ABT-263 enhanced bacterial phagocytosis of macrophages in aged mouse through Beclin-1-dependent autophagy.

BACKGROUND: Sepsis is a critical challenge for the older adults as the immune function is less responsive by aging. Although cell numbers seem preserved in the older adults, macrophages present age-related function decline, which including reduced chemokines, phagocytosis, and autophagy. ABT-263, an inhibitor of the anti-apoptotic protein Bcl-2, is reported had a senolytic effect which can selectively clear the senescent cells in vivo and rejuvenate the aged tissues. METHODS: We treated the aged (12-16 months) and young (4-6 months) C57BL/6 mouse with ABT-263, then gave the animals cecal slurry injection to induce sepsis to observe the effect of senolytic compound ABT-263 on the survival rate of sepsis. Additionally, we isolated peritoneal macrophages from the aged mouse to investigate the cell function and molecular mechanism. 3-methyladenine (3-MA), a phosphatidylinositol 3-kinases (PI3K) inhibitor, and rapamycin, an autophagy-enhancer, were used to block or mimic the autophagy, respectively. RT-PCR and Western Blot were used to detect autophagy related gene and protein changes in sepsis. EGFP-expressing E. coli was used as a marker to evaluate the phagocytic ability of macrophages. RESULTS: The results showed ABT-263 treatment improved the survival rate of sepsis in the aged mouse which related to autophagy, while blocking the autophagy can eliminate this effect. It is revealed that ABT-263 enhanced the phagocytic ability of the peritoneal macrophages by increasing the Trem-2 receptor. Additionally, ABT-263 blocked the binding of Bcl-2 to Beclin-1, thus induced Beclin-1-dependent autophagy. CONCLUSION: ABT-263 enhanced the macrophage function in aged mouse by increasing the Trem-2 receptors and inducing a beclin-1-dependent autophagy, consequently, protected the aged mouse from sepsis.

Tranexamic acid is beneficial for reducing perioperative blood loss in transurethral resection of the prostate.

The aim of this randomized controlled trial was to evaluate the effect of tranexamic acid (TXA) on postoperative blood loss during transurethral resection of the prostate (TURP) for benign prostatic hyperplasia (BPH). A total of 60 patients with BPH and undergoing TURP were randomized into TXA and control groups. Patients were intravenously administered 1 g TXA or placebo (0.9% sodium chloride solution), respectively, after the induction of anesthesia for TURP. Intraoperative and postoperative bladder irrigation volumes and blood loss volumes were compared between the two groups. Coagulation function (measured by prothrombin, activated partial thromboplastin and thrombin time and fibrinogen levels) was measured before the operation and at 4 h post-operation. Complications from thromboembolic events, such as lower-limb and pulmonary embolisms, were also noted. The TXA group had significantly decreased blood loss intraoperatively and at 4 h postoperatively compared with the control group (P<0.05). The 24 h postoperative blood loss and coagulation function of the two groups were not significantly different. No thromboembolic events or other complications occurred in either group. In conclusion, a preoperative single dose of TXA was indicated to reduce perioperative blood loss in TURP without a notable increase in thrombosis risk.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis.

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 °C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Etomidate inhibits nuclear factor-κB through decreased expression of glucocorticoid receptor in septic rats.

The present study aimed to investigate the effect of etomidate administered prior to or following cecal ligation and puncture (CLP) on the expression of glucocorticoid receptor (GR) and lymphocyte apoptosis in septic rats. Right jugular vein catheterization was performed on female Sprague­Dawley rats under isoflurane anesthesia, and CLP surgery was performed to induce sepsis 3 days following catheterization. The rats were randomly divided into five groups. All groups were infused with 2 ml of either etomidate or 5‰ dimethyl sulfoxide (DMSO) at 1 ml/h for 2 h from 6 h post­surgery. The sham group received abdominal sham surgery and infusion with DMSO; the CLP control group received infusion with DMSO. Treatment group A received infusion with 2 mg/kg etomidate; group B received 0.6 mg/kg etomidate following CLP and an infusion of 2 mg/kg etomidate. Group C received 0.6 mg/kg etomidate 24 h prior to CLP and post­surgical etomidate infusion. The 10­day survival rates of the rats in the CLP, A, B and C groups were 60, 50, 55 and 40%, respectively. The serum mRNA expression levels of tumor necrosis factor­α, GR and glucocorticoid­induced leucine zipper were detected by reverse transcription­quantitative polymerase chain reaction, the abundance of inhibitor of nuclear factor (NF)-κB­α was measured by western blotting, and the apoptotic rates of the splenic lymphocytes were determined using flow cytometry. The results suggested that etomidate inhibited NF­κB by decreasing the expression of GR in the septic rats. The increased apoptosis of lymphocytes induced by etomidate may lead to a poor outcome during sepsis.

The pituitary adenylate cyclase-activating polypeptide (PACAP) protects adrenal function in septic rats administered etomidate.

BACKGROUND: Both hyperinflammation during sepsis and etomidate can suppress adrenal function. In this study, we explored whether treatment with pituitary adenylate cyclase-activating polypeptide (PACAP) relieves adrenal suppression in cecal ligation and puncture (CLP)-induced septic rats. MATERIALS AND METHODS: Female Sprague-Dawley rats were randomly divided into five groups (n=7 per group), including the sham group, sepsis group (CLP group), sepsis and etomidate group (CLP+ETO group), PACAP group, and etomidate alone group (ETO group). Rats were sacrificed on the third day of sepsis, and blood and adrenal gland samples were obtained for further testing. RESULTS: The PACAP reduced the apoptosis rate of adrenal cells and peripheral lymphocytes, improving adrenal function, inhibiting the secretion of interferon gamma (IFN-γ) from peripheral lymphocytes, and slightly relieving the suppression of the adrenal function induced by the injection of etomidate in sepsis. CONCLUSION: In septic conditions, the PACAP protects the adrenal gland by regulating peripheral inflammation, which slightly relieves the toxic effects of etomidate on adrenal function.

