The Role And Underlying Mechanism Of Heat Shock Protein 22 In Injury Of HUVECs Induced By Hypoxia/Reoxygenation

Objective:Probe to the role and underlying mechanism of Heat shock protein 22 (HSP22) in injury of HUVECs induced by hypoxia/reoxygenation (H/R).Methods:A RNAi plasmid of HSP22 gene (pGcsi-U6/Neo/GFp/shRNA/HSP22) was constructed and transfected into HUVECs to download the expression of HSP22 which could be up-regulated by stress such as heat and H/R. According to the different intervention method, HUVECs were divided into 4 groups, i.e. Control group, Lipo group, Blank control group (i.e.pGcsi-U6/Neo/GFp/shRNA transfection group) and RNAi group(i.e. pGcsi-U6/Neo/GFp/shRNA/HSP22 transfection group). And these cells were subjected to hypoxia for 24h followed by reoxygenation for 0,3, 6,12h. After intervention of H/R, the rate of HUVECs growth inhibition was assayed by MTT and the expression of IK-Ba, NF-κB, Bcl-2 and Caspase-3 were measured by RT-PCR and Western blot.Results:1.The expression of HSP22 protein in RNAi group was obviously lower than others(0.20±0.02 vs.0.33±0.03 vs.0.45±0.06 vs.0.48±0.05 respectively in reoxyenation for Oh,3,6,12h, P<0.05), and there was no obviously difference among the Control, Lipo and Blank control group (P>0.05)2. The rate of HUVECs growth inhibition in RNAi group were obviously higher than Blank control group at the same time point of reoxygenation (58.5±2.1% vs.41.6±5.4%,62.7±5.4% vs.45.6±3.7%,65.4±8.4% vs.48.5±5.2% and 68.6±6.7% vs.52.9±3.4% respectively in reoxyenation for Oh,3,6,12h, P<0.05)3.The expression of bcl-2 mRNA and protein in RNAi group were obviously lower than Blank control group at the same time point of reoxygenation(1.85±0.06 vs.2.65±0.15,1.66±0.04 vs.2.35±0.08,1.09±0.05 vs.2.05±0.08,0.69±0.04 vs.1.75±0.09 respectively in reoxyenation for Oh,3,6,12h, P<0.05) and (0.54±0.04 vs.1.05±0.05,0.32±0.05 vs.0.75±0.03,0.29±0.02 vs.0.55±0.03,0.13±0.04 vs.0.45±0.07 respectively in reoxyenation for Oh,3,6,12h, P<0.05).4. The expression of Caspase-3 mRNA and protein in RNAi group is obviously higher than the Blank control group at the same time point of reoxygenation. (mRNA expression:2.75±0.04 vs.l.75±0.07,2.96±0.04 vs.1.95±0.06, 3.59±0.05 vs.2.45±0.05,3.59±0.05 vs.2.77±0.09 respectively in reoxyenation for Oh, 3,6,12h, P<0.05)and (protein expression:3.59±0.05 vs.1.75±0.04,6.18±0.24 vs.2.05±0.11,8.82±0.07 vs.2.65±0.12,9.36±0.11 vs.3.05±0.12 respectively in reoxyenation for Oh,3,6,12h, P<0.05).5. The expression of NF-κB mRNA in Blank control group were obviously higher than the RNAi group at the same time point of reoxygenation.(2.54±0.03 vs.2.05±0.02,3.26±0.05vs.2.55±0.04,3.85±0.03 vs.3.12±0.05,4.34±0.06 vs.3.87±0.04, P<0.05). And the expression of Iκ-BαmRNA in Blank control group were obviously lower than the RNAi group at the same time point of reoxygenation (2.95±0.02 vs.3.65±0.04,2.68±3.17 vs.3.05±0.04,1.75±0.03 vs.2.16±0.02,1.62±0.05 vs.1.89±0.04 respectively in reoxyenation for Oh,3,6,12h, P<0.05).Conclusion:1. The expression of HSP22 could be induced by H/R intervention in HUVECs and inhibited by transfection of RNAi plasmid of HSP22 gene. The transfection of Lipo and control plasmid did not influent the express of HSP22 in HUVECs.2. HSP22 might play a protective role in the injury of HUVECs induced by H/R.3. The protective mechanism of HSP22 might involve in the up-regulateing of bcl-2 and down-regulateing of Caspase-3 which contribute to protecting HUVECs away from apoptosis induced by H/R.4. The protective mechanism of HSP22 also might involve in inhibition of inflammation which presented as up-regulateing of IK-Ba and down-regulateing of NF-κB.