Cecal Ligation And Perforation Group Research Articles

Revealing the role of gut microbiota in immune regulation and organ damage in sepsis using 16s rRNA and untargeted metabolomics

To analyze the composition and metabolites of gut microbiota in septic rats by fecal 16s rRNA sequencing and untargeted metabolomics, and to preliminarily explore the effect and potential mechanism of gut microbiota and its metabolites on inflammatory response and multiple organ damage in sepsis. Ten males healthy male Wistar rats were randomly divided into a sham operated group (Sham group) and sepsis model group (CLP group) using a random number table method, with 5 rats in each group. A rat sepsis model was established by cecal ligation and perforation (CLP) method. The animals were sacrificed 24 hours after modeling, the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung and kidney tissues, and the pathological scores were evaluated. Fecal samples were collected, and 16s rRNA high-throughput sequencing and non-targeted metabolomics were used to screen microbiota, metabolites and potential signal pathways that may play an important role in disease outcomes. Spearman correlation analysis was conducted to jointly analyze the gut microbiota and non-targeted metabolism. Compared with the Sham group, the degree of pathological damage to lung and kidney tissues in the CLP group was significantly increased (lung tissue score: 3.60±0.80 vs. 0.00±0.00, kidney tissue score: 2.40±0.80 vs. 0.00±0.00, both P < 0.01), the level of IL-6 and TNF-α in peripheral blood significantly increased [TNF-α (ng/L): 248.12±55.98 vs. 143.28±36.57, IL-6 (ng/L): 260.26±39.47 vs. 116.01±26.43, both P < 0.05], the species diversity of intestinal flora of rats in the CLP group was significantly reduced, the relative abundance of Morganella, Bacteroides and Escherichia-Shigella were significantly increased, and the relative abundance of Lachnospiraceae NK4A136, Ruminococcus, Romboutsia and Roseburia were significantly reduced. In addition, the biosynthesis and bile secretion of phenylalanine, tyrosine, and tryptophan in the gut microbiota of the CLP group were significantly increased, while the biosynthesis of secondary bile acids was significantly reduced. There was a significant correlation between differential metabolites and differential microbiota. Sepsis can cause significant changes in the characteristics of gut microbiota and fecal metabolites in rats, which provides a basis for translational research to seek new targets for the treatment of sepsis.

Positive effects of Xuebijing injection on intestinal microbiota and metabolite spectrum in septic rats

To explore the effect of Xuebijing injection on inflammation in sepsis by regulating intestinal microbiota and its metabolites. A total of 45 male Sprague-Dawley (SD) rats were randomly divided into Sham operation group (Sham group), cecal ligation and perforation (CLP) induced sepsis group (CLP group), and Xuebijing intervention group (XBJ group, 4 mL/kg Xuebijing injection was injected intraperitoneally at 1 hour after CLP), with 15 rats in each group. The survival of rats was observed at 24 hours after operation and sacrificed. Feces were collected for 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS) analysis. At 24 hours after operation, all rats in the Sham group survived, the mortality of rats in the XBJ group was lower than that in the CLP group [47% (7/15) vs. 60% (9/15), P > 0.05]. Compared with the Sham group, the diversity of gut microbiota in the CLP group decreased, the dominant flora changed, and the abundance of inflammation-related flora increased. Xuebijing improved the changes in gut microbiota caused by sepsis, and α diversity showed an increasing trend (Ace index: 406.0±22.5 vs. 363.2±38.2, Chao1 index: 409.7±21.8 vs. 362.4±42.5, both P > 0.05). Restrictive constrained principal coordinate analysis (cPCoA) showed a high similarity in gut microbiota among the same group of rats. The CLP group was dominated by Bacteroidetes, while the Sham and XBJ groups were dominated by Firmicutes. In addition, compared with the CLP group, Xuebijing treatment increased the abundance of beneficial bacteria in septic rats, such as Verrucomicrobia, Akkermansia and Lactobacillus. LC-MS and orthogonal partial least squares discriminant analysis (OPLS-DA) showed that there were 12 main differential metabolites among the three groups, and there were certain correlations between these metabolites, which were related to amino acid and lipid metabolism. Correlation analysis showed a significant correlation between changes in metabolites and microbial communities. Xuebijing can improve the survival rate of septic rats, regulate the composition of intestinal flora and related metabolites, which provides a new pathophysiological mechanism for Xuebijing in the treatment of sepsis.

Effect of Liangxue Huoxue decoction on intestinal flora and NLRP3/caspase-1/GSDMD signaling pathway in mice model of sepsis-induced acute kidney injury

