The Role And Mechanism Of Homocysteine In Sepsis-related Acute Lung Injury

Sepsis is a common clinical syndrome in intensive care units,and about one-fifth of patients in Intensive Care Units(ICU)in mainland my country are diagnosed with sepsis.The prevalence of sepsis around the world continues to rise,and due to the lack of specific treatment methods,the number of deaths is still high,and sepsis has long become an important world health problem.In the novel coronavirus pneumonia epidemic that swept the world at the end of 2019,sepsis caused by severe and critically ill patients was one of the important causes of death in the ICU.The high risk of death and poor prognosis of sepsis are closely related to the occurrence of organ dysfunction,among which acute lung injury/acute respiratory distress syndrome(ALI/Acute respiratory distress syndrome,ADRS)occurs in sepsis patients The earliest,highest incidence,ADRS occurs in about 25%of patients with sepsis,and refractory hypoxemia is the main feature,mainly caused by intrapulmonary shunt and increased physiological dead space.The in-hospital mortality rate of patients with mild ARDS was 34.9%,and that of patients with severe ARDS was 46.1%.Metabolic abnormalities in sepsis lead to blood glucose disturbances,increased fatty acid oxidation and amino acid catabolism,and are closely related to poor patient outcomes.Homocysteine(Hcy)is a sulfur-containing amino acid formed during the mutual conversion of methionine and cysteine,and participates in the methionine cycle.Early elevation of plasma Hcy in sepsis patients is associated with poor prognosis.Hcy can lead to the activation of inflammation-related pathways in the body,resulting in increased expression of inflammation-related genes and activation of immune cells.Induced activation of macrophages is implicated in the pathogenesis of sepsis/septic shock.In the early stages of sepsis,macrophages differentiate into M1 type,clear invading pathogens and necrotic tissue,and release a large number of pro-inflammatory cytokines to promote the body’s defense.However,the effects of Hcy on macrophage function in vivo,the underlying mechanisms,and the underlying pathophysiological implications remain to be elucidated.We first used metabolomics to analyze the changes of serum metabolomics in patients with sepsis-related ARDS,28-day non-surviving patients with sepsis-related ARDS,and rats with septic lung injury,and identified sepsis according to non-targeted metabolomics methods.Associated early biomarkers of ARDS.The correlation between plasma homocysteine levels,inflammatory cytokines,and clinical outcomes in patients with sepsis-related ARDS was then analyzed from clinical cases.At the same time,the effect of homocysteine on the expression of IL-1β in macrophages was explored through in vitro and in vivo experiments.Then,the effect of Hcy on the expression and enzymatic activity of PKM2 in macrophages was detected by extracellular flux analysis technology,the effect of Hcy on the metabolic reprogramming of macrophages was observed,and the molecular mechanism of promoting the secretion of IL-1β by macrophages was investigated.Research.Finally,the therapeutic effect and potential mechanism of PKM2 inhibitor shikonin on sepsis lung injury were explored.To determine its potential and application feasibility in the clinical treatment of sepsis.Part Ⅰ:Metabolomics analysis of potential biomarkers for sepsis-related ARDSObjectiveSepsis-related ARDS has a high mortality rate,and there is currently no effective treatment.Early identification and intervention are the keys to reducing sepsis-related ARDS mortality.To explore the serum metabolomic changes in sepsis-related ARDS to identify early biomarkers of sepsis-related ARDS.Methods1.We enrolled 60 sepsis patients according to the Sepsis 3.0 criteria and the Berlin definition of ARDS,and divided them into control group(Ctrl),sepsis-related ARDS group(S-ARDS),and survival group according to the presence or absence of ARDS and 28-day prognosis.