Effects And Mechanism Of N-n-butyl Haloperidol Iodide On Hypoxia-induced Proliferation Of Vascular Smooth Muscle Cells

N-n-butyl haloperidol iodide (F2) is a new quaternary ammonium salt derivative of Haloperidol, which was synthesized by our laboratory.Our previous studies have shown its protective effects on myocardial ischemia/reperfusion (I/R) injury and hypoxia/reoxygenation(H/R) injury of cardiocytes and endotheliocytes,which is associated with inhibiting egr-1 mRNA transcription and protein overexpression .Hypoxia can induce overproliferation of vascular smooth muscle cells(VSMCs),which further induces a series of vascular proliferous diseases. The purpose of this experiment is to investigate effects of F2 on hypoxia induced proliferation of vascular smooth muscle cells and to explore its relationship with Egr-1.ObjectiveTo investigate the effect and mechanism of F2 on hypoxia-induced proliferation of rat thoracic aortic smooth muscle cells in vitro and show the relationship between the effect of F2 and the overexpression of early growth response gene-1(egr-1) .Methods1 Culture of rat thoracic aortic smooth muscle cells in vitro: cultured by the method of adhesion to wall and observed under the microscope.2 Detection of rat thoracic aortic smooth muscle cells in vitro:detected by the immunohistochemistryα-SM-actin antibody.3 Transfection of egr-1 antisense oligodeoxynucleotides to cultured VSMCs in rat.4 Groups:Passage 3~5 of cultured VSMCs were randomly divided into one of eight groups:control group,hypoxia group, F2 group(1×10 - 6 mol/L), PEG group (1×10 - 6 mol/L),AS-ODN group,S-ODN group,Sc-ODN group and LIP group.5 Passages 3 to 5 of cultured rat thoracic aortic SMCs were used to establish hypoxia models. The viability of cultured VSMCs was detected by MTT assay. The cell cycle of cultured VSMCs was analyzed by flow cytometry(FCM). The expression levels of Egr-1 and PDGF-A proteins were examined by Western blot.Results1 A few of cells grew out of tissue-block about 4 to 7 days late and approached confluence approximately 2 to 3 weeks.A typical"hill-valley"growth pattern was displayed after cells were passaged.2 98% of the cultured smooth muscle cells were positive forα- SM actin.3 FITC labeled antisense oligodeoxynucleotides emited green fluorescence. The location of fluorescence is coincidented with the area of cells under the inverted phasecontrast microscope, which indicated that antisense oligodeoxynucleotides had transfected VSMCs successfully.4 Hypoxia injured VSMCs caused a time-dependent induction of Egr-1 protein, being evident at 1 h, peaking at 2 h, and declining thereafter.5 Compared with the control group, the absorption value,proliferation index and the levels of Egr-1 and PDGF-A of hypoxia group, PEG group,S-ODN group,Sc-ODN group and LIP group were all increased.Compared with the hypoxia group,all these above items of antisense oligodeoxynucleotide group and F2 group were all decreased.ConclusionsF2 inhibts hypoxia-induced proliferation of VSMCs in vitro,which is mediated by Egr-1.