Role Of Ubiquitin Carboxyl-terminal Hydrolase Isozyme L1 In The Regulation Of Mouse Myocardial Fibrosis

Background and Objective: Myocardial fibrosis(MF)is a common pathological manifestation of various cardiac diseases,such as coronary heart disease,hypertensive heart disease,cardiomyopathy and other end-stage diseases.It is the pathological process of excessive proliferation of fibroblasts in Myocardial stroma and imbalance of extracellular matrix deposition and degradation.Myocardial fibrosis can lead to increasing myocardial wall stiffness and decreasing diastolic compliance.In the early stage,diastolic filling is limited,and in the late stage,myocardial systolic function is impaired,which promotes heart failure.On the other hand,the abnormal conduction of myocardial tissue fibrosis often leads to a variety of malignant arrhythmias,or even sudden death.Inhibition of myocardial fibrosis is of great significance in improving the prognosis of various cardiovascular diseases.However,current studies have shown that the causes of myocardial fibrosis are complex,and the mechanism of myocardial fibrosis occurrence and development is not certain.Myocardial fibrosis is a clinical problem that needs to be solved and further studied.Ubiquitin-proteasome system(UPS)is an important mechanism for regulating myocardial protein degradation.The system mainly includes ubiquitin activating enzyme,ubiquitin transshipment enzyme,ubiquitin ligase,26 S proteasome and deubiquitinating enzymes.The ubiquitination process is catalyzed by the sequence of E1,E2 and E3,and deubiquitination enzymes mediate the removal and processing of ubiquitin.Deubiquitination enzyme is the reverse regulation enzyme of ubiquitination enzyme,which can inhibit ubiquitination of protein and further prevent degradation of substrate protein by proteasome.Deubiquitination enzyme seems to be the key regulator of various cell processes such as proliferation,apoptosis,differentiation and inflammation.Ubiquitin carboxy-terminal hydrolase L1(uch-l1)plays an important role in the ubiquitination enzyme family.It not only has the function of the ubiquitination enzyme family,but also acts as a ligase independent of atpase and stabilizes the Ubiquitin monomer in the cell.However,the potential role of ubiquitin carboxyl-terminal hydrolase L1 in the heart remains unclear.Platelet-derived growth factors(PDGFs)are a powerful pro-cell division agent,belonging to the family of growth factors that stimulates cell growth.Previous studies have shown that platelet-derived growth factors PDGFs activate and bind PDGFRs on fibroblast membranes.Phosphorylated PDGFRs further activate intracellular signaling pathways,leading to the proliferation and transformation of fibroblasts into myofibroblasts,which are synthesized in large numbers.Collagen is deposited in the myocardial stroma,thereby promoting myocardial fibrosis.Ang II was induced to establish a mouse myocardial fibrosis model.Mice were treated with UCHL1 specific inhibitor LDN57444,and the blood pressure and cardiac function and structure of the mice were measured.The degree of myocardial fibrosis was measured by Sirius scar staining.The mouse cardiac fibroblasts were cultured in vitro,PDGF-DD was used to induce mouse cardiac fibrosis,and the intracellular PDGF receptor,PI3 K / AKT / NF-?B signaling pathway,m RNA and protein of RB were detected by qRT-PCR and WB.Expression,UCH-L1 specific inhibitors were used to observe changes in genes and proteins of various items and changes in myocardial fibrosis indicators to explore possible molecular mechanisms by which UCH-L1 regulates myocardial fibrosis.Methods: Male mice(6 in each group)were continuously perfused with angiotensin II(Ang II)at a concentration of 1000 ng /(kg � min)for 14 days using a sustained-release pump to establish a model of myocardial fibrosis in mice,and UCHL1 was injected intraperitoneally.The specific inhibitor LDN57444 40 ?g /(kg � day).The blood pressure of mice was measured daily by the non-invasive tail pressure method.M-type echocardiography was used to detect changes in heart function of mice before and after.After 14 days,the mice were sacrificed to remove the heart tissue,and the degree of myocardial interstitial fibrosis(ratio of myocardial collagen area / vascular cavity area ratio)was observed with Sirius Red staining.Primary culture of mouse cardiac fibroblasts,the fibroblasts cultured in vitro were randomly divided into 3 groups: control group(cell +medium),group P(cell + medium + 20 ng.ml-1 PDGF-DD),PL group(cell + medium +20ng.ml-1 PDGF-DD + 40 u M LDN57444).Detection of PI3 K,AKT,I?B,UCH-L1,type I collagen m RNA by real-time PCR.The protein expression levels of PDGFR?,pPDGFR?,pPI3 K,PI3K,pAKT,AKT,UCH-L1,pRB,RB,and type ? collagen were detected by Western blot.Results:(1)Compared with the control group,the mean systolic pressure and mean arterial blood pressure increased significantly and the cardiac structure and function changed in the animals treated with Ang II.Treatment with UCHL1 specific inhibitor LDN57444 could significantly inhibit above Ang II-induced effects,and the severity of myocardial fibrosis decreased markedly(P <0.05).(2)qRT-PCR showed the m RNA expression of UCH-L1 in the P and PL groups was significantly higher than in the control group(P<0.05)and the m RNA expression of type I collagen in the PL group was markedly lower than in the P group(P<0.05).(3)WB showed the protein expression of PDGFR?/p-PDGFR?,pPI3K/PI3 K,UCH L1,pRB/RB,and Col ? in the P group was significantly higher than in the control group(P<0.05),the protein expression of PDGFR?/p-PDGFR?,pPI3K/PI3 K,Col? and pRB/RB in the PL group was markedly lower than in the P group(P<0.05).(4)Chromatin immunoprecipitation assay confirmed NF-?B regulated the transcription of downstream UCH-L1.Conclusions:(1)In animal experiments,the treatment of UCHL1 inhibitor LDN57444 improves Ang II-induced decrease in cardiac function and myocardial fibrosis in mice.(2)PDGF-DD can induce the proliferation of myocardial fibroblasts,promote the synthesis of interstitial collagen,and promote myocardial fibrosis.(3)Based on in vitro cell culture technology,PDGF-DD may affect myocardial fibrosis through the PDGFR? / PI3 K / AKT signaling pathway.(4)PDGF-DD can stimulate m RNA and protein expression of UCH-L1.(5)UCH-L1 participates in regulating the proliferation of mouse cardiac fibroblasts.The mechanism may be the regulation of UCH-L1 expression through the PI3 K,AKT and NF-?B signal axis pathways.UCH-L1 regulates the expression of downstream RB proteins by regulating cells Cycle to achieve the regulation of myocardial fibroblast proliferation.