Hydrogen-rich saline regulates NLRP3 inflammasome activation in sepsis-associated encephalopathy rat model.

Sepsis-associated encephalopathy (SAE) is characterised by long-term cognitive impairment and psychiatric illness in sepsis survivors, associated with increased morbidity and mortality. There is a lack of effective therapeutics for SAE. Molecular hydrogen (H2) plays multiple roles in septic diseases by regulating neuroinflammation, reducing oxidative stress parameters, regulating signalling pathways, improving mitochondrial dysfunction, and regulating astrocyte and microglia activation. Here we report the protective effect of hydrogen-rich saline in the juvenile SAE rat model and its possible underlying mechanisms. Rats were injected intraperitoneally with lipopolysaccharide at a dose of 5 mg/kg to induce sepsis; Hydrogen-rich saline (HRS) was administered 1 h after LPS induction at a dose of 5 ml/kg and nigericin at 1 mg/kg 1 h before LPS injection. H&E staining for neuronal damage, TUNEL assay for detection of apoptotic cells, immunofluorescence, ELISA protocol for inflammatory cytokines and 8-OHdG determination and western blot analysis to determine the effect of HRS in LPS-induced septic rats. Rats treated with HRS showed decreased TNF-α and IL-1ß expression levels. HRS treatment enhanced the activities of antioxidant enzymes (SOD, CAT and GPX) and decreased MDA and MPO activities. The number of MMP-9 and NLRP3 positive immunoreactivity cells decreased in the HRS-treated group. Subsequently, GFAP, IBA-1 and CD86 immunoreactivity were reduced, and CD206 increased after HRS treatment. 8-OHdG expression was decreased in the HRS-treated rats. Western blot analysis showed decreased NLRP3, ASC, caspase-1, MMP-2/9, TLR4 and Bax protein levels after HRS treatment, while Bcl-2 expression increased after HRS treatment. These data demonstrated that HRS attenuated neuroinflammation, NLRP3 inflammasome activation, neuronal injury, and mitochondrial damage via NLRP3/Caspase-1/TLR4 signalling in the juvenile rat model, making it a potential therapeutic agent in the treatment of paediatric SAE.

Melatonin Promotes Mitochondrial Biogenesis and Mitochondrial Degradation in Hepatocytes During Sepsis.

Objective: This study aimed to investigate the protective mechanisms of melatonin in an in vitro model of sepsis-induced hepatocyte injury, specifically focusing on mitophagy and mitochondrial biogenesis. Methods: In this study, we utilized lipopolysaccharide (LPS)-treated AML12 cells to establish an in vitro model of sepsis-induced hepatocyte injury. The effects of melatonin pretreatment were examined through various analyses, including assessments of oxidative stress, inflammation, mitophagy, mitochondrial biogenesis, and adenosine triphosphate (ATP) levels. Results: The results revealed that LPS-treated AML12 cells exhibited elevated levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 protein, intracellular reactive oxygen species (ROS), and lipid peroxidation, specifically malondialdehyde (MDA). Moreover, the levels of key markers associated with mitophagy, including PTEN-induced putative kinase 1 (PINK1), parkin, and LC3, were significantly increased (P < .05). Similarly, markers of mitochondrial biogenesis, such as peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (TFAM), were also significantly increased (P < .05). Conversely, superoxide dismutase (SOD) activity and ATP levels were significantly decreased in LPS-treated AML12 cells compared to the control group (P < .05). However, melatonin pretreatment led to a significant decrease in TNF-α and IL-6 protein levels, intracellular ROS, and MDA levels (P < .05), along with a significant increase in SOD activity, ATP levels, and markers of mitophagy and mitochondrial. Conclusions: Our findings demonstrate that melatonin plays a role in regulating mitochondrial quality control in sepsis-induced hepatocytes. It achieves this result by promoting mitophagy and inducing mitochondrial biogenesis, thereby selectively eliminating dysfunctional mitochondria.

Erratum - Transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) ameliorates sepsis-associated acute kidney injury by maintaining mitochondrial homeostasis and improving the mitochondrial function.

This corrects the article published in European Journal of Histochemistry 2022;66:3412. doi: 10.4081/ejh.2022.3412.

Bovine pulmonary surfactant alleviates inflammation and epithelial cell apoptosis in the early phase of lipopolysaccharide-induced acute lung injury in rats.

