Cecal Ligation Puncture Group Research Articles

#1524 SKLB023 alleviates kidney inflammation and apoptosis via inhibiting TLR4-mediated NF-κB p65 and MAPK signaling pathways in septic AKI mice

Abstract Background and Aims Sepsis is a severe and complex clinical syndrome resulting from the host's inflammatory response to infection. Acute kidney injury (AKI) is one of the major complications of sepsis with high morbidity and mortality, namely sepsis-associated acute kidney injury (S-AKI). Despite current advances, effective drugs for treatment of S-AKI are scarce. SKLB023 is a small molecule compound designed based on 5-benzylidene-thiazolidine-2,4-dione, which has a potent anti-inflammatory effect with favorable efficacy in rheumatoid arthritis. This study aims to evaluate the efficacy of SKLB023 in treating S-AKI and to explore the underlying mechanisms. Method S-AKI was induced by cecal ligation puncture (CLP) and intraperitoneal injection of LPS (10 mg/kg) in male C57BL/6 mice. SKLB023 (25 mg/kg, 50 mg/kg) was administrated by gavage three days in advance and 30 minutes earlier on the day of modeling. Mice death per group was recorded for 100 hours after CLP to evaluate the effect of SKLB023 on the survival rate of S-AKI mice. Other mice were sacrificed 16 hours after model establishment, blood and kidney specimens were collected. Renal function was measured by the detection of serum creatinine (SCr) and blood urea nitrogen (BUN). Kidney pathological injury was evaluated by the expression of kidney injury markers and the assessment of PAS/HE stainings. In vitro experiments were performed with LPS stimulation of TCMK-1 cells and SKLB023 intervention, and the doses were determined by CCK-8 assay. Possible molecular mechanisms involved in SKLB023 against S-AKI were screened by bioinformatics analysis and further validated in vivo and vitro. RT-PCR, western blot, immunofluorescence, and immunohistochemistry were applied to detect renal inflammation and apoptosis. To determine the role of TLR4, TLR4 siRNA and TLR4-KO mice were applied in S-AKI model, and TLR4 expression and the activities of NF-κB p65 and MAPK signaling pathway were assessed. One-way ANOVA was performed for comparison between groups, and logrank analysis was used for comparison of survival curve. P < 0.05 was considered as statistically significant. Results The S-AKI model was successfully established by CLP and LPS injection with obvious renal dysfunction and kidney pathological damage. The mice administrated with SKLB023 had a higher survival rate, lower levels of Scr and BUN, and improved kidney pathological injury than the mice in the CLP group. Transcriptomic analysis identified TLR4-mediated downstream signaling pathways as the potential mechanism of SKLB023 and further experiments in vivo and in vitro proved that SKLB023 regulated the expression of TLR4 and the activities of NF-κB p65 and MAPK signaling pathway in S-AKI mice and LPS-stimulated TCMK-1 cells. Moreover, TLR4 inhibited the expression of inflammatory cytokines including TNF-α, IL-1β, IL-6, iNOS, COX-2, HMGB-1 and alleviated kidney apoptosis in S-AKI mice and LPS-treated TCMK-1 cells. Additionally, knockout of TLR4 remarkably improved inflammation, apotosis, and kidney injury in LPS-treated mice and TCMK-1 cells. Conclusion SKLB023 confers renoprotective effects in S-AKI by modulating inflammation and apoptosis. TLR4 is a key target in S-AKI, and SKLB023 inhibits inflammation and apoptosis by inhibiting the activities of TLR4 mediated NF-κB p65 and MAPK pathways. SKLB023 may be proposed as one of the potential preventive and therapeutic agents for S-AKI.

Inhibiting apoptosis and GSDME-mediated pyroptosis attenuates hepatic injury in septic mice

BackgroundSepsis is characterized by severe inflammation and organ dysfunction resulting from a dysregulated organismal response to infection. Although pyroptosis has been presumably shown to be a major cause of multiple organ failure and septic death, whether gasdermin E (GSDME)-mediated pyroptosis occurs in septic liver injury and whether inhibiting apoptosis and GSDME-mediated pyroptosis can attenuate septic liver injury remain unclear. This study investigated the role of apoptosis and GSDME-mediated pyroptosis in septic liver injury. MethodsAdult male C57BL/6 mice were randomly divided into four groups: sham, cecal ligation puncture (CLP), CLP + Z-DEVD-FMK (a caspase-3 inhibitor, 5 mg/kg), and CLP + Ac-DMLD-CMK (a GSDME inhibitor, 5 mg/kg). Sepsis severity was assessed using the murine sepsis score (MSS). Hepatic tissue damage was observed by the hematoxylin-eosin staining method, the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the levels of malondialdehyde (MDA), the concentrations of interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were measured according to the related kits, and the changes in the hepatic tissue reactive oxygen species (ROS) levels were detected by immunofluorescence (IF). The protein expression levels of cleaved caspase-3, GSDME-N, IL-1β, B-cell lymphoma-2 (Bcl-2), cytochrome C (Cyt-c), and acetaldehyde dehydrogenase 2 (ALDH2) were detected using western blotting. GSDME expression was detected by immunohistochemistry. ResultsCompared with the Sham group, CLP mice showed high sepsis scores and obvious liver damage. However, in the CLP + Z-DEVD-FMK and CLP + Ac-DMLD-CMK groups, the sepsis scores were reduced and liver injury was alleviated. Compared with the Sham group, the serum ALT and AST activities, MDA and ROS levels, and IL-1β and TNF-α concentrations were increased in the CLP group, as well as the protein expression of cleaved caspase-3, GSDME-N, IL-1β, Cyt-c, and GSDME positive cells (P < 0.05). However, the expression levels of Bcl-2 and ALDH2 protein were decreased (P < 0.05). Compared with the CLP group, the CLP + Z-DEVD-FMK and CLP + Ac-DMLD-CMK groups showed low sepsis scores, ALT and AST activities, MDA and ROS levels, decreased IL-1β and TNF-α concentrations, and decreased expression of cleaved caspase-3, GSDME-N, IL-1β protein expression, and GSDME positive cells (P < 0.05). The expression levels of Bcl-2 and ALDH2 protein were increased (P < 0.05). ConclusionApoptosis and GSDME-mediated pyroptosis are involved in the development of sepsis-induced hepatic injury. Inhibition of apoptosis and GSDME-mediated pyroptosis attenuates injury. ALDH2 plays a protective role by inhibiting apoptosis and pyroptosis.