(S)and death group(D);2.A rat model of septic lung injury was established by cecal ligation and puncture(CLP),and 20 SD rats were randomly divided into the control(SC)group and the CLP group(10 rats in each group);3.Untargeted metabolomic screening was performed on serum samples from the control and S-ARDS/D/CLP groups in different groups.Combined multivariate and univariate analyses were used for pairwise comparisons of all groups to identify significant changes in serum metabolite levels in sepsis-related ARDS and sepsis non-survivor groups.Orthogonal partial least squares discriminant analysis(OPLS-DA)showed significant separation between Ctrl and S-ARDS groups,S and D groups,and SC and CLP groups;4.All between-group differential metabolites were identified according to thresholds defined by potential biomarkers.Screening of potential biomarkers for sepsis-related ARDS prognosis by cross pooling of differential metabolites.Results1.Principal component analysis(PCA analysis)was performed on plasma samples from the rat SC group and CLP group,and the results showed significant changes in plasma metabolites in both groups.Homocysteine(Homocysteine),Phenylalanine(Phenylalanine),Lysophosphatidylethanolamine(LysoPEs),Histidine(Histidine),Methionine(Methionine),D-Arginine(D-Arginine)and other 17 metabolites.2.PCA analysis of plasma samples from sepsis patients with and without ARDS showed significant changes in plasma metabolites between the S-ARDS and Ctrl groups.VIP>1 and P<0.01 were used as criteria for screening potential biomarkers.We identified Homocysteine,Methionine,Phenylalanine,Phosphatidylcholines(PCs),Oleoylcarnitine,Stearoylcamitine.25 metabolites.3.PCA analysis of plasma samples from patients in the 28-day survival and death groups of sepsis showed significant changes in plasma metabolites between groups S and D.We used VIP>1 and P<0.01 as screening criteria.A total of homocysteine(Homocysteine),phenylalanine(Phenylalanine),methionine(Methionine),histidine(Histidine),sphingosine(Sphingosine),lysophosphatidylethanolamine(LysoPEs),lysophosphatidyl 20 metabolites including choline(LysoPCs).4.Three metabolites of homocysteine,methionine and phenylalanine were significantly different among the above three groups.It can be used as a potential biomarker to predict the prognosis of sepsis-related ARDS.SummarySepsis leads to abnormal lipid metabolism and amino acid metabolism in the body.Compared with the control group,homocysteine,phenylalanine and methionine were significantly different in the sepsis-related ARDS group and the sepsis non-survivor group,which can be used as potential biomarkers to predict the prognosis of sepsis-related ARDS thing.Part Ⅱ:Homocysteine promotes cell activation and secretion of IL-1βin macrophagesObjectiveThe effect of homocysteine on the expression of IL-1β in macrophages was explored through in vitro and in vivo experiments,which provided a preliminary basis for further research on the molecular mechanism of sepsis-related ARDS.Methods1.Cytokines related to inflammation in peripheral blood of patients with sepsis-related ARDS were screened by PCR Array,real-time PCR(RT-PCR),and enzyme-linked immunosorbent assay(ELISA),and the correlation between the expression level of homocysteine and inflammatory cytokines was verified.relation;2.An animal model of sepsis ALI induced by cecal ligation and puncture was established,and the infiltration of pro-inflammatory macrophages in the lung tissue of CLP rats and the effect of Hcy on macrophage activation and IL-1(3 expression were verified by in vitro and in vivo experiments.Results1.PCR Array screened out that IL-1β,IL-6,IL-10,TNF-α,MCP-1,INF-γ may be highly expressed in patients with sepsis-related ARDS(all P<0.05);2.The expression of IL-1β in the peripheral blood of sepsis-related ARDS has the largest difference(P<0.01),and it is significantly related to the poor prognosis of sepsis-related ARDS patients(P=0.0402);3.There was a significant correlation between plasma Hcy and the expression level of IL-1β(R2=0.62,P<0.01),and Hcy was positively correlated with the