We investigate the impact of bovine pulmonary surfactant (PS) on LPS-induced ALI in vitro and in vivo to improve recognition and prevent mortality in sepsis-induced ALI. Primary alveolar type II (AT2) cells were treated with LPS alone or in combination with PS. Cell morphology observation, CCK-8 proliferation assay, flow cytometry apoptosis assay, and ELISA for inflammatory cytokine levels were performed at different time points after treatment. An LPS-induced ALI rat model was established and treated with vehicle or PS. Lung wet/dry weight ratio, histopathological changes, lung function parameters, and serum inflammatory cytokine levels were examined 6 h after PS treatment. Survival analysis by Kaplan-Meier method. RNA sequencing was conducted to identify LPS-induced differentially expressed genes in rat lungs. Proapoptotic gene expression in rat lungs was determined by Western blot. LPS significantly inhibited cell proliferation while promoting apoptosis of AT2 cells starting 2 h after treatment, accompanied by a significant increase in inflammatory cytokine production; PS reversed these effects. PS decreased the lung wet/dry ratio in septic rats, histological abnormalities, alterations in lung function parameters, and inflammatory cytokines production; while improving the overall survival of rats. LPS-induced differentially expressed genes were closely associated with apoptosis. PS attenuated LPS-induced upregulation of proapoptotic gene expression starting 2 h after treatment in AT2 cells while restoring lung ATPase activity in vivo. Bovine PS alleviates LPS-induced ALI in the early phase, possibly by suppressing inflammation and AT2 cell apoptosis, as a preemptive therapeutic agent for managing sepsis-induced ALI.

Lipopolysaccharide-induced endotoxaemia during adolescence promotes stress vulnerability in adult mice via deregulation of nuclear factor erythroid 2-related factor 2 in the medial prefrontal cortex.

RATIONALE: Sepsis is a severe inflammatory response to infection that leads to long-lasting cognitive impairment and depression after resolution. The lipopolysaccharide (LPS)-induced endotoxaemia model is a well-established model of gram-negative bacterial infection and recapitulates the clinical characteristics of sepsis. However, whether LPS-induced endotoxaemia during adolescence can modulate depressive and anxiety-like behaviours in adulthood remains unclear. OBJECTIVES: To determine whether LPS-induced endotoxaemia in adolescence can modulate the stress vulnerability to depressive and anxiety-like behaviours in adulthood and explore the underlying molecular mechanisms. METHODS: Quantitative real-time PCR was used to measure inflammatory cytokine expression in the brain. A stress vulnerability model was established by exposure to subthreshold social defeat stress (SSDS), and depressive- and anxiety-like behaviours were evaluated by the social interaction test (SIT), sucrose preference test (SPT), tail suspension test (TST), force swimming test (FST), elevated plus-maze (EPM) test, and open field test (OFT). Western blotting was used to measure Nrf2 and BDNF expression levels in the brain. RESULTS: Our results showed that inflammation occurred in the brain 24 h after the induction of LPS-induced endotoxaemia at P21 but resolved in adulthood. Furthermore, LPS-induced endotoxaemia during adolescence promoted the inflammatory response and the stress vulnerability after SSDS during adulthood. Notably, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and BDNF in the mPFC were decreased after SSDS exposure in mice treated with LPS during adolescence. Activation of the Nrf2-BDNF signalling pathway by sulforaphane (SFN), an Nrf2 activator, ameliorated the effect of LPS-induced endotoxaemia during adolescence on stress vulnerability after SSDS during adulthood. CONCLUSIONS: Our study identified adolescence as a critical period during which LPS-induced endotoxaemia can promote stress vulnerability during adulthood and showed that this effect is mediated by impairment of Nrf2-BDNF signalling in the mPFC.

Paediatric sepsis-associated encephalopathy (SAE): a comprehensive review.

