| Objective Essential hypertension is the most common cardiovascular disease with complex pathogenesis.Long-term hypertension will lead to vascular remodeling,which plays a key role in the occurrence and development of hypertension and target organ injury.The dysfunction of vascular smooth muscle cells(VSMCs)is the key mechanism of vascular remodeling.Endothelin-1(ET-1)is mainly synthesized and secreted by endothelial cells.As an important vasoactive substance in the cardiovascular system,it has been considered to be a very strong role in vasoconstriction and stimulate cell proliferation.It is also thought to play an important role in vascular remodeling associated with hypertension.TMEM16 A or Anoctamin-1(ANO1)is one kind of calcium activated chloride channel firstly reported in 2008.It is widely distributed in different blood vessels and regulates vasoconstriction and blood pressure.In spontaneously hypertensive rats(SHR),it is found that overexpression of ANO1 of VSMCs promotes the occurance of hypertension.However,the mechanism responsible for the overexpression of ANO1 is still not clear.In our previous studies,we observed that angiotension II(Ang II)remarkedly stimulated the expression of ANO1 in VSMCs.Similar to Ang II,ET-1 also increased the calcium activated chloride current,thus we hypothesized that ET-1 might stimulate the expession of ANO1 in VSMCs.In this study,we used the primary culture of VSMCs from Wistar rats to observe the effect of ET-1 on ANO1 expression and related signaling pathways.We also observed the effect of ANO1 inhibitor(T16Ainh-A01,A01)on ET-1 induced cell proliferation and possible mechanisms.Content 1.To observe the effect of ET-1 on ANO1 expression and its receptor mechanism.2.LY294002 or PD98059 was pretreated to observe the involvement of AKT and ERK signaling pathways in ET-1 induced expression of ANO1.3.Both MTT assay and PCNA expression were used to observe the effect of ANO1 inhibitor A01 on ET-1 induced cell proliferation.4.To study the possible mechanism of ANO1 inhibitor A01 on the protein expression of ET-1 induced cell cycle regulatory proteins(CDK4,Cyclin D1,p21)and cell phenotypic proteins(α-SMA,OPN).Methods After anesthesia treatment,the thoracic aorta of normal Wistar rats were quickly separated,and the primary culture of VSMCs was carried out by tissue patch method.MTT assay was used to detect the cell survival rate,and Western blot was used to detect the levels of related proteins.Results 1.This experiment aims to observe the effect of ET-1 on ANO1 protein expression.The results were as follows: after treatment with 0.1,1,10,100,1000 nmol/L ET-1 for 24 h,ANO1 expression levels were(1.02 ± 0.05),(1.25 ± 0.06),(0.94 ± 0.06),(0.89 ± 0.04)and(1.00 ± 0.05).The effect of 1 nmol/L ET-1 on ANO1 was the most significant(P<0.05).After 1 nmol/L ET-1 treatment for 1,6,12,24 and 48 h,ANO1 expression levels were(0.88 ± 0.06),(0.90 ± 0.09),(1.05 ± 0.09),(1.26 ± 0.04)and(1.13 ± 0.06).The expression of ANO1 was most significant after 24 h of ET-1 treatment(P<0.01).These results suggest that ET-1 might up-regulate ANO1 protein expression.In subsequent experiments,the cells were treated with 1 nmol/L ET-1 for 24 h.2.To confirm the possible receptor subtype of ET-1,1 μmol/L ZD4054 was added before ET-1 treatment to observe its blocking effect.The results showed as follows: compared with the control group(0.82 ± 0.03),the expression level of ANO1 in ET-1 group was significantly increased(0.96 ± 0.04,P<0.05);compared with ET-1 group,ANO1 protein expression in ET+ZD4054 group was significantly decreased(0.80 ± 0.04,P<0.05).These results suggest that ET-1 might promote ANO1 protein expression by binding with ETA receptor.3.This study aims to verify that ET-1 might up-regulate ANO1 expression through PI3K/AKT pathway.10 μmol/L LY294002(LY)was pretreated to observe the expression levels of AKT and ANO1.AKT protein results: compared with control group(1.06 ± 0.02),p-AKT/AKT protein level was increased to(1.24 ± 0.06,P<0.05)after ET-1 treatment;after LY was added,the protein level of p-AKT/AKT was decreased to(0.80 ± 0.01,P<0.001)compared with ET group.ANO1 protein: compared with control group(0.92 ± 0.08),the level of ANO1 was increased to(1.26 ± 0.03,P<0.05)after ET-1 treatment;after LY was added,the ANO1 protein level was decreased to(0.76 ± 0.08,P<0.01)compared with ET-1 group.These results suggest that the up-regulation of ANO1 protein by ET-1 may be achieved through the activation of