Development of PEGylated serum albumin with multiple reduced thiols as a long-circulating scavenger of reactive oxygen species for the treatment of fulminant hepatic failure in mice

Reactive oxygen species (ROS) are involved in the pathophysiology of fulminant hepatic failure. Therefore, we developed polyethylene glycol-conjugated bovine serum albumin with multiple reduced thiols (PEG-BSA-SH) for the treatment of fulminant hepatic failure. As a long-circulating ROS scavenger, PEG-BSA-SH effectively scavenged highly reactive oxygen species and hydrogen peroxide in buffer solution. PEG-BSA-SH showed a long circulation time in the plasma after intravenous injection into mice. Fulminant hepatic failure was induced by intraperitoneal injection of lipopolysaccharide and d-galactosamine (LPS/d-GalN) into mice. The LPS/d-GalN-induced elevation of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels was significantly inhibited by a bolus intravenous injection of PEG-BSA-SH. Furthermore, the changes in hepatic lipid peroxide and hepatic blood flow were effectively suppressed by PEG-BSA-SH. In contrast, l-cysteine, glutathione, and dithiothreitol, three traditional reduced thiols, had no statistically significant effects on the serum levels of ALT or AST. These findings indicate that PEG-BSA-SH is a promising ROS scavenger and useful in the treatment of fulminant hepatic failure.     

Effect of endotoxin on lipid order and motion in erythrocyte membranes

Electron paramagnetic resonance employing a lipid-specific spin label has been used to investigate the molecular effects of endotoxin on the physical state of bilayer lipids in rat erythrocyte membranes. When added at a concentration as low as 40 μg/ml to whole blood (plasma plus leukocytes present), decreased membrane lipid motion was found in subsequently washed and spin-labeled intact erythrocytes (P < 0.02). However, if endotoxin were added to washed, plasma plus leukocyte-free intact erythrocytes, no change in the motion of the spin label was found, suggesting that plasma-soluble substances and/or leukocytes are required to produce the change in the physical state of lipids. The decreased lipid motion found in these studies is discussed with reference to the known decreased deformability of endotoxin-treated red cells and to the pathogenesis of sepsis.     

基于蛋白组学的脓毒症补体凝血级联通路及标志物KLKB1研究

摘要 目的：通过蛋白质组学方法鉴定脓毒症关键通路及诊断标志物。方法：选取2019年1月至12月西南医科大学附属医院急诊科收治的56例脓毒症患者（脓毒症组）,另取同期50名健康体检志愿者（对照组）。采用随机抽样法分别选取两组中12名脓毒症患者和8名健康体检志愿者,利用非数据依赖模式（DIA）进行血清蛋白数据采集,将数据上传至iDEP在线平台分析脓毒症患者外周血中差异表达蛋白,进一步对这些差异蛋白进行生物信息学分析,包括主成分分析（PCA）、基因本体富集分析（GO）、通路富集分析和蛋白-蛋白相互作用网络（PPI）分析,进而筛选出脓毒症关键蛋白。采用酶联免疫吸附试验（ELISA）对脓毒症组、对照组进行关键蛋白表达验证分析。采用受试者工作特征（ROC）曲线分析关键蛋白对脓毒症的诊断效能。结果：蛋白质组学分析共鉴定出690个蛋白,筛选出171个差异表达蛋白（DEPs）,其中39个蛋白显著下调,132个蛋白显著上调。DEPs富集的核心通路为补体和凝血级联通路。该条通路中的血清激肽释放酶 1（KLKB1）在脓毒症组的表达水平为（121.80±55.63 ng/mL）,显著高于对照组的（68.30±57.11 ng/mL）,差异具有统计学意义（t=4.881,P=0.000）。根据ELISA结果进行脓毒症诊断ROC曲线分析得出,KLKB1蛋白诊断脓毒症的 AUC（95%CI）为0.759（0.594～0.923）。结论：补体和凝血级联通路为脓毒症免疫途径的重要通路,KLKB1具有较好的脓毒症诊断特性,可能是脓毒症潜在的诊断生物标志物。     

The effects of CLP-induced sepsis on proliferation and apoptosis of granulosa and theca cells in rat ovary: A histochemical and ultrastructural study

