OBJECTIVE: To examine the therapeutic effect of Fangji Fuling Decoction (FFD) on sepsis through network pharmacological analysis combined with in vitro and in vivo experiments. METHODS: A sepsis mouse model was constructed through intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS). RAW264.7 cells were stimulated by 250 ng/mL LPS to establish an in vitro cell model. Network pharmacology analysis identified the key molecular pathway associated with FFD in sepsis. Through ectopic expression and depletion experiments, the effect of FFD on multiple organ damage in septic mice, as well as on cell proliferation and apoptosis in relation to the mitogen-activated protein kinase 14/Forkhead Box O 3A (MAPK14/FOXO3A) signaling pathway, was analyzed. RESULTS: FFD reduced organ damage and inflammation in LPS-induced septic mice and suppressed LPS-induced macrophage apoptosis and inflammation in vitro (P<0.05). Network pharmacology analysis showed that FFD could regulate the MAPK14/FOXO signaling pathway during sepsis. As confirmed by in vitro cell experiments, FFD inhibited the MAPK14 signaling pathway or FOXO3A expression to relieve LPS-induced macrophage apoptosis and inflammation (P<0.05). Furthermore, FFD inhibited the MAPK14/FOXO3A signaling pathway to inhibit LPS-induced macrophage apoptosis in the lung tissue of septic mice (P<0.05). CONCLUSION FFD could ameliorate the LPS-induced inflammatory response in septic mice by inhibiting the MAPK14/FOXO3A signaling pathway.
Mice ; Animals ; Mitogen-Activated Protein Kinase 14/metabolism* ; Wolfiporia ; Lipopolysaccharides/pharmacology* ; Sepsis/complications* ; Signal Transduction ; Inflammation/drug therapy* ; Oxygen Radioisotopes

Shenfu Injection(SFI) is praised for the high efficacy in the treatment of septic shock. However, the precise role of SFI in the treatment of sepsis-associated lung injury is not fully understood. This study investigated the protective effect of SFI on sepsis-associated lung injury by a clinical trial and an animal experiment focusing on the hypoxia-inducing factor-1α(HIF-1α)-mediated mitochondrial autophagy. For the clinical trial, 70 patients with sepsis-associated lung injury treated in the emergency intensive care unit of the First Affiliated Hospital of Zhengzhou University were included. The levels of interleukin(IL)-6 and tumor necrosis factor(TNF)-α were measured on days 1 and 5 for every patient. Real-time quantitative polymerase chain reaction(RT-qPCR) was performed to determine the mRNA level of hypoxia inducible factor-1α(HIF-1α) in the peripheral blood mononuclear cells(PBMCs). For the animal experiment, 32 SPF-grade male C57BL/6J mice(5-6 weeks old) were randomized into 4 groups: sham group(n=6), SFI+sham group(n=10), SFI+cecal ligation and puncture(CLP) group(n=10), and CLP group(n=6). The body weight, body temperature, wet/dry weight(W/D) ratio of the lung tissue, and the pathological injury score of the lung tissue were recorded for each mouse. RT-qPCR and Western blot were conducted to determine the expression of HIF-1α, mitochondrial DNA(mt-DNA), and autophagy-related proteins in the lung tissue. The results of the clinical trial revealed that the SFI group had lowered levels of inflammatory markers in the blood and alveolar lavage fluid and elevated level of HIF-1α in the PBMCs. The mice in the SFI group showed recovered body temperature and body weight. lowered TNF-α level in the serum, and decreased W/D ratio of the lung tissue. SFI reduced the inflammatory exudation and improved the alveolar integrity in the lung tissue. Moreover, SFI down-regulated the mtDNA expression and up-regulated the protein levels of mitochondrial transcription factor A(mt-TFA), cytochrome c oxidase Ⅳ(COXⅣ), HIF-1α, and autophagy-related proteins in the lung tissue of the model mice. The findings confirmed that SFI could promote mitophagy to improve mitochondrial function by regulating the expression of HIF-1α.