clinical prognosis of sepsis-related ARDS patients(P=0.0359);4.In vitro and in vivo studies have shown that Hcy is related to the increased infiltration of pro-inflammatory macrophages(M1 type)in the lung tissue of CLP rats(P<0.01),and promotes the expression of IL-1β in macrophages(P<0.01).SummaryPlasma Hcy and IL-1β expression levels are significantly correlated with the poor prognosis of sepsis-related ARDS.Further studies have found that plasma IL-1βand Hcy expression levels in sepsis-related ARDS patients are positively correlated,and Hcy can increase M1 macrophages in lung tissue.Infiltrate and promote the expression of IL-1β.Part Ⅲ:Homocysteine regulates PKM2-dependent metabolic reprogramming to promote IL-1β release from macrophagesObjectiveThe effects of Hcy on the expression and enzymatic activity of PKM2 in macrophages were detected by extracellular flux analysis,and the molecular mechanism of Hcy promoting the secretion of IL-1β in macrophages was discussed.Methods1.Using extracellular flux technology to analyze the energy metabolism of cells,to explore the effect of Hcy on oxidative phosphorylation and glycolysis metabolism in macrophages;2.PKM2 activity was measured by lactate dehydrogenase coupled enzyme assay,and the effect of Hcy on the expression and enzymatic activity of PKM2 in macrophages was analyzed;3.Using western blot,it was verified that Akt-mTOR signaling pathway regulates PKM2 enzymatic activity and mediates Hcy-induced expression of IL-1β in macrophages.Results1.Hcy significantly increased the extracellular acidification rate(ECAR)of macrophages and decreased their cellular oxygen consumption rate(OCR).At the same time,Hcy increases the glycolytic reserve capacity of macrophages and reduces their spare respiratory capacity.ELISA results showed that Hcy led to a decrease in the expression of acetyl-CoA(Acetyl-CoA)(P<0.05)and an increase in the level of lactate dehydrogenase(LDH)compared with the control(P<0.01).These results suggest that Hcy causes a dramatic metabolic reprogramming of macrophages from oxidative phosphorylation to aerobic glycolysis.2.Hcy up-regulated the expression of PKM2 gene and protein in macrophages(all P<0.05).Furthermore,Hcy significantly increased macrophage PKM2 enzymatic activity(P<0.01).3.Hcy activates the Akt-mTOR pathway by increasing the phosphorylation of Akt and mTOR in macrophages.We then used the mTOR inhibitor rapamycin to explore the role of mTOR signaling in the regulation of PKM2.found that low-dose rapamycin significantly reduced Hcy-induced mTOR phosphorylation in macrophages(P<0.05).Rapamycin inhibited Hcy-induced PKM2 protein expression(P<0.05)and reversed the effect of Hcy on PKM2 enzymatic activity in macrophages(P<0.05).Meanwhile,we found that rapamycin reversed Hcy-induced IL-1β secretion(P<0.01).SummaryHcy regulates the expression and activity of PKM2 through the Akt-mTOR pathway,promotes aerobic glycolysis in macrophages,and also promotes macrophage activation and IL-1β secretion.Our findings suggest that PKM2 is a key metabolic regulator of Hcy-induced macrophage activation and may serve as a potential therapeutic target for the treatment of septic lung injury.Part Ⅳ:Shikonin inhibits macrophage activation and ameliorates sepsis-related lung injury by regulating PKM2ObjectiveTo explore the therapeutic effect and potential mechanism of shikonin on septic lung injury.To determine its potential and application feasibility in the clinical treatment of sepsis.Methods1.The energy metabolism of cells was analyzed by extracellular flux technology,and the effect of shikonin on Hcy-induced macrophage oxidative phosphorylation and glycolytic metabolism patterns was investigated;real-time quantitative PCR and ELISA were used to detect the effect of shikonin on Hcy-induced macrophages.The effect of phagocytic IL-1β secretion;2.To construct a macrophage cell line with stable knockout of PKM2,and analyze the effect of silencing PKM2 on the expression of IL-1β in macrophages;3.CLP rats were given shikonin or normal saline intraperitoneally at 24 