BACKGROUND:Chemical-induced acute lung injury is characterized by impaired epithelial regenerative capacity, leading to acute pulmonary edema. Numerous studies have investigated the therapeutic potential of endogenous stem cells with particular emphasis on alveolar type 2 epithelial (AEC2) cells owing to their involvement in lung cell renewal. Sox9, a transcription factor known for its role in maintaining stem cell properties and guiding cell differentiation, marks a subset of AEC2 cells believed to contribute to epithelial repair. However, the role of Sox9+AEC2 cells in the distal lung alveolar cells and the potential roles in chemically induced acute lung injury have never been explored. METHODS:In this study, we generated Sox9flox/flox;SftpcCre-ERT2 mice and examined the effects of Sox9+AEC2 cells on the pathophysiology of epithelial damage during chemical-induced acute lung injury. Subsequently, Sox9-CreERT2 Ai9 mice were used for lineage tracing to elucidate the repair mechanisms. RESULTS:Our findings revealed that Sox9+AEC2 cells endowed with stem cell properties induced cell proliferation during lung injury, predominantly in the damaged alveolar region. This process is accompanied by the regulation of inflammatory responses and orderly differentiation, thereby promoting epithelial regeneration. CONCLUSION:These results provide compelling in vivo genetic evidence supporting the characterization of Sox9+AEC2 cells as bona fide lung epithelial stem cells, demonstrating their multipotency and self-renewal capabilities during lung repair and regeneration. The identification of Sox9+AEC2 cells as crucial contributors to the promotion of epithelial repair underscores their potential as therapeutic targets in chemical-induced acute lung injury.

Purpose: Intra-abdominal infection is a complex pathophysiological process involving multiple systems and organs of the body. Abdominal infections complicated by severe sepsis or septic shock have a high mortality rate of 30-50%. Therefore, novel strategies to treat sepsis are urgently needed. Methods: Andrographolide (AD), the main active ingredient of Andrographis paniculata, reportedly exerts beneficial effects on mice with sepsis. However, its exact mechanism of action in attenuating inflammation due to intra-abdominal sepsis remains unclear to date. Hence, this study aimed to examine the therapeutic effects of AD on cecal ligation and puncture (CLP)-induced sepsis and elucidate the underlying mechanisms. Results: Results showed that AD therapy could significantly improve the 7-day survival rate and alleviate pathological organ injury in mice with CLP. In addition, AD treatment decreased the levels of proinflammatory factors, such as tumor necrosis factor-alpha and interleukin (IL)-6 in the peritoneal cavity fluid and blood and increased the level of anti-inflammatory factor IL-10 in the peritoneal cavity fluid of mice with CLP. Moreover, bacterial counts in the blood and peritoneal lavage fluid were lower in the mice treated with AD than in those untreated. Mechanistically, AD treatment increased the percentage and phagocytic activity of macrophages in the peritoneal cavity. Conclusion: These data showed that AD can improve the survival of mice with intra-abdominal sepsis by enhancing bacterial clearance, as evidenced by the increased percentages and phagocytic activity of macrophages in the peritoneal cavity. This study is the first to demonstrate the protective effects of AD against intra-abdominal sepsis.

Acute respiratory distress syndrome (ARDS) presents as a form of acute respiratory failure resulting from non-cardiogenic pulmonary edema due to excessive alveolocapillary permeability, which may be pulmonary or systemic in origin. In the last 3 years, the coronavirus disease 2019 pandemic has resulted in an increase in ARDS cases and highlighted the challenges associated with this syndrome, as well as the unacceptably high mortality rates and lack of effective treatments. Currently, clinical treatment remains primarily supportive, including mechanical ventilation and drug-based therapy. Mesenchymal stem cell (MSC) therapies are emerging as a promising intervention in patients with ARDS and have promising therapeutic effects and safety. The therapeutic mechanisms include modifying the immune response and assisting with tissue repair. This review provides an overview of the general properties of MSCs and outlines their role in mitigating lung injury and promoting tissue repair in ARDS. Finally, we summarize the current challenges in the study of translational MSC research and identify avenues by which the discipline may progress in the coming years.

