Overexpressed ski efficiently promotes neurorestoration, increases neuronal regeneration, and reduces astrogliosis after traumatic brain injury.

Traumatic brain injury (TBI) survivors suffer from long-term disability and neuropsychiatric sequelae due to irreparable brain tissue destruction. However, there are still few efficient therapies to promote neurorestoration in damaged brain tissue. This study aimed to investigate whether the pro-oncogenic gene ski can promote neurorestoration after TBI. We established a ski-overexpressing experimental TBI mouse model using adenovirus-mediated overexpression through immediate injection after injury. Hematoxylin-eosin staining, MRI-based 3D lesion volume reconstruction, neurobehavioral tests, and analyses of neuronal regeneration and astrogliosis were used to assess neurorestorative efficiency. The effects of ski overexpression on the proliferation of cultured immature neurons and astrocytes were evaluated using imaging flow cytometry. The Ski protein level increased in the perilesional region at 3 days post injury. ski overexpression further elevated Ski protein levels up to 14 days post injury. Lesion volume was attenuated by approximately 36-55% after ski overexpression, with better neurobehavioral recovery, more newborn immature and mature neurons, and less astrogliosis in the perilesional region. Imaging flow cytometry results showed that ski overexpression elevated the proliferation rate of immature neurons and reduced the proliferation rate of astrocytes. These results show that ski can be considered a novel neurorestoration-related gene that effectively promotes neurorestoration, facilitates neuronal regeneration, and reduces astrogliosis after TBI.

Activation of brain lactate receptor GPR81 aggravates exercise-induced central fatigue.

Exercise-induced fatigue is a complex physiological phenomenon and is greatly influenced by central mechanisms in brain. As one of the most abundant circulating carbon metabolites, l-lactate in brain has been considered to be an important supplementary fuel during exercise; however, whether it plays a signaling role in fatigue remains largely obscure. In this study, our results initially revealed that brain l-lactate levels were increased after an exhaustive swimming session in several brain regions including motor cortex, hippocampus, and cerebellum. Then, we examined the specific role of brain lactate receptor, also known as hydroxycarboxylic acid receptor 1 (GPR81), in exercise-induced fatigue. We found that intracerebroventricular injection of either d-lactate (an enantiomer that could mediate activation of GPR81 as l-lactate) or a potent GPR81 agonist 3-chloro-5-hydroxybenzoic acid (CHBA), significantly decreased the swimming time to fatigue. After being subjected to the same weight-loaded swimming for 30 min, no obvious changes of blood lactate levels, gastrocnemius pAMPK/AMPK ratio, and glycogen contents were observed between intracerebroventricular CHBA-injected mice and vehicle-treated ones, which suggested a comparable degree of peripheral fatigue. Meanwhile, there were higher extracellular γ-aminobutyric acid (GABA) levels and lower extracellular glutamate levels and glutamate/GABA ratio in motor cortex of the intracerebroventricular CHBA-injected mice than that of vehicle-treated ones, indicating a greater extent of central fatigue in CHBA-injected mice than that in vehicle animals. Collectively, our results suggested that an increased level of brain l-lactate acts as a signaling molecule via activating GPR81, which in turn exacerbates central fatigue during exercise.

Novel HIF-1-target gene isthmin1 contributes to hypoxia-induced hyperpermeability of pulmonary microvascular endothelial cells monolayers.

Hypoxia-induced pulmonary microvascular endothelial cell (PMVEC) monolayers hyperpermeability is vital for vascular leakage, which participates in vascular diseases, such as acute lung injury (ALI) and high-altitude pulmonary edema (HAPE). We previously observed that PMVEC permeability was markedly elevated in hypoxia when cocultured with primary type II alveolar epithelial cells (AECII) in which isthmin1 (ISM1) was highly upregulated. However, whether the upregulation of ISM1 plays a role in hypoxia-induced PMVEC hyperpermeability is unclear. In this study, we assessed the role of AECII-derived ISM1 in hypoxia-induced PMVEC hyperpermeability with an AECII/PMVEC coculture system and uncovered the underlying mechanism whereby hypoxia stimulates ISM1 gene expression. We found that ISM1 gene expression was upregulated in cultured AECII cells exposed to hypoxia (3% O2) and that AECII-derived ISM1 participated in hypoxia-induced hyperpermeability of PMVEC monolayers, as small interference RNA (siRNA)-mediated knockdown of ISM1 in AECII markedly attenuated the increase in PMVEC permeability in coculture system under hypoxia. In addition, we confirmed that ISM1 was regulated by hypoxia-inducible factor-1α (HIF1α) according to the evidence that silencing of HIF1α inhibited the hypoxia-mediated upregulation of ISM1. Mechanismly, overexpression of HIF1α transcriptionally activated ISM1 gene expression by directly binding to the conserved regulatory elements upstream of the ism1 locus. We identified a novel HIF-1-target gene ISM1, which involves in hyperpermeability of pulmonary microvascular endothelial cell monolayers under hypoxia. Our in vitro cell experiments implied that the upregulated ISM1 derived from alveolar epithelium might be a vital modulator in hypoxia-induced endothelial hyperpermeability and thereby implicates with hypoxic pulmonary-related diseases.