hours,48 hours and 72 hours after the successful establishment of the CLP rat model.Hematoxylin and eosin staining(HE)and Masson staining were used to detect inflammatory infiltration in the lung tissue of CLP rats;RT-PCR and ELISA were used to detect the expression levels of IL-1β in plasma and lung tissue of CLP rats;4.To observe the survival of CLP rats after shikonin treatment.Results1.Shikonin can significantly reduce the activity of PKM2(P<0.05),and also has a significant inhibitory effect on Hcy-induced PKM2 enzyme activity(P<0.05),but it does not affect the expression of PKM2 protein.Subsequently,we examined the effect of shikonin on macrophage metabolism after inhibiting PKM2 activity.The results showed that shikonin significantly inhibited the up-regulation of macrophage ECAR by Hcy and reversed the down-regulation of macrophage OCR by Hcy.The results of RT-PCR and ELISA showed that shikonin finally reversed the Hcy-induced secretion of IL-1β in macrophages(all P<0.01).2.Western blot analysis showed that a stable PKM2 knockout macrophage cell line(PKM2 shRNA)was successfully constructed.The results of ELISA and RT-PCR showed that silencing PKM2 reduced Hcy-induced IL-1β secretion in macrophages(all P<0.01).3.HE and masson staining of lung tissue of CLP rats showed that shikonin treatment could significantly improve the lung injury of septic rats.The expression levels of IL-1β in plasma and lung tissue of CLP rats were significantly decreased after treatment(all P<0.05).At the same time,it was found that shikonin significantly improved the prognosis of CLP rats(P<0.05).SummaryInhibition of PKM2 by shikonin can reverse Hcy-induced secretion of pro-inflammatory cytokine IL-1βin macrophages,improve the degree of septic lung injury,and prolong the survival time of septic rats.Targeting PKM2 may offer a potential therapeutic approach for patients with sepsis-related ARDS.ConclusionsThe metabolomic changes in serum of patients with sepsis-related ARDS,28-day non-surviving patients with sepsis-related ARDS,and rats with septic lung injury were analyzed by metabolomics.Three amino acids,namely Hcy,methionine and phenylalanine,were found to serve as potential prognostic markers in patients with sepsis-related ARDS.Then,the correlation between plasma Hcy levels,inflammatory cytokines and clinical prognosis of patients with sepsis-related ARDS was analyzed from clinical cases,and it was found that plasma Hcy levels in patients with sepsis-related ARDS were significantly higher than those in patients without ARDS.Plasma Hcy levels are positively correlated with poor prognosis in patients with sepsis-related ARDS.Further research found that plasma IL-1β and Hcy expression levels were positively correlated in sepsis-related ARDS patients,and plasma IL-1β levels were closely related to the clinical prognosis of sepsis-related ARDS patients.Then,the effect of Hcy on the expression and enzymatic activity of PKM2 in macrophages was detected by extracellular flux analysis technology,and its effect on the metabolic reprogramming of macrophages was observed.The results suggest that Hcy regulation of PKM2-dependent metabolic reprogramming contributes to macrophage activation and the resulting inflammatory response.PKM2-mediated activation of aerobic glycolysis in macrophages leads to overproduction and release of the inflammatory cytokine IL-1β Given the critical role of PKM2-mediated metabolic reprogramming in regulating inflammation,it may be a therapeutic target for sepsis.We treated septic rats with PKM2 inhibitor shikonin,and found that it can significantly inhibit the PKM2-mediated Warburg effect,reduce the inflammatory response in the lung tissue of septic rats,and improve the prognosis of septic rats.In this study,starting from sepsis plasma metabolism,the role and mechanism of Hcy in sepsis-related ARDS were studied in depth,which provided new ideas for the treatment of sepsis-related ARDS.The study of the treatment of sepsis from the perspective of oxygen glycolysis also has important reference significance.