Phosgene (COCl 2 ) gas is a chemical intermediate of high-volume production with numerous industrial applications worldwide. Due to its high toxicity, accidental exposure to phosgene leads to various chemical injuries, primarily resulting in chemical-induced lung injury due to inhalation. Initially, the illness is mild and presents as coughing, chest tightness, and wheezing; however, within a few hours, symptoms progress to chronic respiratory depression, refractory pulmonary edema, dyspnea, and hypoxemia, which may contribute to acute respiratory distress syndrome or even death in severe cases. Despite rapid advances in medicine, effective treatments for phosgene-inhaled poisoning are lacking. Elucidating the pathophysiology and pathogenesis of acute inhalation toxicity caused by phosgene is necessary for the development of appropriate therapeutics. In this review, we discuss extant literature on relevant mechanisms and therapeutic strategies to highlight novel ideas for the treatment of phosgene-induced acute lung injury.

Alveolar epithelial cells (AECs) play a role in chemically induced acute lung injury (CALI). However, the mechanisms that induce alveolar epithelial type 2 cells (AEC2s) to proliferate, exit the cell cycle, and transdifferentiate into alveolar epithelial type 1 cells (AEC1s) are unclear. Here, we investigated the epithelial cell types and states in a phosgene-induced CALI rat model. Single-cell RNA-sequencing of bronchoalveolar lavage fluid (BALF) samples from phosgene-induced CALI rat models (Gas) and normal controls (NC) was performed. From the NC and Gas BALF samples, 37,245 and 29,853 high-quality cells were extracted, respectively. All cell types and states were identified and divided into 23 clusters; three cell types were identified: macrophages, epithelial cells, and macrophage proliferating cells. From NC and Gas samples, 1315 and 1756 epithelial cells were extracted, respectively, and divided into 11 clusters. The number of AEC1s decreased considerably following phosgene inhalation. A unique SOX9-positive AEC2 cell type that expanded considerably in the CALI state was identified. This progenitor cell type may develop into alveolar cells, indicating its stem cell differentiation potential. We present a single-cell genome-scale transcription map that can help uncover disease-associated cytologic signatures for understanding biological changes and regeneration of lung tissues during CALI.

BACKGROUND:Acute renal failure due to crush syndrome is one of the leading causes of death in disasters. Ischemic Postconditioning (IPC) is a potentially effective strategy to protect against ischemic reperfusion injury, but a few studies noted its protective effect in crush induced acute kidney injury (AKI). Hence, this study investigated the optimal IPC strategy to prevent crush induced AKI and reveal related cellular mechanisms.METHODS:The right lower extremities of rabbits were constantly compressed for 8 h and then performed five cycles of clamping and releasing the femoral artery and vein before depression using a clip. In terms of the duration of clamping and releasing, the animals were randomly divided into 5 groups, Control, IPC-5sec, IPC-30sec, IPC-1min, and IPC-5min groups; 6 rabbits for each group. Biomarkers of inflammation, renal function, renal tubular injury, and muscular injury, apoptosis, and cellular senescence in kidney were detected.RESULTS:Six hours after decompression, the levels of Serum Creatine (SCr), Blood Urea Nitrogen (BUN), K+, and Interleukin-6 (IL-6) in IPC-1min and IPC-5min groups were lower than Control, with a statistically significant difference. The morphological study of Periodic Acid-Schiff (PAS) staining demonstrated that 6 h after decompression, IPC-1min can attenuate renal tubular damage renal tubule. Meanwhile, the level of Neutrophil Gelatinase-Associated Lipocalin (NGAL) in circulation in the IPC-30sec, IPC-1min, and IPC-5min groups was significantly decreased compared with the Control group, 2 h after decompression. On the other hand, the levels of serum Creatine Kinase (CK) and Myoglobin (Mb), and the morphological change of muscular damage detected by hematoxylin and eosin (H&E) staining in IPC-1min-treated group were significantly lower than Control group 6 hours after decompression. Further results of the cellular mechanism showed that the apoptotic markers of Terminal deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) and Caspase3 and the cell senescent markers of senescence-associated β-galactosidase (SA-β-Gal) and nuclear LAMNB1 have changed significantly in the IPC-1min group, compared with the control group.CONCLUSIONS:Performing 5 cycles of 1-min IPC would be a convenient, time-saving, and effective method to prevent crush-induced AKI by attenuating the release of nephrotoxic substances after decompression and downregulation of the expression of apoptosis and cellular senescence biomarkers.