Humans ; Male ; Mice ; Animals ; Leukocytes, Mononuclear ; Mice, Inbred C57BL ; Lung/metabolism* ; Acute Lung Injury/drug therapy* ; Tumor Necrosis Factor-alpha/genetics* ; Sepsis/genetics* ; Hypoxia/pathology* ; Autophagy-Related Proteins ; Body Weight ; Drugs, Chinese Herbal

Reduced chemotactic migration of polymorphonuclear neutrophil (PMN) in sepsis patients leads to decreased bacterial clearance and accelerates the progression of sepsis disease. Quantification of PMN chemotaxis in sepsis patients can help characterize the immune health of sepsis patients. Microfluidic microarrays have been widely used for cell chemotaxis analysis because of the advantages of low reagent consumption, near-physiological environment, and visualization of the migration process. Currently, the study of PMN chemotaxis using microfluidic chips is mainly limited by the cumbersome cell separation operation and low throughput of microfluidic chips. In this paper, we first designed an inertial cell sorting chip to achieve label-free separation of the two major cell types by using the basic principle that leukocytes (mainly granulocytes, lymphocytes and monocytes) and erythrocytes move to different positions of the spiral microchannel when they move in the spiral microchannel under different strength of inertial force and Dean's resistance. Subsequently, in this paper, we designed a multi-channel cell migration chip and constructed a microfluidic PMN inertial label-free sorting and chemotaxis analysis platform. The inertial cell sorting chip separates leukocyte populations and then injects them into the multi-channel cell migration chip, which can complete the chemotaxis test of PMN to chemotactic peptide (fMLP) within 15 min. The remaining cells, such as monocytes with slow motility and lymphocytes that require pre-activation with proliferative culture, do not undergo significant chemotactic migration. The test results of sepsis patients ( n=6) and healthy volunteers ( n=3) recruited in this study showed that the chemotaxis index (CI) and migration velocity ( v) of PMN from sepsis patients were significantly weaker than those from healthy volunteers. In conclusion, the microfluidic PMN inertial label-free sorting and chemotaxis analysis platform constructed in this paper can be used as a new tool for cell label-free sorting and migration studies.
Humans ; Chemotaxis ; Neutrophils/metabolism* ; Microfluidics ; Cell Movement ; Sepsis/metabolism*

Objective To explore the effect and mechanism of penehyclidine hydrochloride (PHCD) on vascular endothelial injury in septic rats. Methods Fifty male SD rats were randomly divided into control group, lipopolysaccharide (LPS) induced sepsis group (model group), low dose PHCD (0.3 mg/kg) group, medium dose PHCD (1.0 mg/kg) group and high dose PHCD (3.0 mg/kg) groups, ten mice for each group. Normal saline was injected into the tail vein of the control group, and 10 mg/kg lipopolysaccharide (LPS) was injected into the tail vein of the rats in other groups to prepare the sepsis rat models. After the models were successfully established, low, medium and high doses (0.3, 1.0, 3.0 mg/kg) of PHCD solution were injected into the tail vein of the rats of corresponding groups. Wet/dry mass ratio (W/D) of lung tissue of rats in each group was measured, and ELISA was used to assay interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6 content and rat plasma angiopoietin 2 (Ang2) content in bronchoalveolar lavage fluid (BALF). HE staining was used to observe the pathological changes of lung tissues. Immunohistochemical staining was used to observe the expression of Ang2 in the right lung tissues. Western blot analysis was performed to detect Ang2 and vascular endothelial cadherin (VE-cadherin) protein in lung tissues. Results Compared with the control group, the W/D ratio of the lung tissues of rats in the model group and the contents of IL-1β, IL-6 and TNF-α in BALF were significantly increased; the lung tissues showed obvious pathological damage, with up-regulation of Ang2 expression and down-regulation of VE-Cadherin expression. Compared with the model group, the W/D ratio of the lung tissues of rats in three PHCD treatment groups and the contents of IL-1β, IL-6 and TNF-α in BALF were significantly reduced; the pathological damage of lung tissue was significantly reduced, with down-regulation of Ang2 expression and up-regulation of VE-cadherin expression. Conclusion PHCD can reduce LPS-induced lung inflammation in rats with sepsis by regulating the Ang2/VE-Cadherin pathway, thereby improving vascular endothelial injury.
