| Objective:To establish a heat stress model of endothelial cells, and to illustrate whether PPAR β/δ was affected by heat stress in HUVECs, and to demonstrate the role of PPAR β/δ in heat pretreated HUVECs’ anti-oxidant injury induced apoptosis.To explore the expression tendency of anti-apoptotic gene Bcl-2, pro-apoptotic gene Bax、P53、APAF-1during the recovering time in heat treated HUVECs, and to illustrate whether PPAR β/δ could moderate the expression of Bcl-2, and understand the specific mechanism.Methods:1) Primary HUVECs were isolated, HUVECs were treated in water bath of different temperature (42℃、43℃、44℃、45℃) for different time(15mhn、20min、25min、30min), cells’survival rate were determined by MTT, we chose the proper water bath temperature and treat time in group which survival rate were proximately50%as a heat interval condition.2) mRNA lever of PPAR β/δ、Bcl-2、P53、APAF-1were detected by RT-PCR during different recover time (1h、3h、6h、12h、24h、48h、72h) in heat treated HUVECs; genes which affected by heat stress apparently were selected, and the protein level of them were detected by Western-blot next; group of HUVECs whose expression of PPAR β/δ reached the maximum during the recovering procedure of heat stress were chose as a typical HUVECs heat stress model.3) HUVECs under heat stress or pretreated by PPAR β/δ agonists GW0742or transfected by PPAR β/δ-shRNA plasmid both under H2O2induced oxidant stress circumstance, apoptosis rate of cells were detected by Hoechst33258dyeing、TUNEL and DNA-ladder gel-electrophoresis.4) Expression of Bcl-2in HUVECs were also detected by RT-PCR and Western-blot after heat stress or pretreated by PPAR β/δ agonist GW0742or transfected by PPAR β/δ-shRNA plasmid.Results:1) HUVECs under43℃,25min water bath show a nearly50%cell survival rate (0.49±0.06), which were chose as a heat stress condition.2) Result of RT-PCR show that PPAR β/δ were up-regulated after heat treated, what’s more it reach the maximum at the48th hours after heat stress(2.26±0.12, P<0.01VS.1.0in Control Group). mRNA of anti-apoptotic gene Bcl-2down-regulated at the earlier stage (3h) of recover time after heat stress (0.81±0.07, P<0.01VS.1.0in Control Group), but up-regulated at the latter stage (24h) of recovering time (1.460±.01, P<0.01VS.1.0in Control Group). mRNA of APAF-1down regulated closely behind heat stress and turned to be normal latter; while expression of P53show little change. Result of Western-blot reveal that protein of PPAR β/δ also reached the peak at the48th after heat stress (3.30±0.13VS.1.0in Control Group, P<0.01), so we chose HUVECs under43℃,25min water bath and recover in normal culture condition for48hours as the typical heat stress cell model. Protein level of Bcl-2and Bax were also detected by Western-blot, to our interesting, ratio of Bcl-2/Bax were down regulated at the earlier stage of recovering time (lh-6h) after heat stress (0.48±0.12in3h Group VS.1.23±0.22in Control Group P<0.01), but distinctly up regulated at the latter stage of recovering time (24h-48h),(1.85±0.10in48h Group VS.1.23±0.22in Control Group P<0.01). Protein level of APAF-1was down regulated at the earlier stage of recovering time (0.46±0.09in3h Group VS.1.0in Control Group, P<0.01), but finally return to be normal by the time of12h after heat stess.3) Both of heat pre-treating or agonist GW0742pre-treating could enhance the survival rate of HUVECs under H2O2induced oxidant stress (28.86±8.38%in heat pretreated group VS.39.61±6.16in H2O2treated simply group,p<0.01),(33.02±5.12%in GW0742pretreated group VS.39.61±6.16in H2O2treated simply group,p<0.05). While HUVECs transfected with PPAR β/δ-shRNA plasmid the apoptosis rate of cells which induced by H2O2were increased apparently (46.11±5.86%VS.39.61±6.16in H2O2treated simply group,p<0.05).4) Consistent with these result, western blot also reveal that heat stress or GW0742could up-regulate the protein expression of Bcl-2in HUVECs,(1.52±0.27in heat pretreated group VS.1.0in Control Group, P<0.01)、(1.98±0.50in GW0742pretreated groupVS.1.0in Control Group, P<0.01); when transfected with PPAR β/δ-shRNA plasmid, protein level of Bcl-2were down regulated in HUVECs.(0.24±0.09VS.1.0in Control Group, P<0.01) But to our interesting, except heat stress (1.53±0.11in heat pretreated group VS.1.0in Control Group,P<0.01) neither GW0742nor PPAR β/δ-shRNA transfection can affect the expression of Bcl-2mRNA.Conclusions:1) We have established a kind of endothelial cells heat stress model, and have demonstrated that after heat stress, HUVECs can recover in a time.2)Heat stress induced PPAR β/δ expression in HUVECs, and it reached the maximum at the48th hours after heat treatment.3) Heat stress up-regulated the expression of Bcl-2in HUVECs, and it show a similar tendency to PPAR β/δ, ratio of Bcl-2/Bax down regulated at the earlier stage of recovering time (1h-6h) after heat stress but up-regulated distinctly at the latter stage of recovering time(24h-48h) in HUVECs. While mRNA of P53show little change in HUVECs after heat stress.4) Heat stress induced PPAR β/δ could enhance the anti-apoptotic characters of heat pretreated HUVECs.5) PPAR β/δplays important roles in regulation of Bcl-2, and PPAR β/δ regulate Bcl-2through an indirect post-transcriptional path way.This research revealed that heat pre-treated endothelial cells can recover from injury in a time, during this procedure the expression of PPAR β/δ were up-regulated, which may protect endothelial cells from oxidant injury, one of the possible mechanism is that PPAR β/δ regulating Bcl-2and moderate the apoptosis pathway. |
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