目的 探讨尿中性粒细胞明胶酶相关脂质运载蛋白(uNGAL)联合尿N-乙酰-β-D-氨基葡萄糖苷酶(NAG)对危重症患者急性肾损伤(AKI)病情及预后的评估价值.方法 选取危重症合并AKI的患者101例(AKI组)和同期收治的非AKI患者27例(对照组).比较2组患者入院后24 h内血肌酐、血钾、血白蛋白和阴离子间隙(AG)、uNGAL、尿NAG、尿蛋白、尿微量白蛋白的差异.AKI组患者根据肾功能损伤严重程度分为Stage 1组(40例),Stage 2组(36例)和Stage 3组(25例),另根据患者出院时存活情况,分为存活组(63例)和死亡组(38例).比较不同严重程度及不同生存状态间上述指标的差异.应用Logistic回归分析危重症患者AKI预后的独立影响因素,受试者工作特征(ROC)曲线评价uNGAL联合尿NAG对危重症患者AKI预后的评估价值.结果(1)与非AKI组相比,AKI组uNGAL、尿NAG、尿蛋白、尿微量白蛋白、血肌酐水平升高,血清白蛋白水平降低(P<0.01).亚组分析结果显示,随着病情的加重,AKI组uNGAL、尿NAG和血肌酐水平均出现明显升高(P<0.05);同时死亡组uNGAL、尿NAG、尿微量白蛋白、尿蛋白及血肌酐高于存活组,血清白蛋白低于存活组(P<0.05).Logistic回归分析显示,uNGAL和尿NAG升高是影响患者预后的独立危险因素,ROC曲线显示uNGAL联合尿NAG预测预后的曲线下面积(0.886)优于uNGAL(0.850)、尿NAG(0.784).结论 uNGAL联合尿NAG检测有助于对危重症患者急性肾损伤的危险分层和预后评估.

Background: Patients with acute respiratory distress syndrome supported with veno-venous extracorporeal membrane oxygenation benefit from higher positive end-expiratory pressure combined with conventional ventilation during the early extracorporeal membrane oxygenation period. The role of incremental positive end-expiratory pressure titration in patients with severe acute respiratory distress syndrome supported with veno-venous extracorporeal membrane oxygenation remains unclear. This study aimed to determine the preferred method for setting positive end-expiratory pressure in patients with severe acute respiratory distress syndrome on veno-venous extracorporeal membrane oxygenation support. Methods: We retrospectively reviewed all subjects supported with veno-venous extracorporeal membrane oxygenation for severe acute respiratory distress syndrome from 2009 to 2019 in the intensive care units in Tianjin Third Central Hospital. Subjects were divided into two groups according to the positive end-expiratory pressure titration method used: P-V curve (quasi-static pressure-volume curve-guided positive end-expiratory pressure setting) group or Crs (respiratory system compliance-guided positive end-expiratory pressure setting) group. Results: Forty-three subjects were included in the clinical outcome analysis: 20 in the P-V curve group and 23 in the Crs group. Initial positive end-expiratory pressure levels during veno-venous extracorporeal membrane oxygenation were similar in both groups. Incidence rates of barotrauma and hemodynamic events were significantly lower in the Crs group (all p < 0.05). Mechanical ventilation duration, intensive care unit length of stay, and hospital length of stay were significantly shorter in the Crs group than the P-V curve group (all p < 0.05). Subjects in the Crs group showed non-significant improvements in the duration of extracorporeal membrane oxygenation support and 28-day mortality (p > 0.05). Conclusion: Respiratory system compliance-guided positive end-expiratory pressure setting may lead to more optimal clinical outcomes for patients with severe acute respiratory distress syndrome supported by veno-venous extracorporeal membrane oxygenation. Moreover, the operation is simple, safe, and convenient in clinical practice.