Sepsis-associated encephalopathy (SAE) is one of the most common types of organ dysfunction without overt central nervous system (CNS) infection. It is associated with higher mortality, low quality of life, and long-term neurological sequelae, its mortality in patients diagnosed with sepsis, progressing to SAE, is 9% to 76%. The pathophysiology of SAE is still unknown, but its mechanisms are well elaborated, including oxidative stress, increased cytokines and proinflammatory factors levels, disturbances in the cerebral circulation, changes in blood-brain barrier permeability, injury to the brain's vascular endothelium, altered levels of neurotransmitters, changes in amino acid levels, dysfunction of cerebral microvascular cells, mitochondria dysfunction, activation of microglia and astrocytes, and neuronal death. The diagnosis of SAE involves excluding direct CNS infection or other types of encephalopathies, which might hinder its early detection and appropriate implementation of management protocols, especially in paediatric patients where only a few cases have been reported in the literature. The most commonly applied diagnostic tools include electroencephalography, neurological imaging, and biomarker detection. SAE treatment mainly focuses on managing underlying conditions and using antibiotics and supportive therapy. In contrast, sedative medication is used judiciously to treat those showing features such as agitation. The most widely used medication is dexmedetomidine which is neuroprotective by inhibiting neuronal apoptosis and reducing a sepsis-associated inflammatory response, resulting in improved short-term mortality and shorter time on a ventilator. Other agents, such as dexamethasone, melatonin, and magnesium, are also being explored in vivo and ex vivo with encouraging results. Managing modifiable factors associated with SAE is crucial in improving generalised neurological outcomes. From those mentioned above, there are still only a few experimentation models of paediatric SAE and its treatment strategies. Extrapolation of adult SAE models is challenging because of the evolving brain and technical complexity of the model being investigated. Here, we reviewed the current understanding of paediatric SAE, its pathophysiological mechanisms, diagnostic methods, therapeutic interventions, and potential emerging neuroprotective agents.

Effects of hydrogen-rich saline in neuroinflammation and mitochondrial dysfunction in rat model of sepsis-associated encephalopathy.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is one of the most common types of sepsis-related organ dysfunction without overt central nervous system (CNS) infection. It is associated with higher mortality, low quality of life, and long-term neurological sequelae in suspected patients. At present there is no specific treatment for SAE rather than supportive therapy and judicious use of antibiotics, which are sometimes associated with adverse effects. Molecular hydrogen (H2) has been reported to play crucial role in regulating inflammatory responses, neuronal injury, apoptosis and mitochondrial dysfunction in adult models of SAE. Here we report the protective effect of hydrogen-rich saline in juvenile SAE rat model and its possible underling mechanism(s). MATERIALS AND METHODS: Rats were challenged with lipopolysaccharide (LPS) at a dose of 8 mg/kg injected intraperitoneally to induce sepsis and hydrogen-rich saline (HRS) administered 1 h following LPS induction at a dose of 5 ml/kg. Rats were divided into: sham, sham + HRS, LPS and LPS + HRS. At 48 h, rats were sacrificed and Nissl staining for neuronal injury, TUNEL assay for apoptotic cells detection, immunohistochemistry, and ELISA protocol for inflammatory cytokines determination, mitochondrial dysfunction parameters, electron microscopy and western blot analysis were studied to examine the effect of HRS in LPS-induced septic rats. RESULTS: Rats treated with HRS improved neuronal injury, improvement in rats' survival rate. ELISA analysis showed decreased TNF-α and IL-1ß and increased IL-10 expression levels in the HRS-treated group. Apoptotic cells were decreased after HRS administration in septic rats. The numbers of GFAP and IBA-1positive cells were attenuated in the HRS-treated group when compared to the LPS group. Subsequently, GFAP and IBA-1 immunoreactivity were decreased after HRS treatment. Mitochondrial membrane potential detected by JC-1 dye and ATP content were decreased in septic rats, which were improved after HRS treatment, while release of ROS was increased in the LPS group reverted by HRS treatment, ameliorating mitochondrial dysfunction. Further analysis by transmission electron microscopy showed decreased number of mitochondria and synapses, and disrupted mitochondrial membrane ultrastructure in the LPS group, while HRS administration increased mitochondria and synapses number. CONCLUSION: These data demonstrated that HRS can improve survival rate, attenuate neuroinflammation, astrocyte and microglial activation, neuronal injury and mitochondrial dysfunction in juvenile SAE rat model, making it a potential therapeutic candidate in treating paediatric SAE.

Transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) ameliorates sepsis-associated acute kidney injury by maintaining mitochondrial homeostasis and improving the mitochondrial function.

