Based On KLK8 Signaling Pathway To Explore The Protective Effect And Mechanism Of Exercise On Sepsis Induced Lung Injury

Objectives:The repair of pulmonary vascular endothelial barrier after sepsis-induced lung injury is crucial for the recovery of lung injury.Tissue Kallikrein related peptide enzyme(tissue Kallikrein-related peptidases,KLKs)is a kind of serine proteinase,participate in the regulation of a variety of physiological processes and is closely related to the occurrence of various diseases.KLKs,however,whether to participate in sepsis during pulmonary vascular endothelial regeneration and vascular repair regulation are still unclear.Relevant studies in the field of exercise and sepsis-induced lung injury agree that regular aerobic exercise has a positive protective effect on sepsis-induced lung injury.Exercise intervention has become a research hotspot in the prevention and treatment of sepsis lung injury.The mechanism of exercise on sepsis lung injury is still being explored.However,the relationship between KLKs and sepsis-induced lung injury and exercise remains unknown.This study focused on kallikrein-related peptidase 8(KLK8)KLK8 to explore the protective effect of exercise on sepsis-induced injury and its related mechanism.(1)In this study,the sepsis model was constructed by intraperitoneal injection of Lipopolysaccharides(LPS),and the exercise group model was constructed by 6 weeks of moderate-intensity exercise.To observe the expression of KLK8,a member of the KLK family,in the lung tissues of sepsis and t investigate the effect of 6 weeks of moderate-intensity aerobic exercise on septic lung tissue injury and the regulatory effect of exercise intervention on KLK8 in septic lung tissue.(2)To verify the effect of KLK8 overexpression or knockdown on lung endothelial cells by cell and animal experiments.(3)RNAseq analysis was used for transcriptome sequencing in cell experiments to screen KLK8 downstream target genes and biological signaling pathways,and to verify relevant signaling pathways that may be regulated by KLK8.(4)To verify the effect mechanism of KLK8 knockdown or blockade and exercise on sepsis-induced lung injury.Methods:Part ⅠLPS-induced changes in KLK family member KLK8 in lung tissue of Sepsis mice and vascular endothelial cells and the effect of 6 weeks of moderate intensity aerobic exercise on lung injury and KLK8 expression levels in septic mice(1)8-week-old C57BL/6 male mice were randomly divided into control group,sepsis group,exercise group and exercise+sepsis group.In the exercise+sepsis group,after 6 weeks of moderate intensity exercise,the mice were tested for maximum exercise intensity,and then the exercise+sepsis model was established by intraperitoneal injection of LPS.(2)The symptoms of mice in sepsis group were monitored every 6 hours and the survival number was counted.Evans blue was used to detect the pulmonary vascular leakage in the control group and some surviving mice.Part of the lung tissue of mice in qRT-PCR to detect lung KLK mRNA expression of the 15 members of the family;Part of the lung tissue of mice using Western blot detection KLK8 protein expression changes of lung tissue;Part of the lung tissue of mice were used in paraffin sections to observe lung histopathological changes and KLK8/CD31 double immunofluorescence staining.(3)Primary pulmonary Endothelial Cells were extracted from 3-week-old C57BL/6 mice,and Mouse Lung Vascular Endothelial Cells(MLVECs)isolated from 3-4 generations of primary endothelial cells were treated with LPS intervention.mRNA levels and protein expression of 15 KLK family members in MLVECs were detected by qRT-PCR and Western blot,respectively.(4)The mouse model of exercise+sepsis group was also monitored every 6 hours for symptoms and survival.Some mice in control group,sepsis group,exercise group and exercise+sepsis group were tested with Evans blue to observe the pulmonary capillary leakage.The protein expression levels of CD31,ZO-1 and ICAM-1 in lung tissues of some mice were detected by Western blot,and the mRNA and protein expression changes of KLK8 in lung tissues were further detected by qRT-PCR and Western blot.Part of mouse lung tissues were sliced in paraffin wax to observe the lung histopathological changes and KLK8 DAB staining.Part ⅡExercise regulates VE-cadherin/Akt/FOXM1 signaling pathway mediated endothelial barrier dysfunction and endothelial cell proliferation by inhibiting KLK8(1)Cell level:Ad-KLK8 and KLK8siRNA were used to induce KLK8 overexpression or inhibition in MLVECs;Animal level:Local KLK8 overexpression was induced by tracheal instillation of Ad-KLK8.KLK8 knockout and tail vein injection KLK8 neutralizing antibodies in mice,inducing KLK8 knockout or blocking in the body.To investigate the effect of KLK8 overexpression or knockdown on LPS-induced lung tissue and MLVECs,and to verify whether KLK8 is involved in sepsis lung injury.