Phosphate and tension homology-induced kinase 1/Parkin signaling mediates cognitive dysfunction in sepsis-associated encephalopathy through activation of hippocampal mitochondrial autophagy

To investigate the effects of gene of phosphate and tension homology (PTEN)-induced putative kinase 1 (PINK1)/Parkin pathway on hippocampal mitophagy and cognitive function in mice with sepsis-associated encephalopathy (SAE) and its possible mechanism. A total of 80 male C57BL/6J mice were randomly divided into Sham group, cecal ligation puncture (CLP) group, PINK1 plasmid transfection pretreatment groups (p-PINK1+Sham group, p-PINK1+CLP group), empty vector plasmid transfection control group (p-vector+CLP group), with 16 mice in each group. The mice in CLP groups were treated with CLP to reproduce SAE models. The mice in the Sham groups were performed laparotomy only. Animals in the p-PINK1+Sham and p-PINK1+CLP groups were transfected with PINK1 plasmid through the lateral ventricle at 24 hours before surgery, while mice in the p-vector+CLP group were transfected with the empty plasmid. Morris water maze experiment was performed 7 days after CLP. The hippocampal tissues were collected, the pathological changes were observed under a light microscope after hematoxylin-eosin (HE) staining, and the mitochondrial autophagy was observed under a transmission electron microscopy after uranyl acetate and lead citrate staining. The expressions of PINK1, Parkin, Beclin1, interleukins (IL-6, IL-1β) and microtubule-associated protein 1 light chain 3 (LC3) were detected by Western blotting. Compared with the Sham group, CLP group mice in Morris water maze experiment had longer escape latency, shorter target quadrant residence time, and fewer times of crossing the platform at 1-4 days. Under the light microscope, the hippocampal structure of the mouse was injured, the neuronal cells were arranged in disorder, and the nuclei were pyknotic. Under the electron microscope, the mitochondria appeared swollen, round, and wrapped by bilayer or multilayer membrane structures. Compared with the Sham group, CLP group had higher expressions of PINK1, Parkin, Beclin1, LC3II/LC3I ratio, IL-6 and IL-1β in hippocampus, indicating that sepsis induced by CLP could activated inflammatory response and caused PINK1/Parkin-mediated mitophagy. Compared with the CLP group, p-PINK1+CLP group had shorter escape latencies, spent more time in the target quadrant and had more number of crossings in the target quadrant at 1-4 days. Under the light microscope, the hippocampal structures of mice was destroyed, the neurons were arranged disorderly, and the nuclei were pyknotic. Under transmission electron microscope, swollen and rounded mitochondria and mitochondrial structure wrapped by double membrane or multilayer membrane structure were observed. Compared with the CLP group, the levels of PINK1, Parkin, Beclin1 and LC3II/LC3 ratio in the p-PINK1+CLP group were significantly increased [PINK1 protein (PINK1/β-actin): 1.95±0.17 vs. 1.74±0.15, Parkin protein (Parkin/β-actin): 2.06±0.11 vs. 1.78±0.12, Beclin1 protein (Beclin1/β-actin): 2.11±0.12 vs. 1.67±0.10, LC3II/LC3I ratio: 3.63±0.12 vs. 2.27±0.10, all P < 0.05], while the levels of IL-6 and IL-1β were significantly decreased [IL-6 protein (IL-6/β-actin): 1.69±0.09 vs. 2.00±0.11, IL-1β protein (IL-1β/β-actin): 1.11±0.12 vs. 1.65±0.12, both P < 0.05], suggesting that overexpression of PINK1 protein could further activate mitophagy and reduce the inflammatory response caused by sepsis. There was no statistically significant difference in the above pathological changes and related indicators between Sham group and p-PINK1+Sham group, CLP group and p-vector+CLP group. PINK1 overexpression can further activate CLP-induced mitophagy by upregulating Parkin, thereby inhibiting inflammation response and alleviate cognitive function impairment in SAE mice.

The role of miR-135b-5p in inhibiting mice acute lung injury (ALI) induced by sepsis and its mechanism

Objective: To investigate the expression status of miR-135b-5p in the sepsis induced acute lung injury (ALI) mice and its effects on inflammatory responses and cell pyroptosis in mice pulmonary tissues. Methods: The cecal ligation puncture (CLP) method was employed to construct sepsis-induced ALI mice models. The C57BL/6 mice were randomly divided into 6 groups with 8 mice in each group. The sepsis mouse models were constructed by performing CLP surgery: mice were anesthetized by intraperitoneal injection of 0.1 mg/kg barbital, the abdomen was cut longitudinally to expose the cecum, the cecum was ligated and perforated with syringe needle, the wound was sutured after extruding part of the intestinal contents. The sham operation group (Sham group) did not undergo any treatment and suture wounds after laparotomy, and no CLP operation was performed. The treatment groups were divided into CLP+NC mimic group, CLP+ miR-135b-5p mimic group, CLP+NC mimic+ Empty vector group, CLP+GSDMD group, and CLP+ miR-135b-5p mimic+GSDMD group. One week before CLP surgery, mice in the treatment group were injected subcutaneously with 200 μ L NC mimic (200 nmol/L), miR-135b-5p mimic (200 nmol/L), Empty vector (100 nmol/L), GSDMD Vector (100 nmol/L), and miR-135b-5p mimic (200 nmol/L), once a day for a week. The euthanasia was performed 24 h after operation by carbon dioxide asphyxiation. The qRT-PCR was utilized to determine miR-135b-5p and GSDMD expressions;HE staing assay was performed to observe the pathological changes of pulmonary tissues. The mice right lung tissues were flushed with 5 ml saline for 3 times, and each flush lasted for 3~5 min to collect the BALF, and the levels of GSDMD, IL-1β and IL-18 in BALF were determined by ELISA. The protein levels of NLRP3, caspase 1 and cleaved GSDMD in mice lung tissues were examined by immunoblotting analysis; Dual-luciferase reporter gene system assay was employed to validate the targeting relationship of miR-135b-5p and GSDMD. Results: Compared with the control group mice, there were a large number of inflammatory cell infiltration, alveolar damage, interstitial edema and alveolar collapse in the lung tissues of the CLP group mice (P<0.01), and the expression levels of the pyroptosis-associated proteins (NLRP3, caspase-1 and GSDMD) were all increased, while miR-135b-5p was down-regulated in the CLP group (P<0.01). Further experiments confirmed that overexpression of miR-135b-5p significantly reduced CLP-induced pyroptotic cell death in mice lung tissues (P<0.01), and dual-luciferase reporter gene system assay confirmed that miR-135b-5p targeted GSDMD for its degradation. Moreover, the rescuing experiments validated that up-regulation of GSDMD abrogated the inhibition effects of miR-135b-5p overexpression on cell pyroptosis in CLP mice lung tissues (P<0.01). Consistently, we verified that miR-135b-5p also suppressed the expression levels of IL-1β and IL-18 in mice BALF via degrading GSDMD (P<0.05). Conclusion: Overexpression of miR-135b-5p attenuated sepsis-induced ALI by inhibiting GSDMD-mediated pyroptotic cell death, and this study provided potential therapeutic target and theoretical foundation for sepsis-induced ALI treatment.