To investigate the effect of Liangxue Huoxue decoction on intestinal flora, intestinal barrier and NOD-like receptor protein 3 (NLRP3)/caspase-1/gasdermin D (GSDMD) pyroptosis signaling pathway in mice model of sepsis-induced acute kidney injury (AKI). The model of AKI was established by cecal ligation and perforation (CLP). Thirty male C57BL/6 mice were randomly divided into sham operation group (Sham group), sepsis group (CLP group) and sepsis+Liangxue Huoxue decoction (CLP+LXHX group), with 10 mice in each group. Mice in Sham group only underwent laparotomy. Two hours before model establishment, mice in CLP+LXHX group were treated with Liangxue Huoxue decoction (6 g/kg) by gavage; mice in Sham group and CLP group were given equal volume of normal saline by gavages. After 24 hours of modeling, all mice were sacrificed under anesthesia, and the colon and kidney tissues and fresh feces in the colon were taken. The pathological changes of kidney and colon were observed by hematoxylin-eosin (HE) staining under light microscope. Real-time polymerase chain reaction (RT-PCR) was used to detect inflammatory factors (interleukins, IL-1β and IL-18) in renal tissue. The expressions of NLRP3, caspase-1 and GSDMD were detected by Western blotting. The changes of intestinal flora in mice were detected by 16S rDNA high-throughput sequencing. Compared with the Sham group, the inflammatory cell infiltration of the kidney tissue was increased and the kidney became vacuolated in CLP group, the mRNA expressions of IL-1β, IL-18, and the protein expressions of NLRP3, caspase-1 and GSDMD were significantly increased in CLP group, the species richness of intestinal microflora decreased significantly, the relative abundance of Enterococcus and Escherichia-Shigella increased significantly, and the relative abundance of Ileibacterium, Alloprevotella, Lachnospiraceae, Klebsiella and Parasutterella increased significantly in CLP group. Compared with CLP group, Liangxue Huoxue decoction can significantly reduce the pathological changes of kidney and colon tissue, reduce the pathological score (1.75±0.43 vs. 3.50±0.50 for kidney tissue, 1.25±0.43 vs. 4.50±0.50 for colon tissue, both P < 0.05), improve the composition of intestinal flora, reduce the relative abundance of Enterococcus and Escherichia-Shigella, and significantly increase the relative abundance of Lactobacillus and Akkermansia. In addition, Liangxue Huoxue decoction can significantly reduce mRNA expressions of IL-1β and IL-18 in kidney tissue [IL-1β mRNA (2-ΔΔCt): 1.59±0.05 vs. 4.61±0.88, IL-18 mRNA (2-ΔΔCt): 1.69±0.17 vs. 2.86±0.63, both P < 0.05] and the protein expressions of NLRP3, caspase-1 and GSDMD (NLRP3/GAPDH: 0.71±0.04 vs. 0.89±0.01, caspase-1/GAPDH: 1.04±0.04 vs. 1.48±0.04, GSDMD/GAPDH: 0.90±0.01 vs. 1.41±0.02, all P < 0.05). Liangxue Huoxue decoction has obvious protective effect on AKI induced by sepsis. It can improve intestinal barrier by regulating intestinal flora, thereby inhibiting the activation of NLRP3/caspase-1/GSDMD signaling pathway in kidney tissue and reducing the expression of proptosis-related inflammatory factors.

A MODIFIED SURGICAL SEPSIS MODEL SATISFYING SEPSIS-3 AND HAVING HIGH CONSISTENCY OF MORTALITY.

Background : Cecal ligation and perforation (CLP) is currently considered the criterion standard model of sepsis; however, there are some deficiencies, such as low clinical relevance, inconsistency in severity grading, and an unknown proportion of CLP animals meeting the requirements of sepsis-3. Methods : Adult rats were randomly divided into the following three groups: modified CLP (M-CLP) group, CLP group, and sham group. The vital organ function of rats was evaluated 24 hours postoperatively by blood pressure, behavioral testing, histopathology, and blood test. Cytokine levels were determined by enzyme-linked immunosorbent assay, and T-cell suppression was assessed by flow cytometry. The stability of the model was evaluated by comparing the survival rates of repeated experiments in all groups from day 1 to day 14. Results : More rats in the M-CLP group met Sepsis-3 criteria than those in the CLP group 24 hours postoperatively (53.1% vs. 21.9%, P = 0.01). Rats in the M-CLP group developed more serious hepatic, pulmonary, and renal dysfunction. Similar to human sepsis, rats in the M-CLP group demonstrated more serious immunosuppression and systemic inflammation compared with the CLP group. In addition, disease development and severity, which was indicated by the stable survival rates of model animals, were more stable in the M-CLP group. Conclusions : More rats could meet Sepsis-3 criteria with this novel surgical procedure, which may reduce the number of animals needed in preclinical sepsis experiments. This stable M-CLP model may contribute to the development of new therapies.

Acetaldehyde dehydrogenase 2 activation attenuates sepsis-induced brain injury through NLRP3 inflammasome regulation

ObjectiveAcetaldehyde dehydrogenase 2 (ALDH2) plays an important part in neuroprotection; however, its effect on sepsis-induced brain injury is nuclear. Our aim is to investigate the potential effect and mechanism of ALDH2 in this condition. MethodsWe established an animal model using cecal ligation and perforation (CLP). Twenty-four rats were divided into sham group (n = 6), CLP group (n = 6), CLP + Alda-1 group (n = 6) and CLP + Cyanamide (CYA) group (n = 6). Vital signs were monitored, and arterial blood gas analysis, hippocampal histological staining and ALDH2 activity analysis were conducted. Western blot analysis and enzyme-linked immunosorbent assays were also carried out. Lipopolysaccharide (LPS)-treated HT22 cells were employed as an in vitro model of sepsis-induced brain injury, with and without pretreatment with Alda-1 or CYA, to further examine the potential mechanisms. Real-time quantitative polymerase chain reaction and western blot were used to determine the levels of pyrin domain-containing 3 (NLRP3) inflammasome. ResultsWe found hippocampal cell injury in the CLP group (p < 0.05), with decreased ALDH2 activity (p < 0.05) and suspected overexpression of NLRP3/caspase-1 axis (p < 0.05). In the group pretreated with Alda-1, there were increased ALDH2 activity (p < 0.05), decreased hippocampal cell damage (p < 0.05), and reduced protein levels of NLRP3, apoptosis-associated speck like protein containing a caspase recruitment domain (ASC), cleaved caspase-1 and Gasdermin D (GSDMD) (p < 0.05). The levels of interleukin 18 (IL-18) and interleukin 1β (IL-1β) were also reduced (p < 0.05). In the group pretreated with CYA, ALDH2 activity was further declined, the cell injury grade increased, and the elevated levels of pyroptosis-related proteins aggravated (p < 0.05). LPS treatment decreased the cell viability and ALDH2 activity of the HT22 cells (p < 0.05), along with increased mRNA levels of the NLRP3 inflammasome, as well as IL-1β and IL-18 (p < 0.05). Western blot further revealed elevated levels of NLRP3, ASC, cleaved caspase-1 and GSDMD (p < 0.05). In the LPS+Alda-1 group, there were increased cell viability (p < 0.05), elevated ALDH2 activity (p < 0.05), and reduced levels of NLRP3 inflammasome and pyroptosis-related proteins (p < 0.05). In the CYA+LPS group, cell viability and ALDH2 activity were further declined (p < 0.05), while levels of NLRP3 /caspase-1 axis were increased (p < 0.05). ConclusionsThe activation of ALDH2 can attenuate sepsis-induced brain injury, hypothetically through regulation of the NLRP3/caspase-1 signaling pathway. Therefore, ALDH2 could potentially be considered as a new therapeutic target for the treatment of sepsis-induced brain injury.