HIF1α-induced upregulation of KLF4 promotes migration of human vascular smooth muscle cells under hypoxia.

Hypoxia-induced vascular smooth muscle cells (VSMCs) migration plays an important role in vascular remodeling and is implicated in vascular diseases, such as atherosclerosis and pulmonary hypertension. We previously observed the increased expression of krüppel-like factor 4 (KLF4) in VSMCs under hypoxia. However, whether the upregulation of KLF4 participates in hypoxia-induced VSMCs migration is still unknown. In this study, we demonstrated that KLF4 was an important player in the process of VSMCs migration under hypoxia since interference of KLF4 by small interfering RNA mostly dampened hypoxia-induced migration of VSMCs. In addition, using luciferase reporter and ChIP assays, we confirmed two hypoxia-inducible factor 1α (HIF1α) binding elements (located at -150 to -163 and -3922 to -3932) in the upstream regulatory region of klf4 locus and identified KLF4 as a novel direct target gene of HIF1α. Our findings unveil a novel regulatory mechanism that involves HIF1α-induced upregulation of KLF4, which plays a vital role in VSMCs migration under hypoxia.

PKC Mediates LPS-Induced IL-1ß Expression and Participates in the Pro-inflammatory Effect of A2AR Under High Glutamate Concentrations in Mouse Microglia.

Pathogens such as bacterial lipopolysaccharide (LPS) play an important role in promoting the production of the inflammatory cytokines interleukin-1 beta (IL-1ß) and tumour necrosis factor-α (TNF-α) in response to infection or damage in microglia. However, whether different signalling pathways regulate these two inflammatory factors remains unclear. The protein kinase C (PKC) family is involved in the regulation of inflammation, and our previous research showed that the activation of the PKC pathway played a key role in the LPS-induced transformation of the adenosine A2A receptor (A2AR) from anti-inflammatory activity to pro-inflammatory activity under high glutamate concentrations. Therefore, in the current study, we investigated the role of PKC in the LPS-induced production of these inflammatory cytokines in mouse primary microglia. GF109203X, a specific PKC inhibitor, inhibited the LPS-induced expression of IL-1ß messenger ribonucleic acid and intracellular protein in a dose-dependent manner. Moreover, 5 µM GF109203X prevented LPS-induced IL-1ß expression but did not significantly affect LPS-induced TNF-α expression. PKC promoted IL-1ß expression by regulating the activity of NF-κB but did not significantly impact the activity of ERK1/2. A2AR activation by CGS21680, an A2AR agonist, facilitated LPS-induced IL-1ß expression through the PKC pathway at high glutamate concentrations but did not significantly affect LPS-induced TNF-α expression. Taken together, these results suggest a new direction for specific intervention with LPS-induced inflammatory factors in response to specific signalling pathways and provide a mechanism for A2AR targeting, especially after brain injury, to influence inflammation by interfering with A2AR.

Adenosine A2A receptor inhibition restores the normal transport of endothelial glutamate transporters in the brain.