The Possible Useful Effectiveness of Sinapic Acid Sepsis-Induced Secondary Organ Damage in Rats

Objectives: In this study, we investigated the possible useful effectiveness of Sinapic acid on rat kidney and lung tissues in an experimental cecal ligation puncture (CLP) model. Methods: CLP model was created for the rats in the CLP group. 20 mg/kg of Sinapic acid was given in the CLP-Sinapic acid group. At the end of the experiment, lung and kidney tissues were collected and biochemical analyzes were evaluated. Results: For the lung and kidney tissue samples; antioxidant levels decreased, and oxidant levels increased in the CLP group. When the immunohistochemical parameters were evaluated, IL-1β, caspase-3, and TNF-α immunopositivity were severe levels in CLP group. But immunopositivity of these parameters have been observed as attenuated in CLP-Sinapic acid group compared to CLP group. Conclusion: The results of our study showed that Sinapic acid has useful effectiveness on the sepsis model caused by CLP in the lung and kidney tissues.

Dose-related effects of dexmedetomidine on sepsis-initiated lung injury in rats

BackgroundSepsis is one of the leading causes of death in intensive care units. Dexmedetomidine is a sedative agent with anti-inflammatory properties. This study is designed to differentiate the impact of two different doses of dexmedetomidine on lung injury induced by sepsis. MethodsAdult male Wistar rats were randomly divided into four groups: sham (n = 6), control (n = 12), 5DEX (n = 12), and 10DEX (n = 12). Cecal ligation puncture (CLP) was applied for sepsis initiation. The 5DEX group received 5 μg.kg-1.h-1 and the 10DEX group received 10 μg.kg-1.h-1 dexmedetomidine intravenous infusions for a 1-hour period. Six hours after CLP, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and intercellular adhesion molecule-1 (ICAM-1) levels were analyzed in blood samples. Twenty-four hours after CLP, lung samples from the remaining rats were collected for the measurement of myeloperoxidase (MPO) activity, histological examination, and TdT- (terminal deoxynucleotidyl transferase) mediated fluorescent-dUTP labeling staining for apoptosis detection. ResultsSerum cytokine release, MPO activity, and apoptosis in the lung were significantly increased in the CLP group compared with the sham and dexmedetomidine groups (p < 0.05). TNF-α, ICAM-1, and MPO were significantly lower in the 10DEX group compared with both 5DEX and control groups, while IL-1β, total injury score, and apoptotic cell count had significantly lower values in both 10DEX and 5DEX groups compared with the control group (p < 0.05). ConclusionDexmedetomidine administration played a protective role against CLP-induced lung injury. High-dose dexmedetomidine was needed for suppressing the leukocyte-mediated lung injury and apoptosis of lung tissue.

Trigonoside II mitigates sepsis-induced myocardial injury via reduction in oxidative stress and regulation of TLR- 4/NF-kB inflammatory pathway

Purpose: To investigate the protective effect of trigonoside II against sepsis-induced myocardial injury in rats, and the mechanism involved.&#x0D; Methods: Adult male Sprague Dawley rats (n = 30) weighing 200 - 230 g (mean weight = 215 ± 15 g) were used for this study. The rats were randomly assigned to 3 groups (10 rats/group): sham, cecal ligation puncture (CLP), and trigonoside II. Rats in the treatment group received trigonoside II at a dose of 2 mg/kg intraperitoneally (i.p.) at 3, 12 and 24 h post-surgery. Sepsis was induced using CLP method. Lactate dehydrogenase (LDH) and creatine kinase (CK-MB) activities, and hemodynamic functions were determined in the rats. The levels of interleukin (IL)-1β and IL-6, and tumor necrosis factor α (TNF-α) were assayed in rat serum. Oxidative stress and myocardial cell apoptosis were determined by measuring malondialdehyde (MDA) levels, while activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and myeloperoxidase (MPO), as well as levels of expression of bax, bcl-2 and caspase-3 were also assessed.&#x0D; Results: Treatment of myocardial injury rats with trigonoside II led to significant reductions in the activities of LDH, CK-MB and MPO, and decreases in levels of IL-1β, IL-6 and TNF-α (p &lt; 0.05). It also significantly reversed the effects of sepsis on rat hemodynamic functions (p &lt; 0.05). Trigonoside IItreatment significantly reduced MDA levels in rat myocardial tissues, but significantly increased SOD and GPx activities (p &lt; 0.05). It significantly down-regulated protein expressions of NF-kB and TLR-4 in myocardial tissues (p &lt; 0.05). The number of apoptotic cells and activity of caspase-3 were significant increased in myocardial tissues of rats in CLP group, when compared with sham group, but were reduced significantly in myocardial tissues of trigonoside II-treated rats (p &lt; 0.05). Similarly, trigonoside II treatment down-regulated the protein expressions of caspase-3 and bax, but upregulated bcl-2 protein expression in the rat myocardial tissues (p &lt; 0.05).&#x0D; Conclusion: The results of this study indicate that trigonoside II confers protection on sepsis-induced myocardial injury via reduction in oxidative stress and regulation of TLR-4/NF-kB inflammatory pathway.&#x0D; Keywords: Cecal ligation puncture, Myocardial injury, Oxidative stress, Sepsis, Trigonoside II

