Cecal Ligation And Puncture Surgery Research Articles

Time-serial expression of toll-like receptor 4 signaling during polymicrobial sepsis in rats.

Sepsis caused by aggressive infection is a severe clinical problem with an increasing incidence worldwide. Toll-like receptors and their common adapter myeloid differentiation factor 88 (MyD88) can activate immune responses by recognizing a foreign microbe’s product. This study aimed to identify the different time expression of TLR four signaling pathway in an experimental rodent model of polymicrobial sepsis. A randomized animal study was investigated in rats with septic peritonitis induced by cecal ligation and puncture (CLP). The expressions of MyD88-dependent pathway biomarkers, including MyD88, nuclear factor-κB (NF-κB), and serum tumor necrosis factor-α (TNF-α), were analyzed and compared to the sham controls at the different time points after CLP surgery. CLP-induced sepsis increased liver MyD88 mRNA expression and protein expression compared to the control groups at 2 h after surgery. The MyD88 mRNA and protein expressions in rats with CLP-induced sepsis marked increased at 4 and 6 h, and their NF-κB activities and serum TNF-α levels also increased at 4 h after CLP surgery (both p < .05). The different serial expression of MyD88-ependent pathway during sepsis may be used as biomarkers during sepsis. These results may provide further helpful information for using pro-inflammatory biomarkers of innate immunity such as MyD88 and TNF-α in clinical sepsis or related abdominal surgical emergency in the future.

Human Amnion Epithelial Cells and Their Derived Exosomes Alleviate Sepsis-Associated Acute Kidney Injury via Mitigating Endothelial Dysfunction.

BackgroundSepsis is characterized by organ dysfunction resulting from a patient’s dysregulated response to infection. Sepsis-associated acute kidney injury (S-AKI) is the most frequent complication contributing to the morbidity and mortality of sepsis. The prevention and treatment of S-AKI remains a significant challenge worldwide. In the recent years, human amnion epithelial cells (hAECs) have drawn much attention in regenerative medicine, yet the therapeutic efficiency of hAECs in S-AKI has not been evaluated.MethodsSeptic mice were induced by cecal ligation and puncture (CLP) operation. hAECs and their derived exosomes (EXOs) were injected into the mice via tail vein right after CLP surgery. The 7-day survival rate was observed. Serum creatinine level was measured and H&E staining of tissue sections were performed 16 h after CLP. Transmission electron microscopy was used to examine the renal endothelial integrity in CLP mice. Human umbilical vein endothelial cells (HUVECs) were treated with lipopolysaccharide (LPS) and EXOs. Zonula occludens-1 (ZO-1) localization was observed by immunofluorescence staining. Expression of phosphor-p65 (p-p65), p65, vascular cell adhesion molecule-1 (VCAM-1), and ZO-1 in the kidney were determined by Western blot.ResultshAECs decreased the mortality of CLP mice, ameliorated septic injury in the kidney, and improved kidney function. More precisely, hAECs suppressed systemic inflammation and maintained the renal endothelial integrity in septic animals. EXOs from hAECs exhibited similar renal protective effects as their parental cells. EXOs maintained endothelial cell adhesion junction in vitro and inhibited endothelial cell hyperactivation in vivo. Mechanistically, EXOs suppressed proinflammatory nuclear factor kappa B (NF-κB) pathway activation in LPS-treated HUVECs and in CLP mice kidneys.ConclusionOur results indicate that hAECs and their derived EXOs may ameliorate S-AKI via the prevention of endothelial dysfunction in the early stage of sepsis in mice. Stem cell or exosome-based therapy targeting endothelial disorders may be a promising alternative for treatment of S-AKI.

Transfusion of fresh washed platelets does not prevent experimental polymicrobial‐induced septic shock in mice

IntroductionThe specific role of platelets during sepsis is not yet fully understood, probably related to the paradox of platelets being potentially beneficial but also deleterious via their thrombotic functions. ObjectiveTo evaluate the impact of thrombocytopenia on septic shock in mice and to investigate whether transfusion of fresh washed platelets, either fully functional or with impaired hemostatic properties, might have beneficial effects. MethodsSeptic shock was induced by cecal ligation and puncture (CLP). Experimental depletion of circulating platelets was induced with a rat anti‐mouse GPIbα monoclonal antibody. Transfusion of either wild‐type washed platelets, platelets treated with the antiplatelet drugs acetylsalicylic acid (ASA) and clopidogrel, or GPIIbIIIa‐deficient washed platelets treated with ASA and clopidogrel was performed 4 h after CLP surgery. ResultsDepletion of circulating platelets negatively affected septic shock, worsening systemic inflammation, coagulopathy, organ damage, and mortality, raising the question of whether a higher platelet count could be protective. Transfusion of fully functional platelets or platelets with combined treatment with ASA and clopidogrel, with or without additional GPIIbIIIa deficiency, afforded an immediate return of circulating platelet counts to their initial values before surgery. However, transfusion of each of the three types of platelets did not prevent arterial hypotension, inflammatory response, coagulopathy, and organ damage during septic shock. ConclusionDepletion of circulating platelets negatively affects septic shock, while transfusion of washed platelets has no significant beneficial effect in mice.