Mitochondrial dysfunction has a role in sepsis-associated acute kidney injury (S-AKI), so the restoration of normal mitochondrial homeostasis may be an effective treatment strategy. Transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) is a main regulator of cell-redox homeostasis, and recent studies reported that NRF2 activation helped to preserve mitochondrial morphology and function under conditions of stress. However, the role of NRF2 in the process of S-AKI is still not well understood. The present study investigated whether NRF2 regulates mitochondrial homeostasis and influences mitochondrial function in S-AKI. We demonstrated activation of NRF2 in an in vitro model: lipopolysaccharide (LPS) challenge of ductal epithelial cells of rat renal tubules (NRK-52e cells), and an in vivo model: cecal ligation and puncture (CLP) of rats. Over-expression of NRF2 attenuated oxidative stress, apoptosis, and the inflammatory response; enhanced mitophagy and mitochondrial biogenesis; and mitigated mitochondrial damage in the in vitro model. In vivo experiments showed that rats treated with an NRF2 agonist had higher adenosine triphosphate (ATP) levels, lower blood urea nitrogen and creatinine levels, fewer renal histopathological changes, and higher expression of mitophagy-related proteins [PTEN-induced putative kinase 1 (PINK1), parkin RBR E3 ubiquitin protein ligase (PRKN), microtubule-associated protein 1 light chain 3 II (LC3 II)] and mitochondrial biogenesis-related proteins [peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α) and mitochondrial transcription factor A (TFAM)]. Electron microscopy of kidney tissues showed that mitochondrial damage was alleviated by treatment with an NRF2 agonist, and the opposite response occurred upon treatment with an NRF2 antagonist. Overall, our findings suggest that mitochondria have an important role in the pathogenesis of S-AKI, and that NRF2 activation restored mitochondrial homeostasis and function in the presence of this disease. This mitochondrial pathway has the potential to be a novel therapeutic target for the treatment of S-AKI.

Continuous Renal-Replacement Therapy in Critically Ill Children: Practice Changes and Association With Outcome.

OBJECTIVES: This study was designed to evaluate practice changes and outcomes over a 10-year period in a large single-center PICU cohort that received continuous renal-replacement therapy. DESIGN: Retrospective study design. SETTING: A multidisciplinary tertiary PICU of a university-affiliated hospital in Guangzhou, China. PATIENTS: All critically ill children who were admitted to our PICU from January 2010 to December 2019 and received continuous renal-replacement therapy were included in this study. MEASUREMENTS AND MAIN RESULTS: A total of 289 patients were included in the study. Of the two study periods, 2010-2014 and 2015-2019, the proportion of continuous renal-replacement therapy initiation time greater than 24 hours was significantly reduced ([73/223] 32.73% vs. [40/66] 60.60%, p < 0.001), the percentage of fluid overload at continuous renal-replacement therapy initiation was lower (3.8% [1.6-7.2%] vs. 12.1% [6.6-23.3%], p < 0.001), the percentage of regional citrate anticoagulation protocol was increased ([223/223] 100% vs. [15/66] 22.7%, p < 0.001), and the ICU survival rate was significantly improved ([24/66] 36.4% vs. [131/223] 58.7%, p = 0.001) in the latter period compared with the former. In addition, subgroup analysis found that survival were higher in patients with continuous renal-replacement therapy initiation time less than 24 hours, regional citrate anticoagulation protocol, and fluid overload less than 10%. CONCLUSIONS: The survival rate of patients received continuous renal-replacement therapy treatment in our center has improved over past 10 years, and some changes have taken place during these periods. Among them, early initiation of continuous renal-replacement therapy, lower fluid overload, and regional citrate anticoagulation method seems to be related to the improvement of outcome. Ongoing evaluation of the practice changes and quality improvement of continuous renal-replacement therapy for critically ill pediatric patients still need attention.

Persistent viral shedding of human adenovirus type 7 in children with severe pneumonia.

To understand host-pathogen interactions and develop effective prevention and control strategies for human adenovirus (HAdV), it is essential to explore the characteristics of HAdV shedding. Hospitalized children <14 years who had severe HAdV pneumonia were tested for HAdV DNA by quantitative real-time PCR in nasopharyngeal aspirate (NPA). A total of 132 children were enrolled, including 102 patients with HAdV type 7 (HAdV-7) infection and 12 patients with HAdV type 3 (HAdV-3) infection. A total of 1372 qualified NPA samples were collected. There was a significant negative correlation between the viral load of HAdV and the course of the disease (Spearman r = -0.547, p = .000). HAdV-7 load decreased at a rate of 0.089 log10 copies/mL per day (95% CI: -0.096 to -0.081; R 2 = 0.332), and the duration of viral shedding was predicted to be 96.9 days (y = 8.624-0.089x). However, HAdV-3 load decreased more quickly (95% CI: - 0.229 to - 0.143; R 2 = 0.403), and the duration of viral shedding was 51.4 days (y = 9.558-0.186x). The median viral load of the HAdV-7 group at weeks 2 and 3, and more than 3 weeks postinfection was higher than that of the HAdV-3 group. No significant differences in the duration of viral shedding were found in different gender, age (>2 vs. ≤2 years), and with or without underlying diseases groups. Viral shedding in children with severe HAdV pneumonia persisted, among which HAdV-7 lasted longer than 3 months and the viral load decreased slowly than HAdV-3.