Targeting NOX4 alleviates sepsis-induced acute lung injury via attenuation of redox-sensitive activation of CaMKII/ERK1/2/MLCK and endothelial cell barrier dysfunction

Increased pulmonary vascular permeability due to endothelial cell (EC) barrier dysfunction is a major pathological feature of acute respiratory distress syndrome/acute lung injury (ARDS/ALI), which is a devastating critical illness with high incidence and excessive mortality. Activation of NADPH oxidase (NOX) induces EC dysfunction via production of reactive oxygen species (ROS). However, the role(s) of NOX isoform(s), and their downstream signaling events, in the development of ARDS/ALI have remained unclear. Cecal Ligation Puncture (CLP) was used to induce preclinical septic ALI in wild-type mice and mice deficient in NOX2 or p47phox, or mice transfected of control siRNA, NOX1 or NOX4 siRNA in vivo. The survival rate of the CLP group at 24 h (26.6%, control siRNA treated) was substantially improved by NOX4 knockdown (52.9%). Mice lacking NOX2 or p47phox, however, had worse outcomes after CLP (survival rates at 0% and 8.3% respectively), whereas NOX1-silenced mice had similar survival rate (30%). NOX4 knockdown attenuated lung ROS production in septic mice, whereas NOX1 knockdown, NOX2 knockout, or p47phox knockout in mice had no effects. In addition, NOX4 knockdown attenuated redox-sensitive activation of the CaMKII/ERK1/2/MLCK pathway, and restored expression of EC tight junction proteins ZO-1 and Occludin to maintain EC barrier integrity. Correspondingly, NOX4 knockdown in cultured human lung microvascular ECs also reduced LPS-induced ROS production, CaMKII/ERK1/2/MLCK activation and EC barrier dysfunction. Scavenging superoxide in vitro and in vivo with TEMPO, or inhibiting CaMKII activation with KN93, had similar effects as NOX4 knockdown in preserving EC barrier dysfunction. In summary, we have identified a novel, selective and causal role of NOX4 (versus other NOX isoforms) in inducing lung EC barrier dysfunction and injury/mortality in a preclinical CLP-induced septic model, which involves redox-sensitive activation of CaMKII/ERK1/2/MLCK pathway. Targeting NOX4 may therefore prove to an innovative therapeutic option that is markedly effective in treating ALI/ARDS.

Effect of inhibiting the activity of double-stranded RNA-dependent protein kinase in sepsis mice

Objective: To observe the effects of 2-aminopurine (2-AP), a double-stranded RNA-dependent protein kinase (PKR) inhibitor, on organ function, plasma inflammatory factor expression and 7 days mortality in sepsis mice induced by cecal ligation puncture (CLP). Methods: Forty specific specific pathogen free C57BL/6 mice were randomly divided into sham group (n=10), CLP group (n=10), CLP+2-AP group (n=10) and 2-AP group (n=10). CLP was used to establish sepsis mice models.Peripheral blood serum was collected 24 hours after operation, alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (Cr), blood urea nitrogen (BUN) and inflammatory factors (IL-1β, IL-10 and TNF-α) were detected; peripheral blood and peritoneal lavage fluid were taken for bacterial clearance detection. Another 60 C57BL/6 mice were selected to observe the 7-day survival rate according to the above groups (n=15). Independent sample t test was used to compare the measurement data between groups. Results: The levels of ALT, AST, Cr and BUN in CLP Group and CLP+2-AP group were significantly higher than those in sham group (all P<0.001). The levels of ALT and AST in CLP+2-AP group were significantly lower than those in CLP Group (t=27.88, 11.33, both P<0.001); the levels of Cr and BUN in CLP+2-AP group were significantly lower than those in CLP Group (t=11.02, 7.15, bothP<0.001). Compared with sham group, the levels of pro-inflammatory (IL-1β and TNF-α) and anti-inflammatory (IL-10) cytokines in CLP group were significantly higher (all P<0.001); the levels of IL-1β and IL-10 in CLP+2-AP group were significantly lower (all P<0.001), but the levels of TNF-α in CLP+2-AP group were not significantly lower (P=0.33). The 7-day survival rate was 100% in sham group, 13.3% in CLP+2-AP group, 86.7% in 2-AP group and 20.0% in CLP+2-AP group. Inhibition of PKR activation slightly improved the trend of 7-days survival rate of CLP model mice (analysis by mantel Cox test, χ(2)=0.0012, P=0.97). Conclusion: In sepsis mice model, inhibition of PKR activity can reduce the expression of inflammatory factors in plasma, decrease bacterial load in blood and abdominal cavity, and protect organ function, which could suggest that inhibition of PKR activity has potential application in sepsis treatment.

Suppressed Prazosin‐Sensitive Cardiovascular Regulation in Central Nervous System During Early Sepsis

