Effect Of ANO1 Inhibitor On Proliferation Of Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats And Its Underlying Mechanism

ObjectiveTMEM16A/Anoctamin-1（ANO1）is a newly-identified calcium-activated chloride channel（CaCC）which is widely distributed in the cardiovascular system.Both in the blood vessels and primarily-cultured vascular smooth muscle cells of spontaneously hypertensive rats（SHR-VSMC）,the protein levels of ANO1 were significantly increased,and the over-expression of ANO1 was found to be involved in the regulation of vasoconstriction and blood pressure.Our previous study found that the proliferation and migration of SHR-VSMC were inhibited by the treatment with ANO1 specific inhibitor T16A（inh）-A01（A01）for 24h.In this study,we observed the effect and mechanism of A01 on the proliferation of SHR-VSMC.Methods and Contents1.The expression of ANO1 in SHR-VSMC was detected by western blot.2.Different concentrations of A01 were incubated with SHR-VSMC for 24h and 72h,and the cell proliferation was estimated by the MTT assay.3.The cell proliferation was also estimated by detecting the PCNA protein level after the treatment with A01（20μM）in SHR-VSMC and WKY-VSMC for 24h.4.The effect of A01 on the cell cycle was observed by flow cytometry.5.To explore the involvement of Erk signaling pathway,A01（20μM）was added into VSMC for 5-60min,or different concentrations of A01 were treated for 30min.The activation of Erk pathway was estimated by the ratio of p-Erk to Erk.6.The effect of A01 on Akt signaling pathway was also observed after A01 treatment.7.The effect of A01 on intracellular calcium level was observed by confocal microscopy and calcium fluorescent dye.Results1.Compared with WKY-VSMC,the protein level of ANO1 was significantly increased in SHR-VSMC（P<0.01）.2.The result of MTT assay showed that the growth rate of SHR-VSMC was significantly accelerated（P<0.01）.Treatment with A01（10-20μM for 24h）suppressed the cell proliferation in SHR（P<0.05）,and this inhibitory effect was enhanced after 72h（P<0.01）.No change was observed in the proliferation of WKY-VSMC after the treatment of A01.3.Compared with the WKY control,the expression of proliferating cell nuclear antigen（PCNA）in SHR-VSMC was significantly higher（P<0.01）,and this elevation could partially attenuated by A01 treatment（20μM,24h）（P<0.05）.4.The results of PI staining showed that the ratio G₁ phase tended to drecrease while the S phase was obviously increased in SHR-VSMC,compared with its WKY control（P<0.05）.After A01 treatment（20μM,24h）,the proportion of S phase was reduced in SHR-VSMC（P<0.05）,while the inhibitory effect of A01 on WKY-VSMC was not obvious.5.The western blot showed that treatment with A01（20μM）suppressed the p-Erk level with the maximal effects at 30-60min（P<0.01）.When different concentrations of A01were incubated with SHR-VSMC for 30min,only 10-20μM A01 inhibited the activation of Erk（P<0.01）.No change was observed in the protein level of p-Erk of WKY-VSMC.6.In contrast,the p-Akt levels were not altered after A01 treatment for 5-60min in SHR-VSMC.The p-Akt expression also did not change after treatment with different concentrations of A01 for 30min.7.The change in intracelluar calcium level([Ca²⁺]_i)was estimated by Ca²⁺fluorescent probe.The results showed that SHR-VSMC had higher[Ca²⁺]_i compared with the WKY control.The[Ca²⁺]_i in SHR-VSMC was immediately suppressed by A01（20μM）treatment（P<0.05）,but it was still higher than that of WKY-VSMC.No change was observed in intracelluar calcium level of WKY-VSMC after A01 treatment.ConclusionsIn conclusion,A01 could inhibit the abnormal proliferation of SHR-VSMC estimated by the MTT assay and the PCNA levels.This inhibitory effect of A01 might be mediated with decreasing the ratio of S phase in cell cycle,inhibiting the activation of Erk pathway and reducing intracelluar calcium levels.