Effects of adenosine N1-Oxide and pioglitazone on inflammatory and antioxidant state in sepsis caused by experimental cecal puncture in rat.

Sepsis is an acute condition caused by the systemic inflammatory response syndrome to an infection. There are very few drugs that could improve the severe conditions in patients with sepsis. Hence, it is important to consider different treatment options. In this survey, we studied the effect of adenosine N1-oxide (ANO) and pioglitazone on rats with cecal ligation and perforation (CLP). They were randomly divided to four groups (n = 10) including Group A: as control group receiving normal saline, Group B: the rats with CLP as surgical control group, Group C: the rats receiving ANO, and Group D: the rats receiving pioglitazone. Interleukin (IL) -6, IL-1β, tumor necrosis factor alpha (TNF-α), nitric-oxide (NO) in serum blood and superoxide dismutase (SOD), catalase (CAT) malondialdehyde, (MDA) and myeloperoxidase (MPO) in liver and spleen homogenates were measured. The amount of antioxidant enzymes in the spleen and liver, and finally cell viability and rats' survival were investigated. The measurement of blood serum nitric-oxide and survival of all groups of rats were also performed. The results indicated that both drugs could cause a decrease in IL-1β, IL-6, TNF-α and NO in rat blood serum and MDA and MPO in the liver and spleen homogenates, however, a significant increase in SOD and CAT in the liver and spleen homogenates in rats that received ANO and pioglitazone was observed compared to rats with CLP group. Cell viability and rats' survival were significantly improved in rats that received ANO and pioglitazone compared to rats with CLP group. Adenosine N1-oxide showed stronger and more effective effects.

Irrigation of peritoneal cavity with cold atmospheric plasma treated solution effectively reduces microbial load in rat acute peritonitis model

Accurate and timely diagnosis of appendicitis in children can be challenging, which leads to delayed admittance or misdiagnosis that may cause perforation. Surgical management involves the elimination of the focus (appendectomy) and the reduction of the contamination with peritoneal irrigation to prevent sepsis. However, the validity of conventional irrigation methods is being debated, and novel methods are needed. In the present study, the use of cold plasma treated saline solution as an intraperitoneal irrigation solution for the management of acute peritonitis was investigated. Chemical and in vitro microbiological assessments of the plasma-treated solution were performed to determine the appropriate plasma treatment time to be used in in-vivo experiments. To induce acute peritonitis in rats, the cecal ligation and perforation (CLP) model was used. Sixty rats were divided into six groups, namely, sham operation, plasma irrigation, CLP, dry cleaning after CLP, saline irrigation after CLP, and plasma-treated saline irrigation after CLP group. The total antioxidant and oxidant status, oxidative stress index, microbiological, and pathological evaluations were performed. Findings indicated that plasma-treated saline contains reactive species, and irrigation with plasma-treated saline can effectively inactivate intraperitoneal contamination and prevent sepsis with no short-term local and/or systemic toxicity.

Lentinan Attenuates Damage of the Small Intestinal Mucosa, Liver, and Lung in Mice with Gut-Origin Sepsis.

This study is aimed at exploring the effects of lentinan on small intestinal mucosa as well as lung and liver injury in mice with gut-origin sepsis. Cecal ligation and perforation (CLP) were used to construct a mouse model of gut-origin sepsis. The mice were randomly divided into six groups: sham operation group (sham), gut-origin sepsis model group (CLP), ulinastatin-positive drug control group (UTI), lentinan low concentration group (LTN-L, 5 mg/kg), lentinan medium concentration group (LTN-M, 10 mg/kg), and lentinan high concentration group (LTN-H, 20 mg/kg). H&E staining was used to detect the pathological damage of the small intestine, liver, and lung. The serum of mice in each group was collected to detect the expression changes of inflammatory cytokines, oxidative stress biomarkers, and liver function indexes. In vitro assessment of bacterial translocation was achieved through inoculated culture media. Western blot and RT-qPCR were used to detect the expression of molecules related to the NF-κB signaling pathway in the small intestine tissues of mice. The results showed that compared with the CLP group, the injury degree of the small intestine, liver, and lung in mice with gut-origin sepsis was improved with the increase of lentinan concentration. In addition, TNF-α, IL-1β, IL-6, and HMGB1 were decreased with the increase of lentinan concentration, but the expression of IL-10 was increased. Lentinan could also reduce the expression of oxidative stress injury indexes and liver function indexes and inhibit bacterial translocation to liver and lung tissues. Further mechanism investigation revealed that lentinan downregulated the expression of the NF-κB signaling pathway molecules (NF-κB, TLR4, and Bax) and upregulated the expression of occludin and Bcl-2. In conclusion, lentinan inhibits the activity of the NF-κB signaling pathway, thus attenuating injuries of small intestinal mucosa and liver and lung in mice with gut-origin sepsis and reducing the inflammatory response in the process of sepsis.