Histones are considered potential risk factors that contribute to the development of septic acute kidney injury (SAKI) by inducing apoptosis and inflammation. This study aimed to explore the protective effects of heparin on septic acute kidney injury through the neutralization of extracellular histones (EH) and to uncover the underlying mechanism. C57BL mice (16 each) were randomly divided into the sham group, the sepsis group (established by cecal ligation and puncture operation, CLP), and the heparin intervention group. Mice in the heparin intervention group received a subcutaneous injection of unfractionated heparin (0.03 IU/g) 4 h after CLP. At 6 h after the operation, nine mice from each group were sacrificed by the removal of the eyeballs to harvest blood samples; the upper half of the right kidney was used as the study sample. Mice renal tubular epithelial cells cultivated in six-well plates were equally divided into five groups. We cultured cells treated with either histone (40 U), histone (40 U) + heparin (25 IU/ml), histone(40U) + lipopolysaccharides (LPS; 10 μg/ml), or histone (40 U) + LPS (10 μg/ml) + heparin (25 IU/ml) for 6 h. For the histone + heparin group and the histone + LPS + heparin group, histone (and LPS) were treated with heparin simultaneously. Mice in the heparin intervention group showed decreased levels of EH4, neutrophil gelatinase-associated lipocalin (NAGL), kidney injury molecule-1 (KIM-1), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6 in the blood serum, longer average 72-h survival rate, significantly decreased kidney tissue edema, and a clearer glomerular structure coupled with decreased protein and mRNA expression levels of kidney apoptosis-related proteins (cleaved Caspase-3/Caspase-3 and Bax/Bcl-2) compared with those in the sepsis group at 6 h after CLP (P < 0.05). Meanwhile, cells in the heparin intervention group exhibited lower expression levels of serum EH4 and inflammatory cytokines, a lower apoptosis rate, and decreased expression of apoptosis-related proteins, both at protein and mRNA levels, than those in the histone-stimulated group at 6 h after stimulation (P < 0.05). Heparin may alleviate apoptosis and inflammation through the neutralization of histones, thus playing a protective role against septic acute kidney injury.

Myocardial cell injury or cardiomyopathy is associated with excessive inflammatory response and apoptosis of cardiac myocytes during sepsis. MicroRNA-23b (miR-23b) is a multifunctional miRNA that is considered to regulate immunosuppression in sepsis. The aim of this study was to examine the effect of miR-23b on cardiomyopathy induced by sepsis and to explore the potential mechanism involved. Sprague-Dawley rats were subjected to cecal ligation and puncture (CLP), and the level of miR-23b at different time points was measured by quantitative real-time polymerase chain reaction (qPCR). Then, we overexpressed miR-23b in vivo and in vitro. The rats were subjected to CLP 7 days after transfection. Cardiac function, inflammatory response, and heart tissues were examined 3 days thereafter. In an in vitro experiment, H9C2 cardiomyoblasts were stimulated with lipopolysaccharide (LPS) after transfection of miR-23b, following which apoptosis and the level of NF-κB were analyzed. The expression of miR-23b was upregulated during polymicrobial sepsis, and transfection of miR-23b lentivirus improved the outcome of sepsis-induced cardiomyopathy by attenuating inflammatory responses and protecting against histopathological damage. In in vitro experiments, elevated miR-23b inhibited excessive apoptosis of cardiomyocytes, which may be because activation of the NF-κB signaling pathway was inhibited by the decreased levels of TRAF6 and IKKβ. Therefore, miR-23b improved sepsis-induced cardiomyopathy by attenuating the inflammatory response, suppressing apoptosis, and preventing NF-κB activation via targeted inhibition of TRAF6 and IκκB. These results indicated that miR-23b may represent a novel therapeutic approach for clinical treatment of sepsis-induced cardiomyopathy.

