| 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(Ang?)is the main active component of the renin-angiotensin system(RAS).Circulation or tissue RAS activation can lead to an increase of Ang? 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 Ang?-induced VSMC proliferation,we first observed the effect of Ang? 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 Ang?-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 Ang? on ANO1 protein expression,the cells were treated with different concentrations of Ang?(1?10?100?500?1000 nmol/L)for 24 h,The ANO1 protein levels after Ang? 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 Ang? showed the maximum effect.After treatment with 100 nmol/L Ang? 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 Ang? 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 Ang? significantly elevated ANO1 protein expression in a dose and time-dependent manner.In subsequent experiments,the cells were incubted with 100 nmol/L Ang? 24 h.2.To explore the receptor mechanism of Ang?-induced expression of ANO1,the specific AT1 R blocker(losartan potassium,LP)and AT2 R blocker(PD123319,PD)were used.In Ang? 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 Ang? 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 Ang?-induced ANO1 up-regulation,20 ?mol/L A01 was pretreated before Ang?.The protein level of ANO1 in the Ang? group was increased to(1.39±0.08),while the protein level in the 20 ?mol/L A01+Ang? 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 Ang?-induced ANO1 protein expression.4.The effect of different concentrations of A01 on the viability of cells that have been treated whith Ang? was detected by MTT method.Cells were treated with different concentrations of Ang?(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 Ang? can promote the proliferation of VSMC.The cell viability in Ang?+A01(1?5?10?20 ?mol/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 ?mol/L A01 significantly inhibited Ang?-induced VSMC proliferation.5.By detecting PCNA,the effect of A01 on Ang?-induced cell proliferation was confirmed.In Ang? group,the PCNA protein level was increased to(1.62±0.08)for 24 h;the PCNA protein in the 20 ?mol/L A01+Ang? 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 Ang?-induced cells.6.After 100 nmol/L Ang? treatment for 24 h,p21 protein level was decreased to(0.70±0.02).In Ang?+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 Ang? 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 Ang?-induced proliferation was due to the activation of PI3K/AKT signaling pathway.After treated with Ang?,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 Ang?.In Western blot analysis,after Ang? 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 Ang? can lead to rapid activation of AKT.Pretreatment with 20 ?mol/L A01 inhibited the AKT activation induced by Ang?(F=24.26,q=7.27~7.62,n=3,P<0.01).The above results suggested that the inhibitory effect of A01 on Ang?-induced proliferation was partially mediated through activation of AKT signaling pathway.8.This experiment was to explore whether the inhibitory effect of A01 on Ang?-induced proliferation was due to activation of ERK signaling pathway.In the MTT assay,the cell viability in Ang? 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 Ang? 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 Ang? can rapidly activate the ERK signaling pathway and the effect can last a longer time(up to 60 min).Pretreatment with 20 ?mol/L A01 inhibited the ERK activation induced by Ang?(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 Ang?-induced proliferation was partially mediated through activation of ERK signaling pathway.Conclusions Ang? promotes ANO1 expression by its combination with AT1 R.ANO1 was suggested to be involved in Ang?-induced poliferation by using its specific inhibitor A01.The inhibitory effect of A01 on Ang?-induced proliferation might be related with upregulation of p21 and p27,and inhibition of both AKT and ERK signalling pathways. |
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