Protective effect and mechanism of Baicalin on acute kidney injury in septic mice

To observe the protective effect and mechanism of different doses of Baicalin (BAI) on acute kidney injury (AKI) in septic mice. According to the random number table, 100 mice were divided into sham operation group (Sham group), cecal ligation and perforation (CLP) induced sepsis model group (CLP group) and BAI pretreatment groups. The mice in BAI pretreatment groups were divided into low-, medium- and high-dose groups (BAI-L+CLP, BAI-M+CLP, BAI-H+CLP groups), with 20 mice in each group. A murine sepsis associated-acute kidney injury (SA-AKI) model was reproduced using CLP. The mice in the Sham group were only opened and closed the abdomen, without ligating or perforating the cecum. The mice in the BAI pretreatment groups were given BAI 25, 50 and 100 mg/kg daily for 3 days, and CLP was performed at 6 hours after administration of BAI at the 3rd day to reproduce sepsis model. The mice in the Sham group and CLP group were given the same amount of distilled water as control. Ten mice were sacrificed at 24 hours after operation to collect orbital blood for renal function determination [serum creatinine (SCr), blood urea nitrogen (BUN), plasma neutrophil gelatinase-associated lipocalin (pNGAL) and plasma kidney injury molecule-1 (pKIM-1)] by enzyme linked immunosorbent assay (ELISA). The kidney tissue was collected to observe the kidney tissue injury under light microscope after hematoxylin-eosin (HE) staining. The TdT-mediated dUTP nick-end labeling (TUNEL) was used to detect the apoptosis of renal tubular epithelial cells. Western blotting was used to detect the expression of cell FLICE like inhibitory protein (c-FLIP) in renal tissue. The remaining 10 mice in each group were used to calculate the survival rate of 7 days after operation. The renal tubular epithelial cells in the CLP group were massively degenerated with necrosis, the renal tubular lumen was significantly expanded, and inflammatory cells were widely infiltrated in the renal interstitium. Furthermore, the renal function deteriorated rapidly. Compared with the CLP group, the renal function of mice pretreated with low dose of BAI was improved, but the difference was not significant. Compared with the CLP group, the renal function in the mice pretreated with medium and high doses of BAI was significantly improved, the SCr, BUN, pNGAL and pKIM-1 were significantly reduced [SCr (μmol/L): 135.16±5.18, 125.70±5.26 vs. 170.42±5.42; BUN (mmol/L): 33.59±1.77, 27.29±1.61 vs. 45.68±1.39; pNGAL (μg/L): 91.29±4.68, 73.40±3.77 vs. 131.50±6.55; pKIM-1 (μg/L): 6.34±0.30, 5.51±0.35 vs. 8.03±0.29; all P < 0.01], the pathological injury of renal tissue was significantly decreased, the apoptotic number of renal tubular epithelial cells was significantly reduced (cells/HP: 16.20±0.49, 13.10±0.66 vs. 29.60±0.49, both P < 0.01), and the expression of c-FLIP protein in renal tissue was significantly increased [c-FLIP protein (c-FLIP/GAPDH): 0.35±0.02, 0.46±0.02 vs. 0.21±0.01, both P < 0.01]. No mouse in the Sham group died within 7 days. Compared with the CLP group, the average survival time of the mice within 7 days in the BAI-L+CLP, BAI-M+CLP and BAI-H+CLP groups was significantly prolonged with a dose-dependent manner (days: 3.5±2.5, 5.4±2.2, 5.9±1.9 vs. 2.1±1.2; Log-Rank test: χ2 = 73.410, P < 0.001). Pretreatment with medium and high doses of BAI can significantly improve the renal function in mice with SA-AKI, decrease the pathological damage and increase the survival of mice, and its mechanism may be related to promoting the increase of c-FLIP protein expression and inhibiting cell apoptosis.

Role and mechanisms of MLKL-NLRP3-mediated necroinflammation in mice with sepsis-induced acute lung injury

To investigate the roles and underlying mechanisms of mixed lineage kinase domain like (MLKL)-mediated inflammatory response induced by NOD-like receptor protein 3 (NLRP3) inflammatory corpuscles in the acute lung injury (ALI) after sepsis. Eighteen BALB/c mice were randomly divided into sham operation group (Sham group), cecal ligation and perforation (CLP)-induced sepsis model group (CLP group) and specific inhibitor Necrostatin-1 intervention group [CLP+Nec-1 group, Necrostatin-1 solution (20 mg/kg) was injected intravenously 10 minutes before modeling], with 6 mice in each group. The mice were sacrificed by neck amputation at the 2nd day after operation, and the serum and lung tissue samples were collected. The morphological changes of lung tissue were observed by hematoxylin-eosin (HE) staining. The water content of lung tissue was detected by dry-wet weight method. The pulmonary vascular permeability was measured by Evans blue (EB) staining. The protein expressions of MLKL and NLRP3 in the lung tissue were detected by Western blotting, and the level of serum interleukin-1β (IL-1β) was detected by enzyme linked immunosorbent assay (ELISA). HE staining showed that the lung morphological structure in Sham group was normal. In CLP group, congestion and edema in the alveolar cavity and interstitium, infiltration of neutrophils and thickening of alveolar wall were observed. The histopathological changes of lung tissue in CLP+Nec-1 group were better than those in CLP group. Compared with Sham group, the water content of lung tissue [(88.00±0.00)% vs. (78.00±0.01)%], pulmonary vascular permeability [EB content (mg/L): 11.82±1.15 vs. 4.00±0.71], the protein expressions of phosphorylated MLKL (p-MLKL) and NLRP3 in lung tissue (p-MLKL/GAPDH: 0.34±0.04 vs. 0.12±0.01,NLRP3/GAPDH: 0.47±0.07 vs. 0.16±0.04), and the level of serum IL-1β (ng/L: 183.56±9.61 vs. 44.14±6.95) in CLP group were all significantly increased (all P < 0.01). Compared with CLP group, the water content of lung tissue [(81.00±0.01)% vs. (88.00±0.00)%], pulmonary vascular permeability [EB content (mg/L): 7.90±0.00 vs. 11.82±1.15], protein expressions of p-MLKL and NLRP3 in lung tissue (p-MLKL/GAPDH: 0.13±0.03 vs. 0.34±0.04, NLRP3/GAPDH: 0.18±0.04 vs. 0.47±0.07), and the level of serum IL-1β (ng/L: 113.81±6.62 vs. 183.56±9.61) were all significantly decreased (all P < 0.01). MLKL-NLRP3-mediated necroinflammation was significantly up-regulatedin the lung tissue of septic mice, which could be attenuated by specific inhibitor Necrostatin-1.