Acute kidney injury (AKI) is a common clinical condition of growing incidence. Patients who suffer severe AKI have a higher risk of developing interstitial fibrosis, chronic kidney disease, and end-stage renal disease later in life. Cellular senescence is a persistent cell cycle arrest and altered gene expression pattern evoked by multiple stressors. The number of senescent cells increases with age and even in small numbers these cells can induce chronic inflammation and fibrosis; indeed, in multiple organs including kidneys, the accumulation of such cells is a hallmark of aging. We hypothesized that cellular senescence might be induced in the kidney after injury and that this might contribute to progressive organ fibrosis. Testing this hypothesis, we found that tubular epithelial cells (TECs) in mice senesce within a few days of kidney injury and that this response is mediated by epithelial Toll-like and interleukin 1 receptors (TLR/IL-1R) of the innate immune system. Epithelial cell-specific inhibition of innate immune signaling in mice by knockout of myeloid differentiation 88 (Myd88) reduced fibrosis as well as damage to kidney tubules, and also prevented the accumulation of senescent TECs. Importantly, although inactivation of Myd88 after injury ameliorated fibrosis, it did not reduce damage to the tubules. Selectively induced apoptosis of senescent cells by two different approaches only partially reduced kidney fibrosis, without ameliorating damage to the tubules. Our data reveal a cell-autonomous role for epithelial innate immunity in controlling TEC senescence after kidney injury, and additionally suggest that early therapeutic intervention is required for effective reduction of long-term sequelae of AKI.

Nephronophthisis (NPHP), the leading genetic cause of end-stage renal failure in children and young adults, is a group of autosomal recessive diseases characterized by kidney-cyst degeneration and fibrosis for which no therapy is currently available. To date, mutations in >25 genes have been identified as causes of this disease that, in several cases, result in chronic DNA damage in kidney tubular cells. Among such mutations, those in the transcription factor-encoding GLIS2 cause NPHP type 7. Loss of function of mouse Glis2 causes senescence of kidney tubular cells. Senescent cells secrete proinflammatory molecules that induce progressive organ damage through several pathways, among which NF-icB signaling is prevalent. Herein, we show that the NF-icB signaling is active in Glis2 knockout kidney epithelial cells and that genetic inactivation of the toll-like receptor (TLR)/IL-1 receptor or pharmacologic elimination of senescent cells (senolytic therapy) reduces tubule damage, fibrosis, and apoptosis in the Glis2 mouse model of NPHP. Notably, in Glis2, Tlr2 double knockouts, senescence was also reduced and proliferation was increased, suggesting that toss of TLR2 activity improves the regenerative potential of tubular cells in Glis2 knockout kidneys. Our results further suggest that a combination of TLR/IL-1 receptor inhibition and senolytic therapy may delay the progression of kidney disease in NPHP type 7 and other forms of this disease.

Sepsis is a life-threatening organ dysfunction syndrome caused by dysregulated host response to infection that leads to uncontrolled inflammatory response followed by immunosuppression. However, despite the high mortality rate, no specific treatment modality or drugs with high efficacy is available for sepsis to date. Although improved treatment strategies have increased the survival rate during the initial state of excessive inflammatory response, recent trends in sepsis show that mortality occurs at a period of continuous immunosuppressive state in which patients succumb to secondary infections within a few weeks or months due to post-sepsis "immune paralysis." Immune cell alteration induced by uncontrolled apoptosis has been considered a major cause of significant immunosuppression. Particularly, apoptosis of lymphocytes, including innate immune cells and adaptive immune cells, is associated with a higher risk of secondary infections and poor outcomes. Multiple postmortem studies have confirmed that sepsis-induced immune cell apoptosis occurs in all age groups, including neonates, pediatric, and adult patients, and it is considered to be a primary contributing factor to the immunosuppressive pathophysiology of sepsis. Therapeutic perspectives targeting apoptosis through various strategies could improve survival in sepsis. In this review article, we will focus on describing the major apoptosis process of immune cells with respect to physiologic and molecular mechanisms. Further, advances in apoptosis-targeted treatment modalities for sepsis will also be discussed.