Rats ; Mice ; Animals ; Male ; Lipopolysaccharides/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Angiopoietin-2/pharmacology* ; Interleukin-6/metabolism* ; Rats, Sprague-Dawley ; Lung ; Acute Lung Injury/metabolism* ; Sepsis/metabolism*

Sepsis is a life-threatening organ dysfunction caused by dysregulated body response to infection. It is also one of the major causes of death in critically ill patients. Over the past few years, despite the continuous improvement in the treatment of sepsis, there is no specific treatment, clinical morbidity and mortality are still rising. Therefore, finding effective methods to treat sepsis and reduce mortality is an urgent clinical problem. Histone modification is an epigenetic modification that produces heritable phenotypic changes without altering the DNA sequence. In recent years, many studies have shown that histone modification is closely related to sepsis. This review discusses the mechanism of histone modification in the pathogenesis of sepsis from the aspects of inflammatory factors, signaling pathways, and macrophage polarization, in aimed to provide reference for the clinical treatment of sepsis.
Humans ; Histone Code ; Sepsis/metabolism* ; Critical Illness ; Macrophage Activation

Sepsis is a life-threatening organ dysfunction caused by infection that lead to dysregulation of the host response. Sepsis and septic shock with a high mortality threaten human health at present, which are important medical and health problems. Early diagnosis and treatment decision-making for sepsis and septic shock still need to be improved. Exosomes are extracellular vesicles with a diameter of 30-150 nm formed by the fusion of multi-vesicle bodies and cell membranes. Exosomes can effectively transport a variety of bioactive substances such as proteins, lipids, RNA, DNA, and participate in the regulation of inflammatory response, immune response, infection and other pathophysiological processes. In recent years, exosomes have become one of the important methods for the diagnosis and treatment of systemic inflammatory diseases. This article will focus on the basic and clinical research of sepsis, and focus on the research progress of exosomes in the diagnosis and targeted therapy of sepsis.
Humans ; Shock, Septic/therapy* ; Exosomes/metabolism* ; Sepsis/therapy* ; Extracellular Vesicles/metabolism* ; RNA/metabolism*

OBJECTIVE: To investigate whether ferroptosis exists in sepsis induced intestinal injury, and to verify the association between ferroptosis in sepsis induced intestinal injury and intestinal inflammation and barrier function by stimulating and inhibiting the nuclear factor E2-related factor 2/glutathione peroxidase 4 (Nrf2/GPX4) pathway. METHODS: Forty-eight SPF grade male Sprague-Darvley (SD) rats with a body weight of 220-250 g were divided into sham operation group (Sham group), sepsis group (CLP group), sepsis+iron chelating agent deferoxamine (DFO) group (CLP+DFO group) and sepsis+ferroptosis inducer Erastin group (CLP+Erastin group) using a random number table method, with 12 rats in each group. The sepsis model was established by cecal ligation and puncture (CLP). The Sham group was only performed with abdominal opening and closing operations. After modeling, the CLP+DFO group received subcutaneous injection of 20 mg/kg of DFO, the CLP+Erastin group was intraperitoneally injected with 20 mg/kg of Erastin. Each group received subcutaneous injection of 50 mg/kg physiological saline for fluid resuscitation after surgery, and the survival status of the rats was observed 24 hours after surgery. At 24 hours after model establishment, 6 rats in each group were selected. First, live small intestine tissue was taken for observation of mitochondrial morphology in smooth muscle cells under transmission electron microscopy and