Protective role of melatonin against testicular damage caused by polymicrobial sepsis in adult rats.

This study aimed to investigate the possible protective effects of melatonin (MEL) against the damage to testicular tissue in rats caused by polymicrobial sepsis as a result of cecal ligation and perforation (CLP). In this study, 21 male Wistar albino rats were used. The rats were randomly divided into three groups (n=7): Sham Control (Group 1), CLP (Group 2), and CLP + MEL (Group 3). Sepsis was created using the CLP method. MEL was administered intraperitoneally in two equal doses of 10 mg/kg at 30 min before and 6 h after perforation. Tissue sections taken from paraffin blocks were stained with hematoxylin and eosin (H and E) and examined histopathologically under a light microscope. Intracellular H2O2 and apoptosis evaluations were carried out using the flow cytometric method. Sepsis caused a significant reduction in all sperm parameters. There was a significant decrease in sperm density, motility and cell numbers with normal morphology (p<0.05). Intracellular H2O2 level and apoptotic cell percentages increased in sperm cells in the CLP group. MEL treatment was found to significantly reduce sperm abnormalities, testicular damage, intracellular H2O2 levels, and apoptosis. This study showed that melatonin administration could be a potential treatment option to reduce acute testicular tissue damage due to sepsis.

MicroRNA miR-126 attenuates brain injury in septic rats via NF-κB signaling pathway

ABSTRACT The purpose of this study was to investigate the impact and mechanism of microRNA miR-126 on brain injury induced by blood-brain barrier (BBB) damage in septic rats. We used cecal ligation and perforation (CLP) to create a rat model of sepsis. The experimental rats were randomly divided into Control group, CLP group, CLP + miR-NC group, CLP + miR-126 group and CLP + miR-126 + NF-κB pathway agonist (PMA) group. MiR-126 expressed in the brain tissue of CLP rats was down-regulated by qRT-PCR. Upregulation of miR-126 in CLP rats could improve brain injury and BBB marker protein level, reduce brain water content, Evans blue extravasation, inflammation, and excessive oxidative stress. This could also result in an inhibition of NF-κB signaling pathway activity. In conclusion, miR-126 overexpression can prevent brain injury caused by BBB damage via the inhibition of NF-κB signaling pathway activity.

Severe systemic inflammation promotes persistent brain metabolic abnormalities

AbstractBackgroundSevere systemic inflammation can spread to the central nervous system, promoting neuronal dysfunction. Individuals who survive to a severe inflammatory episode, have greater chances to develop cognitive impairment. In this context, brain inflammatory changes have been considered a risk factor for Alzheimer’s disease (AD). We previously demonstrated, a significant [18F]FDG hypometabolism 24 h after the induction of severe systemic inflammation by cecal ligation and perforation (CLP), a sepsis model. However, whether severe systemic inflammation causes long‐term effects on brain energy metabolism remains unclear. Here, we aim at Investigating persistent consequences of a severe systemic inflammatory episode on brain energy metabolism. We hypothesized that brain energetic metabolism does not completely recover from a systemic inflammatory episode, which could be a trigger for neurodegeneration.MethodWistar rats (90 days old) were submitted to CLP and, examined in vivo via Micro‐PET [18F]FDG imaging 30 days later. Albumin and cellularity in the cerebrospinal fluid (CSF) were assessed by HPLC and flow cytometry, respectively, as an index of blood brain barrier (BBB) integrity. HPLC was also used for evaluating glutamate levels in CSF and glutamate uptake was evaluated in hippocampal and cortical slices using a radioactive assay.ResultWe found a persistent brain [18F]FDG hypometabolism in the CLP group. A 4‐fold increase in albumin levels was observed, along with a prominent increase in cellularity in the CSF of CLP animals. Additionally, decreased glutamate uptake in cortex (32%) and hippocampus (16%) along with increased levels of glutamate in CSF was found in the CLP group (Figure 1).ConclusionThese results that an episode of severe systemic inflammatory causes persistent brain metabolic disturbances and BBB damage. It is very likely that the long period of BBB disruption, allowing for communication between peripheral mediators/cells and the brain, compromises brain energy metabolism. Together, these findings suggest a link between peripheral inflammation and neurodegeneration.

