Stimulation of angiotensin II type 2 receptor attenuates organ injury in rats with polymicrobial sepsis.

BACKGROUND: Both inflammation and oxidative stress contribute to the pathogenesis of sepsis and its associated organ damage. Angiotensin-(1-7), acting through the Mas receptor and angiotensin II-type 2 receptors (AT2R), could attenuate organ dysfunction and improve survival in rats with sepsis. However, the role of AT2R in inflammation and oxidative stress in rats with sepsis is unclear. Therefore, this study examined the modulatory effects and molecular mechanism of AT2R stimulation in rats with polymicrobial sepsis. METHODS: Male Wistar rats underwent cecal ligation and puncture (CLP) or sham surgery followed by the administration of saline or CGP42112 (a selective, high-affinity agonist of AT2R, 50 µg/kg intravenously) at 3 hours after sham surgery or CLP. The changes in hemodynamics, biochemical variables, and plasma levels of chemokines and nitric oxide were detected during the 24-hour observation. Organ injury was evaluated by histological examination. RESULTS: We found that CLP evoked delayed hypotension, hypoglycemia, and multiple organ injuries, characterized by elevated plasma biochemical parameters and histopathological changes. These effects were attenuated by treatment with CGP42112. CGP42112 significantly attenuated plasma chemokines and nitric oxide production and reduced liver inducible nitric oxide synthase and nuclear factor kappa-B expression. More importantly, CGP42112 significantly improved the survival of rats with sepsis (50% vs. 20% at 24 h after CLP, p < 0.05). CONCLUSION: The protective effects of CGP42112 may be related to anti-inflammatory responses, suggesting that the stimulation of AT2R is a promising therapeutic candidate for the treatment of sepsis.

Antimicrobial peptide cathelicidin LL-37 preserves intestinal barrier and organ function in rats with heat stroke.

Global warming increases the incidence of heat stroke (HS) and HS causes the reduction of visceral blood flow during hyperthermia, leading to intestinal barrier disruption, microbial translocation, systemic inflammation and multiple organ failure. Cathelicidin LL-37 exhibits antimicrobial activities, helps innate immunity within the gut to maintain intestinal homeostasis, and augments intestinal wound healing and barrier function. Thus, we evaluated the effects and possible mechanisms of cathelicidin LL-37 on HS. Wistar rats were placed in a heating-chamber of 42 ̊C to induce HS. Changes in rectal temperature, hemodynamic parameters, and survival rate were measured during the experimental period. Blood samples and ilea were collected to analyze the effects of LL-37 on systemic inflammation, multiple organ dysfunction, and intestinal injury. Furthermore, LS174T and HT-29 cells were used to assess the underlying mechanisms. Our data showed cathelicidin LL-37 ameliorated the damage of intestinal cells induced by HS. Intestinal injury, systemic inflammation, and nitrosative stress (high nitric oxide level) caused by continuous hyperthermia were attenuated in HS rats treated with cathelicidin LL-37, and hence, improved multiple organ dysfunction, coagulopathy, and survival rate. These beneficial effects of cathelicidin LL-37 were attributed to the protection of intestinal goblet cells (by increasing transepithelial resistance, mucin-2 and Nrf2 expression) and the improvement of intestinal barrier function (less cyclooxygenase-2 expression and FITC-dextran translocation). Interestingly, high cathelicidin expression in the ileal samples of inflammatory bowel disease patients was associated with better clinical outcome. These results suggest that cathelicidin LL-37 could prevent heat stress-induced intestinal damage and heat-related illnesses.

Olmesartan Ameliorates Organ Injury and Mortality in Rats With Peritonitis-Induced Sepsis.

INTRODUCTION: Sepsis and related complications lead to high morbidity and mortality in humans and animals. Olmesartan medoxomil (OLM), a nonpeptide angiotensin II type 1 receptor blocker, has antiinflammatory and antioxidative effects in various experimental animal models. The present study aimed to investigate whether OLM protects against sepsis in a clinically relevant model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). METHODS: Sepsis was induced by CLP in anesthetized rats. OLM was administered intraperitoneally 3 h after CLP onset. Hemodynamic, biochemical, and inflammatory parameters were analyzed. RESULTS: The administration of OLM in CLP rats significantly improved their survival rate. Moreover, OLM mitigated CLP-induced hypotension and organ injury (indicated by biochemical parameters), but not tachycardia. OLM significantly reduced the plasma levels of interleukin-6 and nitric oxide. CONCLUSIONS: OLM markedly attenuated CLP-induced hypotension and organ injury, and hence improved survival by inhibiting the inflammatory response and nitrosative stress in this clinically relevant model of sepsis.