目的:了解血浆组蛋白H4浓度是否与脓毒症患者严重程度有关,以及是否可作为判断脓毒症预后的指标.方法:采用前瞻性研究,观察2017年5月-2018年4月天津医科大学总医院急诊医学科50例脓毒症患者.收集脓毒症患者临床资料,采集患者入院24 h及72 h时血浆,通过酶联免疫分析检测血浆组蛋白H4和caspase-3浓度,并对血浆组蛋白H4与24 h及72 h时患者的疾病严重程度(APACHEⅡ评分、SOFA评分)、降钙素原(PCT)、C-反应蛋白(CRP)、乳酸(LAG)等临床指标进行相关性分析.按照患者预后将其分为存活组和死亡组,比较两组血浆组蛋白H4、caspase-3、评分和常用指标的差异,绘制ROC曲线评估血浆组蛋白H4等指标预测脓毒症患者预后的价值.结果:脓毒症患者死亡组24 h血浆组蛋白H4浓度高于存活组[505.80(462.46,657.85)vs408.09 (370.68,483.31),P＜0.05],死亡组与存活组72 h血浆组蛋白H4浓度差异无统计学意义[429.95(354.12,557.70)vs 357.70(331.33,391.16),P＞0.05].存活组血浆组蛋白H4浓度72 h低于24 h[357.70(331.33,391.16)vs 408.09(370.68,483.31),P＜0.05],死亡组血浆组蛋白H4浓度72 h和24 h差异无统计学意义[429.95 (354.12,557.70))vs (505.80(462.46,657.85),P＞0.05].脓毒症患者24 h时血浆组蛋白H4浓度与APACHEⅡ评分值(r=0.747,P＜0.05)和SOFA评分值(r=0.631,P＜0.05)呈正相关.对患者预后进行评估并绘制ROC曲线,血浆组蛋白H4的曲线下面积最高,AUC 0.793,以452.661 pg/mL为截断点时敏感性为88.9％,特异性为66.7％.24 h及72h血浆组蛋白H4浓度均与PCT、CRP和caspase-3呈正相关(r值为0.429,0.306,0.735;0.359,0.354,0.732,P＜0.05),而与PLT计数呈负相关(r值为-0.346,-0.436,P＜0.05),72h血浆组蛋白H4与LDH呈正相关(r=0.47,P＜0.05).结论:入院24h血浆组蛋白H4浓度可反应脓毒症患者疾病严重程度,并作为判断预后的预测指标.

CD4+CD25+ regulatory T cells (Tregs) play an essential role in the suppression of the immune response and prevention of autoimmune reactions. The activation of TLR4, which provides a critical link between the innate and adaptive immune systems, has been implicated in regulating the function of Tregs. Ulinastatin (UTI) is a broad-spectrum protease inhibitor that has been shown to modulate innate immunity and pro-inflammatory signaling in sepsis. In addition, there are reports that UTI may modulate the functional activity of Tregs to influence the inflammatory response in infectious disease. In the present study, we investigated the effect of UTI on the activity of Tregs, which was assessed by measuring the survival and inflammatory responses of mice with cecal ligation and puncture (CLP)-induced sepsis. In addition, we further explored the cellular and molecular mechanisms involved in these effects. The results showed that UTI could enhance survival and attenuate inflammatory responses during CLP-induced sepsis. Moreover, sepsis-induced increases in the quantity and activity of Tregs were attenuated under UTI treatment, but not in TLR4−/− mice. We also found that the functional changes in Tregs could be attributed to the TLR4/NF-κB signaling pathway. Collectively, our results indicated that UTI could ameliorate inflammatory damage by modulating the quantity and function of Tregs via the TLR4/NF-κB signaling pathway. Our study provides theoretical and experimental evidence for the administration of UTI in the treatment of sepsis and other acute critical illnesses.