Sepsis is the main cause of morbidity and mortality in ICU worldwide, with a resulting mortality rate of around 30–40%. The cardiovascular system in sepsis and septic shock is depressed, resulting in fluid‐refractory hypotension and hyporeactivity to vasoactive agents. In blood pressure (BP) neural control, tonic sympathetic nerve activity (SNA) through norepinephrine (NE) release is crucial for BP maintenance. Septic shock has been associated with SNA exacerbation, resulting in pro‐inflammatory activity and increased release of catecholamines. We hypothesized that α1‐adrenergic receptors (α1AR) in central nervous system (CNS) are less sensitive for NE in early sepsis, thus affecting autonomic modulation of cardiovascular function. To evaluate this hypothesis, we assessed BP and heart rate (HR) of anesthetized male rats subjected to cecal ligation puncture (CLP) model of sepsis, which was performed 6 h before the experiments. Control experiments were conducted in naïve rats. These animals received NE, administered by intracerebroventricular (icv; 10 μg/5 μL) or intravenous (iv; 3, 10, and 30 nmol/Kg) routes. In additional experiments, prazosin (5 μg/5 μL, icv), a selective α1AR antagonist, was given 15 min before a second icv administration of NE. In our experiments CLP generated a mortality rate of 50% in 36 h. Systemic administration of NE dose‐dependently increased the mean arterial pressure (MAP) by 9.2 ± 3.3, 74.6 ± 6.6 and 83.5 ± 4.6 mmHg in control group (n = 5), and by 8.9 ± 1.8, 48.9 ± 7.2 (p &lt; 0.05), and 72.1 ± 11.7 mmHg in CLP animals (n = 4) for doses of 3, 10 and 30 nmol/Kg, respectively. When administered by iv route, NE‐increased BP was accompanied by increased HR, with no differences between control and CLP groups. Central microinjection of NE increased MAP and decreased HR of both control and CLP groups. However, responses were significantly depressed in septic animals. For instance, icv injection of NE increased MAP by 53.6 ± 3.3 mmHg in control (n = 5), and 18.9 ± 3.9 mmHg in CLP group (n = 8, p &lt; 0.0001). Prazosin microinjection generated a similar pattern of slight and transitory reduction in BP of both control and CLP animals. In control animals, pretreatment with prazosin reduced NE‐increased MAP to 20.3 ± 2.6 mmHg (n = 5, p &lt; 0.0001). However, icv administered NE maintained its pressor effect by 12.7 ± 1.4 mmHg in CLP subjected rats (n = 6) even after pretreatment with prazosin. Moreover, prazosin reversed the bradycardia induced by icv NE from −41 ± 12 bpm to 11 ± 21 bpm (n = 5, p &lt; 0.05) in control animals, but had no effects in HR of CLP group. Our results disclose that mechanisms involved in tonic SNA stimulation by NE are depressed in CLP‐subjected rats during early sepsis stages, even before the establishment of hyporesponsiveness in peripheral cardiovascular system. The lack of prazosin effects in septic animals reinforces the development of neural sympathetic α1AR dysfunction and abnormal autonomic modulation of cardiovascular function in sepsis.Support or Funding InformationFinancial Support: CNPq (Brazil), with a Ph.D. fellowship to Matsubara, N. K. This study was approved by the Animal Ethics Committee of UFSC (6327180718).

α2A -AR antagonism by BRL-44408 maleate attenuates acute lung injury in rats with downregulation of ERK1/2, p38MAPK, and p65 pathway.

Acute respiratory distress syndrome (ARDS), characterized by acute hypoxic respiratory dysfunction or failure, is a manifestation of multiple organ failure in the lung, and the most common risk factor is sepsis. We previously showed that blocking α2 -adrenoceptor (α2 -AR) could attenuate lung injury induced by endotoxin in rats. α2A -adrenoceptor (α2A -AR), a subtype of α2 -AR plays a key role in inflammatory diseases, but the mechanism remains unknown. Here, we explored the effect of BRL-44408 maleate (BRL), a specific α2A -AR antagonist, on cecal ligation puncture (CLP)-induced ARDS in rats and the underlying mechanism. Preadministration of BRL-44408 maleate significantly alleviated CLP-induced histological injury, macrophage infiltration, inflammatory response, and wet/dry ratio in lung tissue. However, there was no statistical difference in survival rate between the CLP and CLP+BRL groups. Extracellular regulated protein kinase (ERK1/2), p38MAPK, and p65 were activated in the CLP group, and BRL-44408 maleate inhibited the activation of these signal molecules, c-Jun N-terminal kinase (JNK) and protein kinase A (PKA) showed no changes in activation between these two groups. BRL-44408 maleate decreased lipopolysaccharide (LPS)-induced expression of cytokines in NR8383 rat alveolar macrophages and reduced phosphorylation of ERK1/2, p38MAPK, and p65. JNK and PKA were not influenced by LPS. Together, these findings suggest that antagonism of α2A -AR improves CLP-induced acute lung injury and involves the downregulation of ERK1/2, p38MAPK, and p65 pathway independent of the activation of JNK and PKA.

Dexmedetomidine prevents septic myocardial dysfunction in rats via activation of α7nAChR and PI3K/Akt- mediated autophagy

Background and purposeDexmedetomidine (Dex) has been shown to elicit cardio-protective effects in sepsis. The aim of this study was to investigate the role of autophagy in the protective effects of Dex and its possible mechanism in vivo and vitro. Experimental approach6-8-week-old male Wistar rats were performed cecal ligation puncture (CLP) and administered 0.9% saline (CLP group), 50 μg/kg Dex (Dex group), Dex plus chloroquine (20 mg/kg; Dex + CQ group), or 40 μg/kg methyllycaconitin (Dex + MLA group), or 25 μM LY294002 (Dex + LY294002 group). After study, cardiac histology, cardiac function, level of autophagy, cardiomyocytes apoptosis and inflammatory mediators including protein IL-1β, IL-6, and TNF-α were measured. The LPS induced-H9C2 cardiomyocytes were treated with Dex, Dex + CQ and detected for cell apoptosis, autophagy level and cell cycle. Key resultsCLP-induced sepsis resulted in cardiac dysfunction, apoptosis, and inflammatory response. Dex exhibited protective effects on the myocardium by the induction of myocardial autophagy and ameliorated the LPS-induced blockade of autophagic flux in H9C2 cells. CQ was found to significantly inhibit Dex-mediated protection of myocardial apoptosis and inflammation. CLP rats treated with Dex in combination with MLA, an antagonist of α7 nicotinic acetylcholine receptor (α7nAChR), exhibited decreased autophagy and increased inflammation and cell death, identifying α7nAchR was involved in the Dex-mediated pathway. In addition, we found that the PI3K/Akt pathway is involved in Dex-mediated autophagy and convergent with α7nAChR-mediated stimulation of autophagy response. Conclusions and implicationsFor the first time, these data indicate that autophagy is central in Dex-mediated cardio-protection in sepsis. These observations provide the foundation for further study, and may serve as the basis for innovative therapeutic strategies against septic myocardial dysfunction.

