The Mechanisms Of LSD1 On Neointima Formation In Mice And The Intervention Of Clostridium Butyricum CGMCC0313.1

Atherosclerosis(AS)is the leading cause of cardiovascular disease death worldwide at this stage,which is a chronic inflammatory disease characterized by neointima formation.The proliferation and migration of vascular smooth muscle cells(VSMC)contribute to neointima formation.Lysine-specific demethylase 1(LSD1)is an epigenetic modifier widely involved in cell proliferation.However,the role of LSD1 in VSMC proliferation and neointima formation is rarely studied,and the mechanism of LSD1 on neointima formation remains to be further studied.For this reason,this study based on "the relationship between LSD1 and neointima formation ",then focused on "the effects of LSD1 in the process of neointima formation in mice",and explored the potential intervention strategy of preventing or treating neointima formation.Firstly,this study examined whether LSD1 was upregulated in vascular tissue samples from neointima formation in human and mice;Secondly,we knocked down or overexpressed the LSD1 expression by lentivirus infection to explore the role and mechanism of LSD1 on neointima formation and VSMC proliferation in mice;In addition,we used LSD1inhibitor(R)-TML104 to further explore whether LSD1 is a potential target for neointima formation,and explored its impact on neointima formation and VSMC proliferation;Finally,to provide a theoretical basis for the precise intervention of neointima formation,we used Clostridium butyricum CGMCC 0313.1(CB0313.1)as an intervention strategy and investigated the effects of CB0313.1 on LSD1 expression and neointima formation in mice.The main conclusions of this study are as follows:1.LSD1 was upregulated in neointima formationTo explore the relationship between LSD1 and neointima formation,we based our study on clinical samples of patients with neointima formation and the model of neointima formation in mice,tissue immunofluorescence assay,and Western blot assay to evaluate the expression of LSD1 in the vascular tissue of stenotic patients,in vascular tissues of mice and platelet-derived growth factor BB(PDGF-BB)induced proliferative VSMC.The specific results are as follows:(1)Compared with normal human vascular tissue,LSD1 was upregulated in the vascular tissue of stenotic patients(p<0.01);(2)Compared with shamoperated mice,LSD1 was upregulated in vascular tissue in mice(p<0.01);(3)Compared with the blank control group,LSD1 was upregulated in VSMC proliferation(p<0.01).The above results indicated that the expression of LSD1 was correlated with neointima formation.2.LSD1 promoted VSMC proliferation by regulating the expression of p21To explore the effect and mechanism of LSD1 on neointima formation in mice,we based our study on the model of neointima formation in mice,hematoxylin-eosin(H&E)assay,tissue immunofluorescence staining assay,Western blot assay,5-Ethynyl-2’-Deoxyuridine(Ed U)assay and Transwell assay to evaluate the expression of knockdown or overexpression of LSD1 on neointima formation,the proliferation and migration of VSMC in mice.The specific results are as follows:(1)LSD1 knockdown reduced neointimal area and inhibited the expression of Cyclin D1 and proliferating cell nuclear antigen(PCNA)in vivo(p<0.01),LSD1 overexpression promoted VSMC proliferation and migration in mice(p<0.01).(2)LSD1 knockdown significantly inhibited the PDGF-BB-induced the proliferation and migration of VSMC in vitro(p<0.01),and LSD1 overexpression promoted PDGF-BBinduced VSMC proliferation and migration(P<0.01).(3)LSD1 may promoted VSMC proliferation by regulating Histone-3 lysine-4 di-methylation(H3K4me2)to reduce p21 expression.The above results showed that LSD1 promoted neointima formation in mice.3.LSD1 inhibitors inhibited VSMC proliferation and neointima formation in miceTo explore the effect and mechanism of LSD1 on neointima formation in mice,we based our study on the model of neointima formation in mice,H&E assay,tissue immunofluorescence staining assay,Western blot assay,Ed U assay and Transwell assay to evaluate the effect of LSD1 inhibitor resveratrol analogs((R)-4,6-dimethoxy-3-(4-methoxy phenyl)-2,3-dihydro-1H-undone [(R)-TML104])on neointima formation,the proliferation and migration of VSMC in mice.The specific results are as follows:(1)(R)-TML104 reduced the area of injury-induced neointima formation and inhibited the expression of Cyclin D1 and PCNA in vivo(p<0.01).(2)(R)-TML104 significantly inhibited the PDGF-BB-induced proliferation and migration of VSMC in vitro(p<0.01).(3)(R)-TML104 decreased the production of reactive oxygen species(ROS)by increasing the expression of sirtuin 1(SIRT1),and inhibited VSMC proliferation in mice(p<0.01);(4)(R)-TML104 reduced nuclear factor kappa B(NF-κB)acetylation by inhibiting NADPH oxidase 4(NOX4)expression,thereby reducing the production of ROS(p<0.01).The above results showed that(R)-TML104 could have a certain inhibitory effect on neointima formation in mice.4.Clostridium butyricum CB0313.1 inhibited neointima formation in miceTo investigate the effect of Clostridium butyricum CB0313.1 on the LSD1 expression and neointima formation,we based our study on the model of neointima formation,H&E assay,the immunofluorescence assay to observe the effect of Clostridium butyricum on neointima formation,the expression of proliferating cell marker,intestinal tight junction protein and the serum lipopolysaccharide(LPS).The specific results are as follows:(1)Clostridium butyricum CB0313.1 reduced the area of neointima formation and the expression of PCNA and LSD1(p<0.01).(2)Clostridium butyrate CB0313.1 significantly inhibited the neointima formation in mice by increasing butyric acid(p<0.01)(3)Clostridium butyricum CB0313.1 significantly reduced the intestinal barrier damage induced by high-fat diet in mice,significantly increased the expression of intestinal tight junction proteins ZO-1,ZO-2,and Occludin,and significantly decreased the level of serum LPS(p<0.01).The above results showed that Clostridium butyricum CB0313.1 could have a certain inhibitory effect on neointima formation in mice.In summary,this study aims to identify potential therapeutic targets and strategy for neointima formation,and systematically elucidates the mechanism of LSD1 in VSMC proliferation through the use of pharmacological methods.This study aims to explore the mechanism of LSD1 in VSMC proliferation from the perspective that LSD1 can regulate H3K4me2 methylation modification and affect p21 transcription.It is further demonstrated that LSD1 may be a potential target for neointima formation via the study of(R)-TML104.In terms of application,Clostridium butyricum CB0313.1 can reduce LPS translocation by alleviating intestinal barrier damage,and inhibit the neointima formation in mice.This study provides a new idea for LSD1 to become a potential target and provides a theoretical basis for the application of Clostridium butyricum CB0313.1 in the prevention of neointima formation.