(2)MLVECs were treated with Ad-KLK8,and RNAseq analysis was performed to screen differentially expressed genes.(3)Ad-KLK8,Len-FOXM1,Akt agonist SC79,mTOR agonist MHY1485,Len-VE-cadherin and Akt inhibitor AZD5363 were used to treat MLVECs and lung tissues of Ad-KLK8 intratracheally.To verify whether KLK8 is involved in the regulation of the expression of VE-cadherin,Akt and FOXM1 related signals of MLVECs injury and endothelial regeneration and repair.(4)KLK8 knockout+LPS mice;Mice were injected with KLK8 neutralizing antibody+LPS by caudal vein;Exercise+LPS group mice;To investigate the effects of KLK8 knockout or blocking and exercise on the expression ofVE-cadherin,Akt,FOXM1 and endothelial proliferation.To explore the potential mechanism of KLK8 involved in sepsis-induced lung injury and the regulatory effect of exercise on related signaling pathways.Results:Part Ⅰ(1)The expression of KLK8 in lung tissue and MLVECs of LPS-induced sepsis mice was significantly up-regulated(p<0.01).The results of immunofluorescence double staining KLK8/CD31 showed that the percentage of KLK8+/CD31+cells in the total number of CD31+cells in the lung tissues of mice in LPS group was significantly increased(p<0.01).(2)6-week moderate intensity aerobic exercise significantly improved the protein levels of CD31,ZO-1 and ICAM-1 in lung tissue of sepsis mice(p<0.01),reduced the degree of pulmonary vascular leakage and pathological injury of lung tissue(p<0.01),and increased the survival rate of sepsis mice(p<0.01).(3)6-week moderate intensity aerobic exercise decreased the expression levels of mRNA and protein of KLK8 in lung tissue of sepsis mice(p<0.01),and the positive staining of KLK8 DAB in lung tissue of sepsis mice was significantly reduced(p<0.01).Results:Part Ⅱ(1)KLK8 overexpression leads to endothelial cell hyperpermeability and lung injuryThe expression levels of CD31 and ZO-1 and cell activity were significantly decreased,while ICAM-1 protein level and pulmonary vascular endothelial permeability were increased in MLVECs overexpressing KLK8(p<0.01).The expression levels of CD31 and ZO-1 in lung tissue of mice with KLK8 adenovirus intratracheal instillation were significantly decreased,and the high permeability of pulmonary vascular endothelial barrier and lung injury were significantly increased(p<0.01).(2)Knockdown of KLK8 reduced LPS-induced endothelial hyperpermeability,ameliorated lung injury and reduced mortality in miceAfter KLK8siRNA treatment,inhibition of KLK8 ameliorated LPS damage to CD31 and ZO-1(p<0.01),increased cell viability,and decreased ICAM-1 expression level and endothelial permeability of MLVECs(p<0.01).Compared with the LPS group,the expression levels of CD31 and ZO-1 in the lung tissue of KLK8-/-+LPS group were significantly increased(p<0.01),and the expression level of ICAM-1 protein was significantly decreased(p<0.01).KLK8 knockout improved the pulmonary vascular endothelial permeability and the pathological injury degree of lung tissue in sepsis mice.The survival rate of KLK8-/-+LPS group was significantly higher than that of LPS group(p<0.01).(3)KLK8 neutralizing antibody reduced LPS-induced endothelial cell hyperpermeability,ameliorated lung injury and attenuate mortality in miceKLK8 neutralizing antibody significantly improved the expression levels of CD31,ZO-1 and ICAM-1 in lung tissue of mice with LPS-induced sepsis(p<0.01).KLK8 neutralizing antibody mice induced a significant reduction in pulmonary vascular leakage in lung tissue of sepsis mice,the degree of pathological injury in lung tissue and lung injury score were significantly reduced(p<0.01).The survival rate was significantly higher than that of septic mice(p<0.01).(4)RNA-Seq analysis of differentially expressed genes in KLK8 overexpressed MLVECsAdenovirus was used to overexpress KLK8 in MLVECs,and preliminary screening was performed using RNA-Seq analysis.In this study,996 up-regulated and 2368 down-regulated differential genes were identified.Screening of differentially expressed genes using IPA analysis revealed that transcription factor FOXM1 was significantly inhibited in KLK8-overexpressing MLVECs(p<0.01).Consistent with the results of RNAseq analysis,KLK8 overexpression resulted in significant down-regulation of FOXM1mRNA and protein levels in MLVECs.RNAseq data results also show that is regulated by the FOXM1 targeted Aurkb,Birc5,Bub1b,Ccna2,Ccnd1,Ccnel,Ccne2,Ccnf and Cdc20 related genes are significantly decreased.