Pharmacological Inhibition of FAK-Pyk2 Pathway Protects Against Organ Damage and Prolongs the Survival of Septic Mice.

Sepsis and septic shock are associated with high mortality and are considered one of the major public health concerns. The onset of sepsis is known as a hyper-inflammatory state that contributes to organ failure and mortality. Recent findings suggest a potential role of two non-receptor protein tyrosine kinases, namely Focal adhesion kinase (FAK) and Proline-rich tyrosine kinase 2 (Pyk2), in the inflammation associated with endometriosis, cancer, atherosclerosis and asthma. Here we investigate the role of FAK-Pyk2 in the pathogenesis of sepsis and the potential beneficial effects of the pharmacological modulation of this pathway by administering the potent reversible dual inhibitor of FAK and Pyk2, PF562271 (PF271) in a murine model of cecal ligation and puncture (CLP)-induced sepsis. Five-month-old male C57BL/6 mice underwent CLP or Sham surgery and one hour after the surgical procedure, mice were randomly assigned to receive PF271 (25 mg/kg, s.c.) or vehicle. Twenty-four hours after surgery, organs and plasma were collected for analyses. In another group of mice, survival rate was assessed every 12 h over the subsequent 5 days. Experimental sepsis led to a systemic cytokine storm resulting in the formation of excessive amounts of both pro-inflammatory cytokines (TNF-α, IL-1β, IL-17 and IL-6) and the anti-inflammatory cytokine IL-10. The systemic inflammatory response was accompanied by high plasma levels of ALT, AST (liver injury), creatinine, (renal dysfunction) and lactate, as well as a high, clinical severity score. All parameters were attenuated following PF271 administration. Experimental sepsis induced an overactivation of FAK and Pyk2 in liver and kidney, which was associated to p38 MAPK activation, leading to increased expression/activation of several pro-inflammatory markers, including the NLRP3 inflammasome complex, the adhesion molecules ICAM-1, VCAM-1 and E-selectin and the enzyme NOS-2 and myeloperoxidase. Treatment with PF271 inhibited FAK-Pyk2 activation, thus blunting the inflammatory abnormalities orchestrated by sepsis. Finally, PF271 significantly prolonged the survival of mice subjected to CLP-sepsis. Taken together, our data show for the first time that the FAK-Pyk2 pathway contributes to sepsis-induced inflammation and organ injury/dysfunction and that the pharmacological modulation of this pathway may represents a new strategy for the treatment of sepsis.

Large Peritoneal Macrophages and Transitional Premonocytes Promote Survival during Abdominal Sepsis.

Monocytes and macrophages are early sentinels of infection. The peritoneum contains two resident populations: large and small peritoneal macrophages (LPMs and SPMs). While LPMs self-renew, circulating monocytes enter the peritoneum and differentiate into SPMs. We lack information on the dynamics of monocyte-macrophage trafficking during abdominal sepsis, reflecting an important knowledge gap. In this study, we characterize the presence of LPMs, SPMs, and monocytes in the peritoneum of mice following cecal ligation and puncture (CLP)-induced sepsis and sham surgery. LPMs rapidly disappeared from the peritoneum and were scarce at 18-66 h after CLP or sham surgery. By 14 d, LPMs returned for sham mice, but they remained scarce in CLP mice. Depletion of LPMs from the peritoneum of CD11b-DTR mice greatly increased animal mortality. These data imply that LPMs are critical for sepsis survival. Monocytes rapidly infiltrated the peritoneum and were abundant at 18-66 h after CLP or sham surgery. Surprisingly, SPMs only increased at 14 d post-CLP. Therefore, monocytes may defend hosts from acute sepsis mortality without generating SPMs. More monocytes were present in mice predicted to survive sepsis versus mice predicted to die. However, altering monocyte numbers via CCR2 deficiency or adoptive transfer did not significantly affect animal survival. We reasoned that animals destined to survive sepsis may exhibit a different monocyte phenotype, rather than merely enhanced numbers. Indeed, mice predicted to survive possessed more CD31+, CXCR4hi transitional premonocytes in their abdomen. Inhibition of CXCL12-CXCR4 signaling via AMD3100 exacerbated sepsis. These data imply that recruitment of transitional premonocytes to the abdomen promotes sepsis survival.

Tong‑fu‑li‑fei decoction attenuates immunosuppression to protect the intestinal‑mucosal barrier in sepsis by inhibiting the PD‑1/PD‑L1 signaling pathway.

