β-patchoulene alleviates cognitive dysfunction in a mouse model of sepsis associated encephalopathy by inhibition of microglia activation through Sirt1/Nrf2 signaling pathway.

Sepsis associated encephalopathy (SAE) is a common but poorly understood complication during sepsis. Currently, there are no preventive or therapeutic agents available for this neurological disorder. The present study was designed to determine the potential protective effects of β-patchoulene (β-PAE) in a mouse model of SAE and explore the putative mechanisms underpinning the beneficial effects. SAE was induced in C57BL/6 mice by cecal ligation and puncture(CLP). Mice were administrated with β-PAE or saline by intra-cerebral ventricle(i.c.v) injection immediately after CLP surgery. The inhibitory avoidance tests and open field tests were performed at 24h, 48h and 7days after procedures. Cytokines expression, oxidative parameters, microglia polarization and apoptosis in the brain tissue were assessed. Sirt1, Nrf2, HO-1and cleaved-caspase3 expression in hippocampus was determined by western-blotting. Further, serum cytokines expression and spleen lymphocytes apoptosis were evaluated, and survival study was performed. Septic mice suffered severe cognitive decline following CLP as evidenced by decreased memory latency time and lower frequency of line crossing in the behavioral tests. A high dose of β-PAE(1mg/kg) improved the cognitive impairment in SAE mice, which was accompanied by reduced cytokines expression and oxidative stress. Immunofluorescence assay showed that β-PAE inhibited the expression of Iba-1 and iNOS in microglia. The mechanistic study indicated that β-PAE could promote the nuclear expression of Sirt1/Nrf2 and enhance cytoplasmic HO-1 expression. Furthermore, i.c.v administration of β-PAE decreased the expression of serum cytokines and apoptosis in the spleen, thus leading to an improved 7-day survival of septic mice. Finally, blockade of Nrf2 activation with ML385 largely mitigated the protective effects of β-PAE on the cognitive function, neuroinflammation and survival in SAE mice. In this study, we found that β-PAE significantly altered sepsis induced neuroinflammation and microglia activation, thus reversed the cognitive decline and improved the peripheral immune function. The neuroprotective effects were possibly mediated by the activation of Sirt1/Nrf2/HO-1 pathway. β-PAE might serve as a promising therapeutic agent for SAE prevention and treatment.

Tian Y ，Wang L ，Fan X ，Zhang H ，Dong Z ，Tao T ... -  《PLoS One》

β-Patchoulene Preconditioning Protects Mice Against Hepatic Ischemia-Reperfusion Injury by Regulating Nrf2/HO-1 Signaling Pathway.

Hepatic ischemia-reperfusion (I/R) injury is one of the main causes of liver dysfunction after the liver resection and transplantation. Hepatic I/R was characterized by the tissue hypoxia during ischemia phase and oxidative stress and immune response during hypoxia-reoxygenation. The objectives of the present study were to determine the protective effects of β-patchoulene (β-PAE), a novel bioactive agent, in a mice model of hepatic I/R injury and to explore its potential mechanisms. A segmental liver warm I/R injury model was performed by occluding the portal vessels for 1 h followed by 6-h reperfusion. Twenty-four mice were randomly divided into three groups: Sham, I/R, and I/R + β-PAE, with eight mice in each group. Mice were intravenously injected with β-PAE (10 mg/kg) or saline 2 h before surgery, and parameters were measured 6 h after designated treatment. Serum aminotransferase, histologic changes, cytokines expression, and apoptosis were determined. The potential effects of β-PAE on macrophage activation and apoptosis were further evaluated in a hypoxia and reperfusion (H/R) model in vitro. Oxidative stress markers (reactive oxygen species production and malondialdehyde) and cytokines expression were measured by commercial kits. Nrf2/HO-1 and NF-ƘB signaling pathways were determined by Western blotting. Finally, blockade of nuclear factor erythroid 2-related factor 2 (Nrf2) with ML385 was used to confirm the involvement of Nrf2/HO-1 pathway in H/R injury. Hepatic I/R induced apparent tissue injury as evidenced by the increased expression of serum aminotransferase, pro-inflammatory mediators production, hepatocellular apoptosis, and necrosis. β-PAE pretreatment protected mice against I/R-induced injury, which was proved by decreased serum aminotransferase and cytokines production, reduced TUNEL-positive cells, and alleviated histopathological lesion. Immunofluorescence staining showed that β-PAE suppressed the M1 polarization of Kupffer cell induced by I/R injury. Moreover, pretreatment with β-PAE suppressed H/R-induced cytokines expression and apoptosis in cultured macrophage. The mechanistic study demonstrated that β-PAE significantly promoted the nuclear Nrf2 translocation and upregulation of HO-1 while downregulating the NF-ƘB signaling pathway in both in vivo and in vitro experiments. Furthermore, blockade of Nrf2 abolished the protective effects of β-PAE on the inhibition of H/R-mediated oxidative stress, inflammatory response, and apoptosis in vitro. β-PAE preconditioning protects mice against hepatic I/R, which was at least in part through the reversing disequilibrium between Nrf2/HO-1 and NF-ƘB pathways. β-PAE might serve as a promising therapeutic agent in the treatment of hepatic I/R injury.

