Effects Of Elemene On Proliferation Of Human Pulmonary Microvascular Endothelial Cells Induced By Human Airway Fibroblasts And Its Mechanisms

Objective:To investigate the effects of elemene on the proliferation and apoptosis of human pulmonary microvascular endothelial cell(HPMEC)induced by human airway fibroblast(HAFB),and to explore the possible molecular mechanism by detection the expression of Cysteinyl aspartate specific proteinase-3(Caspase-3),B cell lymphoma/ leukemia-2(Bcl-2)and B cell lymphoma/leukemia-2-Associateds X(Bax)proteins in HPMEC,and to provide a new experimental basis for the local application of elemene in the treatment of benign airway stenosis.Methods:(1)Culturing HAFB and HPMEC in vitro.HAFB was randomly divided into five groups:A1:control group,intervention with DMEM;A2:intervention with 30ug/ml elemene;A3:intervention with 60ug/ml elemene;A4:intervention with 90ug/ml elemene;A5:intervention with 120ug/ml elemene.24 hours later,removing out the old culture medium and then adding DMEM to continue cultivating,24 hours later,taking out the culture supernatant of HAFB and keepping in refrigerator.HPMEC was randomly divided into six groups:B1:control group,intervention with DMEM;B2:intervention with the supernatant of conventional cultured HAFB;B3:intervention with supernatant of HAFB treated with 30ug/ml elemene;B4:intervention with supernatant of HAFB treated with 60ug/ml elemene;B5:intervention with supernatant of HAFB treated with 90ug/ml elemene;B6:intervention with supernatant of HAFB treated with 120ug/ml elemene.(2)After culture in different media for 24hours、48hours、72hours,the growth situation and morphological changes of HPMEC was observed under inverted microscope,and the proliferation of HPMEC was detected by MTT method.After culture in different media for 24 hours,the apoptosis rate of HPMEC was detected by Annexin V-FITC/PI double staining flow cytometry.(3)Culturing HPMEC in vitro,randomly dividing it into three groups:G1:control group,intervention with DMEM;G2:intervention with the supernatant of conventional cultured HAFB;G3:intervention with supernatant of HAFB treated with 60ug/ml elemene;After culture for 24 hours,the total protein of HPMEC was extracted and the expression level of Caspase-3,Bcl-2 and Bax protein in HPMEC was detected by western blot method.Results:(1)Compared with the control group,there is no statistical significance(P>0.05)in the effect of the supernatant of conventional cultured HAFB on the proliferative status of HPMEC,and there is no time-dependent(P>0.05).Compared with the control group,the supernatant of HAFB treated with elemene can inhibit the proliferation of HPMEC(P<0.05).The effect of the supernatant of HAFB treated with elemene on the proliferation of HPMEC is dose-dependent(P < 0.05),but not timedependent(P > 0.05).(2)Compared with the control group,after the supernatant of conventional cultured HAFB intervened HPMEC,the morphology of HPMEC did not change significantly.Compared with the control group,the supernatant of HAFB treated with different concentrations of elemene changed the morphology of HPMEC,and with the increase of elemene concentration,the arrangement of HPMEC became irregular and the intercellular space became larger gradually.(3)Compared with the control group,after the supernatant of conventional cultured HAFB intervened HPMEC,the early apoptotic rate of HPMEC increased(P<0.05).Compared with the control group,after the supernatant of HAFB treated with elemene intervened HPMEC,the early apoptosis rate of HPMEC increased significantly(P<0.01),and the higher the concentration of elemene,the higher the early apoptosis rate(P < 0.01).(4)Compared with the control group,after the supernatant of conventional cultured HAFB intervened HPMEC,there is no statistical significance in the expression of Caspase3 in HPMEC(P > 0.05),but the expression of Bcl-2 was down-regulated(P < 0.05),the expression of Bax was down-regulated(P < 0.01).Compared with the control group,after the supernatant of HAFB treated with 60ug/ml elemene intervened HPMEC,the expression of Caspase3 was up-regulated(P< 0.01),the expression of Bax was up-regulated(P< 0.01),the expression of Bcl-2 was down-regulated(P <0.01).Conclusions: HAFB supernatant treated with elemene can inhibit the proliferation of HPMEC by inducing apoptosis of HPMEC.And its molecular mechanism may be related to the up-regulation of Bax、Caspase-3 expression level and the downregulation of Bcl-2 expression level.