Abstract
Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.
Highlights
Sepsis is a systemic host response to an infection, characterized by a misbalance between proinflammatory reactions and anti-inflammatory responses [1]
LPS, sensed by Toll-like receptor 4 (TLR4) on immune cells, activates the inhibitors of kB (IkB) kinase complex and mitogen-activated protein kinase (MAPK) kinases via myeloid differentiation factor 88 (MyD88)-dependent pathway, resulting in phosphorylation of IkB, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK and c-Jun N-terminal kinases (JNK) and the subsequent activation of the transcription factor nuclear factor-kB (NF-kB) and activator protein-1 (AP-1), which induce the expression of cytokines such as tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, IL-12, IL-10 and interferon-c (IFN-c)
Antibodies, including IkBa, ERK, JNK, p38MAPK, NF-kB, IFN regulatory factor 3 (IRF3), tyrosine kinase 2 (Tyk2), signal transducer and activator of transcription 1 (STAT1), phosphorylated IkBa (p-IkBa), phosphorylated JNK (p-JNK), phosphorylated ERK(p-ERK), phosphorylated p38MAPK (p-p38 MAPK), phosphorylated IRF3(p-IRF3), phosphorylated Tyk2 (p-Tyk2), phosphorylated STAT1(p-STAT1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and horseradish peroxidase -labeled secondary antibody were from Cell Signaling Technology, Inc. (Danvers, MA, USA)
Summary
Sepsis is a systemic host response to an infection, characterized by a misbalance between proinflammatory reactions and anti-inflammatory responses [1]. The produced IFN-b causes the phosphorylation of tyrosine kinase 2 (Tyk2) and signal transducer and activator of transcription 1 (STAT1), leading to the inducible nitric oxide synthase (iNOS) and IFN-inducible protein-10 (IP-10) expression [6,7,8]. This progressive release of cytokines and other inflammatory mediators, and severe dysregulation of the inflammatory network eventually lead to the impairment of host defence response, tissue damage, multiple organ dysfunction and even death [9]. The development of novel immunomodulatory strategies to antagonize LPS-induced lethality is important to the treatment of sepsis, especially Gram-negative sepsis
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