The aim of the present study was to investigate the therapeutic effects of Tong-fu-li-fei (TFL) decoction on sepsis-induced injury to the intestinal mucosal barrier and the underlying mechanism. Cecal ligation and puncture (CLP) was used to establish a sepsis model in rats. The post-surgery death of the rats was recorded to calculate the survival rate. A 4-kD fluorescein isothiocyanate (FITC)-dextran assay was used to evaluate the intestinal permeability of the rats. The pathological state of the intestine tissues was detected by hematoxylin and eosin staining and the ultrastructural changes in the endometrium were evaluated by transmission electron microscopy. Enzyme-linked immunosorbent assay was used to determine the concentrations of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the intestinal tissues and cells. The expression levels of SHP-2 and PI3K were detected by reverse transcription-quantitative PCR and western blotting. Sorting by flow cytometry was used to obtain pure dendritic cells (DC), CD8+ T cells and natural killer cells. Western blotting was used to evaluate the expression levels of phosphorylated (p)-AKT and AKT. The results demonstrated that the significantly decreased survival rate caused by CLP surgery was elevated by glutamine (Gln) and TFL treatment. Intestinal permeability was increased by CLP, and greatly suppressed by Gln or TFL treatment. Histopathological changes in the intestinal tissues, such as thinner barrier and atrophied mucosa, and ultrastructure changes such as sharply decreased microvilli and mitochondria dropsy, were observed on sepsis animals; these effects were ameliorated by the introduction of Gln or TFL. The upregulation of SHP-2, PI3K and p-AKT induced by CLP was reversed by TFL. The release of IL-6 and TNF-α was elevated and the expression of SHP-2, PI3K and p-AKT was suppressed in the co-cultural system of DC cells and CD8+ T cells by TFL. Overall, TFL decoction may attenuate immunosuppression to protect intestinal mucosal barrier in sepsis via inhibiting the programmed death1/programmed cell death ligand 1 signal pathway.

Comparison of effects of Xuebijing injection and its component hydroxysafflor yellow A on coagulation and survival rates of septic rats

To explore the effects of Xuebijing injection and its component hydroxysafflor yellow A on coagulation and survival rates of septic rats. (1) Assessment of coagulation: 144 male Sprague-Dawley (SD) rats were divided into four groups by random number table: sham group, cecal ligation and puncture (CLP) induced sepsis model group (CLP group), CLP+Xuebijing group, and CLP+hydroxysafflor yellow A group, with 36 rats in each group. CLP was used for reproducing septic models. The cecum of the rats in the sham group was exposed by laparotomy and then returned to the abdominal cavity without CLP, while the other steps were the same as those in the CLP group. Rats in the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group were injected with Xuebijing (4 mL/kg, twice a day) or hydroxysafflor yellow A solution (0.378 g/L, 298 μg each time, twice a day) through caudal vein after operation. Levels of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fib), and D-dimer in peripheral blood were measured by automatic coagulation analyzer at 6, 12, 24 hours after operation. The enzyme linked immunosorbent assay (ELISA) was applied to determine levels of tissue factor (TF), tissue factor pathway inhibitor (TFPI), and soluble thrombomodulin (sTM) in peripheral blood. (2) Analysis of survival rates: 120 rats were divided into four groups by random number table (the same groups with those in the section of assessment of coagulation), with 30 rats in each group. The Kaplan-Meier survival curve was plotted, and the cumulative survival rates were observed and recorded for 7 days after CLP surgery. (1) Results of coagulation assessment: compared with the sham group, septic rats in the CLP group showed significant dysfunction in coagulation early, as evidenced by prolonged PT at 6 hours after CLP (s: 8.9±0.2 vs. 8.4±0.4, P < 0.01), and significantly increased levels of Fib, D-dimer, TFPI and sTM [Fib (g/L): 2.8±0.3 vs. 2.3±0.1, D-dimer (ng/L): 1.8±0.2 vs. 1.5±0.1, TFPI (ng/L): 131.1±10.9 vs. 102.8±10.5, sTM (μg/L): 27.2±1.2 vs. 19.8±2.9, all P < 0.01]. The coagulation dysfunction became more and more serious at 12 hours after operation, and further deteriorated with time. The use of both Xuebijing and hydroxysafflor yellow A revealed significant improvement in coagulation of septic rats at 6 hours, as shown by shortened PT (s: 8.3±0.2, 8.3±0.1 vs. 8.9±0.2, both P < 0.01), and decreased Fib, D-dimer, TFPI and sTM as compared with those in the CLP group [Fib (g/L): 2.3±0.1, 2.3±0.2 vs. 2.8±0.3; D-dimer (ng/L): 1.5±0.1, 1.5±0.2 vs. 1.8±0.2; TFPI (ng/L): 109.5±10.2, 91.5±5.0 vs. 131.1±10.9; sTM (μg/L): 22.3±1.5, 21.1±1.8 vs. 27.2±1.2; all P < 0.01]. However, there was no significant difference in coagulation function between the two intervention groups. (2) Results of survival rates analysis: the rats in the sham group all survived 7 days after operation. The 7-day cumulative survival rate of the CLP group was only 36.67% (11/30). Compared with the CLP group, the cumulative survival rates were significantly increased in rats of the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group [66.67% (20/30), 66.67% (20/30) vs. 36.67% (11/30), both P < 0.05], but no significant difference was found between the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group. Both Xuebijing and its component hydroxysafflor yellow A appear to be capable of alleviating coagulation disorders and improving survival rates of septic rats effectively, and the effects show no significant difference between them.

Upregulation of miR‑335 exerts protective effects against sepsis‑induced myocardial injury.