(5)FOXM1 overexpression improved endothelial proliferation and endothelial injury in MLVECs inhibited by KLK8 overexpressionFOXM1 overexpression was induced in MLVECs using FOXM1 lentivirus,and the Edu incorporation assay results showed that the proliferation of MLVECs treated with Ad-KLK8 was significantly decreased(p<0.01),and the proliferation of MLVECs was significantly restored by lentivirus-mediated FOXM1 overexpression(p<0.01).The mRNA expression levels of endothelial proliferation related target genes Ccnd1,Cdc20,Ccnf and Bub1b in MLVECs of Len-FOXM1+Ad-KLK8 group were significantly higher than those of Len-Vector+Ad-KLK8 group(p<0.01).Overexpression of FOXM1 increased KLK8-inhibited CD31 and ZO-1 protein levels and cell viability in MLVECs,while decreased ICAM-1 protein level and high permeability of MLVEC s(p<0.01).(6)KLK8 induces endothelial barrier dysfunction by inhibiting VE-cadh erin/Akt/FOXM1 signaling pathway mediated endothelial cell proliferationSingle gene GSEA was used to screen the potential key signaling pathways of KLK8 overexpression to inhibit FOXM1.The results showed that FOXM1 was positively enriched with PI3K/Akt and mTORC1 pathways.In this study,it was observed that the phosphorylation of Akt was significantly reduced in MLVECs overexpressing Ad-KLK8(p<0.01),and the phosphorylation of mTOR and its downstream signaling protein pS6K was not significantly changed.Ad-KLK8 intervention after pretreatment of MLVECs with Akt agonists and mTOR agonists showed that Akt agonists increased Akt phosphorylation and FOXM1 expression,and reversed KLK8-induced FOXM1 downregulation in MLVECs(p<0.01).mTOR agonist MHY1485 increased phosphorylation levels of mTOR and p-S6K,but had no significant effect on FOXM1 expressionTo identify the substrate proteins that KLK8 acts on lung tissue,we examined whether VE-cadherin cleavage is involved in KLK8-induced Akt inactivation.The results showed that the VE-cadherin protein expression level was significantly decreased in MLVECs and lung tissues with KLK8 overexpression(p<0.01).A novel～30 kDa extracellular fragment of VE-cadherin was released into the culture medium,and VE-cadherin N terminal fragment expression was present in the supernatant of MLVECs cells after Ad-KLK8 treatment.Len-VE-cadherin treatment of MLVECs showed that lentivirus-mediated VE-cadherin overexpression significantly increased Akt phosphorylation and FOXM1 levels in KLK8-overexpressed MLVECs(p<0.01).MLVECs were treated with Akt inhibitors,and the results showed that the stimulatory effect of VE-cadherin overexpression on FOXM1 expression was blocked by Akt inhibitors(p<0.01).(7)Knockdown or blocking of KLK8 increased the expression levels of VE-cadherin,Akt and FOXM1 in lung tissue of LPS treated mice,and promoted the regeneration of endothelial cellsUsing KLK8 knockout mice,it was found that KLK8 knockdown significantly reversed VE-cadherin,Akt phosphorylation and FOXM1 expression levels in lung tissues of sepsis mice(p<0.01),and increased the mRNA expression levels of endothelial proliferation related factors Ccnd1,Cdc20,Ccnf and Bub1b(p<0.01).Immunofluorescence double staining of CD31/Ki67 showed endothelial cell proliferation 48 h after LPS administration,and CD31/Ki67 co-localization was significantly increased in lung tissues of KLK8 knockout mice(p<0.01).Using tail vein injection of KLK8 neutralizing antibody,the results showed that KLK8 neutralizing antibody significantly improved VE-cadherin,Akt phosphorylation and FOXM1 expression in lung tissue of septic mice(p<0.01).The mRNA expression levels of endothelial proliferation-related factors Ccnd1,Cdc20,Ccnf and Bub1b were also significantly up-regulated(p<0.01).Sepsis induced significantly increased co-localization of CD31/Ki67 in lung tissue of KLK8 neutralizing antibody mice compared with IgG+LPS group(p<0.01).(8)The expression levels of VE-cadherin,Akt and FOXM1,and the expression of endothelial proliferation-related target genes were significantly increased after 6-week moderate intensity aerobic exercise in lung tissue of sepsis mice6 weeks of moderate-intensity aerobic exercise significantly increased the expression levels of VE-cadherin,Akt and FOXM1 in the lung tissue of sepsis mice(p<0.01).The mRNA expression levels of FOXM1 downstream target genes Ccnd1,Cdc20,Ccnf and Bub1b in lung tissue of mice in Exercise+LPS group were significantly increased compared with LPS group(p<0.01).Conclusions(1)KLK8 expression was significantly up-regulated in lung tissues of mice with LPS induced sepsis and in LPS treated MLVECs.(2)By inhibiting the expression of KLK8,regulating the expression levels of VE-cadherin,Akt and FOXM1,6-week moderate intensity aerobic training can effectively improve the pulmonary endothelial injury in sepsis mice,promote the repair and regeneration of pulmonary vascular endothelial tissue,and improve the lung injury in sepsis mice.