The Mechanism Of SUMO1/HIF-1α Pathway In Regulating Inflammatory Response In Mice With Lower Limb Ischemic Disease

Objective:Acute lower limb ischemia is a common type of peripheral artery disease resulting from sudden decrease of lower limb blood supply,leading to the hypoxic injury of tissue and cells.Therefore,hypoxia-induced pathogenic process is the hallmark of acute lower limb ischemia.As an oxygen-sensing molecule,Hypoxia inducible factor-1α(HIF1α)can regulate angiogenesis,tumorigenesis and inflammation through the activation of various transcription factors.However,the mechanism of HIF-1α induced inflammation in lower limb acute ischemic diseases has not been fully defined.The purpose of this study was to explore the relationship between HIF-1α and inflammation and NLRP3 inflammasome-mediated pyroptosis in acute lower limb ischemic disease,and further explore the specific mechanism of HIF-1α expression changes.The goal is to provide new ideas for treating acute lower limb ischemia.Method:Male C57BL/6J mice aged 6-8 weeks were selected as experimental subjects,and mouse lower limb ischemia model was established by surgical operation.The lower limb blood flow of mice was detected by laser Doppler perfusion imager.The gastrocnemius muscle was taken on the third day after the operation,and the mRN A and protein expressions of HIF-1α,SUMO1,NLRP3,Caspase-1 and GSDMD were detected by qRT-PCR and Western Blot.Serum was collected to detect IL-1β and IL-18 release levels.In vitro anoxic model was constructed.The mRNA and protein levels of HIF-1α,SUMO1 and NLRP3,Caspase-1 and GSDMD were detected by qRT-PCR and Western Blot.The cell supernatant was collected to detect IL-1β and IL-18.In order to investigate the relationship between HIF-1α and N1RP3-mediated inflammation and pyrosis,HIF-1αoverexpression plasmid was constructed and transfected into C2C12 to detect the mRNA and protein expressions of NLRP3 associated inflammatory molecules.PI and EdU were employed to detect pyroptosis and cell proliferation,respectively.CCK8 assay was used to detect cell viability.To determine how HIF-1α was regulated,SUMO1 siRNA was constructed.After SUMO1 is knocked down,Western blot and qRT-PCR were used to analyze HIF-la protein expression and inflammation-related indicators.EdU was used to detect cell proliferation and CCK8 was used to measure cell viability.Bioinformatic analysis and co-immunoprecipitation were used to predict and examine the interaction between HIF-la and SUMO1.HIF-1α was SUMOylation at K477 by point mutation assay.Results:In the animal experiment,the mRNA and protein levels of NLRP3,Caspase1 and GSDMD were significantly increased in the ischemia group compared with the nonischemia group.In the cell experiment,the mRNA and protein levels of NLRP3,Caspase-1 and GSDMD were significantly increased in the hypoxia group compared with the control group,while the cell proliferation and cell vitality were significantly decreased.HIF-1αand SUMO1 are upregulated in ischemic tissue and hypoxic cells.After HIF-αoverexpression.mRNA and protein levels of NLRP3.Caspase-1 and GSDMD were significantly increased,while cell proliferation ability and cell viability were significantly decreased.After SUMO1 knockout,the protein level of HIF-1α,mRNA and protein levels of NLRP3,Caspase-1 and GSDMD were significantly decreased,and the cell proliferation ability and cell vitality were significantly increased.Co-immunoprecipitation revealed an interaction between SUMO1 and HIF-1α,and SUMO modification of HIF-1α at K477.Conclusion:HIF-1α and SUMO1 are involved in the progression of acute lower limb ischemia.HIF-1α can promote the expression of NLRP3 and other inflammation-related molecules,leading to pyroptosis.which may be related to the SUMOylation of HIF-1α,suggesting that SUMO1/HIF-1 a pathway may be a new target for the treatment of inflammation in lower limb ischemic diseases.