Septicemia is associated with excessive inflammation, oxidative stress and apoptosis, causing myocardial injury that results in high mortality and disability rates worldwide. The abnormal expression of multiple microRNAs (miRNAs/miRs) is associated with more severe sepsis-induced myocardial injury (SIMI) and miR-335 has been shown to protect cardiomyocytes from oxidative stress. The present study aimed to investigate the role of miR-335 in SIMI. An SIMI model was established by cecal ligation and puncture (CLP) in mice. An miRNA-335 precursor (pre-miR-335) was transfected to accelerate miR-335 expression and an miR-335 inhibitor (anti-miR-335) was used to inhibit miR-335 expression. CLP or sham surgery was performed on pre-miR-335, anti-miR-335 and wild-type mice and miR-335 expression was determined by reverse transcription-quantitative PCR. Inflammatory factors (TNF-α, IL-6 and IL-10) and troponin (cTNI), brain natriuretic peptide (BNP), creatine kinase (CK), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were assessed using commercial kits. Apoptosis was detected by flow cytometry and cardiac function was assessed using a Langendorff isolated cardiac perfusion system. miR-335 expression was upregulated and an elevation in inflammatory factors and cTNI, BNP, CK, LDH and AST was observed. Compared with the wild-type control group, pre-miR-335 mice treated with CLP exhibited significantly reduced left ventricular development pressure, maximum pressure increased reduction rates, as well as decreased levels of TNF-α, IL-6 and IL-10, myocardial injury and apoptosis; by contrast, these features were amplified in CLP-treated anti-miR-335 mice. In conclusion, the upregulation of miR-335 exerted ameliorative effects on myocardial injury following sepsis and may indicate a novel therapeutic intervention for SIMI.

Geranylgeranyl diphosphate synthase 1 knockdown suppresses NLRP3 inflammasome activity via promoting autophagy in sepsis-induced acute lung injury

BackgroundNOD-like receptor protein 3 (NLRP3) inflammasome activation has emerged as a crucial contributor to sepsis-induced lung injury. Geranylgeranyl diphosphate synthase 1 (GGPPS1) reportedly exerts the pro-inflammatory capability via activation of NLRP3 inflammasome. However, little is known about the role and mechanism of GGPPS1 in sepsis-induced lung injury. MethodsMice underwent cecal ligation and puncture (CLP) surgery to establish the in vivo model of sepsis. The lung injury of mice was assessed by analyzing the histological changes, the lung wet/dry ratio, PaO2/FiO2 ratio, myeloperoxidase (MPO) activity, total protein content, total cell, and polymorphonuclear leukocyte counts. Mouse alveolar macrophages MH-S were exposed to LPS for developing in vitro model of sepsis. The mRNA and protein expression levels of GGPPS1, beclin-1, and autophagy and inflammasome-related genes were detected using quantitative reverse transcription-polymerase chain reaction and western blot assays. Enzyme-linked immunosorbent assay was conducted to determine the levels of interleukin (IL)-1β and IL-18. ResultsWe successfully established sepsis-induced acute lung injury in vivo by CLP surgery. GGPPS1 was upregulated in the lung tissues of CLP-induced septic mice. The activation of autophagy and NLRP3 inflammasome were found in the lung tissues of CLP-induced septic mice. The addition of exogenous GGPP (synthesis products catalyzed by GGPPS1) and autophagic inhibitor 3-MA aggravated sepsis-induced hypoxemia, alveolar inflammatory response, intrapulmonary hemorrhage, and pulmonary edema, as evidenced by increased lung injury score, lung wet/dry weight ratio, MPO activity, total protein content, total cell, and PMNs counts, and decreased PaO2/FiO2 ratio. While NLRP3 inhibitor MCC950 exerted the opposite effects. Additionally, administration of exogenous GGPP could inhibit the activation of autophagy, enhance the activity of NLRP3 inflammasome, and the production of IL-1β and IL-18. Inhibition of autophagy by 3-MA treatment also promoted the activity of NLRP3 inflammasome and the production of IL-1β and IL-18. While MCC950 restrained the activity of NLRP3 inflammasome, but did not affect the activation of autophagy. Notably, the expression of GGPPS1 was unaltered in CLP-induced mice following GGPP, 3-MA, or MCC950 treatment. Moreover, GGPPS1 was upregulated in MH-S cells stimulated with LPS, and GGPPS1 knockdown enhanced the activation of autophagy and inhibited the activity of NLRP3 inflammasome in vitro. Importantly, depletion of GGPPS1 could alleviate LPS-induced inflammatory response by inducing autophagy-dependent NLRP3 inflammasome inhibition. ConclusionGGPPS1 knockdown suppressed NLRP3 inflammasome activity via promoting autophagy and then attenuated sepsis-induced acute lung injury, revealing a novel target for treating sepsis-induced lung injury.