Genetic Polymorphisms of LPCAT1, CHPT1 and PCYT1B and Risk of Neonatal Respiratory Distress Syndrome among a Chinese Han Population.

BACKGROUND: The study of genetic polymorphisms of surfactant-lipids related genes can help to understand individual variability in the susceptibility to development of pulmonary pathologies. The purpose of this study was to evaluate the association of polymorphisms of surfactant-lipids related genes (LPCAT1, CHPT1 and PCYT1B) with the risk/severity of respiratory distress syndrome (RDS) in preterm neonates among the Chinese Han population in Southern China. METHODS: Four hundred and forty-six preterm neonates were enrolled in a case-control study. Six polymorphisms of 3 genes were analyzed by PCR amplification of genomic DNA and genotyping was performed using an improved multiplex ligation detection reaction (iMLDR) technique based on LDR. RESULTS: The GG genotype and G allele of LPCAT1-rs9728 were found less frequently in the RDS group than in the controls (11.5% vs. 22.0% and 38.3% vs. 48.2%, respectively) (p < 0.05). CONCLUSION: This report is the first study to evaluate a direct genetic association between polymorphisms of LPCAT1 and RDS development in Chinese Han preterm infants. Our study raises the possibility that a genetic variation of LPCAT1 could be implicated in the pathophysiology of RDS in preterm neonates. GG genotype and G allele of rs9728 are protective factors for the development of RDS in preterm infants.

UCP2 silencing aggravates mitochondrial dysfunction in astrocytes under septic conditions.

Uncoupling protein 2 (UCP2) plays a positive role in sepsis. However, the role of UCP2 in experimental sepsis in astrocytes remains unknown. The present study was designed to determine whether UCP2 has a protective effect in an experimental sepsis model in astrocytes asnd to clarify the mechanisms responsible for its neuroprotective effects after sepsis. An experimental astrocyte model mimicking sepsis­induced brain injury was established using lipopolysaccharide (LPS) and interferon (IFN)­Î³. Additionally, UCP2 knockdown in astrocytes was achieved by adenovirus transfection. Tumor necrosis factor (TNF)­α and interleukin (IL)­1ß activity, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels were assessed. The mitochondrial ultrastructure was evaluated, and the expression of UCP2 was determined by western blotting. LPS with IFN­Î³ co­stimulation increased the mRNA and protein expression levels of UCP2 in astrocytes, damaged the mitochondrial structure, and accelerated the release of TNF­α and IL­1ß, resulting in a decrease in the MMP, and the excessive generation of ROS. Moreover, sepsis also caused a reduction in ATP production. The knockdown of UCP2 exacerbated astrocyte injury and mitochondrial impairment. In conclusion, both the function and morphology of mitochondria were damaged in an experimental model of sepsis in astrocytes, and knockdown of UCP2 using shRNA exacerbated this impairment, suggesting that UCP2 has a positive effect on astrocytes as determined in an experimental sepsis model.

Upregulation of UCP2 Expression Protects against LPS-Induced Oxidative Stress and Apoptosis in Cardiomyocytes.

Uncoupling protein 2 (UCP2) has a cardioprotective role under septic conditions, but the underlying mechanism remains unclear. This study aimed at investigating the effects of UCP2 on the oxidative stress and apoptosis of cardiomyocytes induced by lipopolysaccharide (LPS). First, LPS increased UCP2 expression in cardiomyocytes in a time-dependent manner. LPS increased the production of lactate dehydrogenase (LDH), reactive oxygen species (ROS), and malondialdehyde (MDA) and decreased the level of superoxide dismutase (SOD). However, UCP2 knockdown increased the LPS-induced cardiac injury and oxidative stress. In addition, LPS damaged the mitochondrial ultrastructure and led to the disruption of mitochondrial membrane potential (MMP), as well as the release of mitochondrial cytochrome c. UCP2 knockdown aggravated mitochondrial injury and the release of mitochondrial cytochrome c. LPS increased the protein levels of Bax and cleaved-caspase-3, decreased the protein level of Bcl-2, and upregulated the protein level of mitogen-activated protein kinase. However, upon UCP2 knockdown, the protein levels of Bax and cleaved-caspase-3 increased even further, and the protein level of Bcl-2 was further decreased. The protein level of phosphorylated p38 was also further enhanced. Thus, UCP2 protects against LPS-induced oxidative stress and apoptosis in cardiomyocytes.