Etomidate Mitigates Lipopolysaccharide-Induced CD14 and TREM-1 Expression, NF-κB Activation, and Pro-inflammatory Cytokine Production in Rat Macrophages.

This study was aimed at investigating the effect of etomidate on the viability of rat macrophages and the function of lipopolysaccharide (LPS)-stimulated macrophages as well as the potential mechanisms. Rat macrophages were isolated and treated with different doses of etomidate for 24 h, and their viability was determined by the CCK-8 assay. Furthermore, macrophages were treated with, or without, 1 µg/ml of LPS, and/or 2.5 or 5 µM etomidate in the presence or absence of a TREM-1 inhibitor (LP17, 100 ng/ml), and the levels of TNF-α, IL-6, CD14, and TREM-1 in the different groups of cells were determined by quantitative RT-PCR, ELISA, and Western blot assays. The levels of NF-κB activation in the different groups of cells were analyzed by an electrophoretic mobility shift assay (EMSA). Etomidate at 31.25 µM or a low dose did not affect the viability of rat macrophages, while etomidate at higher doses reduced the viability of macrophages in vitro. Treatment with 2.5 or 5 µM etomidate or with LP17 alone did not affect the levels of TNF-α, IL-6, CD-14, and TREM-1 in macrophages. Treatment with etomidate significantly mitigated LPS-stimulated TNF-α, IL-6, CD-14, and TREM-1 expression (p < 0.05 for all) and inhibited LPS-induced NF-κB activation in macrophages in vitro. However, treatment with both etomidate and LP17 did not enhance the inhibitory effects in macrophages. Hence, etomidate mitigates LPS-up-regulated pro-inflammatory cytokine production and inhibits LPS-enhanced CD14 and TREM-1 expression and NF-κB activation in macrophages.

Pretreatment with low dose etomidate prevents etomidate-induced rat adrenal insufficiency by regulating oxidative stress-related MAPKs and apoptosis.

Etomidate is frequently used as an anesthetic and sedation agent in the clinic setting. This study determined that a low-dose pre-infusion followed by a continuous dose infusion of etomidate could reduce etomidate-induced adrenal gland insufficiency. Sixty adult male Wistar rats were used, with six rats per group. Based on preliminary experiments, 0.6mg/kg etomidate was selected as the low dose for this study. Oxidative stress and apoptosis-related proteins in the adrenal glands were assayed using Western blot, and serum levels of CORT and 11ß-hydroxylase were detected using ELISA. Pretreatment with a single bolus of low dose etomidate significantly increased the levels of CORT and 11ß-hydroxylase as well as the activities of superoxide dismutase (SOD), catalase (CAT) and glutathioneperoxidase (GPx) in the adrenal glands, but reduced nitric oxide (NO) production when compared to the positive group. Furthermore, Western blot data showed that pretreatment with low dose etomidate increased extracellular signal-regulated kinase1/2 (ERK1/2), CREB and bcl-2 activation, but suppressed the p-p38, c-JunN-terminal kinase (JNK), inducible NO synthase (iNOS), cleaved-caspase3, cleaved-poly-ADP-ribose polymerase (PARP), bax, and AKT activation. The ERK inhibitor PD98059 and the p38MAPK inhibitor SB203580 abolished the protective effect of low dose etomidate pretreatment. These data demonstrated that pretreatment with low dose etomidate attenuated etomidate-induced adrenal insufficiency to rat adrenal glands. Oxidative stress-related MAPKs and apoptosis proteins might be responsible for mediating the etomidate preconditioning effect in rats.

Etomidate increases mortality in septic rats through inhibition of nuclear factor kappa-B rather than by causing adrenal insufficiency.

BACKGROUND: Both hyperinflammation during sepsis and etomidate can suppress adrenal function. In this study, we explored whether pretreatment with etomidate can relieve adrenal suppression and its impact on outcomes of cecal ligation and puncture (CLP)-induced septic rats. MATERIALS AND METHODS: Rats (n = 18 per group) were divided in seven groups, including two control groups and treated with different combinations of a small pretreatment dose (0.6 mg/kg) and a large continuous dose (2 mg/kg/h over 2 h) of etomidate to evaluate the impact of the different administration combinations on the adrenal glands and outcomes in the septic rats. Animals (n = 8 per group) were euthanized at 24 h after CLP and blood samples and adrenal glands were then collected for further measurements. The remaining rats (n = 10 per group) were used to observe the 7-d survival rate post-CLP. RESULTS: The survival rate (30%) was much lower in the group pretreated with a small dose before CLP surgery and followed by a large dose of etomidate than in the other groups. Etomidate decreased serum corticosterone, but not adrenocorticotropic hormone levels in septic rats, and also decreased serum tumor necrosis factor-alpha and interleukin-6 levels. In rat pretreated with a small dose of etomidate, the toll-like receptor-4 expression level in the adrenal glands was decreased and nuclear factor kappa-B (NF-kappa B) translocation was inhibited. CONCLUSIONS: The mortality of septic rats and degree of adrenal injury caused by etomidate are not correlated. The etomidate-induced inhibition of inflammation and NF-kappa B translocation, which was more significant than adrenal suppression, may be responsible for the increased mortality in septic rats.