Mangiferin induces immune responses and evaluates the survival rate in WEHI-3 cell generated mouse leukemia in vivo.

Mangiferin is a naturally occurring polyphenol, widely distributed in Thymeraceae families, and presents pharmacological activity, including anti-cancer activities in many human cancer cell lines. Mangiferin has also been reported to affect immune responses; however, no available information concerning the effects of mangiferin on immune reactions in leukemia mice in vivo. In the present study, we investigated the effects of mangiferin on leukemia WEHI-3 cell generated leukemia BLAB/c mice. Overall, the experiments were divided into two parts, one part was immune responses experiment and the other was the survival rate experiment. The immune responses and survival rate study, 40 mice for each part, were randomly separated into five groups (N = 8): Group I was normal animals and groups II-V WEHI-3 cell generated leukemia mice. Group II mice were fed normal diet as a positive control; group III, IV, and V mice received mangiferin at 40, 80, and 120 mg/kg, respectively, by intraperitoneal injection every 2 days for 20 days. Leukocytes cell population, macrophage phagocytosis, and NK cell activities were analyzed by flow cytometry. Isolated splenocytes stimulated with lipopolysaccharide (LPS) and concanavalin A (Con A) were used to determine the proliferation of B and T cells, respectively, and subsequently were analyzed by flow cytometry. Results indicated that mangiferin significantly increased body weight, decreased the liver and spleen weights of leukemia mice. Mangiferin also increased CD3 T-cell and CD19 B cell population but decreased Mac-3 macrophage and CD11b monocyte. Furthermore, mangiferin decreased phagocytosis of macrophages from PBMC and peritoneal cavity at 40, 80, and 120 mg/kg treatment. However, it also increased NK cell activity at 40 and 120 mg/kg treatment. There were no effects on T and B cell proliferation at three examined doses. In survival rate studies, mangiferin significantly elevated survival rate at 40 and 120 mg/kg treatment of leukemia mice in vivo.

Casticin Inhibits In Vivo Growth of Xenograft Tumors of Human Oral Cancer SCC-4 Cells.

BACKGROUND/AIM: Casticin, one of the active components of Vitex rotundifolia L., presents biological and pharmacological activities including inhibition of migration, invasion and induction of apoptosis in numerous human cancer cells in vitro. This study aimed to assess the effects of casticin on tumor growth in a human oral cancer SCC-4 cell xenograft mouse model in vivo. MATERIALS AND METHODS: Twenty-four nude mice were injected subcutaneously with SCC-4 cells and when palpable tumors reached a volume of 100-120 mm3 the mice were randomly divided into three groups. The control (0.1% dimethyl sulfoxide), casticin (0.2 mg/kg), and casticin (0.4 mg/kg) groups were intraperitoneally injected every two days for 18 days. Tumor volume and body weights were measured every two days. RESULTS: Casticin significantly decreased tumor volume and weight in SCC-4 cell xenograft mice but there was no statistically significant difference between the body weights of control mice and mice treated with 0.2 mg/kg or 0.4 mg/kg casticin. Therefore, the growth of SCC-4 cells in athymic nude mice can be inhibited by casticin in vivo. CONCLUSION: These findings support further investigations in the potential use of casticin as an oral anti-cancer drug in the future.

Low-dose hydralazine improves endotoxin-induced coagulopathy and multiple organ dysfunction via its anti-inflammatory and anti-oxidative/nitrosative properties.

Coagulopathy is the major cause of organ injury as well as a strong predictor of mortality in septic patients. Systemic inflammatory response and redox imbalance are regarded as the major causes of sepsis-induced coagulopathy. There is growing evidence that a vasodilator hydralazine has beneficial effects on heart failure, hypertension, and ischemia/reperfusion injury via its antioxidant and anti-inflammatory properties. However, the effects of hydralazine on sepsis have not been examined. Therefore, we evaluated the effects of low-dose hydralazine on coagulopathy and multiple organ dysfunction in septic rats induced by endotoxin. Sepsis-induced coagulopathy was established by intravenous injection of rats with lipopolysaccharide (LPS). The changes of blood pressure, heart rate, blood glucose, hemostatic variables, prothrombin time, organ function indices, interleukin-6 (IL-6) concentration, and nitric oxide (NO) level were assessed during the experimental period. In addition, the aortas, lungs, livers, and kidneys were dissected to analyze superoxide levels and protein expressions. LPS induced (i) coagulopathy, multiple organ dysfunction, and circulatory failure successfully, and (ii) excessive superoxide, NO, and IL-6 production, accompanied by the overexpression of iNOS and Wnt5a in animals. Treatment of LPS-induced septic rats with low-dose hydralazine not only improved coagulopathy but also ameliorated multiple organ dysfunction. These could be due to attenuation of the overproduction of superoxide, NO, and IL-6, which were attributed to reduction of the overexpression of iNOS and Wnt5a. Thus, these findings indicate that low-dose hydralazine could be a potential therapy for sepsis-induced coagulopathy and multiple organ dysfunction via its anti-inflammatory and anti-oxidative/nitrosative properties.

