The Relationship Between Hypoxia Inducible Factor-1α And NLRP3 Inflammasomes Activation In Hypoxia/reoxygenation

Objective: To study the relationship between hypoxia inducible factor-1α( HIF-1α) and NOD-like receptor3( N LRP3) inflammasomes activation of human umbilical vein endothelial cells( HUVECs) in hypoxia/reoxygenation(H/R).Methods:(1)The HUVECs were cultured in vitro and the H/R model of HUVECs was established.(2)The adequate time of H/R was found out by Western Blot measuring the protein levels of HIF-1α and NLRP3.(3) The cultured cells were intervened in the select time of H/R( hypoxia 16 h / reoxygenation 8h). The study included two parts. In the first part, the cells were randomly divided into four groups: the normal group, the only H/R group, the H/R plus YC-1 group and the H/R plus DMOG group. In the second part, the cells were randomly divided into six groups: the normal group, the only H/R group, the H/R plus negative interference fragment group, the H/R plus HIF-1α si RNA group, the H/R plus control plasmid group and the H/R plus HIF-1α overexpressing plasmid group.(4)The protein and m RNA levels of HIF-1α,NLRP3,ASC,caspase-1 were measured by Western Blot and realtime fluorescence quantitative PCR.ELISA was used to measure the levels of interleukin-18.Results:(1)Western Blot showed that the protein levels of HIF-1α and NLRP3 were significantly higher in hypoxia /reoxygenation, and they peaked at hypoxia 16h/ reoxygenation 8h.(2)Compared with the H/R group, pretreatment with HIF-1α inhibitor YC-1 reduced the protein expressions of HIF-1α,NLRP3,ASC,caspase-1(P<0.05);the m RNA levels of HIF-1α were not changed(P>0.05),while the m RNA levels of NLRP3,ASC,caspase-1 were significantly reduced(P<0.05);the levels of IL-18 werelower(P<0.05). Pretreatment with HIF-1α inducer DMOG increased the protein and m RNA expressions of HIF-1α,NLRP3,ASC,caspase-1(P<0.05) and the levels of IL-18 were higher(P<0.05).(3)The H/R plus HIF-1α si RNA group had lower protein and m RNA levels of HIF-1α,NLRP3,ASC,caspase-1 compared with the only H/R group(P<0.05),and the IL-18 secretions were significantly reduced(P<0.05).The H/R plus HIF-1α overexpressing plasmid group had higher protein and m RNA levels of HIF-1α, NLRP3,ASC,caspase-1 compared with the only H/R group(P<0.05),and the IL-18 secretions were significantly increased(P<0.05).Conclusion:(1) The expressions of HIF-1α and NLRP3 inflammasomes of HUVECs were significantly increased in hypoxia/reoxygenation.(2) Inhibition of HIF-1α can downregulate the expressions of NLRP3 inflammasomes.( 3) Induction of HIF-1α can upregulate the expressions of NLRP3 inflammasomes.(4) HIF-1α can influence the activation of NLRP3 inflammasomes of HUVECs in hypoxia/reoxygenation.