ABSTRACT Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome, remain the leading causes of morbidity and mortality in intensive care units. Ulinastatin (UTI), a serine protease inhibitor, possesses anti-inflammatory properties and has been suggested to modulate lipopolysaccharide (LPS)-induced sepsis; thus, it is now widely used in the treatment of pancreatitis, sepsis, and septic shock. Toll-like receptor 4 (TLR4), an essential LPS signaling receptor, plays a critical role in the activation of innate immunity. The aim of this study was to investigate whether UTI alleviates ALI by attenuating TLR4 expression and to explore the underlying molecular mechanisms involved. Male C56BL/6 mice were administered UTI intravenously 1 h before and 6 h after exposure to LPS by intratracheal instillation. Human lung epithelial (BEAS-2B) cells were incubated with LPS in the presence or absence of UTI. An enzyme-linked immunosorbent assay was used to detect levels of inflammatory cytokines. Western blot analysis was performed to detect changes in TLR4 expression and nuclear factor-κB (NF-κB) activation. UTI significantly protected animals from LPS-induced ALI, decreasing the lung wet/dry weight ratio, ALI score, total cells, neutrophils, macrophages, myeloperoxidase activity, and malondialdehyde content, factors associated with lung histological damage. UTI treatment also markedly attenuated levels of TLR4 and other proinflammatory cytokines. Furthermore, UTI significantly attenuated LPS-induced increases in TLR4 protein expression and NF-κB activation in lung tissues. Similarly, UTI markedly attenuated TLR4 expression and NF-κB activation in LPS-stimulated BEAS-2B cells. These findings indicate that UTI ameliorates LPS-induced ALI by attenuating the TLR4/NF-κB pathway activation.

目的:探讨脓毒症中TLR4 (TLR4)对调节性T细胞功能及活性的影响,并探讨其作用机制.方法:SPF级C57/BL6雄性小鼠(wild type,WT)40只,C57BL/10ScNJNJU(TLR44-)小鼠40只,随机均分为4组,WTSham组,WTCLP组,TLR4-/-Sham组和TLR4-4-CLP.CLP组用盲肠结扎穿孔法制备脓毒症模型.造模后24 h,FCM检测脾脏CD4+CD25+Foxp3+Treg细胞数量、凋亡率及Foxp3和TLR4表达情况,RT-PCR检测Foxp3+mRNA和TLR4mRNA的表达,Western Blot检测rreg细胞内NF-κB、p-NF-κB蛋白表达情况.结果:(1) TLR4-/-CLP组reg细胞凋亡率较WTCLP组明显增多,而细胞数量显著降低(P＜0.05).(2)WT CLP组Treg中Foxp3mRNA及蛋白表达、TLR4mRNA及蛋白水平显著升高(P＜0.05),而在TLR4-/-CLP组中该表达较WTCLP组均显著降低(P＜0.05).(3) WT CLP组Treg细胞NF-κB、p-NF-κB的表达水平明显升高(P＜0.05),而TLR4-/-CLP组较WTCLP组明显降低(P＜0.05).结论:在脓毒症中TLR4可显著影响Treg的功能及其活性,这可能通过TLR4/NF-κB信号通路介导有关.

目的 研究乌司他丁(ulinastatin,UTI)对急性肺损伤(acute lung injury,ALI)小鼠的保护作用,并探讨其机制.方法 雄性SPF级C57/BL小鼠64只,按随机数字表均分为:健康对照组(Sham组)、Sham+UTI组、脂多糖组(LPS组)、LPS+UTI组.气管内滴注LPS建立ALI模型,Sham+UTI组和LPS+UTI组建模前30 min皮下注射UTI 10000 U/kg.监测各组小鼠72 h生存率.建模后24 h取支气管肺泡灌洗液(BALF)和肺组织,检测BALF中炎性细胞、中性粒细胞和巨噬细胞数;测定肺组织湿/干质量比(W/D);观察肺组织病理学变化并计算肺损伤评分;测定肺组织中髓过氧化物酶(MPO)活性及丙二醛(MDA)含量;测定肺组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1(IL-1)、IL-6、IL-2 mRNA水平,检测肺组织中TLR4/NF-κB表达情况.结果 LPS+UTI组小鼠生存率较LPS组明显升高,差异有统计学意义(P<0.05);LPS+UTI组肺组织W/D、肺组织病理评分及肺组织中MPO活性和MDA含量较LPS组明显降低,TNF-α、IL-1、IL-6、IL-2 mRNA水平较LPS组明显降低,且TLR4/NF-κB mRNA表达水平较LPS组显著降低,差异均具有统计学意义(P<0.05或P<0.01).结论 UTI可减轻LPS诱导的ALI小鼠肺组织的炎性反应,提高ALI小鼠生存率,这可能与UTI影响TLR4/NF-κB信号通路有关.

