,andγ-H2Ax protein,fluorescence quantitative PCR was used to detect the m RNA level of GLB1,and immunohistochemistry was used to locate p16^INK4a.Then the effect of D-gal on cardiac function was evaluated:use cardiac echocardiography to detect changes in cardiac function parameters EF,FS,IVSd,IVSs,LVWPd,and LVWPs.Then evaluate the effect of D-gal on cardiac fibrosis:H&E staining and Sirius scarlet fast green staining were used to detect the degree of fibrosis,and fluorescence quantitative PCR and Western blot were used to detect the expression changes of Col-Ⅰand Col-Ⅲm RNA and protein.Next,the D-gal induced senescence cell types were evaluated:immunofluorescence was used to detect the colocalization of p16^INK4a labeled senescent cells with Troponin T labeled cardiomyocytes,Vimentin labeled fibroblasts andα-SMA labeled fibroblasts.Finally,evaluate the effect of D-gal on SASP:fluorescence quantitative PCR to detect the expression levels of TGF-β1,MMP-1,MMP-2,MMP-9,and MMP-13 m RNA,and Western blot to detect MMP-1,MMP-2,MMP-9,and MMP-13 protein expression changes.The results showed that compared with the Young group,the Aged group had significantly higher levels of senescence markers p53,p21,p16^INK4a andγ-H2Ax protein expression,significantly decreased p Rb protein level,and significantly increased GLB1 m RNA level;EF and FS decreased significantly,IVSd,IVSs,and LVPWd increased significantly,LVPWs did not change significantly;pathological section staining showed increased collagen fibers around blood vessels;Col-Ⅰand Col-Ⅲprotein increased in heart tissue,but TGF-β1 m RNA and Col-Ⅰand Col-Ⅲm RNA expression decreased;MMP-1,MMP-2,MMP-9 and MMP-13 m RNA and protein expression decreased.After D-gal was administered to young and aged mice,the expression levels of senescence markers p53,p21,p16^INK4a andγ-H2Ax protein in the heart were significantly increased,p Rb protein level was significantly decreased,and GLB1 m RNA level was significantly increased.Compared with the Young group,Young+D-gal group had significantly lower EF and FS,and increased IVSd,IVSs,and LVPWd,but the difference was not significant;perivascular fibrosis increased;TGF-β1,Col-Ⅰ,and Col-Ⅲm RNA and protein expression increased;MMP-1,MMP-2,MMP-9,and MMP-13m RNA and protein expression decreased.Interestingly,compared with the Aged group,the Aged+D-gal group had significantly higher EF and FS,significantly lower IVSd and LVPWd,and decreased IVSs,but no significant difference;H&E staining and Sirius red/fast green staining showed reduced perivascular fibrosis.TGF-β1,Col-Ⅰ,and Col-Ⅲm RNA and protein levels in cardiac tissue are significantly reduced;most senescent cells express fibroblast markers;MMP-1,MMP-2,MMP-9,and MMP-13 m RNA and protein expression levels increased.In conclusion,D-gal can increase the number of senescent cells in the heart of young and aged mice,and both p53-p21and p16^INK4a-p Rb signaling pathways are involved in this process.D-gal-induced increase in senescent cells regulates the content of collagen in the heart by secreting different levels of TGF-β1 and MMP-1,MMP-2,MMP-9,MMP-13,resulting in cardiac fibrosis in young mice and reduced cardiac fibrosis in aged mice.