Study On The Role Of TMEM16A In Vascular Inflammation In Salt-sensitive Hypertensio

Background and purpose:Hypertension is a major risk factor for cardiovascular disease,chronic kidney disease,and stroke.Previous studies by our research group have confirmed that high-salt diet is an independent risk factor for BP of patients,including clinic and home measurements of blood pressure,but the specific mechanism is unclear.Meanwhile,Vascular smooth muscle cells(VSMCs)can shift from a contractile to a synthetic phenotype in the state of hypertension.The phenomenon is characterized by attenuated expression of contractile proteins along with increased proliferation of VSMCs and functional enhancement of extracellular matrix(ECM)proteins,intercellular adhesion molecule(ICAM)-1 and vascular CAM(VCAM)-1,which can induce numerous white blood cells to infiltrate the vascular wall and trigger the inflammatory response,resulting in vascular damage and remodeling.Transmembrane member 16A(Tmem16A),as a molecular regulator of the calcium-activated chloride channel,is primarily expressed in epithelial cells,smooth muscle cells.It can promote inflammatory responses by increasing proinflammatory cytokine release.Our previous research group has found that TMEM16A can affect the contraction of vascular smooth muscle in hypertensive rats and increase blood pressure.However,little is known about the relationship between salt-sensitive hypertension and the role of TMEM16A in vascular inflammation.Therefore,this experiment aims to explore the role of TMEM16A in high salt-induced vascular inflammatory response,and further elucidate the mechanism of TMEM16A in salt-sensitive hypertension.Methods:1.Establishment of L-NAME/SS mouse model and measurement of blood pressureMale mice aged 8-10 weeks were selected and randomly divided into normal salt diet group(NS),high salt diet group(HS)and HS+Arcrigenin group,the intervention time was 16 weeks.At the same time every week,the blood pressure and heart rate of mice were measured and recorded using a non-invasive tail artery blood pressure measuring instrument.2.Detection of TMEM16A expression in aortic smooth muscle cellsAfter the model was established,the aorta was harvested,and the adventitia and endothelium were peeled off.HE,immunofluorescence,immunohistochemistry,and western blot were used to detect the expression of TMEM16A in aortic smooth muscle of each group.3.Aortic Smooth Muscle Cell CultureMouse aortic vascular smooth muscle cells were cultured in a smooth muscle cell medium containing 2%fetal bovine serum and 1%double antibody.The cells are grown in a 37℃,5%CO2 incubator,and the 2nd to 4th day generation were used to perform next test.4.Inhibition and knockdown of TMEM16A in vitroAortic smooth muscle cells were seeded in 6-well plates,and T16Ainh-A01(T16)was used to inhibit the expression of TMEM16A in vascular smooth muscle cells in vitro and to screen the optimal concentration and time of small interfering RNA knockdown TMEM16A(siTMEM16A).We used western blot to test the expression of TMEM16 in different groups.5.Expression of TMEM16A and the inflammatory phenotype of smooth muscle cellsThe expressions of Vascular adhesion molecule-1(VCAM-1)and Intercellular adhesion molecule-1(ICAM-1)were detected by immunofluorescence in NS,HS,HS+ARC groups,and the expression of NS,HS,HS+ARC,HS+T16,HS by western blot The expression of TMEM16A,VCAM-1 and ICAM-1 in the+siTMEM16A group.6.Aortic smooth muscle cell transcriptome sequencing and screening of differential GenesTrizol was used to extract the total RNA of smooth muscle cells in NS,HS,HS+ARC,and HS+T16 groups,and then RNA sequencing was performed to screen differential genes downstream of TMEM16 based on the sequencing results.7.Verification of differential genes downstream of TMEM16AThe influence of TMEM16A expression on the differential gene expression screened out was detected by western blot,immunofluorescence,and ELISA.8.Macrophage Adhesion AssayMacrophage adhesion experiments were performed in vitro to show the ability of smooth muscle cells in each group to adhere to macrophages.Conclusions:TMEM16A promotes the formation of salt-sensitive hypertension through the TMEM16-ESM1-CXCL16 signaling pathway that affects the inflammatory response of vascular smooth muscle cells.