Excitatory amino acid transporters (EAATs) on cerebral vascular endothelial cells play an important role in maintaining glutamate homeostasis in the brain. The dysfunction of endothelial EAATs is an important reason for the dramatically elevated brain glutamate levels after brain injury, such as traumatic brain injury (TBI). The adenosine A2A receptor (A2AR) plays an important role in regulating the brain glutamate level after brain injury; however, researchers have not clearly determined whether this role was related to its ability to regulate endothelial EAATs. Activation of A2AR in vitro not only decreased the PKA- and glutamate level-dependent strengthening of the interaction between NKA-α1 and the FXYD1 subunit and the subsequent decrease in the activity of Na+/K+-ATPases (NKAs) but also enhanced its interaction with EAATs and ultimately aggravated the reverse transport function of endothelial EAATs under oxygen-glucose deprivation (OGD) conditions. Conversely, inhibition of A2AR restored the normal transport of EAAT. Moreover, A2AR inhibition increased NKA activity and decreased its interaction with EAATs in isolated brain capillaries after TBI, further confirming its role in endothelial EAATs in vivo. Based on our results, A2AR played an important role in regulating endothelial EAAT function, and strategies that restore the normal transport of endothelial EAATs through the inhibition of A2AR might serve as an effective treatment for brain injury.

Hsp90 regulation affects the treatment of glucocorticoid for pancreatitis-induced lung injury.

Glucocorticoids are commonly used for the treatment of pancreatitis and complicated acute lung injury and help to reduce the mortality rates of both. The effect of gene variants in heat shock protein 90 (Hsp90), a key chaperone molecule of the glucocorticoid receptor (GR), on the therapeutic effect of glucocorticoids is unclear. Our study aims to investigate the different susceptibility to glucocorticoid treatment in BALB/c and C57BL/6 mice carrying different Hsp90 genotypes in an animal model of pancreatitis-induced lung injury. Compared with BALB/c mice, C57BL/6 mice have lower mortality rates, decreased water content in their lungs, and a lower level of IL-1 beta in an animal model of acute pancreatitis. C57BL/6 mice show a greater therapeutic effect and increased GR binding activities with glucocorticoid responsive element compared to BALB/c mice after a 0.4 mg/kg dexamethasone (DEX) treatment. Treatment with a higher dose of DEX (4 mg/kg) significantly reduced mortality rates and increased GR-GRE binding activity in both strains of mice, and there was no significant difference between the two strains. DEX did not exert a protective role after geldanamycin, a specific inhibitor of Hsp90, was administered in both strains of mice. Our study revealed that Hsp90 gene variants are responsible for the greater therapeutic effect of DEX in C57BL/6 mice compared to BALB/c mice, which implies that combining DEX treatment with Hsp90 regulation would promote the efficiency of DEX and would be an effective way to alleviate the side effects of hormone therapy.

Comparative evaluation of the wound-healing potency of recombinant bFGF and ski gene therapy in rats.

We previously demonstrated that cellular Sloan-Kettering Institute (c-Ski) played a dual role, both promoting wound healing and alleviating scar formation. However, its mechanism and therapeutic effects are not clear, especially compared with widely used treatments, such as basic fibroblast growth factor (bFGF) administration. However, Ski treatment led to an even shorter healing time and a more significant reduction in scar area than bFGF treatment. The mechanism underlying this difference was related to a reduced inflammatory response, more rapid re-epithelialization, less collagen after healing and a greater reduction in the proportion of alpha-smooth muscle actin and SMemb-positive cells after Ski treatment. These results not only confirm that Ski plays a dual role in promoting healing and reducing scarring but also suggest that Ski yields better treatment effects than bFGF, indicating better potential therapeutic effects in wound repair.

Somatostatin Reduces the Acute Lung Injury of Mice via Increasing the Affinity of Glucocorticoid Receptor.

BACKGROUND/AIMS: Although it has been reported that somatostatin (SOM) upregulated the level of 90-kD heat shock protein (Hsp90), which participates in the inflammatory regulation by its client proteins, such as glucocorticoid receptor (GR), it remains unclear if it has a protective role against acute lung injury (ALI). METHODS: ALI model was established by the injection of oleic acid (OA) into the tail vein of mice. Lung injury was assessed by histological analysis, lung water content and arterial blood gases. The levels of Hsp90 and GR, the binding capacity and the affinity of GR were examined. RESULTS: It was showed that pretreatment with SOM significantly increased Hsp90 levels and alleviated lung injuries in OA-injected mice. Furthermore, SOM increased the GR expression and improved the affinity of the GR in animals with lung injury. However, little alteration was found in the maximum binding capacity of the GR in mice with or without SOM. CONCLUSION: The data indicate SOM exerts a protective effect by increasing Hsp90 abundant and further enhancing the affinity of the GR. The beneficial effects of SOM treatment provide a new strategy for modulation of GR efficiency and alleviation of acute lung injury.

