The Mechanism Of Action And Effect Of The Nppb Gene Via Conditional Knockout In Heart Structure And Function In Septic Mice

BackgroundSepsis can induce myocardial dysfunction and structural alteration,leading to high mortality and poor cardiovascular outcomes in humans.NPPB gene is an essential gene for cardiovascular regulation.Abnormal expression of NPPB gene is indicated to be involved in the progression of myocardial fibrosis and can eventually leads to cardiac dysfunction.However,research about the role of NPPB gene in the regulation of myocardial structure and function in sepsis is limited.In this study,an Nppb conditional knockout mouse model was used to investigate the effect and potential mechanism of myocardial structural and functional damage during sepsis.MethodsThe Nppb floxed mice were generated by CRISPR/Cas9 technology.Inducible cardiomyocyte-specific deletion mice(Nppb^fl/fl;Myh6^cre/ert2)were generated by crossing the Nppb floxed mice and Myh6^cre/ert2 transgenic mice.Genotypes were identified by PCR and DNA sequencing.Tamoxifen was used to induce the loss of Nppb.Male mice of the same age（8 weeks）were divided into four groups according to random number table method:1.Control saline group（Con-Na Cl）;2.Control sepsis group（Con-LPS）;3.CKO saline group（CKO-Na Cl）;4.CKO sepsis group（CKO-LPS）.The sepsis model was induced by intraperitoneal injection of 15mg/kg LPS at a dose of 0.1ml per mouse,while the saline group was intraperitoneally injected with the same dose of normal saline.The general health of mice was observed postinjection and the 5-day mortality of each group after model establishment was recorded and analyzed.The changes of cardiac function in mice were monitored by transthoracic echocardiography within 24h after model establishment.The myocardial structure and collagen deposition were observed by HE and Masson staining at time point 0h,12h and 4w,respectively.The serum levels of BNP,IL-6,TNF-α,and the levels of TGF-β1 and CK in myocardial tissue were detected by ELISA and colorimetric method 24h right after model establishment.The expression levels of Acta2,Vimentin,Pecam1,Tgfb1,Tgfbr1and Tgfbr2 in myocardial tissue were quantified by q PCR.Western blot was used to detect the expression level of TGF-β1,smad2,p-smad2,Notch1 and NICD in myocardial tissue.SPSS 25.0 and Graph Pad Prism 9.0 software were used for data processing and statistical analysis.A value of P<0.05 was considered to be of statistically significance.Results1.DNA identification and sequencing comparison confirmed that the Nppb-CKO mouse model was successfully constructed.2.Mice in Con-LPS group had significantly higher serum BNP level at time point 24h compared to Con-Na Cl group.Also,serum levels of CK,IL-6 and TNF-αwere positively correlated to it.Echocardiography found that,comparing with the baseline,myocardial systolic function at time point 3h,6h and 12h were down-regulated.End MT process was activated.The 5-day mortality was 90%.Myocardial morphology experiment showed several calcifications in surviving mice 4 weeks after model establishment,collagen fiber ratio was significantly higher.3.Mice in CKO-Na Cl group had significantly lower serum BNP level compared to Con-Na Cl group.The serum BNP level did not rise significantly 24h after model establishment in CKO mice.In CKO-LPS group,no significant decline of EF within 24h was observed.FS started to be lower than baseline at 6h after model establishment.End MT process seemed to be suppressed compared to Con-LPS group.5-day mortality was 25%which had been improved significantly compared to Con-LPS group.There was no obvious myocardial lesions or fibrotic changes 4 weeks after model establishment in CKO-LPS group.Conclusions1.The myocardial conditional knockout of Nppb gene had protective effects on the maintenance of early myocardial contractibility such as the stability of myocardial structure and the improvement of prognosis in septic mice.2.Notch1 signaling pathway might be involved in the process of Nppb gene mediating End MT and regulating myocardial structure and function in septic mice.