The Effects Of Notch Signaling And ERK1/2 Pathway In Entresto Inhibiting Vascular Smooth Muscle Cells Proliferation

Objective:Vascular smooth muscle cells（VSMCs）,as the main cellular component of the middle layer of the vascular wall,are involved in maintaining the vascular tension and the integrity of the vascular wall.Changes in the structure and function of VSMCs cause in-stent restenosis（ISR）and other major pathological basis of proliferative vascular diseases.Valsartan/sacubitril（Entresto）,which consists of angiotensinⅡ（AngⅡ）receptor antagonists and sacubitril inhibitors,is mainly used in the treatment of heart failure,but its effect on vascular VSMCs after vascular endothelial injury has not been reported.Notch signaling and ERK1/2 pathway are involved in regulating the proliferation and migration of VSMCs.The objective of this study was to observe the effect of Entresto on the proliferation and migration of VSMCs stimulated by AngⅡin vitro,and to study the role of Notch signaling pathway and ERK1/2 pathway in this pathway,providing new ideas for the prevention and treatment of ISR.Methods:（1）Human aortic smooth muscle cells（HA-VSMCs）were cultured in vitro for subculture and planking.（2）The cultured cells were divided into control group,AngⅡgroup,AngⅡ+Valsartan group and AngⅡ+Entresto group.AngⅡgroup:AngⅡ(1×10^-6mol/L)treated cells with drugs;AngⅡ+Entresto（Valsartan）group:The cells were first treated with 1×10^-5mol/L Entresto（Valsartan）for 1 hour,and then with AngⅡ(1×10^-6mol/L)after fluid change.Control group:cells were not treated with drugs;All groups were cultured for 24h.（3）After drug treatment,the proliferation activity of VSMCs in the above groups was detected by CCK8 to analyze the effect of Entresto on the proliferation of VSMCs.（4）Scratch tests were performed on VSMCs in the above groups after drug treatment.Images were collected at 0h,12h,24h and 36h,respectively,to analyze the effect of Entresto on VSMCs migration.（5）After drug treatment,the m RNA and protein expression levels of PCNA proliferation index and MMP-9 migration index in control group,AngⅡgroup and AngⅡ+Entresto group were detected by q RT-PCR and Western blot,and the effect of Entresto on the proliferation and migration of VSMCs was further analyzed.（6）The m RNA and protein expression levels of Notch1 and Jagged-1 in control group,AngⅡgroup and AngⅡ+Entresto group were detected by q RT-PCR and Western blot after drug treatment,so as to analyze the effect of Entresto on Notch signaling pathway.（7）After drug treatment,the protein expression of p-ERK1/2 in control group,AngⅡgroup and AngⅡ+Entresto group was detected by Western blot,so as to analyze the effect of Entresto on ERK1/2 pathway.Results:（1）Compared with the control group,AngⅡsignificantly increased the proliferative activity of VSMCs by CCK8 method.Compared with AngⅡgroup,the proliferative activity of VSMCs in AngⅡ+Valsartan and AngⅡ+Entresto groups was decreased.Compared with AngⅡ+Valsartan group,the proliferative activity of VSMCs in AngⅡ+Entresto group was lower.（2）The scratch test showed that AngⅡsignificantly increased VSMCs migration compared with the control group.Compared with AngⅡgroup,the migration activity of VSMCs in AngⅡ+Valsartan and AngⅡ+Entresto groups was decreased.The migration activity of VSMCs in AngⅡ+Entresto group was lower than that in AngⅡ+Valsartan group.（3）Compared with the control group,the expression levels of PCNA and MMP-9 in VSMCs of AngⅡgroup were increased;Compared with AngⅡgroup,the expression levels of PCNA and MMP-9 in VSMCs of AngⅡ+Entresto group were decreased.（4）Compared with the control group,the expressions of Notch1 and Jagged-1 in VSMCs of AngⅡgroup were increased;The expression levels of Notch1 and Jagged-1 in VSMCs of AngⅡ+Entresto group were decreased compared with that of AngⅡgroup.（5）Compared with the control group,the expression of p-ERK1/2 of VSMCs in AngⅡgroup was increased;Compared with AngⅡgroup,the expression of p-ERK1/2 in VSMCs in AngⅡ+Entresto group was decreased.Conclusion:1.Entresto can inhibite the proliferation and migration of AngⅡ-induced VSMCs,and the inhibitory effect is stronger than that of Valsartan group;2.Entresto can reduce the expression of Jagged-1,Notch1 and ERK1/2pathways in Notch signaling pathway;3.These effects may be related with activation of Notch1/Jagged-1 and ERK1/2 signaling pathway.