| Objective Vascular remodeling(VR)is the key pathological basis for the development of hypertension and target organ damage,in whcih the abnormal proliferation of vascular smooth muscle cells(VSMC)play a central role.Angiotensin II(AngⅡ)is the main active component of the renin-angiotensin system(RAS).Circulation or tissue RAS activation can lead to an increase of AngⅡ concentration.By promoting VSMC proliferation and migration,it promotes vascular remodeling in hypertension.ANO1 is a newly-discovered calcium-activated chloride channel with a wide distribution in various vascular tissues.It has been proved that ANO1 is involved in the regulation of vasoconstriction and blood pressure,but there are few reports on ANO1 and vascular remodeling.In this experiment,to explore the role of ANO1 in AngⅡ-induced VSMC proliferation,we first observed the effect of AngⅡ on ANO1 expression and explored the receptor mechanism.Secondly,by using ANO1 inhibitor T16 A inh-A01(A01),we observed the effect of A01 on AngⅡ-induced VSMC proliferation and explored its related mechanisms.Methods The thoracic aorta of normal rats were taken to culture primary cells of VSMC by the tissue patch method.The MTT method was used to assess the cell viability,and the western blot was used to determine the protein levels.Results 1.To observe the effect of AngⅡ on ANO1 protein expression,the cells were treated with different concentrations of AngⅡ(1、10、100、500、1000 nmol/L)for 24 h,The ANO1 protein levels after AngⅡ treatment were elevated to(1.09±0.03),(1.17±0.02),(1.46±0.07),(1.21±0.01)and(1.17±0.01)(F=17.03,q=5.74~12.28,n=4,P<0.05-0.01).So 100 nmol/L AngⅡ showed the maximum effect.After treatment with 100 nmol/L AngⅡ at different times(1、6、12、24、48 h),ANO1 levels were(1.13±0.08),(1.25±0.15),(1.80±0.08),(1.74±0.06)and(1.65±0.04)respectively,indicating that ANO1 expression was increased significantly after AngⅡ treatment for 12-48 h(F=15.05,q=7.36~9.08,n=4,P<0.05-0.01).The above results suggested that AngⅡ significantly elevated ANO1 protein expression in a dose and time-dependent manner.In subsequent experiments,the cells were incubted with 100 nmol/L AngⅡ 24 h.2.To explore the receptor mechanism of AngⅡ-induced expression of ANO1,the specific AT1 R blocker(losartan potassium,LP)and AT2 R blocker(PD123319,PD)were used.In AngⅡ group,ANO1 protein level was increased to(1.45±0.08),while this elevation was blocked by LP instead of PD(F=9.68,q=5.31~5.87,n=3,P<0.05).This indicated that AngⅡ promoted the expression of ANO1 in VSMC by binding with AT1 R.3.To observe the effect of ANO1 inhibitor T16 A inh-A01(A01)on AngⅡ-induced ANO1 up-regulation,20 μmol/L A01 was pretreated before AngⅡ.The protein level of ANO1 in the AngⅡ group was increased to(1.39±0.08),while the protein level in the 20 μmol/L A01+AngⅡ treated group was decreased to(1.09±0.04)(F=14.88,q=5.65~7.37,n=6,P<0.01).The above results showed that ANO1 inhibitor A01 significantly inhibited AngⅡ-induced ANO1 protein expression.4.The effect of different concentrations of A01 on the viability of cells that have been treated whith AngⅡ was detected by MTT method.Cells were treated with different concentrations of AngⅡ(1、5、10、100、500、1000 nmol/L)for 24 h,and the cell survival rates were(1.05±0.05),(1.09±0.03),(1.29±0.07),(1.61±0.03),(1.66±0.06)and(1.66±0.06)(F=30.02,q=5.53~12.17,n=6,P<0.05-0.01),suggesting that 10-1000 