Study On The Mechanism Of The Interaction Between Notchl1 And TGF-?1/Smad3 Signaling Pathway Inhibiting Myocardial Fibrosis

Chapter 1 IntroductionIt is myocardial infarction(MI)is a major cause of death.Myocardial fibrosis after a myocardial infarction is a critical step in heart repair,but excessive fibrosis can lead to heart disease and even death.Therefore,restricting post-mi fibrosis is important for the prevention and treatment of heart disease.Approximately 60-70% of the total heart cells are cardiac fibroblasts(CFs).Under normal conditions,CFs remains silent,secreting extracellular gels that maintain structural integrity and the normal function of the heart.After MI,CFs is activated,amplified and transformed into myofibroblasts,which are highly expressed in the apoptosis-sma,resulting in excessive collagen deposition in the myocardium of fibrosis.The transformation from CF to myofibroblast(CMT)is the initial step of myocardial fibrosis after MI,and the targeting of CMT can provide new hope for preventing myocardial fibrosis.Transforming growth factor ?1(TGF-?1)is a key regulatory factor for cardiac repair and fibrosis after MI.During MI,the level of TGF-?1 increased significantly,and it promoted CMT through TGF-?1/Smad3(Small mother against decapentaplegics)signaling pathway,while inhibiting TGF-?1/Smad3 signaling pathway could inhibit CMT,and improve cardiac function after MI.Notch1 signaling has been associated with tissue fibrosis under various conditions,such as scleroderma,pulmonary fibrosis,renal fibrosis,liver fibrosis and cardiac fibrosis.In particular,the Notch1 signaling plays an important role in heart repair.After cardiac ischemia,the Notch1 signaling is activated to induce angiogenesis to improve blood flow supply,stimulate CFs amplification,reduce the generation of reactive oxygen in myocardium,inhibit the apoptosis of cardiac cells,and alleviate the ischemia reperfusion injury(IRI).Interestingly,recent studies have reported an interaction of the signals between Notch1 and TGF-?1/Smad3 in tissue fibrosis such as pulmonary fibrosis and renal fibrosis.However,it is still unclear whether Notch1 signaling can interact with TGF-?1/Smad3 signaling to regulate myocardial fibrosis.In this study,we aimed to study the interaction of these two important signaling pathways during myocardial fibrosis and identify new therapeutic targets for heart disease.Chapter 2 The regulation of Notch1 and TGF-?1 signaling pathway in myocardial fibrosisObjective: Adenovirus vectors used for overexpression or underexpression of N1 ICD were constructed to explore whether the effect of TGF-?1 on CFs in mice was antagonised by the Notch1 signaling.Adenovirus vectors for Smad3 overexpression or knockdown expression were constructed to investigate whether the effect of TGF-?1 on CFs in mice was mediated by Smad3.Methods: The effect of TGF-?1 on the expression of fibroblast labeled Vimentin(Vimentin),disk domain receptor 2(DDR2),spf-sma and Tensin(Tensin)was observed by CFs of mice treated with TGF-?1.The influence of tgf-g1 on the Notch1 signaling in CFs of mice was studied by Western blotting analysis.CCK8 and Brd U assay were used to determine the effects of TGF-?1 treatment on CFs amplification.The effects of TGF-?1 on CFs invasion were measured by Transwell assay.Whether TGF-?1 treatment and overexpression or underexpression of N1 ICD,Smad3 has an effect on CFs adhesion and collagen I secretion.Results: Western blotting analysis showed that TGF-?1 decreased expressions of Notch1,N1 ICD,DII1 and DII4 in CFs.CCK8 and Brd U experiments showed that TGF-?1 treatment increased the amplification of CFs.Knockdown N1 ICD without TGF-?1 stimulation increases the amplification of CFs,while overexpression of N1 ICD significantly reduces the effect of TGF-?1 on CFs.The results of Transwell experiment indicated that TGF-?1 increased CFs invasion.Knockdown N1 ICD expression without TGF-?1 treatment enhances CFs invasion,while overexpression of N1 ICD inhibits CFs invasion induced by TGF-?1 treatment.TGF-?1 treatment increases CFs adhesion.In the absence of TGF-?1 treatment,knockdown N1 ICD expression enhances CFs adhesion,while overexpression of N1 ICD inhibits CFs adhesion induced by TGF-?1 treatment.Similarly,TGF-?1 treatment increases the secretion of type I collagen in CFs.Knockdown N1 ICD expression increases