| Background and Objective:Myocardial fibrosis is a common pathological manifestation of various cardiovascular diseases to the end stage.The activation of the renin-angiotensin-aldosterone system(RAAS)is the main cause of myocardial fibrosis,of which Angiotensin Ⅱ(Ang Ⅱ)and aldosterone are the main effectors of RAAS.Its main pathological features are excessive proliferation,migration,collagen deposition and abnormal distribution of myocardial interstitial fibroblasts,and there is currently no targeted treatment method with high cure rate.Neddylation-like ubiquitination modification is a new type of post-translational modification,which requires the joint action of E1 activating enzyme(NAE),E2 binding enzyme(UBC12,UBE2F),E3 ligase(a dozen kinds,including DCN1)and de NEDDase.NEDD abnormalities have been shown to be closely related to various diseases such as cancer,neurodegenerative diseases and congenital heart disease.DCN1 is a type of E3 ligase.DCN1 directly interacts with UBC12(one of E2)on the overlapping surface of the E1 binding site.Crystal structure analysis showed that the methionine at the N-terminus of UBC12 was acetylated and inserted into the hydrophobic vesicle of DCN1 together with the downstream amino acid residues to promote Cullins-like ubiquitination modification.The binding of DCN1-UBC12 can be destroyed by small molecule inhibitors,designed to selectively inhibit the ubiquitin-like modification of Cullin3,causing the accumulation of substrate Nrf2.The DCN1-UBC12 interaction is a promising research target.The effects of small molecule compounds targeting DCN1-UBC12 interaction on immune response and inflammatory factors have been reported,and it has been confirmed that myocardial fibrosis is closely related to oxidative stress inflammation.However,the effect and mechanism of targeting DCN1-UBC12 interaction inhibitors on myocardial fibrosis is not clear,and it is worth further discussion.Methods:This topic is dividedinto two parts.First,the cardiac pressure load induced myocardial fibrosis by aortic arch constriction(TAC),and whether the expression of DCN1 protein is involved in aortic constriction(TAC)-induced myocardial fibrosis is initially discussed.Secondly,based on the establishment of a stable screening system for DCN1-UBC12 in vitro enzyme levels in this laboratory,a better selection of small molecule compounds targeting DCN1-UBC12 was selected.By extracting neonatal rat myocardial fibroblasts and Ang Ⅱ-induced myocardial fibrosis model,molecular biology techniques were used to explore the anti-cardiac fibrosis effect and mechanism of compounds targeting DCN1-UBC12 interaction.Part Ⅰ: To explore whether DCN1 is involved in aortic coarctation(TAC)-induced myocardial fibrosis(1)Select healthy C57BL/6 mice and divide them into sham operation group(Sham),aortic arch constriction 4W group(TAC 4W)and aortic arch constriction 8W group(TAC 8W)group,detect cardiac function and calculate left ventricular ejection fraction(LVEF%),left ventricular short axis shortening rate(LVFS%),left ventricular late systolic posterior wall thickness(LVPWs),left ventricular end diastolic posterior wall thickness(LVPWd),lung weight/body weight(LW/BW)and lung weight /Tibia length(LW/TL).(2)Extract the whole heart protein and detect the expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ,DCN1,Nrf2,NQ-1 protein by Western Blot method.In the other part of the heart,paraformaldehyde was fixed and paraffin section immunofluorescence was used to detect the expression of DCN1 protein.Part Ⅱ: To explore the effect and mechanism of inhibitors acting on DCN1-UBC12 against myocardial fibrosis(1)Newborn neonate rats born with SD rats of 1-3 days were selected,isolated,extracted and cultured neonatal rat myocardial fibroblasts(CFs),and the cell purity and activity were identified by immunofluorescence with Vemintin antibody.Through MTT method,immunofluorescence experiment,scratch experiment,Transwell experiment,Western Blot to detect whether the compound targeting DCN1-UBC12 can effectively alleviate the myocardial fibrosis induced by Ang Ⅱ,and related mechanism pathway protein Nrf2,NQ-1 changes.