Tao T ，Ye B ，Xu Y ，Wang Y ，Zhu Y ，Tian Y ... -  《-》

Hydrogen gas alleviates blood-brain barrier impairment and cognitive dysfunction of septic mice in an Nrf2-dependent pathway.

Sepsis-associated encephalopathy (SAE) is a cognitive impairment caused by sepsis and is related to increased morbidity and mortality. Damage to the blood-brain barrier (BBB) has been proved to be one of the important causes of SAE. Molecular hydrogen (H2) is a promising method for the treatment of SAE, yet the underlying mechanism is not clear. This study was designed to demonstrate whether H2 can alleviate SAE by protecting the BBB, and whether it is protected by Nuclear factor erythroid-2-related factor 2 (Nrf2) and its downstream signaling pathways. Either a sham or a cecal ligation and puncture (CLP) procedure was applied to female wild-type (WT) and Nrf2-knock-out (Nrf2-/-) C57BL/6J mice. H2 (2%) was given for 60 min starting at 1 h and 6 h after the sham or CLP procedure. In addition, bEnd.3 cells cultured with medium which contained LPS, Saline, DMSO or ML385 (a Nrf2 inhibitor) were also used in the research. The 7-day survival rates were recorded. The Morris water maze was used to determine cognitive function. Pro-inflammatory and anti-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), HMGB1, and IL-10), antioxidant enzymes, and oxidation products [superoxide dismutase (SOD), chloramphenicol acetyltransferase (CAT), malondialdehyde (MDA), and (8-iso-PGF2α)] were determined by enzyme-linked immunosorbent assay (ELISA). Brain water content, Dextran tracer, and Evans blue extravasation were used to detect the damage of the BBB. Western blot analysis was used to detect β-catenin, phosphorylated β-catenin, adhesion-linked protein VE-cadherin, and associated tight junction protein ZO-1. We found that H2 can improve survival in septic mice, decrease escape latency and platform crossing times, decrease pro-inflammatory cytokines and oxidative product levels in the mouse cortex, and increase the expression of anti-inflammatory factors in WT, but not Nrf2-/-, mice. Moreover, H2 can also decrease brain water content, extravascular dextran, extravascular Evans blue dye, and β-catenin level, and increase ZO-1 and VE-cadherin expressions in WT mice, but not in Nrf2-/- mice. Our result shows that H2 can protect the BBB by decreasing its permeability, thereby reducing SAE and improving cognitive function, which is mediated through Nrf2 and its downstream signaling pathways.

Yu Y ，Feng J ，Lian N ，Yang M ，Xie K ，Wang G ，Wang C ，Yu Y ... -  《-》

Blockade of IL-17A/IL-17R Pathway Protected Mice from Sepsis-Associated Encephalopathy by Inhibition of Microglia Activation.