Sepsis constitutes a serious life-threatening syndrome associated with complications of deregulated inflammatory response against endotoxin/lipopolysaccharide (LPS)-mediated severe infection. Toll-like receptor 4 (TLR4) plays a critical role in the activation of innate immunity through recognition of LPS. However, the impact of TLR4 signaling on the development of sepsis-induced immune dysfunction remains unclear. The aim of this study was to investigate the effect of TLR4 on regulatory T cells (Tregs) and its potential mechanism. To simulate sepsis, male C57BL/6 (wild-type) and C57BL/10ScNJNJU (TLR4-/-) mice were subjected to cecal ligation and puncture (CLP). After 24h, pro- and anti-inflammatory cytokine secretion, neutrophil and macrophage lung and liver infiltration were assessed to evaluate the sepsis-induced inflammatory response. The quantity and apoptotic rate of Tregs were measured. The expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and forkhead/winged helix transcription factor p3 (Foxp3) were analyzed. Cytokine (i.e., TNF-α, IL-2, IL-10, and IL-4) secretion by Tregs in the cell suspensions and the suppressive activity on CD4+CD25- T cell proliferation were also determined in vitro. At 24h after the CLP procedure, the wild-type mice exhibited increased Treg levels and expression, and secreted inflammatory factors in the serum were markedly overproduced. However, the TLR4-/- mice attenuated the increased Treg expression and inflammatory factor overproduction. These results indicate that in a model of post-septic mice, TLR4 deficiency improves immune paralysis by attenuating Treg activity and restoring a pro-inflammatory cytokine balance. Thus, modulation of the TLR4 activity may be useful in preventing immune dysfunction in sepsis.

Objective To investigate the effect of ulinastatin on the apoptosis of regulatory T cells (Tregs) and cytokine secretion in septic mice. Methods A total of 60 male mice were randomized into the sham group, model group and ulinastatin group, 20 mice in each group. The model of sepsis was reproduced by cecal ligation and perforation, the mice in the sham group received celiotomy without ligation and puncture, and the mice in the ulinastatin group were given 100000 U/kg ulinastatin by tail intravenous injection at 30 min after operation. The apoptosis rates of regulatory T cells were detected by flow cytometry, and the level of interleukin-2 (IL-2), IL-4 and interferon γ were determined by enzyme-linked immunosorbant assay. Results The apoptosis rates of Tregs in the sham group, model group and ulinastatin group showed significant differences [(20.1 ± 1.0)%, (10.9 ± 0.8)%, (13.7 ± 0.8)%, F=10.236, P=0.030], and the apoptosis rates of Tregs in the ulinastatin group were much lower than those in the sham group, and much higher than those in the model group (all P<0.05). Meanwhile, the levels of IL-2 [(352 ± 76), (172 ± 59), (249 ± 64) ng/L] and interferon γ [(177 ± 25), (56 ± 14), (109 ± 18) ng/L] in the model group and ulinastatin group decreased obviously as compared with the sham group, and it decreased most in the model group (all P<0.05). The levels of IL-4 in the model group and ulinastatin group were much higher than those in the sham group, and were highest in the model group [(46 ± 15), (328 ± 81), (242 ± 62) ng/L, all P<0.05]. Conclusion Ulinastatin may induce the apoptosis of Tregs, improve the levels of IL-2 and interferon γ, and reduce the levels of IL-4.