Sepsis is defined as a systemic inflammatory response to infection. This study is aimed to evaluate the effects of experimental sepsis on the proliferation and apoptosis of granulosa and theca cells in the rat ovary.28-day-old immature Wistar-Albino female rats were treated with pregnant mare serum gonadotrophin to develop the first generation of preovulatory follicles. Sepsis was induced by cecal ligation and puncture (CLP). Following in vivo 5-Bromo-2-deoxyuridine (BrdU) labeling, animals were sacrificed and ovaries were embedded in paraffin and Epon. Besides electron microscopic evaluation, BrdU, cleaved caspase-3, p27 immunostaining, and TUNEL labeling were performed.In CLP-operated animals, cleaved caspase-3 immunoreactivity was significantly increased in Graafian follicles. TUNEL and BrdU labeling in the ovarian follicles were not statistically different between CLP and sham-operated rats. In septic animals, p27 immunoreactivity was increased significantly in the nuclei of oocytes and decreased in the cytoplasm of granulosa and theca cells in multilaminar primary follicles compared to the sham group. In ultrastructural evaluation, increased apoptosis was observed in theca interna and granulosa cells in both the early and late stages of follicles in the CLP group.In conclusion, experimentally-induced sepsis leads to apoptosis in ovarian follicles at advanced stages of development. Our data suggest that although sepsis may not cause a potential threat to developing follicles at least in the short term, more severe damage may occur during advanced stages of follicle development.     

Classification of the Gut Microbiota of Patients in Intensive Care Units During Development of Sepsis and Septic Shock

The gut microbiota of intensive care unit (ICU) patients displays extreme dysbiosis associated with increased susceptibility to organ failure, sepsis, and septic shock. However, such dysbiosis is difficult to characterize owing to the high dimensional complexity of the gut microbiota. We tested whether the concept of enterotype can be applied to the gut microbiota of ICU patients to describe the dysbiosis. We collected 131 fecal samples from 64 ICU patients diagnosed with sepsis or septic shock and performed 16S rRNA gene sequencing to dissect their gut microbiota compositions. During the development of sepsis or septic shock and during various medical treatments, the ICU patients always exhibited two dysbiotic microbiota patterns, or ICU-enterotypes, which could not be explained by host properties such as age, sex, and body mass index, or external stressors such as infection site and antibiotic use. ICU-enterotype I (ICU E1) comprised predominantly Bacteroides and an unclassified genus of Enterobacteriaceae, while ICU-enterotype II (ICU E2) comprised predominantly Enterococcus. Among more critically ill patients with Acute Physiology and Chronic Health Evaluation II (APACHE II) scores > 18, septic shock was more likely to occur with ICU E1 (P = 0.041). Additionally, ICU E1 was correlated with high serum lactate levels (P = 0.007). Therefore, different patterns of dysbiosis were correlated with different clinical outcomes, suggesting that ICU-enterotypes should be diagnosed as independent clinical indices. Thus, the microbial-based human index classifier we propose is precise and effective for timely monitoring of ICU-enterotypes of individual patients. This work is a first step toward precision medicine for septic patients based on their gut microbiota profiles.     

Identification of key genes and novel immune infiltration-associated biomarkers of sepsis

Sepsis is the major cause of mortality in the intensive care unit. The aim of this study was to identify the key prognostic biomarkers of abnormal expression and immune infiltration in sepsis. In this study, a total of 36 differentially expressed genes were identified to be mainly involved in a number of immune-related Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. The hub genes (MMP9 and C3AR1) were significantly related to the prognosis of sepsis patients. The immune infiltration analysis indicated a significant difference in the relative cell content of naive B cells, follicular Th cells, activated NK cells, eosinophils, neutrophils and monocytes between sepsis and normal controls. Weighted gene co-expression network analysis and a de-convolution algorithm that quantifies the cellular composition of immune cells were used to analyse the sepsis expression data from the Gene Expression Omnibus database and to identify modules related to differential immune cells. CEBPB is the key immune-related gene that may be involved in sepsis. Gene set enrichment analysis revealed that CEBPB is involved in the processes of T cell selection, B cell–mediated immunity, NK cell activation and pathways of T cells, B cells and NK cells. Therefore, CEBPB may play a key role in the biological and immunological processes of sepsis.     

LPS诱导脓毒症小鼠脾组织microRNA表达的筛选研究

目的采用Small RNA测序技术检测脓毒症小鼠24 h脾组织microRNA(miRNA)的表达变化,并从非编码基因层面分析脓毒症脾功能损伤的可能机制。方法将30只SPF级健康BALB/C小鼠随机分为对照组和脓毒症组,每组15只。脓毒症组采用LPS 10 mg/kg腹腔注射进行建模,对照组仅予等量生理盐水腹腔注射。两组术毕均行肌肉注射平衡液5 ml/kg,并于术后24 h各组随机取5只小鼠的脾组织提取RNA,采用Small RNA测序技术进行miRNA检测,然后应用生物信息学软件分析脓毒症小鼠脾组织的miRNA表达差异及靶基因预测。结果小鼠建模后24 h,与对照组相比,脓毒症小鼠脾脏组织已知及未知miRNA表达上调数分别为39个、3个,下调数分别为16个、3个。其中已知miRNA表达上、下调倍数最大的前5个基因分别为Rno-miR-217-5p,rno-mir-216a-5p,rnomiR-216b-5p,rno-miR-375-3p,rno-mir-216b-3p。下调倍数前5的miRNA:Rno-miR-138-5p,rno-miR-451a-3p,rno-miR-138-1-3p,rno-miR-202-5p,rno-miR-592。结论LPS诱导脓毒症小鼠可出现miRNA表达量的改变,其上、下调倍数最显著的miRNA可能对预测LPS诱导的脓毒症脾功能损伤有一定指导意义。     

