Research Regarding The Mechanism Of PGC1α Regulating Autophagy-induced Inflammatory Impairment Of The Vascular Endothelial Cells In Kawasaki Disease

Kawasaki disease is a systemic immune vascular inflammation of small and medium-sized blood vessels in infants and young children,which is the main cause of acquired heart disease in children.At present,the pathogenesis is still unclear.Immune activation and endothelial cell damage are recognized as the basic changes.Some studies have shown that mitochondria mediate the signal cascade of immune cell activation and immune response,but there are few studies on the role and mechanism of mitochondria in immune vasculitis of Kawasaki disease.The function of vascular endothelial cells is involved in the development and prognosis of Kawasaki disease,and the dysfunction of endothelial cells is closely related to the state of mitochondria.Our previous study found that there was significant damage in mitochondria of vascular endothelial cells in mice with KD-like vasculitis,which proved that mitochondria were involved in the occurrence and development of Kawasaki disease-like immune vasculitis.As the core regulatory molecule of mitochondrial function,PGC1α participates in the regulation of mitochondrial oxidative stress,energy metabolism and biogenesis.It can represent the function of mitochondria to some extent,and its abnormal expression is closely related to vascular diseases.Therefore,the main purpose of this study is to establish an immune vasculitis model to study the role and mechanism of PGC1α in Kawasaki disease-like vascular injury,so as to provide new ideas and potential targets for the diagnosis and treatment of Kawasaki disease.Part Ⅰ Expression and clinical significance of PGC1αin Kawasaki diseaseObjective:To investigate the changes of PGC1α in Kawasaki disease and to analyze the correlation between the changes and clinical events such as coronary artery injury in Kawasaki disease.Methods:The peripheral blood of normal group,fever control group and Kawasaki patients in acute and subacute stage were collected,mononuclear cells(PBMC)were isolated,and the morphology of cells and organelles were observed by electron microscope.The mRNA expression of PGC1α in acute phase was detected by fluorescence quantitative PCR(RT-qPCR),and the protein expression was detected by WesternBlot(WB),and the correlation with coronary artery damage was analyzed.The mouse model of coronary arteritis was induced by intraperitoneal injection of Candida albicans cell wall extract(CAWS)4mg for 2 rounds for 5 days.The body weight of mice was monitored,the heart and coronary artery inflammation were detected by immunohistochemistry,and the level of PGC1α in PBMC was detected by WB and Real-time methods.Results:(1)In the acute stage of Kawasaki disease,PBMC mitochondria were damaged,azurophilic granules leaked,vacuolated mitochondria and autophagosomes were seen,which were more than those in normal group and febrile control group,but the damage recovered in subacute stage,but some mitochondrial vacuolation could still be seen.(2)Compared with the control group,the transcriptional and protein levels of PGC1αin PBMC of patients with Kawasaki disease were significantly down-regulated.(3)The transcriptional level of PGC1α in PBMC of patients with KD was significantly lower in the coronary artery injury group than in the non-coronary artery injury group(p<0.01).(4)Transcriptional and protein levels of PGC1α were down-regulated in PBMCs of immune vasculitis mice at 14-dayConclusion:(1)Mitochondrial damage exists in both acute and subacute stages of PBMC in Kawasaki disease,and it is most significant in acute phase.(2)The levels of PGC1α transcription and protein in PBMC of mice with acute Kawasaki disease and vasculitis were significantly lower than those of the control group.