| Sepsis is a clinical syndrome characterized by a host’s dysregulated response to infection,leading to life-threatening organ dysfunction.In the past decade,the incidence of sepsis has reached 437 cases per 100,000 people,with a hospital mortality rate of 26%,imposing a huge disease burden on the global population.Recent studies have found that septic patients with myocardial injury are more prone to induce multiple organ dysfunction,and have a worse prognosis.Epidemiological surveys show that septic patients with concomitant myocardial dysfunction account for about 40%-50%of the total population,with a mortality rate as high as 70%.Prevention,early diagnosis,and intervention of sepsis-induced myocardial disease can help reduce sepsis mortality and improve patient prognosis.In the normal physiological process,iron is an active component that participates widely in metabolism and biological functions,such as metabolic regulation,and has a significant impact on many cellular life activities.However,the absorption,transportation,and storage of iron must be strictly monitored because this element essential to living organisms.High levels of iron can lead to increased oxidative stress in the host,causing damage to cells and tissues,making it particularly important to clarify changes in iron metabolism in sepsis.To explore the causal relationship between iron,sepsis,and myocardial disease,we used Mendelian randomization analysis and a whole-genome association study strategy.In addition,we conducted experiments on high-iron and low-iron diets in a mouse sepsis model to observe their effects on sepsis and cardiac function and to detect changes in circulating iron parameters,inflammatory markers,and ferroptosis in cardiac tissue.Furthermore,we used a sepsis myocardial cell injury model to explore the mechanisms of sepsis-induced myocardial injury.Subsequently,we evaluated the correlation between iron parameter changes and disease progression and cardiac function between septic myocardial disease and non-septic myocardial disease patients.Additionally,since gut microbiota plays an important role in the pathogenesis of sepsis,such as participating in circulating immunity,metabolism,and other processes through the metabolites of intestinal flora and microbial translocation,we further investigated differences in gut microbiota and evaluated the relationship between changes in gut microbiota and iron parameters,disease progression,and cardiac function.Through these studies,we hope to provide new theoretical basis and practical guidance for the prevention,early diagnosis,and treatment of sepsis-induced myocardial disease.Our research results are expected to reveal the inherent connection between iron metabolism and the occurrence and development of sepsis-induced myocardial disease and provide scientific evidence for a better understanding of the pathogenesis of sepsis-induced myocardial disease.Meanwhile,through the study of gut microbiota,we may discover potential biomarkers associated with sepsis-induced myocardial disease,providing new targets for clinical treatment.Chapter 1:Mendelian Randomization Study of Iron and Sepsis-induced Cardiomyopathy and the Impact of Iron on Sepsis-induced CardiomyopathyObjective:Iron homeostasis is crucial for normal cardiac function,and mounting evidence suggests that iron dysregulation is a shared feature of many subtypes of cardiovascular disease.To further explore the potential mechanisms by which iron is involved in sepsis and sepsis-associated cardiac dysfunction,we first used Mendelian randomization(MR)analysis to explore the potential causal relationship between them based on the latest genetic association analysis databases for iron status,sepsis,and cardiomyopathy.In this study,the mouse model of sepsis induced by lipopolysaccharide(LPS)was used to investigate the effect of iron on cardiac function in sepsis mice and its potential pathogenesis.Methods:1.Conduct a Two-sample bidirectional Mendelian randomization(MR)study to explore potential causal relationships between iron-related parameters(serum iron,ferritin,transferrin saturation,transferrin,transferrin receptor 1),sepsis,and cardiomyopathy.2.Approximately 8-week-old,male C57BL/6J wild-type mice are selected and divided into four groups for the study:blank control group,lipopolysaccharide(LPS)group,LPS+high iron diet group,and LPS+low iron diet group.3.Record mouse mortality and assess changes in mouse cardiac function using a small animal high-frequency ultrasound system.4.Use relevant assay kits to assess changes in malondialdehyde(MDA)and reduced glutathione(GSH)/oxidized glutathione(GSSG)in cardiac tissue.5.Finally,use methods such as RT-qPCR and Western