the secretion of type I collagen without TGF-?1 treatment,while overexpression of N1 ICD inhibits the secretion of type I collagen induced by TGF-?1 treatment.If not treated by TGF-?1,Smad3 overexpression or underexpression does not affect the biological behavior of CFs.However,knockdown Smad3 expression significantly reduced TGF-?1 induced amplification,invasion,adhesion and secretion of type I collagen.Conclusions: The effect of TGF-?1 on CFs in mice was opposite to the Notch1 signaling.The effect of TGF-?1 on CFs in mice was mediated by Smad3.Chapter 3 Notch1 obstructs TGF-?1/Smad3 signaling transduction and inhibits myocardial fibrosisObjective: To clarify the interference of TGF-?1/Smad3 and Notch1 signaling in the management of biological behavior of CFs,and to explore the molecular mechanism of TGF-?1/Smad3 and Notch1 signaling antagonism in CFs of mice.The luciferase reporter gene of gamma-sma promoter was constructed and the luciferase was determined to better understand how TGF-?1/Smad3 and the Notch1 signaling antagonized the regulation of CMT.Methods: Immunoprecipitation of N1 ICD and Smad3 in CFs was studied.Immunofluorescence staining experiments confirmed the co-localization of N1 ICD and Smad3 in CFs nucleus.Western-blottingting detected the protein expression of Vimentin,DDR2,and prism-sma and Tensin,which are markers of fibroblasts.CCK 8 and Brd U experiment,Transwell experiment,cell invasion detection such as N1 ICD knock and Smad3 expression and lower expression to the biological behavior of CFs,proliferation,invasion and the influence of the adhesion and ? collagen secretion.The effect of overexpression and underexpression of N1 ICD and Smad3 on luciferase activity was detected by luciferase.Results: Immune coprecipitation experiments show that interaction between N1 ICD and Smad3 and immunofluorescence staining revealed N1 ICD and Smad3 in CFs intranuclear positioning.By the way,the expression of N1 ICD waveform by fibroblasts markers protein(Vimentin)and DDR2 m RNA expression,but this has been the Smad3 antagonism Vimentin m RNA expression of Vimentin and DDR2.N1 ICD downgrades,but this is expression on low Smad3 antagonism.On the other hand,the m RNA expression of the myofibroblasts and Tensin protein(Tensin)was down-regulated by N1 ICD overexpression,but this was antagonised by Smad3 overexpression.In addition,the m RNA expression of gamma-sma and Tensin was up-regulated by N1 ICD knock,but this was antagonised by Smad3 knock against Western blotting analysis showed that fibroblasts markers and muscle fibroblasts marker protein expression changes of the same pattern,in CFs N1 ICD too expression inhibits N1 ICD expression on low invasion of adhesion promotes the proliferation and secretion of collagen,they have respectively been Smad3 low expression and Smad3 knock the antagonism N1 ICD expression significantly inhibits the TGF-?1 induced luciferase activity,inhibitory effect been Smad3 expression On the other hand,knockdown N1 ICD expression can significantly enhance the activity of luciferase induced by TGF-?1,which is inhibited by Smad3 expression knockdown.Conclusions: Antagonistic effects of TGF-?1/Smad3 and Notch1 signaling in CFs of mice;TGF-?1/Smad3 and Notch1 signaling regulate CFs biological behavior in reverse.TGF-?1/Smad3 and Notch1 signaling antagonically regulate transcription of ?-SMA in CFs.Chapter 4 Validation of Notch1 regulating myocardial fibrosis through inhibiting TGF-?1/Smad3 signaling In vivoObjective: To investigate the regulatory effects of TGF-?1/Smad3 and Notch1 signaling on myocardial fibrosis in mouse MI model.Methods: The MI model of rats was established by 8w large male sprague-dawley(SD)rats to ligate the left anterior descending artery(LAD),and the effect of Smad3 and N1 ICD overexpression and knock low expression on cardiac function recovery,CMT and collagen synthesis was detected by Western-blotting.Results: Overexpression of N1 ICD significantly improved cardiac function recovery after heart attack,weakened CMT,and inhibited collagen synthesis.In contrast,N1 ICD expression knockdown significantly worsened cardiac function damage after a heart attack,enhanced CMT,and increased collagen synthesis.Conclusions: TGF-?1/Smad3 and Notch1 signaling antagonically modulated myocardial fibrosis in mouse models of myocardial infarction.