Astragaloside IV Attenuates Polymicrobial Sepsis-Induced Cardiac Dysfunction in Rats via IKK/NF-κB Pathway

To evaluate the protective effects of Astragaloside IV (AST) in a rat model of myocardial injury induced by cecal ligation and puncture (CLP). The model of sepsis-induced cardiac dysfunction was induced by CLP. Using a random number table, 50 specific pathogen free grade of Sprague Dawley rats were randomized into 5 groups: the sham group (sham), the model group (CLP, 18 h/72 h) and AST group (18 h/72 h). Except the sham group, the rats in other groups received CLP surgery to induce sepsis. CLP groups received intragastric administration with normal saline after CLP. AST groups received intragastric administration with AST solution (40 mg/kg) once a day. The levels of inflammatory mediators and oxidative stress markers in the serum of the septic rats were determined via enzyme-linked immunosorbent assay (ELISA) at different time point, such as interleukin 6 (IL-6), IL-10, high mobility group box-1 protein B1 (HMGB-1), superoxide dismutase (SOD), and malondialdehyde (MDA). Cardiac function was determined by echocardiography. Moreover, changes in myocardial pathology were evaluated using hematoxylin and eosin staining. The levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were analysed to determine the status of CLP-induced myocardium. In addition, the apotosis of myocardial cells was analysed by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). The protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), IκB kinase α (IKKα), nuclear factor kappa B p65 (NF-κB p65) were detected by Western blot analysis. Moreover, survival rate was investigated. AST improved the survival rate of CLP-induced rats by up to 33.3% (P<0.05). The cardioprotective effect of AST was observed by increased ejection fraction, fractional shortening and left ventricular internal diameter in diastole respectively (P<0.01 or P<0.05). Subsequently, AST attenuated CLP-induced myocardial apoptosis and the ratio of Bcl-2/Bax in the myocardium, as well as the histological alterations of myocardium (P<0.01 or P<0.05); the generation of inflammatory cytokines (IL-6, IL-10, HMGB-1) and oxidative stress markers (SOD, MDA) in the serum was significantly alleviated (P<0.01 or P<0.05). On the other hand, AST markedly suppressed CLP-induced accumulation of IKK-α and NF-κB p65 subunit phosphorylation (P<0.01 or P<0.05). AST plays a significant protective role in sepsis-induced cardiac dysfunction and survival outcome. The possible mechanism of cardioprotection is dependent on the activation of the IKK/NF-κB pathway in cardiomyocytes.

MiR-128-3p reduced acute lung injury induced by sepsis via targeting PEL12.

ObjectiveAcute lung injury (ALI) caused by sepsis is clinically a syndrome, which is featured by damage to the alveolar epithelium and endothelium. In this study, we employed mice models of cecal ligation and puncture (CLP) and primary mice pulmonary microvascular endothelial cells (MPVECs) in vitro to investigate the effect of miR-128-3p in ALI caused by sepsis.MethodsmiR-128-3p agomir or randomized control were injected into adult male C57BL/6 mice 1 week before the CLP surgery. We used miR-128-3p agomir or scrambled control to transfect MPVECs and then employed lipopolysaccharide (LPS) stimulation on the cells. Pellino homolog 2 (PELI2) was predicted to be a direct target of miR-128-3p via luciferase reporter assay. MPVECs were cotransfected with lentiviral vector that expressed PELI2 (or empty vector) as well as miR-128-3p-mimics 1 day before LPS stimulation in rescue experiment. Transcriptional activity of caspase-3, cell apoptosis rate, and the expression levels of miR-128-3p, interleukin-1β (IL-1β), interleukin-6 (IL-6), and PELI2 were analyzed.ResultsCompared with the sham group, the lung of mice in the CLP group showed pulmonary morphological abnormalities, and the expression of IL-6 and IL-1β, caspase-3 activity, and apoptosis rate were significantly upregulated in the CLP group. Inflammatory factor levels and apoptosis rate were also significantly induced by LPS stimulation on MPVECs. Upregulation of miR-128-3p effectively inhibited sepsis-induced ALI, apoptosis as well as inflammation. miR-128-3p also played a role in antiapoptosis and anti-inflammation in MPVECs with LPS treatment. PEL12 upregulation in MPVECs alleviated miR-128-3p-induced caspase-3 activity inhibition and pro-inflammatory factor production.ConclusionsmiR-128-3p enabled to alleviate sepsis-induced ALI by inhibiting PEL12 expression, indicating a novel treatment strategy of miR-128-3p for sepsis-induced ALI.

Alpinetin Attenuates Persistent Inflammation, Immune Suppression, and Catabolism Syndrome in a Septic Mouse Model

Patients who survive the acute phase of sepsis can progress to persistent inflammation, immunosuppression, and catabolism syndrome (PICS), which usually results in extended recovery periods and multiple complications. Alpinetin is a flavonoid isolated from Alpinia katsumadai Hayata that has been demonstrated to have anti-inflammatory, antibacterial, and antioxidant activities. The aim of this study was to investigate whether the administration of alpinetin could attenuate PICS in a septic mouse model. Mice were randomly divided into four groups: the (1) sham-operated group, (2) sham+alpinetin (1 mg/kg intravenously infused for once per day after sham operation), (3) cecal ligation and puncture (CLP), and (4) CLP+alpinetin (50 mg/kg intravenously infused for once per day after CLP). Eight days after sham operation or CLP surgery, mice were euthanized for subsequent examination. Alpinetin significantly improved the survival of septic mice. Also, it attenuated the CLP-induced persistent inflammation, immunosuppression, and catabolism syndrome. The level of plasma proinflammatory cytokines and apoptosis of T lymphocytes were obviously decreased by alpinetin as well. Moreover, oxidative stress in the organs was compelling lower in the alpinetin-treated CLP mice. In this clinically relevant model of sepsis, alpinetin ameliorates CLP-induced organ dysfunction and improves the likelihood of survival, possibly through suppressing the inflammatory response, oxidative stress, and apoptosis. These findings suggested that alpinetin could be a potential novel therapeutic approach to prevent sepsis-induced PICS.