Protective effects of PNU‑282987 on sepsis‑induced acute lung injury in mice.

The cholinergic anti‑inflammatory pathway is considered an attractive approach for the alleviation of inflammatory diseases. Sepsis is characterized by systemic inflammation and widespread organ injury, especially that in the lung. In the present study, we explored the effects of an α7nAChR agonist, PNU‑282987, on sepsis‑induced lung injury and investigated the mechanisms of PNU‑282987 in response to lipopolysaccharide (LPS) stimulation in peritoneal macrophages. Sepsis was induced in C57BL/6 mice via cecal ligation puncture (CLP). Fifty mice were randomly divided into five groups: The sham group treated with vehicle, the sham group treated with PNU‑282987, the CLP group treated with vehicle, and the CLP group treated with PNU‑282987 (1mg/kg) 1h before or 2h after surgery. All mice were sacrificed at 12 or 24h after CLP. Both pre‑ and post‑CLP treatment with PNU‑282987 significantly attenuated sepsis‑induced lung injury and the release of IL‑6 in the bronchoalveolar lavage fluid (BALF). Pre‑treatment with PNU‑282987 also inhibited sepsis‑increased TNF‑α and IL‑6 production, while post‑CLP treatment only inhibited IL‑6 production in the lung tissue. Neither pre‑ nor post‑CLP treatment with PNU‑282987 affected IL‑6 release in the serum. Furthermore, pretreatment with PNU‑282987 resulted in reductions in TNF‑α and IL‑6 release in a dose‑ and time‑dependent manner and decreased the phosphorylation levels of p38, JNK and ERK under LPS conditions in peritoneal macrophages. Our results demonstrate that activation of α7nAChR alleviates sepsis‑induced lung injury; this effect is associated with the suppression of inflammatory responses via the MAPK pathway, suggesting that α7nAChR is a potential therapeutic target for the treatment of sepsis.

Development of cardiac dysfunction in sepsis‐surviving rats

ObjectivesSepsis is a life‐threatening syndrome induced by an infection and is associated with physiological, pathological and biochemical systemic changes. It is one of the major causes of death in critical care units and a public health concern around the world. A particular occurrence in sepsis is the development of cardiovascular dysfunction. Notably, cardiovascular diseases have been described as an important risk factor among those who survived sepsis (ANNANE et al., 2007; YENDE et al., 2014). We have hypothesized that depressed cardiac function is one of the long‐lasting effects in sepsis.Material and methodsTo investigate our hypothesis, we evaluated the cardiac function from male Wistar rats subjected to the cecal ligation puncture (CLP) model at 6, 24, 72 h, and 30 days after the surgery. Age matched sham‐operated animals were used as control. For this, the animals were maintained under anesthesia and a pressure‐volume catheter was inserted into the left ventricle chamber through the carotid artery. This approach allowed the assess of thirteen volume dependent cardiac parameters: cardiac output, CO; heart rate, HR; stroke volume, SV; end diastolic volume, EDV; end diastolic pressure, EDP; ventricular stroke work, SW; the rate of ventricle pressure rise and decrease, dP/dtmax and dP/dtmin, respectively; the pressure at dP/dtmax, P@dP/dtmax, relaxation time constant, TAU; ejection fraction, EF; the rate of ventricle volume rise, dV/dtmax; and end systolic volume; ESV. These parameters were analyzed before and after intravenous injection of 30 nmol/Kg dobutamine.ResultsAt 6 h and 24 h after the CLP surgery, the CO, EF, and SW were significantly reduced in the CLP groups when compared with control animals. For instance, the CO, EF, and SW were reduced from 30.4 ± 1.9 mL/min, 43.6 ± 2.2%, and 6097 ± 469 mmHg*μl in sham‐operated animals to 12.4 ± 1.2 mL/min, 24.4 ± 3.1%, and 2561 ± 597 mmHg*μl in the CLP 24 h group, respectively. Importantly, as measured under basal condition, all cardiac parameters at 72 h after the CLP were back to levels found in sham‐operated animals or even increased (i.e. both dV/dtmax and SW were around 70 and 57% higher in the CLP group). Nevertheless, when evaluated at 30 days after the CLP, the sepsis‐surviving animals presented decreased CO (20.85 ± 5.01 mL/min), EF (41.3 ± 4.8%), SW (6481 ± 1252 mmHg*μl), and EDV (169.3 ± 7.7 μl), compared with control animals (38.4 ± 4.1 mL/min, 57.8 ± 2.3%, 10551 ± 725 mmHg*μl, and 226.6 ± 12.0 μl, respectively). On the other hand, both EDP and Tau were increased in sepsis‐surviving rats around 57 and 70% respectively, compared with control animals. In addition, dobutamine, an inotropic adrenergic agent, had its effect on intraventricular pressure of sepsis‐surviving animals reduced aroud 58% compared with control animals.DiscussionAltogether these data indicate that cardiac injury, characterized by both diastolic and systolic dysfunction is present in early (6 and 24 h), but not late (72 h) sepsis, and reappeared in the sepsis‐surviving rats at 30 days after the CLP surgery. The translation of our results for humans may allow the development of new strategies for the management of cardiac function in those who survived the septic insult.Support or Funding InformationFAPESC, SC, Brazil (TR2012000367) and CNPq, Brazil (448738/2014)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cyclosporine A attenuates cardiac dysfunction induced by sepsis via inhibiting calcineurin and activating AMPK signaling.