blot to evaluate changes in cardiac-related genes or protein molecules.Results:1.Genetic variations predicting higher transferrin saturation levels were associated with increased risk of any cardiomyopathy,hypertrophic cardiomyopathy,obstructive hypertrophic cardiomyopathy,and hypertrophic cardiomyopathy with heart failure after FDR correction.Transferrin also showed a decreased risk for hypertrophic cardiomyopathy,obstructive hypertrophic cardiomyopathy,and hypertrophic cardiomyopathy with heart failure after FDR correction.The differences in the other two groups were not statistically significant after correction.In the reverse analysis,iron status was not affected by cardiomyopathy.2.Observing the mortality of mice after LPS or physiological saline intervention at 1day,2 day,7 day,14day,it was found that the mortality changed within 7 day;the LPS combined with high iron diet intervention group had the highest mortality rate,which occurred mainly between 1-7 day.The low iron diet combined group could increase the survival rate of mice compared to the LPS group.3.Changes in inflammation levels such as IL-1β,IL-6,and TNF-α levels increased significantly in both the LPS group and the LPS combined with high iron diet group,and the combined group showed a more significant increase.The level of IL-10 in the LPS combined with high iron diet group decreased significantly,indicating that a high iron diet could significantly increase the inflammatory level of mice,and a low iron diet combined group could effectively reduce the systemic inflammatory status.4.Ejection fraction(EF)and fractional shortening(FS)in the LPS group were significantly lower than those in the CON group(P<0.001),indicating that sepsis induced cardiac dysfunction.After high iron diet intervention,these values further decreased to 21.31%and 11.68%(P<0.05),while the losses of ejection fraction and fractional shortening in the low iron diet group were to 60.29%and 31.54%,respectively.In addition,the high iron diet combined with LPS intervention impaired diastolic function,such as isovolumic relaxation time and E/A ratio(the ratio of the peak blood flow rate during early diastolic left ventricular relaxation(E wave)to the peak blood flow rate during late diastolic relaxation induced by atrial contraction(A wave)),while the low iron diet could mitigate the damage to diastolic function induced by LPS.5.We performed RT-qPCR on mRNA related to fibrosis,and found that the levels of COL I,COLⅢ,TGFβ,and SMAα in the heart tissue of the LPS group were significantly higher than those in the CON group and the low iron diet combined group,and this phenomenon was more pronounced in the high iron diet combined group.At 14 days of ultrasound in mice,ejection fraction and short axis shortening rate of combined high iron group were still significantly decreased,while isovolumic diastolic time and E/A ratio were not significantly different.6.For the ferroptosis characteristic indicators(PTGS2,GPX4),it was found that compared to the control group,the expression of the ferroptosis protein PTGS2 in the myocardial tissue of the LPS group was increased,while the expression of GPX4 was decreased.The intervention of a high iron diet in combination with LPS also increased the expression of PTGS2 and decreased the expression of GPX4.However,this phenomenon was partially reversed in the group given a low iron diet.The levels of oxidative stress-related indicators in the myocardial tissue,such as GSH,MDA,and GPX activity,were measured.LPS intervention decreased the content of GSH in the myocardial tissue,and the intervention of a high iron diet in combination with LPS further decreased the content of GSH.In contrast,the low iron diet group restored the content of GSH.This phenomenon was also observed in the activity of GPX.Both LPS intervention and the intervention of a high iron diet in combination with LPS increased the content of MDA in the tissue,while the low iron diet partially reversed this phenomenon.Conclusion:A Mendelian randomization study suggests that increasing iron levels can increase the risk of sepsis and heart disease in humans.The animal model found that the cardiovascular function of septic mice was impaired,and the increase in iron further damaged the heart function of septic mice.The mechanism by which it affects the cardiovascular system can induce ferroptosis in cardiac myocytes by exacerbating the imbalance of oxidative and antioxidant capacity induced by LPS.Chapter 2:The Mechanisms of Iron Metabolism Disorder and Ferroptosis in Lipopolysaccharide-induced Myocardial DamageObjective:Cardiomyocytes are rich in mitochondria,but the effect of mitochondrial iron metabolism on sepsis-induced