Senkyunolide I protect against lung injury via inhibiting formation of neutrophil extracellular trap in a murine model of cecal ligation and puncture

BackgroundSenkyunolide I (SEI), a component of a Chinese herb named Ligusticum Chuanxiong hort, which is included in the formulation of Xuebijing Injection, a medication used to treat sepsis in China. Our previous study showed that SEI was protective against sepsis-associated encephalopathy and the present study was performed to investigate the role of SEI in sepsis-induced lung injury in a murine model of cecal ligation and puncture (CLP). MethodsSEI (36 mg/kg in 200 μl) or vehicle was administered immediately after CLP surgery. The lung injury was assessed 24 h later by histopathological tests, protein concentration in the bronchoalveolar lavage fluid (BALF), neutrophil recruitment in the lung tissue (myeloperoxidase fluorescence, MPO), pro-inflammatory cytokines and oxidative responses. Platelet activation was detected by CD42d/GP5 immunofluorescence and neutrophil extracellular trap (NET) were determined by immunofluorescence assays and enzyme linked immunosorbent assay (ELISA) of MPO-DNA. In vitro experiments were performed to detect the level of MPO-DNA complex released by SEI-treated neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) or co-cultured with platelets from CLP mice. ResultsSEI administration relieved the injury degree in CLP mice according to the histopathological tests (P < 0.05 compared with DMSO + CLP group). Protein level in the BALF and neutrophil infiltration were remarkably reduced by SEI after CLP surgery (P < 0.05 compared with DMSO + CLP group). TNF-α, IL-1β and IL-6 were decreased in the plasma and lung tissues from CLP mice treated with SEI (P < 0.05 compared with DMSO + CLP group). The phosphorylation of JNK, ERK, p38 and p65 were all inhibited by SEI (P < 0.05 compared with DMSO + CLP group). Immunofluorescence of MPO showed that neutrophil number was significantly lower in SEI treated CLP mice than in vehicle treated CLP mice (P < 0.05). The CD42d/GP5 staining suggested that platelet activation was significantly reduced and the NET level in the lung tissue and plasma was greatly attenuated by SEI treatment (P < 0.05 compared with DMSO + CLP group). In vitro experiments showed that the MPO-DNA level stimulated by PMA was significantly reduced by SEI treatment (P < 0.05 compared with DMSO treatment). Co-culture neutrophils with platelets from CLP mice resulted in higher level of MPO-DNA complex, while SEI partly reversed such effects of platelet on NET formation. ConclusionsSEI was protective against lung injury induced by CLP in mice. The NET formation was significantly reduced by SEI treatment, which might be involved in the mechanism of the protective effect.

Multi-Organ Transcriptome Dynamics in a Mouse Model of Cecal Ligation and Puncture-Induced Polymicrobial Sepsis.

PurposeDuring sepsis, an excessive inflammatory immune reaction contributes to multi-organ dysfunction syndrome (MODS), a critical condition associated with high morbidity and mortality; however, the molecular mechanisms driving MODS remain elusive.MethodsWe used RNA sequencing to characterize transcriptional changes in the early phase of sepsis, at 6, 12, 24 hour time points in lung, kidney, liver, and heart tissues, in a cecal ligation and puncture (CLP)-induced polymicrobial sepsis murine model.ResultsThe CLP surgery induced significant changes (adj. p-value<0.05) in expression of hundreds of transcripts in the four organs tested, with the highest number exceeding 2,000 differentially expressed genes (DEGs) in all organs at 12 hours post-CLP. Over-representation analysis by functional annotations of DEGs to the Reactome database revealed the immune system, hemostasis, lipid metabolism, signal transduction, and extracellular matrix remodeling biological processes as significantly altered in at least two organs, while metabolism of proteins and RNA were revelaed as being liver tissue specific in the early phase of sepsis.ConclusionRNA sequencing across organs and time-points in the CLP murine model allowed us to study the trajectories of transcriptome changes demonstrating alterations common across multiple organs as well as biological pathways altered in an organ-specific manner. These findings could pave new directions in the research of sepsis-induced MODS and indicate new sepsis treatment strategies.