Ouabain induces apoptotic cell death in human prostate DU 145 cancer cells through DNA damage and TRAIL pathways.

Ouabain, a cardiotonic steroid and specific Na+ /K+ -ATPase inhibitor, has a potential to induce cancer cell apoptosis but the mechanisms of apoptosis induced by ouabain are not fully understand. The aim of this study was to investigate the cytotoxic effects of ouabain on human prostate cancer DU 145 cells in vitro. Cell morphological changes were examined by phase contrast microscopy. Cell viability, cell cycle distribution, cell apoptosis, DNA damage, the production of ROS and Ca2+ , and mitochondrial membrane potential (ΔΨm ) were measured by flow cytometry assay. Results indicated that ouabain induced cell morphological changes, decreased total cell viability, induced G0/G1 phase arrest, DNA damage, and cell apoptosis, increased ROS and Ca2+ production, but decreased the levels of ΔΨm in DU 145 cells. Ouabain also increased the activities of caspase-3, -8, and -9. Western blotting was used for measuring the alterations of apoptosis-associated protein expressions in DU 145 cells and results indicated that ouabain increased the expression of DNA damage associated proteins (pATMSer1981 , p-H2A.XSer139 , and p-p53Ser15 ) and ER-stress-associated proteins (Grp78, ATF6ß, p-PERKThr981 , PERK, eIF2A, GADD153, CaMKIIß, and caspase-4) in time-dependently. Furthermore, ouabain increased apoptosis-associated proteins (DR4, DR5, Fas, Fas Ligand, and FADD), TRAIL pathway, which related to extrinsic pathway, promoted the pro-apoptotic protein Bax, increased apoptotic-associated proteins, such as cytochrome c, AIF, Endo G, caspase-3, -8, and -9, but reduced anti-apoptotic protein Bcl-2 and Bcl-x in DU 145 cells. In conclusion, we may suggest that ouabain decreased cell viability and induced apoptotic cell death may via caspase-dependent and mitochondria-dependent pathways in human prostate cancer DU 145 cells.

Levosimendan mitigates coagulopathy and organ dysfunction in rats with endotoxemia.

BACKGROUND: In patients with severe sepsis, pro-inflammatory cytokines and subsequent activation of tissue factors trigger a cascade of events that lead to coagulation dysfunction and multiple organ failure. It has been shown that levosimendan has protective effects against tissue injury caused by endotoxin. The purpose of this study was to evaluate the effects of levosimendan on consumptive coagulopathy and organ dysfunction in an endotoxemic animal model induced by lipopolysaccharide (LPS). METHODS: Forty-six male adult Wistar rats were randomly divided into four groups: 1) control group (n = 10), an intravenous infusion of 5% dextrose 1.2 mL/kg for 20 min and 0.03 mL/kg/min for 4 h; 2) the levosimendan-treated control group (n = 12), an intravenous levosimendan infusion (24 µg/kg for 20 min plus 0.6 µg/kg/min for 4 h); 3) the LPS group (n = 12), an intravenous LPS (4 mg/kg) infusion followed by dextrose administration; and 4) the levosimendan-treated LPS group (n = 12), an intravenous LPS infusion followed by levosimendan treatment. Various parameters of hemodynamics, biochemistry, hemostasis and inflammatory response were examined during the experimental period. RESULTS: The administration of levosimendan significantly attenuated (i) consumptive coagulopathy displayed by thromboelastography, (ii) the decreases of platelet count and plasma fibrinogen level, (iii) injury in the lung, liver and kidney, and (iv) the rise in plasma interleukin-6 in rats treated with LPS. CONCLUSION: The treatment of LPS rats with levosimendan was found to reduce organ injury and coagulopathy. These protective effects may be attributed to the anti-inflammatory effects of this drug.

Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions.