Polyinosinic-polycytidylic acid has therapeutic effects against cerebral ischemia/reperfusion injury through the downregulation of TLR4 signaling via TLR3.

Recent reports have shown that preconditioning with the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) protects against cerebral ischemia/reperfusion (I/R) injury. However, it is unclear whether poly(I:C) treatment after cerebral I/R injury is also effective. We used mouse/rat middle cerebral artery occlusion and cell oxygen-glucose deprivation models to evaluate the therapeutic effects and mechanisms of poly(I:C) treatment. Poly(I:C) was i.p. injected 3 h after ischemia (treatment group). Cerebral infarct volumes and brain edemas were significantly reduced, and neurologic scores were significantly increased. TNF-α and IL-1ß levels were markedly decreased, whereas IFN-ß levels were greatly increased, in the ischemic brain tissues, cerebral spinal fluid, and serum. Injuries to hippocampal neurons and mitochondria were greatly reduced. The numbers of TUNEL-positive and Fluoro-Jade B(+) cells also decreased significantly in the ischemic brain tissues. Poly(I:C) treatment increased the levels of Hsp27, Hsp70, and Bcl2 and decreased the level of Bax in the ischemic brain tissues. Moreover, poly(I:C) treatment attenuated the levels of TNF-α and IL-1ß in serum and cerebral spinal fluid of mice stimulated by LPS. However, the protective effects of poly(I:C) against cerebral ischemia were abolished in TLR3(-/-) and TLR4(-/-)mice. Poly(I:C) downregulated TLR4 signaling via TLR3. Poly(I:C) treatment exhibited obvious protective effects 14 d after ischemia and was also effective in the rat permanent middle cerebral artery occlusion model. The results suggest that poly(I:C) exerts therapeutic effects against cerebral I/R injury through the downregulation of TLR4 signaling via TLR3. Poly(I:C) is a promising new drug candidate for the treatment of cerebral infarcts.

Toll-like receptor 2/4 heterodimer mediates inflammatory injury in intracerebral hemorrhage.

OBJECTIVE: Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced secondary brain injury. However, the upstream events that initiate inflammatory responses following ICH remain elusive. Our previous studies suggested that Toll-like receptor 4 (TLR4) may be the upstream signal that triggers inflammatory injury in ICH. In addition, recent clinical findings indicated that both TLR2 and TLR4 may participate in ICH-induced brain injury. However, it is unclear how TLR2 functions in ICH-induced inflammatory injury and how TLR2 interacts with TLR4. METHODS: The role of TLR2 and TLR2/TLR4 heterodimerization in ICH-induced inflammatory injury was investigated in both in vivo and in vitro models of ICH. RESULTS: TLR2 mediated ICH-induced inflammatory injury, which forms a heterodimer with TLR4 in both in vivo and in vitro models of ICH. Hemoglobin (Hb), but not other blood components, triggered inflammatory injury in ICH via assembly of TLR2/TLR4 heterodimers. MyD88 (myeloid differentiation primary response gene 88), but not TRIF (Toll/IR-1 domain-containing adaptor protein inducing interferon-beta), was required for ICH-induced TLR2/TLR4 heterodimerization. Mutation of MyD88 Arg196 abolished the TLR2/TLR4 heterodimerization. INTERPRETATION: Our results suggest that a novel TLR2/TLR4 heterodimer induced by Hb initiates inflammatory injury in ICH. Interfering with the assembly of the TLR2/TLR4 heterodimer may be a novel target for developing effective treatment of ICH.

The glucocorticoid dexamethasone inhibits U937 cell adhesion and neutrophil release via RhoA/ROCK1-dependent and independent pathways.