The aim of the present study was to investigate whether cyclosporine A (CSA) improved cardiac dysfunction at an early stage of sepsis. Male Wistar rats were randomly divided into the following three groups: the sham‑operated control group, the cecal ligation puncture (CLP) procedure‑induced sepsis group and the CSA intervention group. Cecal ligation was performed to generate a sepsis model. At different time points (2, 6, 12, 24 and 72h) following sepsis induction, blood pressure, cardiac function, and non‑esterified free fatty acid (NEFA) levels in the plasma and myocardia were measured, and the expression levels of components associated with the AMP‑activated protein kinase (AMPK)‑acetyl CoA carboxylase (ACC)‑carnitine palmitoyl transferase 1 (CPT1) signaling pathway were compared among the three groups. Sepsis induced a decrease in blood pressure and cardiac function at 24h following sepsis induction in the CLP group, and CSA treatment ameliorated these pathophysiological alterations. In addition, rats in the CLP group exhibited significant increases in calcineurin activity and NEFA accumulation in the heart when compared with those in the sham group. These effects were attenuated by CSA treatment. Mechanistically, the activity of the AMPK‑ACC‑CPT1 pathway was enhanced by CSA treatment. The present study revealed that CSA treatment increases cardiac function at an early stage of sepsis in rats. This treatment partially suppresses calcineurin activity while activating the AMPK‑TCC‑CPT1 pathway.

Regulation of Sirtuin 3-Mediated Deacetylation of Cyclophilin D Attenuated Cognitive Dysfunction Induced by Sepsis-Associated Encephalopathy in Mice

The present study aimed to investigate cognitive dysfunction in the hippocampus induced by sepsis-associated encephalopathy (SAE) via acetylation of cyclophilin D (CypD) and opening of mitochondrial permeability transition pore. It also explored whether activating sirtuin 3 (SIRT3) can mediate deacetylation of CypD and prevent the development of SAE. Male mice were randomly assigned to six groups: sham group, cecal ligation puncture group, CypD siRNA transfection (CypD-si) group, CypD control siRNA transfection (CypD-c) group, SIRT3 overexpression vector pcDNA3.1 (SIRT3-p) group, and SIRT3 empty vector pcDNA3.1 (SIRT3-v) group (n = 18). The CypD-si and CypD-c groups were transfected with CypD siRNA and CypD control siRNA, respectively. The SIRT3-p and SIRT3-v groups were injected with SIRT3 pcDNA3.1 and vector pcDNA3.1, respectively. The learning and memory function was assessed using the learning version of the Morris water maze test. Then, cell apoptosis and the levels of CypD, acetylated CypD, SIRT-3, interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and caspase-3 in the hippocampus were determined. The levels of CypD and acetylation of CypD increased in the hippocampus induced by SAE. Increasing SIRT3 and decreasing CypD can attenuate cognitive impairment and neuroapoptosis, and protect the integrity of mitochondrial membrane from damage and restore the protein expressions of IL-6, TNF-α, and caspase-3. Activating SIRT3-mediated deacetylation of CypD attenuated learning and memory dysfunction induced by SAE.

Rosiglitazone protects acute kidney injury in septic rats

To investigate the effects of rosiglitazone on the structure of the kidney of rats in sepsis with acute kidney injury. Sepsis models were established using male Sprague-Dawley rats by cecal ligation puncture (CLP). 96 healthy rats were randomly divided into 4 groups: cecal ligation puncture and rosiglitazone group (group CLP+ ROSI), cecal ligation puncture group (group CLP), Sham and rosiglitazone group (group Sham+ ROSI), sham group (group Sham). Six rats were sacrificed at 0, 6, 12, 24 h after CLP respectively in each group. The general status of rats was observed and H&E staining was applied to detect renal pathological histology changes. Myeloperoxidase(MPO) was measured in kidney tissues. Expression of the ligand of peroxisome proliferators-activated receptor γ (PPARγ), and B-cell lymphoma-2(Bcl-2) were also determined by Western blot analysis. The increased MPO activity of group CLP+ ROSI was significantly lower than that of group CLP at 12 h and 24 h[(4.07±0.16)vs(4.62±0.08); P12=0.01, P24=0.006]. The MPO activity of group CLP was significantly higher than that of group Sham at 6, 12 and 24 h[(4.62±0.08)vs(1.30±0.06); P6=0.00, P12=0.00, P24=0.00]. The MPO activity of group CLP+ ROSI was significantly higher than that of group Sham+ ROSI at 6, 12 and 24 h[(4.07±0.16)vs(1.27±0.06); P6=0.00, P12=0.00, P24=0.00]. Compared with group Sham and group Sham+ ROSI, in rats of group CLP and group CLP+ ROSI the symptoms of sepsis and organ dysfunction were observed. The symptoms of sepsis and organ dysfunction of group CLP+ ROSI were significantly reduced than that of group CLP. Expression of PPARγ and Bcl-2 were determined by Western blot analysis, the expression of PPARγ and Bcl-2 of group CLP+ ROSI was significantly lower than that of group CLP. Rosiglitazone, as the ligand of PPARγ, reduced the inflammatory response in sepsis, decreased the apoptosis of kidney cell, reduced kidney injury, and protected acute kidney injury in rats of sepsis.

The Potential Role of Hemopexin and Heme Oxygenase-1 Inducer in a Model of Sepsis

Background and Aims. Sepsis can evoke disseminated intravascular coagulation, resulting in multiple organ failure and death. Heme oxygenase-1 (HO-1) and hemopexin (HPx) can mediate cytoprotective mechanisms against these deleterious effects. This study aims to determine a role for HO-1 and HPx in coagulopathy induced by septic inflammation and define whether they can enhance the production of anti-inflammatory cytokine IL-10. Materials and Methods. 48 healthy male albino rats were divided equally into 4 groups: control group: animals subjected to laparotomy and bowel manipulation; CLP group: severe sepsis induced by cecal ligation puncture (CLP); CLP + hemin group: animals received single intraperitoneal injection of hemin (50 µmol/kg) 12 h before sepsis induction; CLP + HPx group: animals received single HPx dose (150 µg/rat, i.v.) 30 min before sepsis induction. Survival rates were calculated. Prothrombin time (PT), activated partial thromboplastin time (APTT), and activated protein C (APC), liver HO-1, serum, and liver IL-10 levels were measured, 48 hrs after sepsis induction. Liver and lung were excised for histopathological examination. Results. Hemin and HPx administration upregulated liver HO-1 and IL-10. They prolonged PT, PTT and increased APC. They reduced the inflammatory infiltrate and thrombosis in liver and lung parenchyma. However, hemin was superior in controlling coagulopathy and HO-1 production, while HPx was more potent stimulant of IL-10 expression. Conclusions. Hemin and HPx have a potential beneficial effect in severe sepsis regarding coagulopathy and inflammation.