脓毒症患儿血清SAA、PCT、CRP水平与预后的关系及其诊断价值分析

摘要 目的：探讨脓毒症患儿血清淀粉样蛋白A（SAA）、降钙素原（PCT）、C反应蛋白（CRP）与预后的关系,并分析三者对脓毒症的诊断价值。方法：纳入我院于2016年8月～2020年6月期间收治的脓毒症患儿60例开展回顾性研究,作为脓毒症组,选取同期于我院进行体检的健康儿童40例作为对照组,比较两组血清SAA、PCT、CRP水平。根据脓毒症患儿1个月内的生存、死亡情况,分成生存组（n=42）、死亡组（n=18）,比较两组临床资料及血清SAA、PCT、CRP水平,经COX回归模型分析脓毒症患儿死亡的危险因素。绘制受试者工作特征（ROC）曲线分析血清SAA、PCT、CRP对脓毒症的诊断价值。结果：脓毒症组血清SAA、PCT、CRP水平显著高于对照组（P＜0.05）。死亡组器官障碍数量＞2个、脓毒性休克患儿占比分别为55.56%、44.44%,显著高于生存组的19.05%、9.52%（P＜0.05）;死亡组入院后1 h内使用抗菌治疗患儿占比为38.89%,显著低于生存组的69.05%（P＜0.05）;死亡组血清SAA、PCT、CRP水平高于生存组（P＜0.05）。COX多因素分析结果显示,器官障碍数量＞2、脓毒性休克及血清SAA、PCT、CRP水平升高是脓毒症患儿死亡的危险因素（P＜0.05）,而入院后1 h内使用抗菌治疗是脓毒症患儿死亡风险的保护性因素（P＜0.05）。血清SAA、PCT、CRP单独及三者联合诊断脓毒症的曲线下面积（AUC）分别为0.808、0.780、0.761、0.912。结论：脓毒症患儿血清SAA、PCT、CRP明显升高,三者升高均为脓毒症患儿死亡的危险因素,且对脓毒症具有一定诊断价值。     

脓毒症患者血清H2S、GDF-15、PTX-3水平与凝血功能、炎症指标及病情评分的相关性分析

摘要 目的：探讨脓毒症患者血清硫化氢（H₂S）、生长分化因子-15（GDF-15）、穿透素-3（PTX-3）水平与其凝血功能、炎症指标及病情评分的相关性。方法：随机选取我院2018年2月～2020年2月收治的脓毒症患者52例作为脓毒症组,另选取我院同期收治的脓毒症休克患者46例作为休克组以及同期于我院进行体检的健康者50例作为对照组。检测三组血清H₂S、GDF-15、PTX-3水平以及凝血功能、炎症指标,其中凝血功能指标包括血小板计数（PLT）、部分凝血活酶时间（APTT）、凝血酶原时间（PT）、纤维蛋白原（FIB）。炎症指标包括降钙素原（PCT）、C反应蛋白（CRP）。采用急性生理学和慢性健康状况评分系统Ⅱ（APACHEⅡ）、快速序贯器官功能（qSOFA）评分对脓毒症组、休克组患者病情进行评分。分析血清H2S、GDF-15、PTX-3与患者凝血功能、炎症指标及病情评分的相关性。结果：脓毒症组、休克组的血清H₂S、PLT均低于对照组,且休克组低于脓毒症组（P＜0.05）。脓毒症组、休克组的血清GDF-15、PTX-3、APTT、PT、FIB、PCT、CRP均高于对照组,且休克组高于脓毒症组（P＜0.05）。脓毒症组APACHEⅡ、qSOFA评分均显著低于休克组（P＜0.05）。血清H2S与PLT呈正相关（P＜0.05）,与APTT、PT、FIB、PCT、CRP、APACHEⅡ评分、qSOFA评分呈负相关（P＜0.05）。血清GDF-15、PTX-3与PLT呈负相关（P＜0.05）,与APTT、PT、FIB、PCT、CRP、APACHEⅡ评分、qSOFA评分呈正相关（P＜0.05）。结论：脓毒症患者的血清H₂S明显下降,而血清GDF-15、PTX-3增高,三者与凝血功能、炎症以及病情评分均存在密切关联,这可能是影响脓毒症进展的重要原因之一。