Effects of S100B neutralization on the long-term cognitive impairment and neuroinflammatory response in an animal model of sepsis

The nervous system is one of the first systems to be affected during sepsis. Sepsis not only has a high risk of mortality, but could also lead to cerebral dysfunction and cognitive impairment in long-term survival patients. The receptor for advanced glycation end products (RAGE) can interact with several ligands, and its activation triggers a series of cell signaling events, resulting in the hyperinflammatory condition related to sepsis. Recent studies show that elevated levels of S100B (RAGE ligand) are associated with the pathophysiology of neurodegenerative disorders. They also participate in inflammatory brain diseases and may lead to an increased activation of microglia and astrocytes, leading to neuronal death. This study aimed to determine the effect of S100B inhibition on the neuroinflammatory response in sepsis. Sepsis was induced in Wistar rats by cecal ligation and perforation (CLP). There were three groups: Sham, CLP, and CLP +10 μg/kg of monoclonal antibody (Anti-S100B) administered intracerebroventricularly. The animals were killed 30 days after sepsis following behavioral evaluation by open field, novel object recognition, and splash test. The hippocampus, prefrontal cortex, and amydgala were used for the determination of S100B and RAGE proteins by western blotting and for the evaluation of cytokine levels and verification of the number of microglial cells by immunohistochemistry. On day 30, both the Sham and CLP + anti-S100B groups were capable of recovering the habitual memory in the open field task. Regarding novel object recognition, Sham and CLP + anti-S100B groups increased the recognition index during the test session in comparison to the training session. There was a significant increase in the time of grooming in CLP + anti-S100B in comparison to the CLP group. There was a modulation of cytokine levels and immunohistochemistry showed that the CLP + anti-S100B group had a decrease in the number of microglial cells only in the hippocampus. These results helped to understand the role of S100B protein in the pathophysiology of sepsis-associated encephalopathy and could be helpful to further experimental studies regarding this subject.

Uncoupling protein 2 overexpression alleviates sepsis-induced myocardial injury via inhibiting reactive oxygen species production and inflammation

To investigate whether the overexpression of uncoupling protein 2 (UCP2) can protect myocardium from sepsis by inhibiting the production of reactive oxygen species (ROS) and inflammatory response. Forty Sprague-Dawley rats were divided into four groups according to random number table method (n = 10): sham transfection and sham surgery group (Sham group), sham transfection and cecal ligation and perforation (CLP) group (CLP group), simple adeno-associated virus (AAV) transfection surgery group (AAV group), and UCP2 overexpression surgery group (UCP2 group). In UCP2 group, UCP2 adeno-associated virus (AAV-UCP2; titer 1×1012 v.g/mL, 10 μL per site, 60 μL in total) was injected into myocardium, and CLP was performed 3 weeks later. In AAV group, the myocardium was transfected with AAV virus and CLP was performed 3 weeks later. Twenty-four hours after modeling, whether the model was successfully prepared was evaluated. The transfection effect of AAV virus on the frozen sections of myocardial tissue was observed under fluorescence microscope, the expression of UCP2 protein was detected by Western blotting, ROS production was detected by dihydroethidine (DHE) staining, and serum myocardial markers and inflammatory cytokines were detected by enzyme linked immunosorbent assay (ELISA). Twenty-four hours after CLP, the rats showed stiff hair, increased secretions from eyes, nose and mouth, and symptoms of pyuria, loose stools, and dyspnea. After laparotomy, the cecum showed purple and black, and there was purulent exudation around the intestinal cavity. The virus was successfully transfected on frozen section under the fluorescence microscope (the site of the transfection was green fluorescence), and further Western blotting revealed that the expression of UCP2 in the CLP group was higher than that in the Sham group (UCP2/β-tubulin: 1.53±0.06 vs. 1, P < 0.01). Compared with the AAV group, UCP2 expression was further increased in the UCP2 group (UCP2/β-tubulin: 1.96±0.22 vs. 1.59±0.07, P < 0.01). Under the fluorescence microscope, ROS production in the CLP and AAV groups were found significantly increased compared with that in the Sham group; when UCP2 was overexpressed, ROS production were significantly decreased compared with the CLP and AAV groups (A value: 1.03±0.10 vs. 1.81±0.13, 1.67±0.08, both P < 0.01). ELISA showed that compared with the Sham group, the levels of lactate dehydrogenase (LDH), creatine kinase (CK), cardiac troponin I (cTnI), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly increased in the CLP and AAV groups; when UCP2 was overexpressed, the above myocardial enzymes and inflammatory cytokines secretion were significantly decreased compared with the CLP group and AAV group [LDH (ng/L): 48.97±1.04 vs. 56.85±1.36, 57.08±1.54; CK (ng/L): 235.23±20.33 vs. 306.34±25.93, 304.76±25.29; cTnI (ng/L): 199.79±18.27 vs. 241.88±14.32, 243.33±23.79; TNF-α (ng/L): 385.71±20.09 vs. 488.92±26.92, 489.03±33.37; IL-6 (ng/L): 121.12±7.61 vs. 159.07±17.65, 157.61±15.13; all P < 0.01]. Kaplan-Meier survival curve showed that the survival rate of rats 36 hours after CLP was only 30.0%. When UCP2 overexpressed, the survival rate was significantly higher than that of the CLP group and AAV group (60.0% vs. 30.0%, 30.0%, both P < 0.05). There was no significant difference between the AAV group and CLP group. UCP2 overexpression can reduce myocardial injury and improve the survival rate of septic rats by reducing ROS production and inhibiting inflammatory reaction in septic myocardium.

The role of lipoprotein-associated phospholipase A2 in acute kidney injury of septic mice.

