Using Enhanced Green Fluorescence Protein-expressing <em>Escherichia Coli</em> 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 ...

The second hospital of Dalian Medical University

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.

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

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 11β-hydroxylase were detected using ELISA. Pretreatment with a single bolus of low dose etomidate significantly increased the levels of CORT and 11β-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. 

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

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. 

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

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.

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

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 Sprague‑Dawley 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 post‑surgery. 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 post‑surgical etomidate infusion. The 10‑day 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 factor‑α, GR and glucocorticoid‑induced leucine zipper were detected by reverse transcription‑quantitative polymerase chain reaction, the abundance of inhibitor of nuclear factor (NF)-κB‑α was measured by western blotting, and the apoptotic rates of the splenic lymphocytes were determined using flow cytometry. The results suggested that etomidate inhibited NF‑κB 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.

Jun-yu Xiong

Anesthesiology Department

The second hospital of Dalian Medical University

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

Jun-yu Xiong

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Anesthesiology Department

The second hospital of Dalian Medical University

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

Anesthesiology Department