cardiomyopathy is not clear.Mitochondrial iron homeostasis is an important factor in maintaining normal myocardial cell function.In animal and cell models of sepsis,disruption of iron metabolism can lead to myocardial dysfunction and ferroptosis.Considering the importance of mitochondrial iron metabolism,we explored the changes in cytoplasmic and mitochondrial iron metabolism and the mechanism of inducing ferroptosis in a cell model of sepsis.Methods:1.Treat HL-1 cardiomyocytes with a series of lipopolysaccharide(LPS)concentration gradients(0-20 μg/ml)for 24 hours.2.Evaluate the situation of cardiomyocyte ferroptosis by detecting ferroptosis-related markers PTGS2 and GPX4,and applying ferroptosis-specific inhibitor Ferrostatin-1.3.Use immunofluorescent JC-1,Liperfluo,and DCFH-DA probes to evaluate changes in cardiomyocyte mitochondrial membrane potential,lipid peroxidation,and reactive oxygen species.4.Use FerroOrange and Mito-FerroGreen probes to evaluate changes in ferrous ions in the cytoplasm and mitochondria.5.Assess changes in cardiomyocyte autophagy by detecting BECN1,LC3BII,and LC3BI proteins.6.Evaluate changes in key proteins affecting cardiomyocyte iron metabolism,such as FTH1,FTMT,and MFRN1.7.To explore the role of oxidative stress in ferroptosis,we treated with deferoxamine(DFO),N-acetylcysteine(NAC),and Mito-TEMPO(a mitochondrial antioxidant).8.By detecting the nuclear receptor coactivator 4(NCOA4)and applying ubiquitination inhibitor MG132,we assessed the degradation method of mitochondrial ferritin.9.Finally,we used lentivirus to overexpress mitochondrial ferritin in cardiomyocytes and evaluated the effect of mitochondrial ferritin on mitophagy with the mitophagy activator CCCP.Results:In this study,a septic cardiomyopathy model was established using LPS,and the effects of LPS on cardiomyocytes were observed.After LPS intervention,the expression of PTGS2 mRNA gradually increased while GPX4 expression gradually decreased,suggesting that cardiomyocytes may undergo ferroptosis.Ferroptosis inhibitors could reverse the decrease in cardiomyocyte survival rate,indicating that cardiomyocytes did undergo ferroptosis.Further observation found that LPS also affected the iron metabolism of cardiomyocytes,increasing the levels of iron ions in the cytoplasm and mitochondria.Overexpression of FTMT(ferritin mitochondrial)could reduce the level of ferrous ions and partially reverse cardiomyocyte death.In addition,LPS can also activate autophagy in cardiomyocytes,which is involved in the iron metabolism pathway in cardiomyocytes.NAC can reduce the level of autophagy in cardiomyocytes and increase the survival rate of cardiomyocytes just like TEMPO.However,DFO intervention failed to reduce the level of autophagy.And no significant improvement was seen in the survival rate of cardiomyocytes when NAC and TEMPO were combined with inhibitor compare with when autophagy was inhibited alone.Further experiments showed that FTMT was reduced by NCOA4-mediated autophagy degradation rather than ubiquitination mediated protein degradation.In addition,LPS could increase the expression of mitochondrial iron transporters,indicating that LPS was involved in the transport and storage of iron in cardiomyocyte mitochondria.The expression of PINK1 and Parkin increased,indicating the activation of mitochondrial autophagy.Overexpression of FTMT could reduce the expression of PINK1 and Parkin,indicating that FTMT could inhibit autophagy.The activation of mitochondrial autophagy inducer CCCP increased the expression of iron death markers in cardiomyocytes and decreased cardiomyocyte viability,indicating that FTMT could also affect cardiomyocyte ferroptosis through the regulation of mitochondrial autophagy.Conclusion:LPS can induce disturbances in the transport and storage of iron in cardiomyocytes,while also affecting the iron metabolism of mitochondrial.This leads to disturbances in cardiomyocyte iron metabolism and ferroptosis.Inhibiting disturbances in mitochondrial iron metabolism can partially reverse LPS-induced disturbances in cardiomyocyte iron metabolism and ferroptosis.Chapter 3:The Relationship between Iron Metabolism and Gut Microbiota Dysbiosis,Sepsis-induced CardiomyopathyObjective:The gut is the most important site for iron absorption and metabolism.It had been confirmed that the microbiome plays a role in sepsis,but few studies have reported the relationship of gut microbiota and iron metabolism in septic cardiomyopathy.This part of the study aims to explore the changes and correlation of gut microbiota and iron metabolism in septic cardiomyopathy.Methods:This is a single-center observational study that selected patients with sepsis admitted to the Department of