Constitutive functional HIF-2α was recently identified in cancer and stem cell lines under normoxia. In this study, BEAS-2B, a bronchial epithelial cell line, was shown to constitutively express active HIF-2α under normoxia and exhibit markers of pluripotency including Oct-4, Nanog, and sphere formation. Oct-4 expression was reduced after knockdown of HIF-2α under normoxia. Global enrichment analysis of HIF-2α demonstrated the diverse functions of HIF-2α under normoxia. Bioinformatics analysis of the enriched loci revealed an enhancer role of HIF-2α binding sites, involvement of HIF-2α interacting proteins, and enriched de novo motifs which suggest the diverse role of HIF-2α in pseudohypoxia. The low ratio of the discovered loci overlapping with those revealed in cancer cell lines 786-O (16.1%) and MCF-7 (15.9%) under hypoxia indicated a prevailing non-canonical mechanism. Hypoxia had positive, marginal or adverse effects on the enrichment of the selected loci in ChIP-PCR assays. Deletion of the N-terminal activation domain (N-TAD) of HIF-2α disrupted the reporting activity of two of the loci annotated to ELN and ANKRD31. Hypoxia incurring abundance variation of HIF-2α may misrepresent the N-TAD functions as canonical hypoxia inducible features via C-TAD activation. Elucidation of the pseudohypoxia functions of constitutive HIF-2α is useful for resolving its role in malignancy and pluripotency.

Levosimendan attenuates multiple organ injury and improves survival in peritonitis-induced septic shock: studies in a rat model.

INTRODUCTION: The aim of this study was to investigate the effects of levosimendan on rodent septic shock induced by cecal ligation and puncture (CLP). METHODS: Three hours after peritonitis-induced sepsis, male Wistar rats were randomly assigned to receive an intravenous infusion of levosimendan (1.2 µg/kg/min for 10 min and then 0.3 µg/kg/min for 6 h) or an equivalent volume of saline and vehicle (5% dextrose) solution. RESULTS: The levosimendan-treated CLP animals had significantly higher arterial pressure and lower biochemical indices of liver and kidney dysfunction compared to the CLP animals (P < 0.05). Plasma interleukin-1ß, nitric oxide and organ superoxide levels in the levosimendan-treated CLP group were less than those in CLP rats treated with vehicle (P < 0.05). In addition, the inducible nitric oxide synthase (iNOS) in lung and caspase-3 expressions in spleen were significantly lower in the levosimendan-treated CLP group (P < 0.05). The administration of CLP rats with levosimendan was associated with significantly higher survival (61.9% vs. 40% at 18 h after CLP, P < 0.05). At postmortem examination, the histological changes and neutrophil filtration index in liver and lung were significantly attenuated in the levosimendan-treated CLP group (vs. CLP group, P < 0.05). CONCLUSIONS: In this clinically relevant model of septic shock induced by fecal peritonitis, the administration of levosimendan had beneficial effects on haemodynamic variables, liver and kidney dysfunction, and metabolic acidosis. (1) Lower levels of interleukin-1ß, nitric oxide and superoxide, (2) attenuation of iNOS and caspase-3 expressions, and (3) decreases of neutrophil infiltration by levosimendan in peritonitis-induced sepsis animals suggest that anti-inflammation and anti-apoptosis effects of levosimendan contribute to prolonged survival.

Adjuvant potential of selegiline in attenuating organ dysfunction in septic rats with peritonitis.

Selegiline, an anti-Parkinson drug, has antioxidant and anti-apoptotic effects. To explore the effect of selegiline on sepsis, we used a clinically relevant animal model of polymicrobial sepsis. Cecal ligation and puncture (CLP) or sham operation was performed in male rats under anesthesia. Three hours after surgery, animals were randomized to receive intravenously selegiline (3 mg/kg) or an equivalent volume of saline. The administration of CLP rats with selegiline (i) increased arterial blood pressure and vascular responsiveness to norepinephrine, (ii) reduced plasma liver and kidney dysfunction, (iii) attenuated metabolic acidosis, (iv) decreased neutrophil infiltration in liver and lung, and (v) improved survival rate (from 44% to 65%), compared to those in the CLP alone rats. The CLP-induced increases of plasma interleukin-6, organ superoxide levels, and liver inducible nitric oxide synthase and caspase-3 expressions were ameliorated by selegiline treatment. In addition, the histological changes in liver and lung were significantly attenuated in the selegiline -treated CLP group compared to those in the CLP group. The improvement of organ dysfunction and survival through reducing inflammation, oxidative stress and apoptosis in peritonitis-induced sepsis by selegiline has potential as an adjuvant agent for critical ill.

Hyperoncotic albumin attenuates lung and intestine injuries caused by peritonitis-induced sepsis in rats.