Hepatoprotective effect of anemoside B4 against sepsis-induced acute liver injury through modulating the mTOR/p70S6K-mediated autophagy

Sepsis triggers liver dysfunction with high morbidity and mortality. Here, we elucidated the effect of anemoside B4 on sepsis in cecal ligation and puncture (CLP)-induced mouse model and LPS-induced primary hepatocytes. Following CLP surgery, septic mice were intraperitoneally injected with anemoside B4 (50 or 100 mg/kg). Anemoside B4 improved septic mouse survival rate, decreased serum AST and ALT levels and attenuated liver histopathologic damages. Western blot analysis showed that anemoside B4 elevated the expression of Beclin-1, LC3II/LC3I, Atg3, Atg5, and Atg7, and reduced p62, suggesting the restoration of autophagy flux in liver. More autophagic vesicles were observed in liver after anemoside B4 treatment using transmission electron microscopy. Using ELISA and commercial enzyme kits, we found that anemoside B4 decreased serum TNF-α, IL-6, and IL-1β levels and increased CAT, SOD and GSH activities. TUNEL staining and western blot revealed that anemoside B4 suppressed cell apoptosis, along with decreased Bax, leaved caspase-3, cleaved PARP, but increased Bcl-2. Consistent with in vivo findings, anemoside B4 inhibited apoptosis, inflammatory response, and oxidative stress and enhanced autophagy in LPS-induced primary hepatocytes. Importantly, these cellular processes were possibly mediated by mTOR/p70S6K signaling, as reflected by the offset of 3-MA in the immunosuppression of anemoside B4.

Anti-Septic Potential of 7-α-Obacunyl Acetate Isolated from the Toona sinensis on Cecal Ligation/Puncture Mice via Suppression of JAK-STAT/NF-κB Signal Pathway.

PurposeSepsis is a life-threatening clinical syndrome and characterized by an inflammatory and innate immune response to infections. The current study was aimed to evaluate the anti-sepsis effect of 7-α-Obacunyl acetate (7-OBA), the abundant constituent isolated from Toona sinensis (Meliaceae), in cecal ligation and puncture (CLP)-induced mice and to investigate the related molecular mechanisms.MethodsThe CLP operation was performed to establish the sepsis mice model, and the survival rate and temperature were measured after 7-OBA treatment (7.5, 15, and 30 mg/kg; i.p.). Inflammatory cytokines levels of TNF-α, IL-1β, IL-6, and IL-10 were detected by ELISA kits, and the kidney, liver, and heart function were measured using an automatic biochemistry analyzer. Effects of 7-OBA on NF-κB and JAK2-STAT3 signaling pathways were determined by Western blot analysis in a lipopolysaccharide (LPS) stimulated RAW264.7 cells model.Results7-OBA treatment significantly increased the survival rate (p<0.05 and p<0.01) and normalized temperature (p<0.05 and p<0.01) of sepsis mice. The levels of pro-inflammatory cytokines like TNF-α, IL-1β, and IL-6 in serum were obviously decreased, whereas the anti-inflammatory cytokines of IL-10 were increased. CLP-induced increases of the main markers of kidney, liver, and heart function in mice (p<0.01) were also obviously reversed by 7-OBA. The anti-sepsis effect of 7-OBA might be associated with regulation of nuclear factor kappa-B (NF-κB) and Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) signal pathways.ConclusionOur investigation indicated that 7-OBA can be developed as an effective agent for treating/curing sepsis in the future.

Tea Polyphenols Prevent Sepsis-Induced Lung Injury via Promoting Translocation of DJ-1 to Mitochondria.

BackgroundSepsis is the systemic inflammatory response syndrome caused by infection, which commonly targets on the lung. Tea polyphenols (TP) have many pharmacological activities, but their role in sepsis induced lung injury remains unclear.ResultsInjection of TP after cecal ligation and puncture (CLP) operation elevated the survival rate in a concentration dependent manner. TP treatment improved alveoli structure injury under CLP operation. CLP surgery increased the expression of inflammatory factors IL1β, IL6, and TNFα expression, which was reversed by TP injection. In addition, CLP operation promoted apoptosis and senescence in tissues and cells during lung injury, while TP administration removed the damaged role of CLP on lung tissues and cells. Furthermore, CLP operation or LPS (lipopolysaccharide) treatment induced dysfunction of mitochondria in lung tissues and cells, but TP contributed to recover mitochondria function, which exhibited as inhibition of ROS production inhibition and increase of ATP content and Mitochondrial membrane potential (MMP). Interestingly, DJ-1 was inhibited by CLP operation but promoted by TP treatment. Overexpression of DJ-1 reversed the injury of LPS on L2 cells and recovered mitochondria normal function. And silencing of DJ-1 in rats or alveolar epithelial cells blocked the protection effect of TP.ConclusionOur research revealed that TP protected against lung injury via upregulating of DJ-1 to improve mitochondria function, which contributed to the prevention and treatment of sepsis induced lung injury.

Dexmedetomidine Protects Against Septic Liver Injury by Enhancing Autophagy Through Activation of the AMPK/SIRT1 Signaling Pathway.