(2)Construct si RNA targeting DCN1,and Western Blot again tested whether the compound targeting DCN1-UBC12 could effectively alleviate Ang Ⅱ-induced fibrosis,as well as changes in the related pathway proteins Nrf2 and NQ-1.(3)Preliminary evaluation of pharmacodynamics at the cellular level,screening of new DCN1-UBC12 compounds and losartan,DC-2(published small molecule compounds targeted to DCN1-UBC12)and screening by Western Blot experiment evaluation A new DCN1-UBC12 compound with similar structure but no targeted positive control compound compared the inhibitory effects of Ang Ⅱ-induced myocardial fibrosis.(4)Preliminary evaluation of the safety of DCN1-UBC12 targeted compounds in mice.Acute toxicity test was used.Compound 2 g/kg DN-2 and the same volume of corn oil were used as a control group.The body weight of mice was recorded during the experiment.Cardiac function was tested after 14 days and 14 days,and important organs were stained with Hematoxylin-Esoin 14 days later.The safety of DCN1-UBC12-targeted compounds in mice was preliminarily evaluated Results:Part Ⅰ: Myocardial hypertrophy occurred 4W after TAC,and cardiac function decreased in 8W after TAC;the protein expression of DCN1 was involved in aortic constriction(TAC)-induced myocardial fibrosis(1)After aortic arch narrowing in mice,the left ventricular ejection fraction(LVEF%)in the TAC 4W group was reduced by 24.58% compared with the Sham group,and the TAC 8W group was decreased by 43.60% compared with the Sham group.The left ventricular short axis shortening rate(LVFS%)was 19.41% at 8 weeks of TAC surgery(TAC 8W group)and 41.26% at the Sham group,indicating a decrease in cardiac function in mice.The posterior wall thickness of the left ventricle(LVPWs)and the posterior wall thickness of the left ventricle(LVPWd)in the TAC 4W group increased significantly.The ratio of lung weight/weight(LW/BW)and lung weight/tibia length(LW/TL)of TAC 8W was significantly higher than that of Sham group.(2)Eight weeks after aortic arch narrowing(TAC 8W),compared with Sham group,the α-SMA,Collagen Ⅰ,and Collagen Ⅲ of myocardial tissue in mice increased significantly,with statistical differences.At the same time,TAC 8W group and Sham The expression of DCN1 protein in the group increased by about 1.5 times,and the expression of its downstream regulatory proteins Nrf2 and NQ-1 decreased significantly.Part Ⅱ: The inhibitor DN-2 targeting DCN1-UBC12,by inhibiting the activity of DCN1-UBC12,regulates the Nrf2 signaling pathway,and alleviates the activation,proliferation,migration,and collagen secretion of myocardial fibroblasts induced by Ang Ⅱ.(1)The isolated,extracted and cultured CFs are identified by Vemintin antibody,the cell purity is greater than 95%,and they have good activity to meet the experimental needs.The MTT method,immunofluorescence test,scratch test,Transwell test,and Western Blot detection showed that Ang Ⅱ can induce the occurrence of myocardial fibrosis,and targeting DCN1-UBC12 inhibitor DN-2 can effectively alleviate the Ang Ⅱ-induced myocardial fibrosis.Related pathway proteins Nrf2 and NQ-1 also have corresponding changes.(2)si RNA experiments verified that DN-2 regulates the Nrf2 signaling pathway by inhibiting DCN1 activity to participate in Ang Ⅱ-induced myocardial fibrosis.(3)The activity of DN-2 at the cellular level is comparable to that of the positive control drug losartan,and it has better protection against fibrosis than the published DC-2 small molecule compound.(4)In the acute toxicity experiment,compared with the control group,compound DN-2 did not cause the death of the mice,and there was no significant change in the cardiac function of the mice,and the HE staining of the important organs was not different.DN-2 has good safety in animals.Conclusion:Targeting DCN1-UBC12 small molecule compound DN-2 can effectively alleviate the activation,proliferation,migration and collagen secretion of myocardial fibroblasts induced by Ang Ⅱ.The mechanism may be that DN-2 can further regulate by inhibiting the DCN1-UBC12 protein-protein interaction The Nrf2 signaling pathway alleviates the occurrence of fibrosis. |
