Myeloid-specific Knockout Of SNX16 Accelerates Cardiac Fibrosis Induced By Isoproterenol In Mice

Objective:Cardiac fibrosis is caused by myocardial infarction,myocarditis,hypertension and other pathological factors,and eventually results in serious cardiac malfunction and even heart failure.Macrophages and neutrophils infiltration often leads to the proliferation and activation of myocardial fibroblasts and excessive deposition of extracellular matrix after myocardial injuries.In the early stage of fibrosis,macrophages mainly show the pro-inflammatory M1 type,which releases pro-inflammatory cytokines such as IL-1β,IL-6,TNF-α,and aggravates the inflammatory process;in the later stage,the anti-inflammatory M2 macrophages increase and produce IL-10,TGF-βand other anti-inflammatory cytokines and growth factors.Sorting Nexin 16（SNX16）is a member of the sorting Nexin protein family,which is a component of inclusion bodies and plays an important role in cellular endocytosis,protein sorting,and cell signal transduction.Research shows that SNX16 plays an important role in macrophage-mediated inflammation.This project intends to explore the role of myeloid-specific knockout of SNX16 on isoproterenol-induced cardiac fibrosis and its related mechanisms.Experimental Methods:1.Preparation and genotyping of myeloid-specific SNX16 knockout mice:1)Myeloid-specific knockout SNX16 mice SNX16^fl/flLyz2^cre（+）（F/F+）and the control group mice SNX16^fl/flLyz2^cre（-）（F/F-）were obtained by mating SNX16^fl/fl mice,with myeloid-specific expressing Lyz2cre mice;2)mouse DNA was extracted for PCR genotype identification,and the protein and RNA levels of SNX16 were detected with bone marrow and primary peritoneal macrophages samples.2.Preparation and analysis of ISO-induced mouse cardiac fibrosis model:1)Four F/F+mice and F/F-mice at 8-12 weeks were divided into the experimental group（ISO）and the control group（saline）.Isoproterenol（ISO）was subcutaneously injected 20mg/kg/day for 7 consecutive days to induce mouse cardiac fibrosis model;2)Vevo3100echocardiography was used to detect the cardiac function in mice before and after after the ending of the experiments;3)Routine blood analysis was performed in blood from the eyeballs after Inhalation anesthesia with isoflurane;4)Mouse hearts were collected,photograghed and fixed for sectioning and HE Masson staining to detect the degree of cardiac fibrosis in mice;5)Western Blot and RT-PCR were used to detect cardiac fibrosis-related genes such asα-SMA,Collagen Ⅰ and Collagen Ⅲ and inflammatory factors CCR2,CCL2 and IL-1β.3.ISO-induced macrophage inflammation:1)Primary mouse peritoneal macrophages were harvested 5 days post intraperitoneal injection of 3%fluid gum,from F/F+mice and F/F-mice.2)SNX16 knockout and control group macrophages were treated with 10μM ISO or saline for 24 h,respectively,and the expressions of inflammatory factors CCR2,IL-6 and TNF-αwere detected after ISO treatment by Western Blot and RT-PCR.4.The effects of macrophages on fibroblast activation:The supernatants from ISO-stimulated macrophages were collected and added to cardiac fibroblasts,for 24 h,the fibroblast activation markersα-SMA and Collagen I were detected in the treated fibroblasts by Western Blots.Results1.The myeloid-specific knockout SNX16 mice（F/F+）were successfully constructed,and the expression of SNX16 was decreased by 50%-70%in myeloid macrophages.2.Myeloid-specific knockout SNX16 significantly accelerated ISO-induced myocardial injury,ventricular hypertrophy,myocardial fibrosis,increased total numbers of peripheral blood neutrophils,and increased secretion of myocardial macrophage M1-type inflammatory factors CCR2,CCL2 and IL-1β;decreased expression of M2 macrophage CD206 and IL-10 in mice.3.Myeloid-specific knockout of SNX16 significantly increased the expression of M1-type pro-inflammatory cytokines CCR2,IL-6,and TNF-αin macrophages after ISO stimulation.4.Myeloid-specific knockout of SNX16 promoted fibroblast activation via the paracrine factors from macrophages induced by ISO.Conclusion:Myeloid-specific knockout of SNX16 significantly accelerated ISO-induced cardiac fibrosis via increasing the secretion of pro-inflammatory cytokines from macrophages to reinforce cardiac fibroblast activation and extracellular matrix accumulation.