UCP2 ameliorates mitochondrial dysfunction, inflammation, and oxidative stress in lipopolysaccharide-induced acute kidney injury.

OBJECTIVE: UCP2 is involved in the maintenance of mitochondrial function, immune response and regulation of oxidative stress under physiological or pathological conditions. The aim of this study was to investigate the effects of UCP2 on mitochondrial dysfunction, inflammation, and oxidative stress in septic acute kidney injury (AKI). METHODS: We established LPS-induced AKI model in mice and HK-2 cells. In vivo, the UCP2 inhibitor genipin was used to downregulate UCP2 in mouse kidneys. In vitro, UCP2 overexpression or knockdown was achieved by LV5-UCP2 or si-UCP2 transfection, respectively, to characterize the mechanisms of UCP2 in septic AKI. Indicators of renal injury, cell apoptosis, inflammation, oxidative stress, and mitochondrial dysfunction were assessed. RESULTS: Compared to the control group, LPS treatment increased UCP2 expression in vitro and in vivo. In vitro, UCP2 overexpression protected HK-2 cells from LPS-induced injury by suppression of apoptosis, inflammation, oxidative stress, MMP loss and ROS production, increase of ATP production and mtDNA content, and amelioration of damage to the mitochondrial ultrastructure. Additionally, inhibition of UCP2 expression by si-UCP2 resulted in decreased HK-2 cell resistance to LPS toxicity, as shown by increased apoptosis, inflammation, mitochondrial dysfunction and oxidative stress. In vivo, UCP2 downregulation aggravated the LPS-induced renal injury, inflammation, macrophages infiltration, mitochondrial dysfunction, and oxidative stress. CONCLUSION: UCP2 may protect LPS-induced AKI by ameliorating mitochondrial dysfunction, anti-inflammation, and antioxidative activities, ultimately inhibiting tubule epithelial cell apoptosis, and that increasing the UCP2 content in mitochondria constitutes a new therapeutic approach for septic AKI.

A New Neural Pathway from the Ventral Striatum to the Nucleus Basalis of Meynert with Functional Implication to Learning and Memory.

The cholinergic neurons in the nucleus basalis of Meynert (NBM) are among the first group of neurons known to become degenerated in Alzheimer's disease, and thus the NBM is proposed to be involved in learning and memory. The marginal division (MrD) of the striatum is a newly discovered subdivision at the ventromedial border of the mammalian striatum and is considered to be one part of the ventral striatum involved in learning and memory. The present study provided evidence to support the hypothesis that the MrD and the NBM were structurally connected at cellular and subcellular levels with functional implications in learning and memory. First, when wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was stereotaxically injected into the NBM, fusiform neurons in the MrD were retrogradely labeled with WGA-HRP gray-blue particles and some of them were double stained in brown color by AchE staining method. Thus, cholinergic neurons of the MrD were shown to project to the neurons in the NBM. Second, in anterograde tract-tracing experiments where WGA-HRP was injected to the MrD, the labeled WGA-HRP was found to be anterogradely transported in axons from the MrD to the synaptic terminals with dendrites, axons, and perikaryons of the cholinergic neurons in the NBM when observed under an electronic microscope, indicating reciprocal structural connections between the MrD and the NBM. Third, when bilateral lesions of the MrD were injured with kainic acid in rats, degenerative terminals were observed in synapses of the NBM by an electronic microscope and severe learning and memory deficiency was found in these rats by the Y-maze behavioral test. Our results suggest reciprocal cholinergic connections between the MrD of the ventral striatum and the NBM, and implicate a role of the MrD-NBM pathway in learning and memory. The efferent fibers of cholinergic neurons in the NBM mainly project to the cortex, and severe reduction of the cholinergic innervation in the cortex is the common feature of Alzheimer's patients. The newly discovered cholinergic neural pathway between the MrD of the ventral striatum and the NBM is supposed involved in the memory circuitries of the brain and probably might play a role in the pathogenesis of the Alzheimer's disease.