nmol/L AngⅡ can promote the proliferation of VSMC.The cell viability in AngⅡ+A01(1、5、10、20 μmol/L)was(1.12±0.01),(1.09±0.01),(0.84±0.01)and(0.78±0.01)(F=57.96,q=19.69~22.85,n=6,P<0.01),showing that 10-20 μmol/L A01 significantly inhibited AngⅡ-induced VSMC proliferation.5.By detecting PCNA,the effect of A01 on AngⅡ-induced cell proliferation was confirmed.In AngⅡ group,the PCNA protein level was increased to(1.62±0.08)for 24 h;the PCNA protein in the 20 μmol/L A01+AngⅡ group was decreased to(1.06±0.01)(F=18.47,q=7.97~8.85,n=3,P<0.01).The above results further suggested that A01 can significantly inhibited the proliferation of AngⅡ-induced cells.6.After 100 nmol/L AngⅡ treatment for 24 h,p21 protein level was decreased to(0.70±0.02).In AngⅡ+A01 treatment group,p21 protein level was increased to(1.04±0.01)(F=17.44,q=8.22~9.36,n=3,P<0.01).The changes of p27 protein were similar to p21 in each group.These results suggested that AngⅡ promoted cell cycle progression by inhibiting the expression of p21 and p27 proteins,while these changes were counteracted by A01 7.This experiment was to explore whether the inhibitory effect of A01 on AngⅡ-induced proliferation was due to the activation of PI3K/AKT signaling pathway.After treated with AngⅡ,the cell survival rate was elevated to(1.62±0.05) by the MTT assay.However,this effect could be counteracted by LY294002(a PI3 K inhibitor)pretreatment(F=44.56,q=13.64~13.95,n=6,P<0.01),indicating that PI3K/AKT signaling pathway could participate in cell proliferation induced by AngⅡ.In Western blot analysis,after AngⅡ treatment for different times(5、15、30、60、180 min),p-AKT/AKT levels were(1.57±0.01),(0.96±0.05),(0.63±0.02),(0.66±0.06)and(0.65±0.04)(F=57.14,q=12.02,n=3,P<0.01),suggesting that AngⅡ can lead to rapid activation of AKT.Pretreatment with 20 μmol/L A01 inhibited the AKT activation induced by AngⅡ(F=24.26,q=7.27~7.62,n=3,P<0.01).The above results suggested that the inhibitory effect of A01 on AngⅡ-induced proliferation was partially mediated through activation of AKT signaling pathway.8.This experiment was to explore whether the inhibitory effect of A01 on AngⅡ-induced proliferation was due to activation of ERK signaling pathway.In the MTT assay,the cell viability in AngⅡ group rose to(1.62±0.05).However,this effect could be counteracted by PD98059(PD,a MEK inhibitor)pretreatment(F=41.86,q=12.29~13.05,n=6,P<0.01).In Western blot analysis,after AngⅡ treatment for different times(5、15、30、60、180 min),the p-ERK/ERK ratios were(2.82±0.24),(2.35±0.01),(1.89±0.09),(1.69±0.01)and(1.44±0.05)(F=35.72,q=6.31~16.72,n=4,P<0.05-0.01),indicating that AngⅡ can rapidly activate the ERK signaling pathway and the effect can last a longer time(up to 60 min).Pretreatment with 20 μmol/L A01 inhibited the ERK activation induced by AngⅡ(F=21.89,q=7.49~8.85,n=4,P<0.05-0.01).The above results suggested that the inhibitory effect of A01 on AngⅡ-induced proliferation was partially mediated through activation of ERK signaling pathway.Conclusions AngⅡ promotes ANO1 expression by its combination with AT1 R.ANO1 was suggested to be involved in AngⅡ-induced poliferation by using its specific inhibitor A01.The inhibitory effect of A01 on AngⅡ-induced proliferation might be related with upregulation of p21 and p27,and inhibition of both AKT and ERK signalling pathways. |
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