Change Of Cerebral Vascular Smooth Muscle Expression Pattern And The Role Of GJA1 Promotes Proliferation Of Smooth Muscle And Contraction In High Salt Environment

BackgroundThe prevalence of hypertension in China is increasing.At least 2 out of every 10 adults are high blood pressure.Hypertension is the most important risk factor for stroke,heart disease and kidney disease.High-salt diet is the most important risk factor for the development of hypertension.Vascular smooth muscle cells（VSMCs）are the main cellular components that constitute the structure of blood vessel walls and maintain vascular tone.The shrinking of vascular cavities and thickening of the wall induced by the hyperproliferative VSMCs play an important role in the pathogenesis of hypertension.Vascular function is largely determined by the cytoplasmic Ca²⁺concentration in smooth muscle cells（SMCs）.Gap-junction（GJ）channels composed of connexin（Cx）proteins are crucial in coordinating Ca²⁺signalling and vessel function,setting the balance between vasodilation and vasoconstriction.The gap junction alpha-1protein GJA1,also known as connexin 43（Cx43）,is a component of gap junctions that allow gaps between cells to link between cells to regulate cell death,proliferation and differentiation.At the same time,GJA1 plays a key role in regulating the proliferation of VSMCs and the development of atherosclerotic diseases.Therefore,by analysising the sequencing results of cerebrovascular expression profiles in high-salt induced hypertensive rats identify key proteins GJA1.This study attempts to explore the role and mechanism of GJA1 in regulating the proliferation of VSMCs in high-salt environment by simulating high-salt environment in vitro,and provides a theoretical basis for drug development for the treatment of hyper-salt induced hypertensive vasculopathy.Objective1.To investigate the effect and mechanism of high-salt environment on the proliferation of primary rat cerebral VSMCs;2.To observe the role of GJA1 in agonist-induced vasoconstriction in high-salt environment.Method1.High-throughput sequencingIn the early stage,we obtained spontaneously hypertensive rats through high-salt diet.Three rats in the control group and the hypertensive group were used to perform sequencing of cerebral vascular smooth muscle expression profiles,and the sequencing results were analyzed.2.Cell culture and identificationSPF SD male rats amouting about 2-3,weighting about 230 g,CO₂ asphyxiation death.The rats’brain was quickly dissected in a sterile environment,placed in sterile PBS,the rats’brain basal cerebral vessels were isolated,endothelium was gently wiped off with a fine toothpick,then was cutted into 2 mm long blood vessels with scissors in a clean bench.The vessels were cultured for 4-7 days in 1640 with 20%fetal bovine serum（FBS）and 1%penicillin streptomycin.Until the tissue pieces were completely adhered and a small amount of cells were crawled around the tissue block can be exchanged.VSMCs were identified with specificα-SM-actin monoclonal antibody by immunofluorescence,and cells in passage 3-7 with good growth status were selected for subsequent experiments;3.Western blot Western Blot was used to detect the expression levels of p-GJA1 and GJA1 proteins in VSMCs;4.siRNA transient transfectionSpecific GJA1 siRNAs were transfected into VSMCs with lipo3000.GJA1 protein expression level was detected and cells were harvested for the following experiments at48 h after siRNA transfection;5.Cell proliferation10³-10⁴ cells were harvested in 96-well plates.200 ul medium was added in each well.After cell were processed for 48 h,the medium was replaced with 100 ul fresh 1640medium,10 ul CCK-8 solution was added in each well,then the plate was incubated in the incubator for 2 h.The absorbance at 450 nm was detected with microplate reader;6.Vascular tension measurementThe SD rats were sacrificed by CO₂ asphyxiation,and the cerebral vessels were incubated with Scramble,GJA1 siRNA for 24 hours.The vasoconstriction intensity induced by ET1 and U4 under different culture conditions were detected by DMT 620M vascular tension analyzer.7.Statistical analysisAll datas in this study were presented as means±standard error（mean±S.E.M.）.The significance was analyed using Student’s unpaired t test by Sigma Plot 12.5 software,and the experimental results were statistically analyzed.A value of P<0.05 was considered statistically significant.Result（1）The CytoHubba software plug in Cytolscape was successfully found the top ten genes in the center,GJA1 was one of the core genes;（2）Primary rat vascular smooth muscle cells were successfully cultured.The results ofα-SM-actin monoclonal antibody immunofluorescence identification showed cytoplasmic were stained,and the nucleus were clearly visibled;（3）The expression levels of p-GJA1 was increased in the high-salt cultured VSMCs at48 h,while GJA1 was unchanged,and cells proliferation were enhanced;（4）Gja1 specific siRNAs transfection for 48h significantly decreased the expression levels of GJA1 and cells proliferation;（5）The results of vascular tension showed that the contraction agent mediated vasoconstriction ability were enhanced after 24 h in high-salt environment,contraction agent mediated vasoconstriction were weakened at 24 h after siRNA transfection compared with Scramble siRNA in normal-salt and high-salt environment.Conclusion1.The expression of p-GJA1 protein was increased and the proliferation of VSMCs were inhanced in high salt environment;2.Specific siRNA knockdown of GJA1 protein expression inhibited the proliferation of VSMCs;3.In high salt environment,vasoconstriction was significantly enhanced,specific siRNA knocked down GJA1 protein significantly inhibited the cerebral vasoconstriction enhancement by high-salt.