Mechanisms Of GATA4 Mediated TMAO Promoting Vascular Aging In Chronic Kidney Disease

Background:Studies have shown that patients with chronic kidney disease(CKD)have significantly increased cardiovascular disease(CVD)morbidity and mortality compared with normal people.The cause and mechanism of vascular aging in patients with CKD remains unclear.Vascular aging is an independent risk factor for the development of CVD.Its pathological changes mainly involve the senescence of endothelial cells and smooth muscle cells.Therefore,strengthening the study of vascular aging is of great significance for the prevention and treatment of CVD.Trimethylamine-N-oxide(TMAO),a choline-derived intestinal flora metabolite,is mainly excreted by the kidney and is associated with cardiovascular risk.At present,little is known about the relationship and the mechanism between TMAO and vascular aging.In patients with CKD,due to impaired renal function,whether high plasma levels of TMAO affect vascular aging has aroused our attention.A striking feature of senescence is the senescence-associated secretory phenotype(SASP),a pro-inflammatory response associated with senescence.GATA4 functions as a key switch in the senescence regulatory network,resulting in NF-?B activation and SASP induction.Our study investigates the effects of TMAO on vascular aging and the role of the GATA4 signaling pathway in TMAO-induced senescence.Objective:To investigate whether TMAO plays a role in cell senescence and vascular aging in vitro and in vivo.It was also explored whether TMAO plays a role in promoting inflammation and aging by promoting the expression of GATA4,which in turns activates the transcription factor NF-?B to initiate the SASP.Methods:1.Primary human umbilical vein endothelial cells(HUVEC)and human vascular smooth muscle cells(HVSMC)were cultured and treated with TMAO at concentrations of 50,100 and 200 ?M for 3 days to establish of induced senescence cell model.The cell migration and angiogenesis function of HUVECs after TMAO treatment were detected by scratch test and tube formation test.The animal model of CKD was surgically induced(5/6 nephrectomy).Rats with chronic kidney disease were intraperitoneally injected with TMAO at a dose of 0.33 ?mol/100 g body weight/day for 3 weeks.The activity of the ?-galactosidase(SA-?-gal)and the expression of the senescence-associated genes P53,P21 and P16 were used to identify cellular senescence and vascular aging.2.The mRNA and protein levels of the transcription factor GATA4 in HUVECs and vascular were detected by qRT-PCR and WB.The protein level of the transcription factor NF-?B was detected by WB.qRT-PCR was used to detect the mRNA levels of SASP such as IL-1?,IL-6,IL-8,IL-18 and TNF-?.3.The expression of the transcription factor GATA4 in HUVECs was silenced by small interfering RNA,and were treated with TMAO for 3 days.The activity of the ?-galactosidase(SA-?-gal)and the expression of the senescence-associated genes P53,P21 and P16 were used to identify cellular senescence.The expression level of each gene in Method 2 was detected.The cell migration and angiogenesis function were detected by scratch test and tube formation test.Results:1.TMAO promoted HUVECs and HVSMCs senescence and vascular aging(P<0.05)and impaired HUVECs function(P<0.05).TMAO can increase the protein levels of GATA4 and NF-?B in cells and vascular(P<0.01),and promote the secretion of SASP such as IL-1?,IL-6,TNF-?,IL-8 and IL-18(P<0.05).2.Silencing the transcription factor GATA4 inhibits the pro-inflammatory and aging effects of TMAO(P<0.01)and improves the migration and angiogenesis capacity during TMAO-induced HUVECs senescence(P<0.01).Conclusions:TMAO increases protein levels of GATA4,which in turn activates the transcription factor NF-?B to initiate the SASP and facilitate senescence.