(3)The transcription level of PGC1α in PBMCs of patients with Kawasaki disease in the acute phase was significantly lower in the coronary artery injury group than in the non-coronary artery injury group.Part Ⅱ Role of PGC1α and autophagy in vascular endothelial inflammatory injury in Kawasaki diseaseObjective:In view of the phenomena and conclusions of the first part,the role of PGC1α and autophagy in the inflammatory injury of vascular endothelial cells in Kawasaki disease was further explored.Methods:The role and significance of PGC1α and autophagy in immune cell-related vascular injury in Kawasaki disease in patients with Kawasaki were investigated in a model of coronary endothelial cell inflammatory injury and on vascular tissue in mice with immune vasculitis.1.In vitro cell experiments:PBMC primary cells from patients with Kawasaki disease were collected as stimulators to stimulate human coronary artery endothelial cells(HCAEC)to construct an immune vascular endothelial cell injury model.A normal cell control group(Mock group,without stimulation);a normal child control group(Normal group,given PBMC stimulation of normal children);a fever control group(Fever group,given PBMC stimulation of fever children);Kawasaki disease acute phase PBMC stimulation The Kawasaki disease group(KD group,given PBMC stimulation in the acute phase of Kawasaki disease).The detection methods are as follows:(1)The cellular localization of PGC1α(red)and mitochondrial marker protein TOM20(green)in HCAECs was detected by immunofluorescence.(2)HCAECs were stimulated with PBMCs in each group for different time,and the changes of cell morphology were observed,and the content of PGC1α in HCAECs was detected by WB method.(3)The changes of autophagy-related protein light chain 3(LC3)were detected by immunofluorescence and WB;(4)The levels of tumor necrosis factor alpha(TNF-α)and interleukin 6(IL-6)were detected by flow cytometry,and the levels of intercellular adhesion molecule(ICAM)and vascular cell adhesion molecule(VCAM)were detected by enzyme-linked immunosorbent assay(ELISA).2.Study on the expression of PGC1α and autophagy in mice with immune vasculitis:set up a CAWS-induced immune vasculitis mouse model group(CAWS group)and a control group of mice injected with PBS intraperitoneally(WT group).Arteries,common iliac arteries,internal and external iliac arteries,common carotid arteries,thoracic aorta and abdominal aorta and other vascular tissues were used for experiments.(1)The ultrastructure of vascular endothelial cells was observed by electron microscope;(2)the expression level of PGC1α was detected by immunohistochemistry and WB;(3)the content of LC3 was detected by immunofluorescence and WB.Results:1.In vitro cell inflammatory injury experiments:(1)HCAEC immunofluorescence staining indicated that PGC1α and mitochondrial marker protein TOM20 co-localized.(2)In the Kawasaki disease-like immune vascular endothelial injury cell model,WB indicated that PGC1α was significantly down-regulated in endothelial cells after 48 hours(compared with the normal control group,p<0.01).(3)In the KD-like immune vascular endothelial injury cell model,immunofluorescence showed that the brightness of LC3 was significantly higher than that of the normal control group,and WB showed that the expression of LC3 in endothelial cells increased(compared with the normal children group,p<0.01).(4)In the KD-like immune vascular endothelial injury cell model,the expressions of pro-inflammatory factors TNF-α(compared with normal children,p<0.001)and IL-6(p<0.001)in vascular endothelial cells increased;The expression of cytokines ICAM(compared with normal children,p<0.001)and VCAM increased(p<0.001).2.Study on the expression of PGC1α and autophagy in mice with immune vasculitis:(1)Electron microscope observed the vascular endothelial cells of mice with immune vasculitis,and found that compared with the normal control group,the endothelial cells were shed from the basement membrane,and localized disintegration,the normal structure of mitochondria disappeared,swelling was obvious,and many vacuolar mitochondria were seen.(2)Both immunohistochemistry and WB indicated that the level of PGC1α in the vascular tissue of mice with immune vasculitis decreased(compared with the normal control group,p<0.01).(3)Both WB and immunofluorescence indicated that the content of LC3 in the vascular tissue of the mice with immune vasculitis increased(p<0.01 compared with the control group).Conclusion:(1)PGC1α in HCAEC is located in mitochondria.(2)In the model of HCAEC damage induced by PBMC in Kawasaki disease,the expression of PGC1αdecreased,while the autophagy flux increased,TNFα,IL6,ICAM and VCAM increased significantly.