AIMS: The aim of the present study was to investigate the role of the Ras homolog family member A (RhoA)/Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) signaling pathway in the inhibition of inflammatory responses by the glucocorticoid dexamethasone (Dex). METHODS: The inhibitory effects of Dex and Rho-kinase inhibitor fasudil (Fas) on phorbol ester-induced release of O2(-) and MPO from neutrophils and on U937 mononuclear cell adhesion were examined along with the expression and activity levels of RhoA and ROCK1. RESULTS: High doses of Dex rapidly inhibited the release of O2(-) and myeloperoxidase (MPO) from neutrophils and the adhesion of U937 cells, while Fas was only found to inhibit U937 cell adhesion. Additionally, Dex suppressed ROCK1 activity. However, Dex had no effects on ROCK1 or RhoA expression levels or on RhoA activity. Neither the glucocorticoid receptor antagonist mifepristone (RU-486) nor the protein synthesis inhibitor cycloheximide (CHX) was able to suppress the effects of Dex (p>0.05). CONCLUSIONS: The present findings indicate that Dex suppressed neutrophil release through ROCK1-independent mechanisms and inhibited the adhesion of U937 mononuclear cells through ROCK1-dependent non-genomic mechanisms that did not involve RhoA.

Ski ameliorates synovial cell inflammation in monosodium iodoacetate-induced knee osteoarthritis.

Knee osteoarthritis (KOA) is one of the most common degenerative diseases and is characterized by cartilage degeneration, synovial inflammation, joint stiffness and even loss of motor function. In the clinical treatment of arthritis, conventional analgesic and anti-inflammatory drugs have great side effects. We have evaluated the possibility of the endogenous transcription regulator Ski as an anti-inflammatory alternative in OA through experimental studies in animal models and in vivo and in vitro. Male Sprague‒Dawley rats were injected with monosodium iodoacetate (MIA) into the knee joints to induce symptoms identical to those of human OA. We isolated knee synovial tissue under sterile conditions and cultured primary synovial cells. In vitro, Ski inhibits the proinflammatory factors IL-1ß, IL-6 and TNF-α mRNA and protein expression in lipopolysaccharide (LPS)-stimulated fibroblast-like synoviocytes (FLSs) and U-937 cells. In addition, Ski attenuates or inhibits OA-induced synovial inflammation by upregulating the protein expression of the anti-inflammatory factor IL-4 and downregulating the protein expression of downstream molecules related to the NF-κB inflammatory signaling pathway. In vivo, Ski downregulated proinflammatory factors and p-NF-κB p65 in KOA synovial tissue and alleviated pain-related behaviors in KOA rats. These experimental data show that Ski has strong anti-inflammatory activity. Ski is an endogenous factor, and if used in the clinical treatment of OA, the side effects are small. However, the anti-inflammatory mechanism of Ski must be further studied.

Identification of a Hippocampus-to-Zona Incerta Projection involved in Motor Learning.

Motor learning (ML), which plays a fundamental role in growth and physical rehabilitation, involves different stages of learning and memory processes through different brain regions. However, the neural mechanisms that underlie ML are not sufficiently understood. Here, a previously unreported neuronal projection from the dorsal hippocampus (dHPC) to the zona incerta (ZI) involved in the regulation of ML behaviors is identified. Using recombinant adeno-associated virus, the projections to the ZI are surprisingly identified as originating from the dorsal dentate gyrus (DG) and CA1 subregions of the dHPC. Furthermore, projection-specific chemogenetic and optogenetic manipulation reveals that the projections from the dorsal CA1 to the ZI play key roles in the acquisition and consolidation of ML behaviors, whereas the projections from the dorsal DG to the ZI mediate the retrieval/retention of ML behaviors. The results reveal new projections from the dorsal DG and dorsal CA1 to the ZI involved in the regulation of ML and provide insight into the stages over which this regulation occurs.

Ski, a modulator of wound healing and scar formation in the rat skin and rabbit ear.

We recently demonstrated that Ski is a novel wound healing-related factor that promotes fibroblast proliferation and inhibits collagen secretion. Here, we show that increasing local Ski expression by gene transfer not only significantly accelerated wound healing by relieving inflammation, accelerating re-epithelialization and increasing formation of granulation tissue, but also reduced scar formation by decreasing collagen production in rat dermal wounds. Similarly, ski gene transfer accelerated wound healing, reduced the protuberant height and volume of scars and increased collagen maturity in a hypertrophic scar model in the rabbit ear. Conversely, reducing Ski expression in the wound by RNA interference resulted in significantly slower wound healing and increased scar area in rat dermal wounds. We demonstrated that these effects of Ski are associated with transforming growth factor-ß-mediated signalling pathways through both Smad2/3-dependent and Smad-independent pathways. Together, our results define a dual role for Ski in promoting wound healing and alleviating scar formation, identifying a new target for therapeutic approaches to preventing scar hyperplasia and accelerating wound healing.