BackgroundThis experiment aimed to investigate the role and mechanism of lipoprotein-associated phospholipase A2 (Lp-PLA2) in kidney injury in septic mice induced by cecal ligation and perforation (CLP).MethodsMale BALB/c mice were randomly divided into two groups: sham-operation group (Sham group) and septic group (CLP group). The septic model was simulated by cecal ligation and puncture method, but only cecal ligation was used for the sham operation group. The whole serum and renal tissue samples of the mice were collected 24 hours after modeling in both groups. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissue, the renal injury score was recorded, and the creatinine (Cr) and blood urea nitrogen (BUN) levels were detected by automatic biochemical analyzer, while the serum Lp-PLA2 level was detected by enzyme-linked immunosorbent assay (ELISA). The 7-day survival rate and the survival curve of the two groups were statistically analyzed.ResultsCompared with the Sham group, the pathological score of renal injury in the CLP Group was higher, the level of Lp-PLA2 in serum was significantly increased (all P<0.01), and the expression of Lp-PLA2 in renal tissue was significantly elevated (all P<0.01). Furthermore, the 7-day survival rate of the Sham group was 90%, while that of CLP group was 25%.ConclusionsThe expression level of Lp-PLA2 in blood and kidney tissue of septic mice was increased and correlated with prognosis. However, the predictive value of Lp-PLA2 for prognosis in septic mice needs further study.

The protective effect of PPARγ in sepsis-induced acute lung injury via inhibiting PTEN/β-catenin pathway.

The present study aims to reveal the molecular mechanism of peroxisome proliferator-activated receptor γ (PPARγ) on sepsis-induced acute lung injury (ALI). To do that, the rat injury model was established using cecal ligation and perforation (CLP) method, followed by different treatments, and the rats were divided into Sham group, CLP group, CLP + rosiglitazone (PPARγ agonist) group, CLP + GW9662 (PPARγ inhibitor) group, CLP + bpV (phosphatase and tensin homolog (PTEN) inhibitor) group, CLP + GW9662 + bpV group. Compared with Sham group, the mRNA and protein expression levels of PPARγ were down-regulated, the inflammation levels were elevated, and the apoptosis was increased in CLP group. After treatment with rosiglitazone, the protein expression level of PPARγ was significantly up-regulated, the phosphorylation level of PTEN/β-catenin pathway was decreased, the PTEN/β-catenin pathway was inhibited, the lung injury, inflammation and apoptosis were reduced. The opposite effect was observed after treatment with GW9662. Besides, bpV inhibited PTEN/β-catenin pathway, and relieved the lung tissue injury. The overexpression of PPARγ reduced inflammatory response and inhibited apoptosis in sepsis-induced ALI. Furthermore, PPARγ relieved the sepsis-induced ALI by inhibiting the PTEN/β-catenin pathway.

Ligands of Alpha‐1 Adrenergic Receptors: Common Signaling Pathway, but Different Sensitivity to Nitric Oxide in Sepsis

We have shown that arteries present differential responses to α1 adrenergic receptors (α1ARs) agonists in sepsis (Bernardelli et al., Can J Physiol Pharmacol, 94, 1227–36, 2016). Indeed, in animals subjected to the cecal ligation and perforation (CLP) model of sepsis, activation of α1ARs by norepinephrine (NE) results in a small degree of hyporesponsiveness, compared with the responses generated by phenylephrine (PE). It is well known that increased amounts of nitric oxide (NO) are produced in sepsis. NO plays a crucial role in septic vasoplegia, so we hypothesized that α1ARs agonists could differentially modulate the activity of NO synthases (NOS). Here, endothelium‐intact aortic rings from male Wistar rats subjected to CLP were studied in organ baths at 6 h after the septic insult. Control experiments were conducted with arteries from naïve animals. The effects of increasing concentrations (1 nM–300 mM) of PE and NE were evaluated in preparations previously incubated with L‐NAME (a non‐selective inhibitor of NOS; 100 μM), 1400W (a selective inhibitor of the inducible isoform of NOS; 10 μM), or S‐Methyl‐L‐thiocitrulline (a selective inhibitor of the neuronal isoform of NOS; 10 μM). We also investigated the influence of sodium nitroprusside (SNP; 0.1 μM, during 30 min) in the responses of control arteries to PE and NE. Aortic sections were also evaluated in experiments using the fluorescent BODIPY® FL prazosin (QAPB) (250 nM) and DAF‐FM (10 μM). Incubation with L‐NAME increased the contractile effects of PE and NE in both the control and CLP group. On the other hand, both 1400W and S‐methyl‐L‐thiocitrulline had no influence in the effects of PE and NE in control preparations, and increased the responses to PE (but not to NE) from 1.31 ± 0.06 g in control to 2.24 ± 0.11 and 2.21 ± 0.14 g, respectively, in the CLP group (p &lt; 0.05). The fluorescence of QAPB in aortic sections did not differ between the groups, regardless of the previous incubation with PE or NE, suggesting that there were no significant differences in the density of α1ARs between the groups. Besides, neither PE nor NE were able to increase NO production, as detected by the NO sensitive fluorescence probe DAF‐FM. Interestingly, incubation with SNP reduced the maximal contractile effect of NE and PE by 35 and 65%, respectively (p &lt; 0.05). These results suggest that neither an impaired interaction of PE and NE with α1ARs nor a differential modulation of NO production account for the distinct degree of vascular hyporeactivity observed. Nevertheless, our findings reveal remarkable differences between the intracellular signaling activated by PE and NE, regarding their sensitivity to NO. The better comprehension of the behavior of α1ARs can allow the development of ligands with enhanced efficacy for the management of vasoplegia and hypotension in sepsis.Support or Funding InformationFinancial Support: CNPq (Brazil), with a Ph.D. fellowship to Bernardelli, A. K. This study was approved by the Animal Ethics Committee of UFSC (6327180718).