(3)The expression of PGC1α in vascular tissue of vasculitis mice decreased while autophagy increased.Part Ⅲ Research regarding the mechanism of PGC1α regulating autophagy-induced inflammatory impairment of the vascular endothelial cells in Kawasaki diseaseObjective:In this section,we further explore the regulatory relationship between PGC1α and autophagy and its role and mechanism in vascular endothelial inflammatory injury in KD.Methods:1.Cell inflammatory injury experiment:Collect KD patient PBMC primary cell culture(csf-PBMC)as a stimulator to stimulate HCAEC to build a KD-like immune vascular endothelial cell inflammatory injury model.(1)Experimental study of HCAEC after knockout of PGC1α:Cas9 virus constructs a knockout cell line of HCAEC(KO-PGC1α).Set up HCAEC group,HCAEC model group,KO-PGC1α group and KO-PGC1α model group.The expression of LC3 in HCAEC was detected by immunofluorescence and WB;the content of pro-inflammatory factors TNF-α and IL-6 in HCAEC was detected by flow cytometry;the content of chemokine ICAM and VCAM was detected by ELISA.(2)Experimental study of HCAEC cells after overexpression of PGC1α:The overexpression of PGC1α cell line was constructed by lentivirus transfection of exogenous PGC1α(Flag-PGC1α).Set HCAEC group,HCAEC model group,Flag-PGC1α group and Flag-PGC1α model group.The expression of LC3 in HCAEC was detected by immunofluorescence and WB;the content of TNF-α and IL-6 in HCAEC was detected by flow cytometry;the content of ICAM and VCAM was detected by ELISA.2.Effect of intervention autophagy on immune vasculitis in mice:mice with immune vasculitis of Kawasaki disease were given 3-MA(1Omg/kg),model group(CAWS group,intraperitoneal injection of CAWS),intervention autophagy group(CAWS+3-MA group,intraperitoneal injection of CAWS+3-MA),normal control group(WT group,intraperitoneal injection of PBS).The morphology of vascular endothelium was observed by electron microscope and the infiltration degree of vascular inflammation was observed by HE staining.Results:1.Inflammatory injury test:(1)Knockout PGC1α HCAEC cell experiment.①LC3 in KO-PGC1α model group increased significantly(compared with HCAEC model group,P<0.01).②Proinflammatory cytokine TNF-α in endothelial cells of KO-PGC1α model group(compared with HCAEC model group,p<0.01),IL-6 increased significantly(p<0.01),Chemokine ICAM(p<0.001)and VCAM(p<0.001)increased significantly.(2)HCAEC cell experiment after overexpression of PGC1α.①LC3 was not significantly increased in model Flag-PGC1α group(comared with model HCAEC group,p<0.01).②TNF-α and IL-6 levels in Flag-PGC1αmodel group(compared with HCAEC model group,p<0.001)decreased(p<0.001),ICAM(p<0.001)and VCAM(p<0.001)also decreased.(3)The effect of autophagy on HCAEC after PGC1α knockout:After pretreatment with 3-MA,TNF-α and IL-6 levels in the KO-PGC1α model group(compared with the KO-PGC1α model group without pretreatment,p<0.01),ICAM(p<0.001)and VCAM(p<0.001)also decreased.2.Effects of autophagy intervention on immune vasculitis in mice:(1)compared with the model group,the infiltration of perivascular inflammatory cells in the 3-MA intervention group was reduced.(2)Compared with the model group,the connection between vascular endothelial cells and basement membrane of mice in 3-MA intervention group was closer,the shedding phenomenon was reduced,and the number of vacuolated mitochondria was reduced.Results:Reduced PGC1α might aggravate immune-associated vascular inflammation by promoting autophagy activation in KD.1.After PGC1α was knocked out/overexpressed,pro-inflammatory factors and chemokines were up-regulated/down-regulated in immune-inflammatory vascular endothelial cells,indicating the inflammatory damage/protective effect of PGC1α knocked out/overexpressed on endothelial cells.2.Immunoinflammatory coronary artery endothelial cells with PGC1α knockout were up-regulated to express pro-inflammatory factors and chemokines,and autophagy inhibitors(3-MA)could inhibit these inflammatory mechanisms,suggesting that over-autophagy plays a pro-inflammatory role in immunoinflammatory vascular endothelial cells.3.In vivo experiments have verified that the intervention of autophagy can relieve the inflammatory injury of vascular endothelium.