BACKGROUND: Hyperoncotic albumin may be a therapeutic option to improve tissue perfusion and organ injury in sepsis. To clarify the hypothesis and its mechanism, hyperoncotic albumin was administered to the rats in a polymicrobial sepsis-peritonitis model. MATERIALS AND METHODS: Peritonitis was induced by a surgery of cecal ligation and puncture (CLP) in 27 male Wistar rats. For control purposes, sham operations without ligating and puncturing the cecum were performed in 20 rats. Three hours later, rats were randomized to receive intravenously 3 mL/kg of 5% albumin, 25% albumin, or normal saline. All the hemodynamic and biochemical parameters were measured during the 18-h observation. RESULTS: In septic rats, 25% albumin attenuated hypotension, vascular hyporeactivity to norepinephrine, and the elevated serum levels of lactate dehydrogenase and blood urea nitrogen. However, these improvements were not noted in CLP rats after 5% albumin treatment. In addition, 25% albumin decreased metabolic acidosis and improved the CLP-induced hypoperfusion in the intestine and kidney. Superoxide levels in the aorta and lung and the protein expression of inducible nitric oxide synthase in the lung were also attenuated by 25% albumin in CLP rats. Microscopic findings confirmed that 25% albumin attenuated the substantial swelling and cell infiltration in the intestine and lung caused by CLP. CONCLUSIONS: In this sepsis rat model, 25% albumin reduced macro- and microhemodynamic changes and attenuated intestine and lung injuries in peritonitis-induced sepsis.

Effects of small-volume hypertonic saline on acid-base and electrolytes balance in rats with peritonitis-induced sepsis.

Our previous study has demonstrated that hypertonic saline (HS) given at 3 h after cecal ligation and puncture (CLP) surgery alleviates circulatory failure, multiple organ dysfunction syndrome, and mortality rate in rats. However, only few data exist on the application of HS in acid-base and electrolyte imbalance of sepsis. In addition, early one-dose HS administration seems to have only modest improvement on mortality rate. Thus, we evaluated the effects of HS on acid-base equilibrium and electrolyte balance in CLP-induced sepsis model and further compared with the effects of two- and one-dose HS administration. Male Wistar rats received CLP or sham operation followed by the administration of saline or HS (7.5% NaCl, 4 mL/kg, intravenously at 3 and 9 h after laparotomy or CLP). The changes in hemodynamics, biochemical variables, blood gas, electrolytes, organ histology, and plasma levels of nitric oxide (NO) and interleukin 1ß (IL-1ß) were examined during the 18-h observation. Hypertonic saline given either at 3 h (one-dose administration) or at 3 and 9 h (two-dose administration) after CLP attenuated circulatory failure, multiple organ dysfunction syndrome, metabolic acidosis, hyperkalemia, neutrophil infiltration, and 18-h mortality. Moreover, both one- and two-dose HS administrations significantly diminished plasma NO and IL-1ß levels in CLP rats. However, only the two-dose HS administration significantly improved hyponatremia and hypocalcemia in septic rats. Beneficial effects of HS in septic rats may be attributed to not only reducing plasma levels of NO and IL-1ß, but also improving metabolic acidosis and electrolyte imbalance. In addition, two-dose HS administration could reverse electrolyte imbalance caused by CLP.

Beneficial effects of hyperoncotic albumin on liver injury and survival in peritonitis-induced sepsis rats.

Liver injury/dysfunction developing in patients with sepsis may lead to an increased risk of death. Small-volume resuscitation with hyperoncotic albumin (HA) has been proposed to restore physiologic hemodynamics in hemorrhagic and septic shock. We evaluated whether HA resuscitation could alleviate the development of liver injury/dysfunction in rats with polymicrobial sepsis induced by cecal ligation and puncture (CLP). The male Wistar rats received 0.9% saline or HA (25%, 3 mL/kg intravenously) at 3 h after CLP or sham operation. All hemodynamic and biochemical variables were measured during the 18-h observation. After 18 h of CLP, the septic rats developed circulatory failure (i.e., hypotension, tachycardia, and poor tissue perfusion), liver injury (examined by biochemical variables and histologic studies), and a higher mortality. Hyperoncotic albumin not only ameliorated the deterioration of hemodynamic changes but also attenuated neutrophil infiltration and cell death in the liver of septic animals. The septic rats treated with HA had a higher survival when compared with those with 0.9% saline treatment. Moreover, the increased plasma IL-1ß, plasma IL-6, plasma nitrite/nitrate concentrations, liver iNOS expression, and liver superoxide levels in CLP rats were attenuated after administration of HA. Thus, HA may be regarded as a potential therapeutic agent in the early treatment of septic shock to prevent or reduce subsequent liver failure.