The arresting phase determines the total healing time of a locally irradiated skin wound in swine.

OBJECTIVE: Radiation is an important cause of delayed wound healing, and there still exist many questions regarding the patterns and mechanisms of wound healing. This study investigated the characteristics of wound healing after varying doses of local radiation and explored possible causes of the delay in healing caused by radiation. METHODS: A full-thickness dorsal longitudinal skin tissue, 2 cm in diameter, was excised after local irradiation on one side of the back of swine, and the other side was wounded as a control. The size of the wound area was recorded every two days after injury. Pathological changes, proliferating cell nuclear antigen (PCNA, immunohisto- chemistry) and apoptosis levels (TUNEL assay) were measured at different time points after wounding. RESULTS: The course of wound healing can be divided into four phases, namely: the arresting phase, the healing priming phase, the fast healing phase, and the healed phase. Although the total wound healing time was closely correlated to the dose of irradiation (R(2) equal to 0.9758), it was more dependent on the length of the arresting phase (R(2) equal to 0.9903) because once the arresting phase ended, the wound healed at a similar speed regardless of radiation doses. Pathological analysis showed that compared with the control side there were more necrotic tissues, slower epithelial crawling, as well as fewer blood vessels and cellular components in the irradiated side at the arresting phase, while other phases revealed no significant difference concerning these measurements. Immunohistochemistry showed that the irradiated wounds had significantly less PCNA-positive and more TUNEL-positive labeling of cells in the arresting phase than in other phases. Moreover, the changes were positively related to the radiation doses, but there was no obvious difference in cell proliferation or apoptosis among the healing priming phase, fast healing phase or healed phase, whether on the control side or on the irradiated side. CONCLUSIONS: After local irradiation, the length of the arresting phase determines the wound healing time. Increased apoptosis and decreased cell proliferation might be an important reason for the formation of the arresting phase.

Simultaneous determination of fourteen ß2-agonist enantiomers in food animal muscles by liquid chromatography coupled with tandem mass spectrometry.

Illegal drug residues in animal derived foods are closely related to human's life and health. Studies on illegal drug residues and the metabolism, such as ß2-agonists in animals have attracted more and more attention. In most cases, ß2-agonists are suppliedand used astheracemate. The metabolic process and distribution of the two enantiomers in animal tissues are different. Therefore, it is very necessary to develop a simple and fast method for chiral resolution of these drugs in animal tissues. In this paper, a reliable resolution and determination method was presented using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for fourteen enantiomers of seven ß2-agonist racemates, clenbuterol (CLE), salbutamol (SAL), cimaterol (CIM), terbutaline (TER), clorprenaline (CLO), tulobuterol (TUL), penbuterol (PEN) in pork, beef, and lamb muscle samples. The samples were added the internal standard solution (IS) and extracted in the alkaline medium with acetonitrile. The further sample purification was accomplished through MCX solid phase extraction cartridge. Chromatographic chiral separation was carried out on a VancoShell chiral column (100 mm × 4.6 mm, 2.7 µm) with an isocratic mobile phase consisting of methanol and 10 mmol mL-1 ammonium formate aqueous solution (85:15, v/v). Under the optimized conditions, the resolution (R) of CIM was 2.0, CLE and PEN were 1.5, the others were all greater than 1.0. Enantiomeric determination was performed in the positive electrospray ionization mode using multiple reaction monitoring (MRM). The correlation coefficient (r) in the range of 0.2-25.0 µg L-1 was above 0.993. The average recoveries at the three spiking levels ranged from 95.3% to 117.7% with the relative standard deviation (RSD) lower than 15%. The limit of detection (LOD) and the limit of quantification (LOQ) of ß2-agonist enantiomers was 0.2 µg kg-1 and 0.5 µg kg-1 respectively. The method was successfully applied in the analysis and evaluation of ß2-agonist enantiomers in positive food animal muscle samples, CLE, SAL, TEB and CIM enantiomers were detected. The concentrations of the corresponding enantiomers were in the range of 1.06-17.3 µg kg-1, the lowest enantiomer fraction (EF) value was 0.42, and the highest value was 0.69. The work is expected to provide a method for chiral separation and enantiomeric determination of the further study of pharmacology, toxicity and residue elimination of ß2-agonist enantiomers.