determination of reactive oxygen species (ROS). Then, blood was collected from the abdominal aorta and euthanized. The remaining 6 rats were sacrificed after completing blood collection from the abdominal aorta, and then small intestine tissue was taken. Western blotting was used to detect the expression of intestinal injury markers such as Claudin-1 and ferroptosis related proteins GPX4 and Nrf2. Observe the pathological changes of small intestine tissue using hematoxylin-eosin (HE) staining and complete Chiu score; Detection of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, IL-6) levels in serum using enzyme-linked immunosorbent assay (ELISA). The levels of serum iron ions (Fe³⁺), malondialdehyde (MDA), and D-lactate dehydrogenase (D-LDH) were measured. RESULTS: (1) Compared with the Sham group, the 24-hour survival rate of rats in the CLP group and CLP+Erastin group significantly decreased (66.7%, 50.0% vs. 100%, both P < 0.05), while there was no significant difference in the CLP+DFO group (83.3% vs. 100%, P = 0.25). (2) Western blotting results showed that compared with the Sham group, the expressions of GPX4 and Claudin-1 in the small intestine tissue of the CLP group, CLP+DFO group, and CLP+Erastin group decreased significantly, while the expression of Nrf2 increased significantly (GPX4/β-actin: 0.56±0.02, 1.03±0.01, 0.32±0.01 vs. 1.57±0.01, Claudin-1/β-actin: 0.60±0.04, 0.96±0.07, 0.41±0.01 vs. 1.40±0.01, Nrf2/β-actin: 0.88±0.02, 0.72±0.01, 1.14±0.01 vs. 0.43±0.02, all P < 0.05). Compared with the CLP group, the expressions of GPX4 and Claudin-1 were significantly increased in the CLP+DFO group, while the expression of Nrf2 was significantly reduced. In the CLP+Erastin group, the expressions of GPX4 and Claudin-1 further decreased, while the expression of Nrf2 further increased (all P < 0.05). (3) Under the light microscope, compared with the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group showed structural disorder in the small intestinal mucosa and submucosal tissue, significant infiltration of inflammatory cells, and destruction of glandular and villous structures. The Chui score was significantly higher (3.25±0.46, 2.00±0.82, 4.50±0.55 vs. 1.25±0.45, all P < 0.05). (4) Under transmission electron microscopy, compared with the Sham group, the mitochondria in the other three groups of small intestinal smooth muscle cells showed varying degrees of volume reduction, increased membrane density, and reduced or disappeared cristae. The CLP+Erastin group showed the most significant changes, while the CLP+DFO group showed only slight changes in mitochondrial morphology. (5) Compared to the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group had serum levels of TNF-α, IL-1β, IL-6, MDA, D-LDH, and ROS in small intestine tissue were significantly increased, while the serum Fe³⁺ content was significantly reduced [TNF-α (ng/L): 21.49±1.41, 17.24±1.00, 28.66±2.72 vs. 14.17±1.24; IL-1β (ng/L): 108.40±3.09, 43.19±8.75, 145.70±11.00 vs. 24.50±5.55; IL-6 (ng/L): 112.50±9.76, 45.90±6.52, 151.80±9.38 vs. 12.89±6.11; MDA (μmol/L): 5.61±0.49, 3.89±0.28, 8.56±1.17 vs. 1.86±0.41; D-LDH (kU/L): 39.39±3.22, 25.38±2.34, 53.29±10.53 vs. 10.79±0.52; ROS (fluorescence intensity): 90 712±6 436, 73 278±4 775, 110 913±9 287 vs. 54 318±2 226; Fe³⁺ (μmol/L): 22.19±1.34, 34.05±1.94, 12.99±1.08 vs. 51.74±11.07; all P < 0.05]. Compared with CLP group, the levels of TNF-α, IL-1β, IL-6, MDA, D-LDH and ROS in CLP+Erastin group were further increased, and the content of Fe³⁺ was further decreased, the CLP+DFO group was the opposite (all P < 0.05). CONCLUSIONS Ferroptosis exists in the intestinal injury of septic rats, and stimulating or inhibiting ferroptosis through the Nrf2/GPX4 pathway can effectively intervene in the inflammatory state and intestinal mechanical barrier of the body.