Critical Care Medicine of Subei People’s Hospital from February to September 2022.Blood and fecal samples were collected from the patients included in the study on days 1,3,and 7.Blood samples were used to detect levels of iron metabolism,and fecal samples were subjected to 16S rDNA sequencing to evaluate gut microbiota diversity.The patients included to the study underwent echocardiography within 72 hours,and were divided into sepsis cardiomyopathy and non-sepsis cardiomyopathy groups accord to echocardiography.Clinical data of the two groups of patients,including age,gender,body mass index(BMI),Sequential Organ Failure Assessment(SOFA)score,Acute Physiology and Chronic Health Evaluation Ⅱ(APACHE Ⅱ)score,and other basic information were collected.The gut microbiota diversity,iron metabolism levels,28-day mortality rate,length of ICU stay,total hospital stay,and intensity of use of vasoactive drugs were compared between the two groups of patients.Results:A total of 48 patients were enrolled during the study period,including 23 patients in the septic cardiomyopathy group and 25 patients in the non-septic cardiomyopathy group.Comparison of baseline characteristics between the two groups showed that there was no statistical significance(P>0.05)for age,SOFA score,APACHE Ⅱ score,Calcitonin Ⅰ(cTnⅠ),B-type natriuretic peptide(BNP),calcitoninogen(PCT),and interleukin-6(IL-6)in the two groups.However,there were statistical differences(P<0.05)in the percentage of central venous pressure(CVP),and lactate(Lac)levels between the two groups,and all were higher in the septic cardiomyopathy group than in the non-septic cardiomyopathy group.Analysis of iron metabolism levels in the two groups showed that there was a statistical difference in serum ferritin(SF)levels between the two groups on day 1 and day 3 of enrollment(P<0.05),and that ferritin levels were higher in the septic cardiomyopathy group than in the non-septic cardiomyopathy group.Other iron metabolism levels including serum iron(SI),,serum transferring(TRF),transferrin saturation(TSAT),and total iron binding capacity(TIBC)on days 1,3,and 7 were not statistically significant(P>0.05).Analysis of the richness and diversity of the gut microbiota in the two groups showed that the ACE index and Chao1 index were statistically different between the two groups(P<0.01).while the Shannon index and Simpson index were not statistically different(P>0.05).We further assessed time-related changes in gut microbiota alpha diversity in both groups and found a statistically significant decrease in Simpson’s index on day 7 compared to day 1 in the septic cardiomyopathy group(P<0.05).Beta diversity of gut microbiota was analyzed in both groups and PCoA analysis showed a significant difference(P<0.01).We compared the composition of the gut microbiota at different taxonomic levels in the two groups of patients,and at the portal level,the abundance of Actinobacteria(P=0.018)and unidentified Bacteria(P=0.024)was lower in the septic cardiomyopathy group.At the family level,the abundance of Aeromonadaceae was lower in the septic cardiomyopathy group(P=0.023).At the genus level,Citrobacter was more abundant in septic cardiomyopathy(P=0.007).At the species level,Bacteroides_nordii(P=0.037)and[Clostridium]_celerecrescens(P=0.026)were more abundant in septic cardiomyopathy.By Linear discriminant analysis Effect size(LEfSe)analysis,we identified Enterobacter and Klebsiella_quasipneumoniae as possible gut microbe specific for septic cardiomyopathy(LDA score=4.2747,P=0.003).Using Spearman’s rank correlation analysis of clinical indicators and gut microbiota,we found that Bacteroides_thetaiotaomicron was positively correlated with BNP,SI,and TSAT(P<0.05).bacteroides_fragilis was negatively correlated with cTnⅠ,TRF,TIBC were negatively correlated(P<0.05).prevotella_disiens and prevotella_timonensis were negatively correlated with SF(P<0.05).The secondary observations of this study,including the patients’ 28-day morbidity and mortality rate,length of ICU stay,total length of stay,mechanical ventilation rate,continuous renal replacement therapy utilization rate,intensity of vasoactive drug use,and 24-hour fluid use in the enrolled group were not statistically different.Conclusion:Our study suggests that ferritin may have predictive value for early identification of septic cardiomyopathy,while Enterobacteriaceae may be the gut microbiota specific to septic cardiomyopathy.Furthermore,alterations in gut microbiota diversity may influence changes in iron metabolism and ultimately induce the development of septic cardiomyopathy,and larger studies are needed to validate this in the future. |
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