Role and mechanism of splenic myeloid-derived suppressor cells in sepsis-induced adrenal injury in mice

To investigate the role and mechanism of splenic myeloid-derived suppressor cells (MDSCs) in sepsis-induced adrenal injury (SAI). Thirty male C57 mice aged 6-8 weeks were randomly divided into normal control group (n = 5), sham operation group (Sham group, n = 5), sepsis model group [cecal ligation and perforation (CLP) group, n = 10] and sepsis+splenectomy group (CLPS group, n = 10). The sepsis model of mice was reproduced by CLP method. In Sham group, only the cecum was opened and separated, then closed, without CLP. In CLPS group, the spleen was removed before CLP. In normal control group, no challenge was given. After 24 hours, the rats were sacrificed by anesthesia, and peripheral blood, spleen, bone marrow, and bilateral adrenal glands were harvested. The pathological of adrenal gland was assessed by hematoxylin-eosin (HE) staining under optical microscope. The ratio of MDSCs in peripheral blood, spleen and bone marrow was determined by flow cytometry. The expressions of MDSCs surface antigen CD11b, Gr-1 and interleukins (IL-6, IL-1β) mRNA in adrenal tissue were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Western Blot was used to detect the expressions of mammalian rapamycin target protein (mTOR) pathway related proteins including total mTOR (T-mTOR), phosphorylation of mTOR (p-mTOR) and caspase-3. The adrenal cortex and medulla of the normal control group and Sham group were intact and the structure was clear under optical microscope, while in the CLP group, the adrenal gland showed edema, cortical hemorrhage and cell edema. Compared with the CLP group, the adrenal tissue injury was significantly reduced in the CLPS group. Compared with the normal control group and Sham group, MDSCs ratio in the peripheral blood was significantly increased and significantly reduced in the spleen in the CLP group, but there was no significant difference in bone marrow, the expression levels of CD11b, Gr-1, IL-6, IL-1β mRNA and caspase-3 protein were increased significantly and p-mTOR protein expression was significantly decreased in adrenal tissue, there was no significant difference in the expression of T-mTOR protein. Compared with the CLP group, in the CLPS group, the MDSCs ratio in the peripheral blood was significantly decreased (0.143±0.011 vs. 0.324±0.023, P < 0.01), the expression levels of CD11b, Gr-1, IL-6 , IL-1β mRNA and caspase-3 protein in adrenal gland were significantly decreased [CD11b mRNA (2-ΔΔCt): 2.90±0.56 vs. 5.74±0.13, Gr-1 mRNA (2-ΔΔCt): 2.71±0.14 vs. 4.59±0.46, IL-6 mRNA (2-ΔΔCt): 2.44±0.64 vs. 5.17±1.04, IL-1β mRNA (2-ΔΔCt): 3.58±0.52 vs. 4.44±0.26, caspase-3 protein (caspase-3/GAPDH): 0.05±0.01 vs. 0.13±0.02, all P < 0.01], the p-mTOR protein expression was significantly increased (p-mTOR/GAPDH: 0.61±0.11 vs. 0.27±0.04, P < 0.01). The spleen is the major source of MDSCs in SAI. Splenectomy can attenuate SAI by reducing mobilization of MDSCs and activating the mTOR signaling pathway.

Recombinant CC16 inhibits NLRP3/caspase-1-induced pyroptosis through p38 MAPK and ERK signaling pathways in the brain of a neonatal rat model with sepsis

BackgroundSepsis is a critical disease associated with extremely high mortality. Some severe forms of sepsis can induce brain injury, thus causing behavioral and cognitive dysfunction. Pyroptosis is a type of cell death that differs from apoptosis and plays an important role in the occurrence and development of infectious diseases, nervous system-related diseases. A recent study has found that there is pyroptosis in the hippocampus of sepsis-induced brain injury, but its mechanism and treatment scheme have not been evaluated.MethodsWe established immediately a septic rat model by cecal ligation and perforation (CLP) after administration with recombinant club cell protein (rCC16) and/or U46619 in different groups. The clinical performance, survival percentage, vital signs, and neurobehavioral scores were monitored at different time points. Cortical pathological changes were also examined. The expression of cortical nucleotide-binding domain leucine-rich repeat-containing pyrin domain-containing 3 (NLRP3), caspase-1, (p)-p38 mitogen-activated protein kinase (MAPK), and (p)-extracellular signal-related kinase (ERK) was detected by western blotting and immunofluorescence analysis. The levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha in the cortical supernatant were detected by enzyme-linked immunosorbent assay.ResultsCompared with the sham group, the clinical performance, survival percentage, vital signs, and severe cortical pathological changes in the CLP group were worse; NLRP3, caspase-1, and inflammatory factor levels were increased; and phosphorylation of p38 MAPK and ERK was also increased. Meanwhile, multiple indicators were deteriorated further after administration of U46619 in CLP rats. The clinical performance of CLP rats, however, was better after rCC16 administration; cortical pathological changes were attenuated; and NLRP3, caspase-1, and inflammatory factor levels and the phosphorylation of signaling pathway proteins (p38 MAPK and ERK) were reduced. Interestingly, the CLP rats showed the opposite changes in all indicators after administration with both rCC16 and U46619 when compared with those administered rCC16 alone.ConclusionsIn sepsis, rCC16 inhibits cortical pyroptosis through p38 MAPK and ERK signaling pathways. Meanwhile, rCC16 has a protective effect on newborn rats with sepsis, but it is not clear whether its mechanism is directly related to pyroptosis.