Rats ; Male ; Animals ; NF-E2-Related Factor 2 ; Tumor Necrosis Factor-alpha ; Ferroptosis ; Reactive Oxygen Species ; Actins ; Claudin-1 ; Interleukin-6 ; Sepsis/metabolism* ; Iron

OBJECTIVE: To explore the prognostic value of early multiple detection indicators in combination with sequential organ failure assessment (SOFA) in sepsis patients. METHODS: A retrospective analysis was conducted. Patients with sepsis admitted to the department of critical care medicine of Huanggang Central Hospital of Yangtze University from May 2020 to May 2022 were selected as the research subjects. Coagulation indicators, inflammatory factors, blood routine, liver and kidney function, and blood gas analysis were collected at admission. Organ dysfunction was assessed based on the SOFA score within 24 hours after admission. Patients were divided into a survival group and a death group according to the outcome of 28 days in ICU. Differences in the above indicators between the two groups were compared. Multifactorial Logistic regression analysis was used to analyze prognostic factors of 28-day mortality in sepsis patients. Receiver operator characteristic curve (ROC curve) was drawn to analyze the predictive performance of various indicators, the SOFA score, and the combine model for the 28-day outcome in patients with sepsis. RESULTS: A total of 101 patients with sepsis were enrolled, 56 patients survived and 45 patients died. Compared to the survival group, patients in the death group were older, the proportion of patients with septic shock was larger, the SOFA score, and the proportion of pulmonary infection were higher, the prothrombin time (PT) and activated partial thromboplastin time (APTT) were significantly prolonged, the prothrombin activity (PTA) was significantly shortened, and antithrombin (AT) was significantly decreased, the levels of hypersensitivity C-reactive protein (hs-CRP), blood urea nitrogen (BUN), total bilirubin (TBil), and lactic acid (Lac) were significantly increased, while the platelet count (PLT) was significantly decreased. Multifactorial Logistic regression analysis showed that pulmonary infection [odds ratio (OR) = 0.010, 95% confidence interval (95%CI) was 0.001-0.164, P = 0.001], AT (OR = 0.944, 95%CI was 0.910-0.978, P = 0.002), hs-CRP (OR = 1.008, 95%CI was 1.001-1.015, P = 0.017), Lac (OR = 1.619, 95%CI was 1.195-2.193, P = 0.002), and SOFA score (OR = 1.363, 95%CI was 1.076-1.727, P = 0.010) were independent prognostic factors for 28-day mortality in patients. A combined model was constructed using pulmonary infection, AT, hs-CRP, Lac, and SOFA score. ROC curve analysis showed that the area under the ROC curve (AUC) for the combine model in predicting sepsis prognosis was 0.936 (95%CI was 0.869-0.975, P < 0.001), which was higher in value compared to single indicators (AUC of AT, hs-CRP, Lac, and SOFA score were 0.775, 0.666, 0.802, 0.796, respectively, all P < 0.05). CONCLUSIONS The predictive ability of the SOFA score for sepsis patient outcomes is limited. The combine model combining infection site, AT, hs-CRP, and Lac shows better predictive ability.
Humans ; Organ Dysfunction Scores ; Retrospective Studies ; C-Reactive Protein ; ROC Curve ; Sepsis/metabolism* ; Prognosis ; Anticoagulants ; Antithrombin III ; Intensive Care Units

OBJECTIVE: To explore the role of endoplasmic reticulum ryanodine receptor 1 (RyR1) expression and phosphorylation in sepsis- induced diaphragm dysfunction. METHODS: Thirty SPF male SD rats were randomized equally into 5 groups, including a sham-operated group, 3 sepsis model groups observed at 6, 12, or 24 h following cecal ligation and perforation (CLP; CLP-6h, CLP-12h, and CLP-24h groups, respectively), and a CLP-24h group with a single intraperitoneal injection of KN- 93 immediately after the operation (CLP-24h+KN-93 group). At the indicated time points, diaphragm samples were collected for measurement of compound muscle action potential (CMAP), fatigue index of the isolated diaphragm and fitted frequencycontraction curves. The protein expression levels of CaMK Ⅱ, RyR1 and P-RyR1 in the diaphragm were detected using Western blotting. RESULTS: In the rat models of sepsis, the amplitude of diaphragm CMAP decreased and its duration increased with time following CLP, and the changes were the most obvious at 24 h and significantly attenuated by KN-93 treatment (P < 0.05). The diaphragm fatigue index increased progressively following CLP (P < 0.05) irrespective of KN- 93 treatment (P>0.05). The frequency-contraction curve of the diaphragm muscle decreased progressively following CLP, and was significantly lower in CLP-24 h group than in CLP-24 h+KN-93 group (P < 0.05). Compared with that in the sham-operated group, RyR1 expression level in the diaphragm was significantly lowered at 24 h (P < 0.05) but not at 6 or 12 following CLP, irrespective of KN-93 treatment; The expression level of P-RyR1 increased gradually with time after CLP, and was significantly lowered by KN-93 treatment at 24 h following CLP (P < 0.05). The expression level of CaMKⅡ increased significantly at 24 h following CLP, and was obviously lowered by KN-93 treatment (P < 0.05). CONCLUSION Sepsis causes diaphragmatic dysfunction by enhancing CaMK Ⅱ expression and RyR1 receptor phosphorylation in the endoplasmic reticulum of the diaphragm.