Sepsis-associated encephalopathy (SAE) is a poorly understood condition that leads to long-term cognitive impairment and increased mortality in survivors. Recent research revealed that IL-17A/IL-17R might serve as a checkpoint in microglia-mediated neuroinflammation. The present study was designed to determine the specific role of IL-17A-mediated microglia activation in the development of SAE. A mouse model of SAE was induced by cecal ligation and puncture (CLP), and behavior performance was evaluated by the inhibitory avoidance test and the open field test. Cytokine expression and microglia activation in brain tissue were determined at 6 h, 12 h, 24 h, 48 h, and day 7 post surgery. Further, septic mice were intracerebral ventricle- (i.c.v.-) injected with recombinant IL-17A, anti-IL-17A ab, anti-IL-17R ab, or isotype controls to evaluate the potential effects of IL-17A/IL-17R blockade in the prevention of SAE. Septic peritonitis induced significant impairment of learning memory and exploratory activity, which was associated with a higher expression of IL-17A, IL-1β, and TNF-α in the brain homogenate. Fluorescence intensity of Iba-1 and IL-17R in the hippocampus was significantly increased following CLP. Treatment with recombinant IL-17A enhanced the neuroinflammation and microglia activation in CLP mice. On the contrary, neutralizing anti-IL-17A or anti-IL-17R antibodies mitigated the CNS inflammation and microglia activation, thus alleviating the cognitive dysfunction. Furthermore, as compared to the sham control, microglia cultured from CLP mice produced significantly higher levels of cytokines and expressed with higher fluorescence intensity of Iba-1 in response to IL-17A or LPS. Pretreatment with anti-IL-17R ab suppressed the Iba-1 expression and cytokine production in microglia stimulated by IL-17A. In conclusion, blockade of the IL-17A/IL-17R pathway inhibited microglia activation and neuroinflammation, thereby partially reversing sepsis-induced cognitive impairment. The present study suggested that the IL-17A/IL-17R signaling pathway had an important, nonredundant role in the development of SAE.

Ye B ，Tao T ，Zhao A ，Wen L ，He X ，Liu Y ，Fu Q ，Mi W ，Lou J ... -  《-》

An Fgr kinase inhibitor attenuates sepsis-associated encephalopathy by ameliorating mitochondrial dysfunction, oxidative stress, and neuroinflammation via the SIRT1/PGC-1α signaling pathway.

Sepsis-associated encephalopathy (SAE) is characterized by diffuse brain dysfunction, long-term cognitive impairment, and increased morbidity and mortality. The current treatment for SAE is mainly symptomatic; the lack of specific treatment options and a poor understanding of the underlying mechanism of disease are responsible for poor patient outcomes. Fgr is a member of the Src family of tyrosine kinases and is involved in the innate immune response, hematologic cancer, diet-induced obesity, and hemorrhage-induced thalamic pain. This study investigated the protection provided by an Fgr kinase inhibitor in SAE and the underlying mechanism(s) of action. A cecal ligation and puncture (CLP)-induced mouse sepsis model was established. Mice were treated with or without an Fgr inhibitor and a PGC-1α inhibitor/activator. An open field test, a novel object recognition test, and an elevated plus maze were used to assess neurobehavioral changes in the mice. Western blotting and immunofluorescence were used to measure protein expression, and mRNA levels were measured using quantitative PCR (qPCR). An enzyme-linked immunosorbent assay was performed to quantify inflammatory cytokines. Mitochondrial membrane potential and morphology were measured by JC-1, electron microscopy, and the MitoTracker Deep Red probe. Oxidative stress and mitochondrial dysfunction were analyzed. In addition, the regulatory effect of Fgr on sirtuin 1 (SIRT1) was assessed. CLP-induced sepsis increased the expression of Fgr in the hippocampal neurons. Pharmacological inhibition of Fgr attenuated CLP-induced neuroinflammation, the survival rate, cognitive and emotional dysfunction, oxidative stress, and mitochondrial dysfunction. Moreover, Fgr interacted with SIRT1 and reduced its activity and expression. In addition, activation of SIRT1/PGC-1α promoted the protective effects of the Fgr inhibitor on CLP-induced brain dysfunction, while inactivation of SIRT1/PGC-1α counteracted the benefits of the Fgr inhibitor. To our knowledge, this is the first report of Fgr kinase inhibition markedly ameliorating SAE through activation of the SIRT1/PGC-1α pathway, and this may be a promising therapeutic target for SAE.

Liu Y ，Yang H ，Luo N ，Fu Y ，Qiu F ，Pan Z ，Li X ，Jian W ，Yang X ，Xue Q ，Luo Y ，Yu B ，Liu Z ... -  《Journal of Translational Medicine》