PI3K/AKT pathway.4.This study was to confirm that ET-1 might induce up-regulation of ANO1 protein through MEK/ERK pathway.20 μmol/L PD98059(PD)was added for pretreatment to observe the expression levels of ERK and ANO1.ERK protein: compared with control group(0.90 ± 0.06),p-ERK/ERK protein level was increased to(1.11 ± 0.05,P<0.05)after ET-1 treatment;after PD was added,the p-ERK/ERK protein level was decreased to(0.60 ± 0.04,P<0.001)compared with ET-1 group.ANO1 protein: compared with control group(0.84 ± 0.02),the level of ANO1 protein was increased to(1.11 ± 0.08,P<0.05)after ET-1 treatment;after PD was added,ANO1 protein level was decreased to(0.82 ± 0.08,P<0.05)compared with ET-1 group.These results suggest that the up-regulation of ANO1 protein by ET-1 might be mediated by activating MEK/ERK pathway.5.In this study,MTT method was used to observe the effect of ANO1 inhibitor A01 on the survival rate of VSMCs treated with ET-1.The survival rates treated with 0.1,1,10,100 and 1000 nmol/L ET-1 for 24 h were(1.09 ± 0.06),(1.40 ± 0.08),(0.91 ± 0.03),(0.95 ± 0.02)and(1.04 ± 0.03).1 nmol/L ET-1 showed most effective in promoting the cell proliferation(P<0.001).After treatment with 1,5,10,20 μmol/L A01 and 1 nmol/L ET-1 for 24 h,the cell survival rates were decreased to(1.20 ± 0.06),(1.16 ± 0.06),(1.03 ± 0.04),(0.88 ± 0.04).10 and 20 μmol/L A01 had significant inhibitory effects on ET-1 induced proliferation,and 20 μmol/L A01 had the strongest inhibitory effect(P<0.001).Therefore,20 μmol/L A01 was selected for subsequent experiments.6.The detection of PCNA protein further proved that A01 inhibit the proliferation of ET-1-treated cells.Compared with the control group(0.87 ± 0.04),the expression level of PCNA was increased to(1.20 ± 0.01,P<0.001)after treatment with ET-1.After A01 pretreatment,compared with ET-1 group,the expression of PCNA protein in ET+A01 group was decreased to(0.98 ± 0.05,P<0.05).These results further suggest that A01 can inhibit the proliferation of VSMCs.7.This study observed the influence of A01 on the expression of several cell cycle regulatory proteins(CDK4,Cyclin D1 and p21).CDK4 results: compared with the control group(0.76 ± 0.09),the CDK4 protein level in ET was increased to(1.07 ± 0.03,P<0.05).After the treatment of A01,compared with ET group,CDK4 protein level in ET+A01 group was decreased to(0.81 ± 0.06,P<0.05).Cyclin D1 expression was similar to CDK4: compared with the control group(0.74 ± 0.04),the Cyclin D1 protein levelin ET was increased to(1.10 ± 0.05,P<0.05).When A01 was added,Cyclin D1 protein level in ET+A01 group was decreased to(0.76 ± 0.05,P<0.05)compared with ET-1 group.The expression of p21 is opposite to those of the above two proteins: compared with the control group(0.94 ± 0.04),the expression of p21 protein in ET group was decreased to(0.54 ± 0.06,P<0.01).When A01 was added,p21 protein level in ET+A01 group was increased to(0.88 ± 0.05,P<0.05)compared with ET group.These results suggest that A01 can inhibit expression of CDK4 and Cyclin D1 proteins but promote the expression of p21 protein,thus inhibiting cell cycle progression and cell proliferation induced by ET-1.8.This experiment mainly detected the effect of A01 on two phenotypic proteins induced by ET-1(α-SMA,OPN).Compared with the control group(1.14 ± 0.05),the expression of α-SMA in ET was significantly decreased to(0.80 ± 0.04,P<0.01).After the addition of A01,the α-SMA level in ET+A01 group was significantly increased to(1.08 ± 0.04,P<0.05)compared with ET group.The expression of OPN is opposite to α-SMA: compared with the control group(0.78 ± 0.05),the expression level of OPN in ET was significantly increased to(1.01 ± 0.04,P<0.05).When A01 was added,OPN expression level in ET+A01 group was significantly decreased to(0.77 ± 0.05,P<0.05)compared with ET group.These results suggest that A01 can regulate the phenotype transformation of VSMCs from secretion type to contraction type,thus inhibiting cell proliferation induced by ET-1.Conclusions In conclusion,ET-1 can induce the expression of ANO1 of VSMCs.This effect might be mediated by its binding of ET-1 with ETA receptor and subsequent activation of PI3K/AKT and MEK/ERK pathways.The ANO1 specific inhibitor A01 showed the significant inhibitory effect in ET-1 treated cells,suggesting that ANO1 might be involved in ET-1 induced proliferation of VSMCs by regulating cyclins and phenotypic transformation of VSMCs. |
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