Rats ; Male ; Animals ; Diaphragm/metabolism* ; Ryanodine Receptor Calcium Release Channel/metabolism* ; Rats, Sprague-Dawley ; Phosphorylation ; Muscle Contraction/physiology* ; Endoplasmic Reticulum ; Sepsis/metabolism*

OBJECTIVE: To investigate the effect of circulating exosomes (EXO) on T cell function in patients with sepsis. METHODS: Plasma EXO were obtained by ultracentrifugation from 10 patients with sepsis admitted to the emergency intensive care unit of Guangdong Provincial People's Hospital Affiliated to Southern Medical University. Transmission electron microscopy observation, nanoparticle tracking analysis (NTA), and Western blotting were used to detect EXO markers to identify their characteristics. Furthermore, peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of 5 healthy volunteers, primary T cells were sorted by magnetic beads and expanded in vitro. After 24 hours of intervention with different doses (0, 1, 2.5, 5, 10 mg/L) of circulating EXO in patients with sepsis, T-cell activity was assessed using a cell counting kit-8 (CCK-8). The expression of T cell activation indicators CD69 and CD25 were observed using flow cytometry. Additional evaluations were performed on immunosuppressive indicators including the expression of programmed cell death 1 (PD-1) in CD4⁺ T cells and the proportion of regulatory T cell (Treg). RESULTS: The identification results confirmed that the successful isolation of EXO from the plasma of sepsis patients. The expression level of circulating EXO in sepsis patients was higher than that in healthy control group (mg/L: 48.78±5.14 vs. 22.18±2.25, P < 0.01). After 24 hours of intervention with 5 mg/L of plasma EXO from sepsis patients, T cells activity began to show suppression [(85.84±0.56)% vs. (100.00±0.00)%, P < 0.05]. As the dosage increased, after 24 hours of intervention with 10 mg/L of EXO, T cells activity was significantly suppressed [(72.44±2.36)% vs. (100.00±0.00)%, P < 0.01]. Compared with the healthy control group, after T cells intervention with plasma EXO from sepsis patients, the expression of early activation marker CD69 was significantly reduced [(52.87±1.29)% vs. (67.13±3.56)%, P < 0.05]. Meanwhile, there was an upregulation of PD-1 expression in T cells [(57.73±3.06)% vs. (32.07±0.22)%, P < 0.01] and an increase in the proportion of Treg [(54.67±1.19)% vs. (24.60±3.51)%, P < 0.01]. However, the expression of the late activation marker CD25 remained stable [(84.77±3.44)% vs. (85.93±2.32)%, P > 0.05]. CONCLUSIONS Circulating EXO in sepsis patients induce T cell dysfunction, which may be a novel mechanism lead to immunosuppression in sepsis.
Humans ; Leukocytes, Mononuclear ; Exosomes/metabolism* ; Programmed Cell Death 1 Receptor/metabolism* ; T-Lymphocytes, Regulatory/metabolism* ; Sepsis/metabolism*