Effects And Mechanisms Of Urea Inducing Pulmonary Arterial Hypertension In Broiler Chickens

Pulmonary arterial hypertension(PAH)is a fatal disease that occurs in rapidly growing chickens,causing significant economic losses to the broiler breeding industry.The pathological features of PAH in broiler chickens is pulmonary vascular remodeling,including tunica media hypertrophy and the formation of plexiform lesions(PLs).However,the exact causes and mechanisms of PAH are not fully understood.Urea is the end product of the urea cycle,and elevated urea levels can lead to endothelium dysfunction and endothelial progenitor cell dysfunction.In our previous observations,we found increased urea levels in the lungs of PAH chickens,but its role in the pathogenesis of PAH remains unclear.This study aims to investigate the effects and mechanisms of intratracheal urea infusion on pulmonary vascular remodeling,providing new insights for the prevention and treatment of PAH in broiler chickens.Part 1: The mechanism of urea-induced pulmonary vascular remodeling via intratracheal instillation.18-day-old AA broiler chickens were selected and treated intracheal instillation with 100 μL urea(1.2 g/L)once every 3 days,a total of 3 times.The control group treated with PBS.On the third day after the last intracheal instillation,lungs were collected for pathological morphological analysis.Immunohistochemistry was used to detect the expression of endothelial nitric oxide synthase and arginase 2.The results showed that intratracheal infusion of urea significantly promoted the plexiform lesion formation and vascular remodeling,upregulated the expression of arginase 2(ARG2)and inhibited endothelial nitric oxide synthase(e NOS)in pulmonary vascular endothelial.The results suggested that intratracheal instillation of urea could induce pulmonary vascular remodeling and promote PAH.By constructing a pulmonary artery smooth muscle cells(PASMCs)urea treated model,CCK-8 was used to detect proliferation ability,scratch test was used to detect migration ability,Western blot was used to detect the expression of contraction and synthetic phenotype proteins.The results showed that urea treatment significantly promoted the proliferation of PASMCs,but did not induce the migration and phenotype transformation.The above results suggested that urea could induce tunica media hypertrophy by promoting the PASMCs proliferation.Part 2: Urea promotes the transformation of eEPCs into macrophages and induces the formation of PLs by activating Nrf2.By constructing a urea treated model of early endothelial progenitor cells(eEPCs),Western blot was used to detect the protein levels of TGF-β1,α-SMA,Vimentin and Snail in eEPCs,immunofluorescence was used to detect the cell positivity of eEPCs and macrophage phenotype markers.The results showed that urea did not induce End MT in eEPCs but could induce transformation of eEPCs into macrophages.The E.coli phagocytosis test was used to detected cell phagocytosis ability.q PCR was used to detect the m RNA levels of inflammatory factors.The results showed that urea induced phagocytosis and pro-inflammatory phenotype in eEPCs.NO fluorescence probe method was used to detect NO production.In vivo Matrigel plug assay was used to detect angiogenesis ability of EPCs.The results showed that urea inhibited NO production and vasculogenesis ability of eEPCs,suggesting that urea could induce eEPCs dysfunction.Western blot was used to detect the Nrf2 pathway and determine the mechanism of urea induced phenotypic transformation of eEPCs.The results showed that urea could induce phenotypic transformation of eEPCs by activating Nrf2.Immunohistochemistry revealed the expression of KUL01 in PLs,and the nuclear translocation of Nrf2 was evident in PLs,suggesting that there had a relationship between PLs formation,Nrf2 activation,and the transformation of eEPCs into macrophages.The above results indicated that urea activated Nrf2 to induce the transformation of eEPCs into macrophages,participating in PLs formation.Part 3: Intratracheal instillation of urea promotes specific microbiota changes in broiler chickens.The contents of the jejunum were collected from the PBS group and the urea-treated group of broiler chickens,and the intestinal microbiota was identified using 16 S r RNA sequencing.Compared to the control group,the abundance of Tyzzerella＿3,a genus associated with pro-inflammatory reactions and oxidative stress,increased in the intestinal tract of broiler chickens in the urea-treated group.Conversely,the abundance of genera(Virvallis,Papillibacter,and Barnesiella)associated with anti-inflammatory short-chain fatty acid production decreased.Lung tissues were collected,Western blot was used to detect the expression of inflammatory cytokines TNF-α and IL-1β.Malondialdehyde(MDA)assay kit was used to detedt the level of MDA.The results showed that urea promoted the production of inflammatory cytokines and MDA.The above results suggest that urea may induce lung inflammation and oxidative stress by altering the specific intestinal microbiota.The microbiota may play a role in the progression of PAH.The above results indicate that local urea generation in the lungs can induce PASMCs proliferation and promote the transformation of eEPCs into macrophage-like cells,leading to pulmonary vascular remodeling and PLs formation.Changes in the gut microbiota may play a role in the progression of PAH.