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

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Highlights

  • We firstly showed that long time infusion of etomidate-induced oxidative adrenal insufficiency.

  • Adrenal insufficiency decreased the levels of CORT and 11β-hydroxylase.

  • Adrenal insufficiency also increased NO production.

  • Pretreatment with small dose of etomidate has antioxidative and anti-adrenal insufficiency activities.

  • MAPKs and apoptosis proteins involved mediating the preconditioning effect.

Abstract

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, 0.6 mg/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.

Introduction

Etomidate is a frequently used short acting intravenous anesthetic agent for either general anesthesia or sedation in hemodynamically unstable emergency department and intensive care units (Zed et al., 2006). Compared to other general anesthetic agents, etomidate minimally affects hemodynamics and has a cerebral protective effect (Jackson, 2005). However, long-term use of etomidate can significantly suppress corticosteroid synthesis in the adrenal cortex (Dorr et al., 1984), limiting its application in the clinic. Our previous study showed that high dose etomidate reduced HL-60 cell viability by increasing NO production and induced immunocyte injury. Preconditioning with 500 μM etomidate attenuates these detrimental effects (Zhang et al., 2010). Nevertheless, the underlying molecular events remain to be fully elucidated.

Oxidative stress and immune toxicity in animals are considered as two of the most important responses to intravenously applied medicine (Ashry et al., 2010, Jin et al., 2014). Other studies have indicated that general anesthetics, including propofol and etomidate, have the potential to induce oxidative stress and immunotoxicity (Wang et al., 2011, Zhang et al., 2010). Indeed, oxidative stress can result in cell death due to several different mechanisms, including increased formation of reactive oxygen species (ROS), mitochondrial dysfunction, or activation of apoptosis-related death signaling (Klaunig et al., 2011, Pereira and Oliveira, 2000). Intracellular antioxidant mechanisms involve antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) (Dantas Ada et al., 2015, Sun et al., 2015). Inducible nitric oxide synthase (iNOS), a member of the NOS protein family, catalyzes L-arginine and leads to formation of nitric oxide (NO). Moreover, mitogen-activated protein kinases (MAPKs) play an important role in various physiologic processes, such as cell growth, differentiation, and apoptosis (Kim and Choi, 2010). Recent studies have reported that members of the MAPK family, such as p38 kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase1/2 (ERK1/2), are involved in cytotoxicity (Lee et al., 2012). Also, MAPKs play a crucial role in many of the signaling transduction pathways associated with apoptosis of cells (Fukui et al., 2009). AKT activation can phosphorylate and regulate functions of many cellular proteins in apoptosis, such as CREB, bcl-2, bax, caspase-3 and PARP (Fulda, 2015, Juraver-Geslin and Durand, 2015). In addition, a previous study reported that inhibition of MAPK pathways could be used to treat human diseases (Li et al., 2014). Thus, in this study, we performed animal experiments to determine whether preconditioning with a low dose of etomidate could reduce the detrimental effects of subsequent high doses of etomidate on the adrenal gland. We detected serum levels of corticosteroid (CORT), SOD, CAT, GPx as parameters for adrenal gland function. In addition, we determined whether oxidative stress and the MAPK and Akt signaling pathways were involved in these processes.

Section snippets

Materials

Pure etomidate (99%) was obtained from the Enhua Pharmacological Group (Xuzhou, Jiangsu, China). A p38 MAPK inhibitor SB203580 (Catalog #13067), Erk1/2 inhibitor PD98059 (#10006726) and JNK inhibitor SP600125 (#10010466) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). The following rabbit antibodies were purchased from Bioworld Technology, Inc. (Louis Park, MN, USA): CREB (#BS1624), Akt (#BS1810), JNK1/2 (#BS3631), ERK (#BS1112), phosphor-CREB (#BS4053), phospho-Akt (#BS4007),

Effect of etomidate on serum CORT concentration

Changes in serum CORT concentration in each group were analyzed. First, we assessed four different concentrations (0.2, 0.6, 1.3, and 2 mg/kg) of etomidate following continuous treatment with etomidate (1.2 mg/kg/h) for 6 h to find the optimal pretreatment dose. The etomidate dose-dependent changes in CORT levels were assessed (13.62 ± 1.26, 15.18 ± 1.41, 13.05 ± 1.43, and 12.44 ± 1.36 ng/mL, respectively; Fig. 2A). Thus, pretreatment with 0.6 mg/kg etomidate had a better effect on CORT levels compared to

Discussion

Long-term use of etomidate can significantly suppress corticosteroid synthesis in the adrenal gland. During etomidate infusion, serum concentrations of cortisol, cortisone and aldosterone decrease, whereas concentrations of 11-deoxycorticosterone, 11-deoxycortisol, progesterone and 17-hydroxyprogesterone increase (Dorr et al., 1984). These clinical data indicate that etomidate inhibits adrenal steroid synthesis primarily by blocking the activity of CYP11B1, also known as 11β-hydroxylase or

Conclusions

In summary, our current data demonstrates that pretreatment with a single low dose of etomidate attenuated etomidate-induced damage to rat adrenal glands. These finding may be mediated by MAPK, Akt and oxidative stress proteins. Specifically, pretreatment with a single low dose of etomidate may restore antioxidant defense, thereby reducing NO production and preventing apoptosis. Furthermore, the protective effects were associated with an increase in ERK1/2 and CREB expression as well as bcl-2

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgement

This work was supported in part by a grant to J.Y.X. from the National Natural Science Foundation of China (#81171791).

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