Simultaneous determination of praziquantel and its main metabolites in the tissues of black goats and their residue depletion.

Praziquantel (PZQ) is a pyrazino-isoquinoline compound with broad spectrum of activity against parasitic trematodes and cestodes, and a key veterinary drug in the parasitic disease control field. However, PZQ residues caused by non-conforming or excessive use in food-producing animals may pose a serious threat to human health. Herein, a simple, sensitive and reproducible LC-MS/MS method was developed for the simultaneous determination of praziquantel and trans- and cis-4-hydroxypraziquantel in black goat tissues to guide the reasonable use of PZQ. The mean recoveries for three target analytes were 71.2 ∼ 117.6%, and the limits of quantification were 1.0 µg/kg. Twenty-five healthy black goats were administered a single dose of praziquantel tablets at a dose of 35 mg/kg of body weight for residue elimination study, The results revealed that praziquantel and 4-hydroxypraziquantel were rapidly depleted in goat tissues and the elimination half-lives did not exceed 1 day in all tissues except for muscle and lung. It provides guidance for the establishment of maximum residue limit of praziquantel in goat.

Inhibition of HIF-1α-AQP4 axis ameliorates brain edema and neurological functional deficits in a rat controlled cortical injury (CCI) model.

Traumatic brain injury (TBI) is an important cause of death in young adults and children. Till now, the treatment of TBI in the short- and long-term complications is still a challenge. Our previous evidence implied aquaporin 4 (AQP4) and hypoxia inducible factor-1α (HIF-1α) might be potential targets for TBI. In this study, we explored the roles of AQP4 and HIF-1α on brain edema formation, neuronal damage and neurological functional deficits after TBI using the controlled cortical injury (CCI) model. The adult male Sprague Dawley rats were randomly divided into sham and TBI group, the latter group was further divided into neutralized-AQP4 antibody group, 2-methoxyestradiol (2-ME2) group, and their corresponding control, IgG and isotonic saline groups, respectively. Brain edema was examined by water content. Hippocampal neuronal injury was assessed by neuron loss and neuronal skeleton related protein expressions. Spatial learning and memory deficits were evaluated by Morris water maze test and memory-related proteins were detected by western blot. Our data showed that increased AQP4 protein level was closely correlated with severity of brain edema after TBI. Compared with that in the control group, both blockage of AQP4 with neutralized-AQP4 antibody and inhibition of HIF-1α with 2-ME2 for one-time treatment within 30-60 min post TBI significantly ameliorated brain edema on the 1st day post-TBI, and markedly alleviated hippocampal neuron loss and spatial learning and memory deficits on the 21st day post-TBI. In summary, our preliminary study revealed the short-term and long-term benefits of targeting HIF-1α-AQP4 axis after TBI, which may provide new clues for the selection of potential therapeutic targets for TBI in clinical practice.

High glutamate concentration reverses the inhibitory effect of microglial adenosine 2A receptor on NLRP3 inflammasome assembly and activation.

NLRP3 inflammasome plays a crucial role in the innate immune system. Our group previously reported that the microglial adenosine 2A receptor (A2AR) regulates canonical neuroinflammation, which is affected by the glutamate concentration. However, the regulatory effect of A2AR on NLRP3 inflammasome and the effects of glutamate concentration remain unknown. Therefore, we aimed to investigate the regulatory effect of microglial A2AR on NLRP3 inflammasome assembly and activation as well as the effects of glutamate concentration on the inflammasome assembly and activation. Experiments were conducted on magnetically sorted primary microglia from P14 mice. The results showed that pharmacological A2AR activation ameliorated NLRP3 activation under no or low glutamate concentrations, but this effect was reversed by high glutamate concentrations. Moreover, the mRNA levels of NLRP3 inflammasome-related genes were not affected by A2AR activation or the glutamate concentration. We further demonstrated that A2AR activation inhibited the interaction between NLRP3 and caspase 1 under no or low glutamate concentrations while promoting their interaction under high glutamate concentrations. The oligomerization of ASC also showed a similar trend. In conclusion, our findings proved that the high glutamate concentration could reverse the inhibition of A2AR on NLRP3 inflammasome activation by modulating its assembly, which provides new insights into the regulatory effect of A2AR on neuroinflammation under different pathological conditions.