Genome-wide CRISPR-Cas9 viability screen reveals genes involved in TNF-α-induced apoptosis of human umbilical vein endothelial cells.

Tumor necrosis factor α (TNF-α), a pivotal cytokine in sepsis, protects the host against pathogens by promoting an inflammatory response while simultaneously inducing apoptosis of the vascular endothelium. Unfortunately, inhibitors targeting certain components of the TNF-α signaling pathway to reduce cellular apoptosis have failed to translate into clinical applications, partly due to the adverse effects of excessive immunosuppression. In an attempt to discover potential targets in the TNF-α signaling pathway to modulate moderate inflammation and apoptosis during the development of sepsis, we performed a pooled genome-wide CRISPR/Cas9 knockout screen in human umbilical vein endothelial cells (HUVECs). Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), B-cell lymphoma 2 (BCL2), Bcl2-associated death promoter (BAD), and NLR family member X1 (NLRX1) deficiencies were identified as the effective genetic suppressors of TNF-α cytotoxicity on a list of candidate regulators. CRISPR-mediated NLRX1 knockout conferred cellular resistance to challenge with TNF-α, and NLRX1 could be induced to colocalize with mitochondria following TNF-α stimulation. Thus, our work demonstrates the advantage of genome-scale screening with Cas9 and validates NLRX1 as a potential modulator of TNF-α-induced vascular endothelial apoptosis during sepsis.

GDF­15 prevents LPS and D­galactosamine­induced inflammation and acute liver injury in mice.

Growth differentiation factor­15 (GDF­15) is a transforming growth factor (TGF)­ß superfamily member with a poorly characterized biological activity, speculated to be implicated in several diseases. The present study aimed to determine whether GDF­15 participates in sepsis­induced acute liver injury in mice. Lipopolysaccharide (LPS) and D­galactosamine (D­GalN) were administered to mice to induce acute liver injury. Survival of mice, histological changes in liver tissue, and levels of inflammatory biomarkers in serum and liver tissue were evaluated following treatment with GDF­15. The underlying mechanism was investigated by western blotting, ELISA, flow cytometry, and reverse transcription­quantitative polymerase chain reaction using Kupffer cells. The results demonstrated that GDF­15 prevented LPS/D­GalN­induced death, increase in inflammatory cell infiltration and serum alanine aminotransferase and aspartate aminotransferase activities. In addition, GDF­15 treatment reduced the production of hepatic malondialdehyde and myeloperoxidase, and attenuated the increase of interleukin (IL)­6, tumor necrosis factor (TNF)­α, and IL­1ß expression in serum and liver tissue, accompanied by inducible nitric oxide synthase (iNOS) inactivation in the liver. Similar changes in the expression of inflammatory cytokines, IL­6, TNF­α and IL­1ß, and iNOS activation were observed in the Kupffer cells. Further mechanistic experiments revealed that GDF­15 effectively protected against LPS­induced nuclear factor (NF)­κB pathway activation by regulating TGFß­activated kinase 1 (TAK1) phosphorylation in Kupffer cells. In conclusion, GDF­15 reduced the activation of pro­inflammatory factors, and prevented LPS­induced liver injury, most likely by disrupting TAK1 phosphorylation, and consequently inhibiting the activation of the NF­κB pathway in the liver.

Weight and the vitamin K expoxide reductase 1 genotype primarily contribute to the warfarin dosing in pediatric patients with Kawasaki disease.

