Role Of Rev-erb? In Neointima Hyperplasia After Vascular Endothelial Injury

Objective: 1.To investigate the concrete role of Rev-erb? in neointima hyperplasia after vascular injury.2.To investigate the concrete role of Rev-erb? in proliferation of vascular smooth muscle cell.3.To determine whether Nlrp3 is an important target for Rev-erb? to inhibit the proliferation of vascular smooth muscle cell(VSMC).Methods: 1.In vivo experiment: The model of vascular injury was established by wire injury of carotid artery.And the specific agonist(SR9011)and antagonist(SR8278)were used to interfere the activity of Rev-erb? in vascular tissue.(1)The neointimal hyperplasia of carotid artery was detected by hematoxylin-eosin staining.(2)The proliferation of VSMC was detected by immunohistochemical staining.(3)The expression of Rev-erb? in carotid artery was detected by qRT-PCR and Western blot.2.In vitro experiment: Proliferation was induced via incubation of VSMC with 100 nmol/L angiotensin ?(Ang ?)for 24 hours in vitro.The activity of Rev-erb? in VSMC was interfered by agonist(SR9011)and antagonist(SR8278).Respectively.Specific small interfering RNA(siRNA)was delivered to knock down the NOD-like receptor family,pyrin domain containing 3(Nlrp3)levels in VSMC.(1)The expression of Rev-erb? and Nlrp3 in VSMC was detected by qRT-PCR and Western blot.(2)The proliferation rate of cells were measured by cell counting kit-8(CCK-8)assay.(3)The proliferation of VSMC was detected according to the positive expression rate of Ki-67 by immunofluorescence staining.Results: 1.Rev-erb? was expressed in both carotid artery tissue and VSMC.2.In vivo experiments,Compared with sham operation group,Intima-media ratio and positive expression rate of Ki-67 increase in endothelial injury group(P<0.01).And the expression of Rev-erb? reduced in endothelial injury group(P<0.01).3.Intima-media ratio decrease after endothelial injury when Rev-erb? was activated by SR9011(P<0.01).While intima-media ratio increase after endothelial injury when Rev-erb? was inhibited by SR8278(P<0.01).At the same time,The results of immunohistochemistry also suggest that the positive expression rate of Ki-67 decrease after endothelial injury when Rev-erb? was activated by SR9011(P<0.01).While the positive expression rate of Ki-67 increase after endothelial injury when Rev-erb? was inhibited by SR8278(P<0.01).4.About in vitro experiments,Compared with control group,We confirmed that absorbance of CCK-8 and positive expression rate of Ki-67 increase in Ang ? group(P<0.01).And compared with control group,the expression of Rev-erb? in Ang ? group was lower by the results of PCR and Western blot(P<0.01).5.SR9011 was used to activate Rev-erb?,Compared with Ang ? group,The absorbance of CCK-8 and positive expression rate of Ki-67 decrease in Ang ?+SR9011 group(P<0.05).While SR8278 was used to inhibite Rev-erb?,Compared with Ang ? group,The absorbance of CCK-8 and positive expression rate of Ki-67 increase in Ang ?+SR9011 group(P<0.05).6.Compared with Ang ? group,The expression of Nlrp3 mRNA and protein decrease after Rev-erb? was activated by SR9011(P<0.05).While The expression of Nlrp3 mRNA and protein increase after Rev-erb? was inhibited by SR8278(P<0.05).Compared with Ang ? group,the positive expression rate of Ki-67 increase when knock down the Nlrp3(P<0.01).Compared with Ang ?+ SR8278 group,the positive expression rate of Ki-67 reduced in Ang ?+ SR8278+ si-Nlrp3 group(P<0.01).And there was no significant change between SR8278+ si-Nlrp3 group and si-Nlrp3 group(P>0.05).Conclusion: 1.The vascular expression of Rev-erb? declines during neointimal hyperplasia after endothelial injury,and activating Rev-erb? inhibits intimal hyperplasia while inhibiting Rev-erb? promotes intimal hyperplasia.2.The expression of Rev-erb? decrease in proliferative VSMC.Activating Rev-erb? inhibits VSMC proliferation,inhibiting Rev-erb? promotes VSMC proliferation.3.Nlrp3 is an important target for Rev-erb? to inhibit the proliferation of VSMC.