Effects of rosiglitazone on levels of TNFα in septic rats with acute kidney injury

To investigate the influence of rosiglitazone on tumor necrosis factor-α (TNFα) of rats in sepsis with acute kidney injury. Sepsis models were established using male Sprague-Dawley rats by cecal ligation puncture (CLP). Ninety six healthy rats were randomly divided into 4 groups: cecal ligation puncture and rosiglitazone group (group CLP+ROSI), cecal ligation puncture group (group CLP), Sham and rosiglitazone group (group Sham+ROSI), Sham group (group Sham). Blood samples of 6 rats in each group were collected at postoperative 0, 6, 12, 24 h, to detect TNFα levels in serum. Observe and compare the mobility of rats in each group and the level of serum creatinine and H&E staining was applied to detect renal pathological histology changes. Statistics was analyzed by SPSS 13.0 software application of independent samples t test. The creatinine level of Group CLP was increased with the extension of time, the creatinine level of group CLP was significantly higher than that of group Sham at 12 h and 24 h (P(12) = 0.047, P(24) = 0.001). The creatinine level of group CLP+ROSI was significantly higher than that of group Sham+ROSI at 12 h and 24 h (P(12) = 0.033, P(24) = 0.039). The increased creatinine level of group CLP +ROSI was significantly lower than that of group CLP at 12 h and 24 h (P(12) = 0.04, P(24) = 0.006). The TNFα concentration of group CLP was significantly higher than that of group Sham at 6 h, 12 h and 24 h (P < 0.05). The TNFα concentration of group CLP+ROSI was significantly higher than that of group Sham+ROSI at 6 h, 12 h and 24 h (P < 0.05). The increased TNFα concentration of group CLP+ROSI was significantly lower than that of group CLP at 6 h, 12 h and 24 h (P(6) = 0.01, P12 = 0.02, P(24) = 0.01). Compared with group Sham and group Sham+ROSI, in rats of group CLP and group CLP+ROSI the symptoms of sepsis and organ dysfunction were observed. The symptoms of sepsis and organ dysfunction of group CLP+ROSI were significantly reduced than that of group CLP. Rosiglitazone can inhibit the expression of TNFα in serum of septic rats, reduce the inflammatory reaction, reduce kidney injury, improve the renal function. Rosiglitazone on septic rats with acute kidney injury has a protective effect.

Effects of rosiglitazone on pulmonary injury of septic rats induced by cecal ligation puncture

Objective To observe the effects of rosiglitazone,an agonist of peroxisome proliferator-activated receptor γ (PPARγ),on pulmonary injury in septic rats induced by cecal ligation puncture(CLP).Methods Eighty healthy male Wistar rats were randomly divided into 5 groups:sham group (group SHAM),cecal ligation puncture group (group CLP).rosiglitazone group (group ROSI),in which rats were intravenous injected with rosiglitazone 0.3 mg/kg 30 min before CLP.GW9662 group (group GW),in which rats were intravenous injected with GW9662 0.3 mg/kg 30 m in before CLP.GW9662 plus rosiglitazone group (group GW+ROSI),in which rats were injected intravenously with GW9662 0.3 mg/kg 30 min before CLP and then with rosiglitazone 0.3mg/kg 15 min before CLP.Eight rats were sacrificed at 6 and 24 h after CLP respectively in each group.Plasma was collected for the measurement of the level of plasma tumor necrosis factor-α (TNF-α) by enzyme linked immunosorbent assay (ELISA) and lung tissues were harvested for the detections of lung wet/dry (W/D) ratio,lung myeloperoxidase (MPO) activity,and lung tissue high mobility group protein B1 (HMGB1) mRNA expression by real-time PCR.Results The lung W/D ratio,MPO activity,plasma TNF-α level and the lung tissue HMGB1 mRNA expression of group CLP,group ROSI,group GW and group GW+ROSI were significantly higher than that of group SHAM.The lung W/D ratio,MPO activity,plasma TNF-α level and the lung tissue HMGB1 mRNA expression of group CLP [(5.31±0.14),(3.15±0.47) U/g,(15.064±0.448) μg/L and (0.758± 0.130)],group ROSI [(4.33±0.21),(2.21±0.31) U/g,(3.334±0.212) μg/L and (0.422±0.124)],group GW[(4.93±0.14),(3.13± 0.28) U/g,(15.001±0.343)μg/L and (0.732±0.198)] and group GW+ROSI[(4.87±0.13),(3.08±0.35) U/g,(14.866±0.412) μg/L and (0.763±0.017)] at 6 hafter CLP were significantly higher than that of the group SHAM[(3.98±0.17),(0.67±0.43) U/g,(0.452±0.033) μg/L and (0.244±0.053)](P＜0.05).At 24 h after CLP,the lung W/D ratio,MPO activity,plasma TNF-α level and the lung tissue HMGB1 mRNA expression of group CLP[(10.23±0.19),(3.61±-0.82) U/g,(1.542±0.193) μg/L and (2.413±0.211)],group ROS[(6.39±0.18),(2.17±1.02) U/g,(0.947±0.281) μg/L and (1.517±0.076)],group GW[(10.08±0.26),(3.45±0.36) U/g,(1.513±0.210).μg/L and (2.385±0.323)] and group GW+ROSI[(10.15±0.18),(3.53±0.32) U/g,(1.524±0.180) μg/L and (2.404±0.135)] were significantly higher than that of the group SHAM[(4.02±0.20),(0.68±0.34) U/g,(0.429±0.021) μg/L and (0.232±0.032)] (P＜0.05).The lung W/D ratio,MPO activity,plasma TNF-α level and the lung tissue HMGB1 mRNA expression of group ROSI were significantly lower than that of group CLP,group GW and group GW+ROSI respectively (P＜0.05).Conclusions Rosiglitazone can decrease the plasma TNF-α level and the lung tissue HMGB1 mRNA expression level and attenuate the pulmonary edema and inflammation in septic rats induced by CLP.It may have protective function against pulmonary injury. Key words: Sepsis; Pulmonary injury; Rosiglitazone