INTRODUCTION: Warfarin therapy is recommended in children with giant coronary artery aneurysms (GCAAs) after Kawasaki disease (KD). Large individual variability makes it difficult to predict the warfarin dose. Polymorphisms in the vitamin K expoxide reductase 1 (VKORC1) and cytochrome P4502C9 (CYP2C9) genes have been reported to influence the warfarin dose. We investigated the effects of the VKORC1 and CYP2C9 genotypes on the warfarin dose in pediatric patients with giant CAAs after KD. We attempted to create a dosing algorithm. MATERIALS AND METHODS: The clinical and genetic data of patients were documented. VKORC1 (rs 9923231) and CYP2C9 *3 (rs 1057910) were genotyped using TaqMan real-time polymerase chain reaction. A linear regression analysis was performed to evaluate the contribution of clinical and genetic factors to the warfarin maintenance dose. RESULTS: Forty-seven patients were enrolled. Patients with the CT or CC genotype of VKORC1 had a relatively higher warfarin dose than did those with the TT genotype (p < 0.05). Three patients with CYP2C9*1/*3 had a lower warfarin dose than did those with the wild CYP2C9*1/*1 genotype, but the difference did not reach significance (p > 0.05). Weight and the VKORC1 genotype predominantly contributed to the warfarin dose, with 33.0% and 11.2% of variability, respectively. The observed warfarin dose was correlated with the predicted dose based on the algorithm used in our study (r = 0.45, p < 0.01). CONCLUSIONS: Weight and the VKORC1 genotype primarily determined the warfarin dose in Chinese pediatric patients with KD. Further studies are warranted to verify the findings of our study.

Effects of interleukin-6 and IL-6/AMPK signaling pathway on mitochondrial biogenesis and astrocytes viability under experimental septic condition.

OBJECTIVE: Interleukin-6 (IL-6) is a neuromodulation factor with extensive and complex biological activities. IL-6 has been reported to activate AMPK, while AMPK regulates mitochondrial biogenesis and autophagy. The aim of this study was to investigate the role of IL-6 in mitochondrial biogenesis using astrocytes under experimental septic condition and examined how IL-6/AMPK signaling pathway affected this process. METHODS: The primary cultures of cerebral cortical astrocytes were randomly allocated into six groups: control group, LPS+IFN-γ group, IL-6 group (LPS+IFN-γ+IL-6), C group (LPS+IFN-γ+IL-6+Compound C), siRNA group (LPS+IFN-γ+IL-6+IL-6R siRNA) and siRNA+C group (LPS+IFN-γ+IL-6+IL-6R siRNA+ Compound C). All groups were stimulated for 6 h. Cytokines and reactive oxygen species (ROS) analyses, detection of adenosine triphosphate (ATP), mtDNA content and cell viability, evaluation of the mitochondrial ultrastructure and volume density, western blots of proteins associated with mitochondrial biogenesis and phospho-adenosine monophosphate activated protein kinase (p-AMPK) were performed respectively. RESULTS: Compared with LPS+IFN-γ group, IL-6 group had milder ultrastructural damage of mitochondria, higher mtDNA content and mitochondrial volume density, higher expression of proteins associated with mitochondrial biogenesis (PGC-1α, NRF-1 and TFAM) and p-AMPK, and thus higher cell viability, whereas blocking IL-6/AMPK signaling pathway, the protective effect of IL-6 has been diminished, compared with IL-6 group. CONCLUSION: IL-6 enhances mitochondrial biogenesis in astrocytes under experimental septic condition through IL-6/AMPK signaling pathway.

Insulin alleviates mitochondrial oxidative stress involving upregulation of superoxide dismutase 2 and uncoupling protein 2 in septic acute kidney injury.

The aim of the present study was to explore the effects and mechanisms of insulin on mitochondrial oxidative stress in septic acute kidney injury (AKI). Male Sprague Dawley rats were divided randomly into four groups: Control group, sham surgery group, cecal ligation and puncture (CLP) group, and CLP plus insulin group. Blood specimens and kidney tissues were obtained at 12 and 24 h after surgery as separate experiments. Analyses of histology and indicators of renal injury [blood urea nitrogen (BUN) and serum creatinine (CRE) and neutrophil gelatinase-associated lipocalin (NGAL)], mitochondrial function [adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP)], oxidative stress [inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS) and nitric oxide (NO)], endogenous antioxidant systems [superoxide dismutase (SOD) and glutathione (GSH)] as well as the expression of uncoupling protein (UCP), PINK1 protein (a major mediator of mitophagy), PGC1α protein (a major regulator of mitochondrial biogenesis) were performed. Compared with CLP group, the CLP plus insulin group had milder histological damage, higher levels of ATP and MMP as well as lower levels of BUN, serum CRE and NGAL, intrarenal iNOS, mitochondrial ROS and total NO. Moreover, the CLP plus insulin group demonstrated increased expression of SOD2 and UCP2. In contrast, insulin administration suppressed mitophagy meanwhile did not upregulate total GSH and induce mitochondrial biogenesis following CLP. These findings indicated that the upregulation of SOD2 and UCP2 may be involved in insulin protecting against mitochondrial oxidative stress in septic AKI.