Background: Liver injury is one of the serious complications of sepsis. Previous studies suggested that dexmedetomidine (DEX) could alleviate cecal ligation and puncture (CLP)-induced liver injury. However, it is unclear whether the protective effect of DEX on sepsis-induced liver injury is related to autophagy. Methods: Mice (n = 105) were randomly divided into the following groups: (i) CON group (Sham); (ii) CLP group (CLP-induced liver injury + saline); (iii) CLP + DEX group (CLP-induced liver injury + DEX). Mouse models of sepsis-induced liver injury were established using CLP. DEX or normal saline was administered by intraperitoneal injection at 0, 2, and 4 h after CLP surgery. The mortality rate within 120 h was calculated. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and inflammatory cytokines were measured at 6, 12, and 24 h in each group. Hematoxylin and eosin staining assay was carried out to detect the morphological changes of mouse liver cells in each group. The levels of autophagy-associated proteins LC3II, Beclin-1, p62, and LAMP-2 were detected in three groups of mice using western blotting. The expression of LC3II was detected using immunofluorescence. Transmission electron microscopy (TEM) of liver tissue was used to observe autophagosomes and autophagosome–lysosomes. Lastly, the effect of DEX on the AMPK/SIRT1 pathway-associated protein levels were detected using western blotting. Meanwhile, we used L0-2 cells infected with mRFP-GFP-LC3 adenovirus to further analyze the role of SIRT1 in DEX-induced autophagy in liver injury model in vitro. Results: DEX significantly improved the survival rate of septic mice at the early stage and ameliorated the pathology of sepsis-induced liver injury. The level of autophagy-associated proteins, phosphorylated (p)-AMPK/AMPK, and SIRT1 in the liver of CLP-induced sepsis mice peaked at 12 h post-CLP and decreased significantly at 24 h. In the CLP + DEX group, the levels of autophagy-associated proteins, p-AMPK/AMPK, and SIRT1 increased, whereas inflammatory cytokines decreased at 24 h. The autophagosome structure was clearly observed at different time points in the CLP + DEX group. In the in vitro hepatocyte injury model, the SIRT1 inhibitor significantly increased intracellular ROS levels and reversed the effect of DEX on autophagy flux. Conclusion: We demonstrated a novel mechanism in which DEX protects against CLP-induced liver injury. DEX enhances autophagy, which alleviates the inflammatory responses in CLP-induced liver injury by regulating the SIRT1/AMPK pathway.

Bacterial sepsis increases hippocampal fibrillar amyloid plaque load and neuroinflammation in a mouse model of Alzheimer's disease

BackgroundSepsis, a leading cause for intensive care unit admissions, causes both an acute encephalopathy and chronic brain dysfunction in survivors. A history of sepsis is also a risk factor for future development of dementia symptoms. Similar neuropathologic changes are associated with the cognitive decline of sepsis and Alzheimer's disease (AD), including neuroinflammation, neuronal death, and synaptic loss. Amyloid plaque pathology is the earliest pathological hallmark of AD, appearing 10 to 20 years prior to cognitive decline, and is present in 30% of people over 65. As sepsis is also more common in older adults, we hypothesized that sepsis might exacerbate amyloid plaque deposition and plaque-related injury, promoting the progression of AD-related pathology. MethodsWe evaluated whether the brain's response to sepsis modulates AD-related neurodegenerative changes by driving amyloid deposition and neuroinflammation in mice. We induced polymicrobial sepsis by cecal ligation and puncture (CLP) in APP/PS1–21 mice, a model of AD-related β-amyloidosis. We performed CLP or sham surgery at plaque onset (2 months of age) and examined pathology 2 months after CLP in surviving mice. ResultsSepsis significantly increased fibrillar amyloid plaque formation in the hippocampus of APP/PS1–21 mice. Sepsis enhanced plaque-related astrocyte activation and complement C4b gene expression in the brain, both of which may play a role in modulating amyloid formation. CLP also caused large scale changes in the gut microbiome of APP/PS1 mice, which have been associated with a pro-amyloidogenic and neuroinflammatory state. ConclusionsOur results suggest that experimental sepsis can exacerbate amyloid plaque deposition and plaque-related inflammation, providing a potential mechanism for increased dementia in older sepsis survivors.

The Protective Effect of PK-11195 on Cognitive Impairment in Rats Survived of Polymicrobial Sepsis.

Sepsis is an organ dysfunction caused by a host's unregulated response to infection, causing long-term brain dysfunction with microglial activation, the release of inflammatory components, and mitochondrial changes. Neuroinflammation can increase the expression of the 18-kD translocator protein (TSPO) in the mitochondria, leading to the activation of the microglia and the release of inflammatory components. The antagonist PK-11195 can modulate TSPO and reduce microglial activation and cognitive damage presented in an animal model of sepsis. The aim of this was to evaluate the effects of PK-11195 on long-term brain inflammation and cognitive impairment in an animal model of sepsis. Wistar rats, 60 days old, were submitted to cecal ligation and puncture (CLP) surgery, divided into groups control/saline, control/PK-11195, sepsis/saline, and sepsis/PK-11195. Immediately after surgery, the antagonist PK-11195 was administered at a dose of 3 mg/kg. Ten days after CLP surgery, the animals were submitted to behavioral tests and determination of brain inflammatory parameters. The sepsis/saline group presented cognitive damage. However, there was damage prevention in animals that received PK-11195. Besides, the sepsis increased the levels of cytokines and M1 microglia markers and caused oxidative damage. However, PK-11195 had the potential to decrease inflammation. These events show that the modulation of neuroinflammation during sepsis by PK-11195, possibly related to changes in TSPO, improves mitochondrial function in the animals' brains. In conclusion, the antagonist PK-11195 attenuated brain inflammation and prevented cognitive impairment in animals subjected to sepsis.