| Part One Impact of cigarettes smoke extract on pulmonary vascular endothelial cell apoptosis and expression of Notch signalingObjective: To investigate the impact of CSE on HPMEC apoptosis and whether CSE can affect the expression of Notch signaling.Methods: Cultured HPMEC were exposed to CSE at various concentrates(0-5%) and for different durations(0h-24h). Flow cytometry with double staining(Annexin V-FITC/PI) was used to detect the apoptosis rate of HPMEC. Then, HPMECs were divided into control group and CSE group. The expression of Notch1, Notch2, Notch3, Notch4, Hes1, Hey2 m RNA and protein in CSE group compared to the control group were detected by Real-time PCR and Western Blot respectively. Statistical analyses were performed using SPSS version 17.0. P<0.05 was considered significant.Results:(1) With 0.5% CSE, the HPMEC apoptosis was increased obvious compared to control group(P<0.05). In the range of 0.5~2.5%CSE, with the increasing concentration of CSE, the apoptosis rate increased gradually. However, if the concentration was more than 2.5%, the necrosis rate would be too high.(2) With 1%CSE, the apoptosis rates of HPMEC increased as incubation times increasing. However, if the intervention time was longer than 12 h, the necrosis rate would increase, rather than apoptosis rate.(3) With 1%CSE treatment for 6h, Notch1/4 m RNA was obviously higher than control group(P<0.05). And along with the extending of intervening time, Notch1/4 m RNA decreased gradually. When intervened for 24 h, no matter m RNA or protein of Notch1/4 were obviously lower than control group(P<0.05).(4) With 1%CSE treatment for 6h, Notch2 m RNA had no significant difference with control group(P>0.05). And along with the extending of intervening time, Notch2 m RNA increased gradually. When intervened for 24 h, no matter m RNA or protein of Notch2 were obviously higher than control group(P<0.05).(5) With 1%CSE treatment for 6h, the expression of Hes1 and Hey2 m RNA had no significant difference with control group(P>0.05). And along with the extending of intervening time, Hes1 and Hey2 m RNA decreased gradually. When intervened for 24 h, no matter m RNA or protein of Hes1 and Hey2 were obviously lower than control group(P<0.05).Conclusions:(1) CSE induced HPMEC apoptosis in a dose and time dependent manner.(2) CSE could inhibit Notch1/4, and induce Notch2.(3) CSE may be involved in HPMEC apoptosis through affecting the expression of Notch signaling.Part Two Protective effect of Notch1 in CSE-induced pulmonary vascular endothelial cell apoptosisObjective: To investigate whether Notch1 can play a protective effect on CSE-induced pulmonary vascular endothelial cell apoptosis.Methods: Notch1 over-expression plasmids were constructed and packaged by lentivirus. Notch1 over-expression lentivirus were adopted to transfect into HPMEC. 1%CSE was used to incubate HPMEC for 24 h. Then, HPMECs were divided into five groups: control group, overexpression group, negative lentivirus group, CSE + over-expression group, CSE + negative lentivirus group. The expression of Notch1 m RNA and protein were detected by Real-time PCR and Western Blot respectively. Flow cytometry with double staining(Annexin V-FITC/PI) was used to detect the apoptosis rate. Statistical analyses were performed using SPSS version 17.0. P<0.05 was considered significant.Results:(1) The expression of Notch1 was increased in over-expression group compared to control group and negative lentivirus group(P<0.05). It had no significance between control group and negative lentivirus group(P>0.05). After CSE intervention, no matter over-expression group or negative lentivirus group, the expression of Notch1 was decreased(P<0.05). Moreover, Notch1 expression in CSE + over-expression group was still higher than CSE + negative lentivirus group(P<0.05).(2) The cell apoptosis rate was similar between over-expression group and control group(P>0.05), both of the two groups were lower than negative lentivirus group(P<0.05). After CSE intervention, nomatter over-expression group or negative lentivirus group showed an increase in cell apoptosis(P<0.05). Furthermore, the cell apoptosis rate in CSE + over-expression group was still lower than CSE + negative lentivirus group(P<0.05).Conclusions: The overexpression of Notch1 could inhibit HPMEC apoptosis induced by CSE.Part Three Notch1 inhibited pulmonary vascular endothelial cell apoptosis through ERK pathwayObjective: To explore whether Notch1 affect CSE-induced pulmonary vascular endothelial cell apoptosis through ERK pathway.Methods: First, HPMECs were divided into control group and CSE group; Second, HPMECs were divided into five groups: control group, over-expression group, negative lentivirus group, CSE + over-expression group, CSE + negative lentivirus group; Third, HPMECs were divided into four groups: control group, CSE group, PD98059 group, CSE + PD98059 group. The expression of ERK1, ERK2, Notch1 m RNA and protein were detected by Real-time PCR and Western Blot respectively. Flow cytometry with double staining(Annexin V-FITC/PI) was used to detect the apoptosis rate. Statistical analyses were performed using SPSS version 17.0. P<0.05 was considered significant.Results:(1) With 1%CSE treatment for 6h, ERK1 m RNA had no significant difference with control group(P>0.05). And along with the extending ofintervening time, ERK1 m RNA increased gradually. When intervened for 24 h, no matter m RNA or protein of ERK1 were obviously higher than control group(P<0.05).(2) With 1%CSE treatment for 6h, ERK2 m RNA was higher than control group(P<0.05). And along with the extending of intervening time, ERK2 m RNA increased gradually. When intervened for 24 h, no matter m RNA or protein of ERK2 were obviously higher than control group(P<0.05).(3) The expression of ERK1 and ERK2 were increased in overexpression group compared to control group and negative lentivirus group(P<0.05). There was no significance between control group and negative lentivirus group(P<0.05). After CSE intervention, no matter over-expression group or negative lentivirus group, the expression of ERK1 and ERK2 were increased(P<0.05). Moreover, ERK1 and ERK2 expression in CSE + over-expression group were still higher than CSE + negative lentivirus group(P<0.05).(4) The Notch1 expression in CSE group, PD98059 group, as well as CSE+PD98059 group decreased obviously than control group(P<0.05). However, the differences among the three group had no significant(P>0.05).(5) Compared to control group, the apoptosis rate in CSE group increased significantly(P<0.05). However, when combined with PD98059, the apoptosis rate was much lower than CSE group(P<0.05).Conclusions: CSE could affect ERK pathway by inhibiting Notch1, then lead toHPMEC apoptosis. |
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