Potential Applications of Remote Limb Ischemic Conditioning for Chronic Cerebral Circulation Insufficiency

Chronic cerebral circulation insufficiency (CCCI) refers to a chronic decrease in cerebral blood perfusion, which may lead to cognitive impairment, psychiatric disorders such as depression, and acute ischemic stroke. Remote limb ischemic conditioning (RLIC), in which the limbs are subjected to a series of transient ischemic attacks, can activate multiple endogenous protective mechanisms to attenuate fatal ischemic injury to distant organs due to acute ischemia, such as ischemic stroke. Recent studies have also reported that RLIC can alleviate dysfunction in distant organs caused by chronic, non-fatal reductions in blood supply (e.g., CCCI). Indeed, research has indicated that RLIC may exert neuroprotective effects against CCCI through a variety of potential mechanisms, including attenuated glutamate excitotoxicity, improved endothelial function, increased cerebral blood flow, regulation of autophagy and immune responses, suppression of apoptosis, the production of protective humoral factors, and attenuated accumulation of amyloid-β. Verification of these findings is necessary to improve prognosis and reduce the incidence of acute ischemic stroke/cognitive impairment in patients with CCCI

Integrated GWAS and transcriptomic analysis reveal the candidate salt-responding genes regulating Na+/K+ balance in barley (Hordeum vulgare L.)

Salt stress is one of the main abiotic stresses affecting crop yield and quality. Barley has strong salt tolerance, however, the underlying genetic basis is not fully clear, especially in the seedling stage. This study examined the ionic changes in barley core germplasms under the control and salt conditions. Genome-wide association study (GWAS) analysis revealed 54 significant SNPs from a pool of 25,342 SNPs distributed in 7 chromosomes (Chr) of the Illumina Barley 50K SNP array. These SNPs are associated with ion homeostasis traits, sodium (Na+) and potassium (K+) content, and Na+/K+ ratio representing five genomic regions on Chr 2, 4, 5, 6, and 7 in the leaves of worldwide barley accessions. And there are 3 SNP peaks located on the Chr 4, 6, and 7, which could be the “hot spots” regions for mining and identifying candidate genes for salt tolerance. Furthermore, 616 unique candidate genes were screened surrounding the significant SNPs, which are associated with transport proteins, protein kinases, binding proteins, and other proteins of unknown function. Meanwhile, transcriptomic analysis (RNA-Seq) was carried out to compare the salt-tolerant (CM72) and salt-sensitive (Gairdner) genotypes subjected to salt stress. And there was a greater accumulation of differentially expressed genes(DEGs) in Gairdner compared to CM72, mainly enriched in metabolic pathway, biosynthesis of secondary metabolites, photosynthesis, signal transduction,emphasizing the different transcriptional response in both genotypes following salt exposure. Combined GWAS and RNA-Seq analysis revealed 5 promising salt-responding genes (PGK2, BASS3, SINAT2, AQP, and SYT3) from the hot spot regions, which were verified between the salt-tolerant and salt-sensitive varieties by qRT-PCR. In all, these results provide candidate SNPs and genes responsible for salinity responding in barley, and a new idea for studying such genetic basis in similar crops

A review of stress-induced hyperglycaemia in the context of acute ischaemic stroke: Definition, underlying mechanisms, and the status of insulin therapy

The transient elevation of blood glucose produced following acute ischaemic stroke (AIS) has been described as stress-induced hyperglycaemia (SIH). SIH is common even in patients with AIS who have no previous diagnosis of diabetes mellitus. Elevated blood glucose levels during admission and hospitalization are strongly associated with enlarged infarct size and adverse prognosis in AIS patients. However, insulin-intensive glucose control therapy defined by admission blood glucose for SIH has not achieved the desired results, and new treatment ideas are urgently required. First, we explore the various definitions of SIH in the context of AIS and their predictive value in adverse outcomes. Then, we briefly discuss the mechanisms by which SIH arises, describing the dual effects of elevated glucose levels on the central nervous system. Finally, although preclinical studies support lowering blood glucose levels using insulin, the clinical outcomes of intensive glucose control are not promising. We discuss the reasons for this phenomenon

Cerebral Ischemic Postconditioning Plays a Neuroprotective Role through Regulation of Central and Peripheral Glutamate

Following cerebral ischemia/reperfusion (I/R) injury, a series of pathophysiological processes are stimulated in both the central nervous system (CNS) and the periphery, including, but not limited to, the peripheral immune and endocrine systems and underregulation of the neuroendocrine-immune network. Glutamate (Glu) is an important excitatory neurotransmitter in the CNS; its excitotoxicity following cerebral ischemia has been a focus of study for several decades. In addition, as a novel immunoregulator, Glu also regulates immune activity in both the CNS and periphery and may connect the CNS and periphery through regulation of the neuroendocrine-immune network. Ischemic postconditioning (IPostC) is powerful and activates various endogenous neuroprotective mechanisms following cerebral I/R, but only a few studies have focused on the mechanisms associated with Glu to date. Given that Glu plays an important and complex pathophysiological role, the understanding of Glu-related mechanisms of IPostC is an interesting area of research, which we review here

Astrocytic gap junction inhibition by carbenoxolone enhances the protective effects of ischemic preconditioning following cerebral ischemia

Abstract Background Stroke is the second leading cause of death worldwide and the most common cause of adult-acquired disability in many nations. Thus, attenuating the damage after ischemic injury and improving patient prognosis are of great importance. We have indicated that ischemic preconditioning (IP) can effectively reduce the damage of ischemia reperfusion and that inhibition of gap junctions may further reduce this damage. Although we confirmed that the function of gap junctions is closely associated with glutamate, we did not investigate the mechanism. In the present study, we aimed to clarify whether the blockade of cellular communication at gap junctions leads to significant reductions in the levels of glutamate released by astrocytes following cerebral ischemia. Methods To explore this hypothesis, we utilized the specific blocking agent carbenoxolone (CBX) to inhibit the opening and internalization of connexin 43 channels in an in vitro model of oxygen-glucose deprivation/re-oxygenation (OGD/R), following IP. Results OGD/R resulted in extensive astrocytic glutamate release following upregulation of hemichannel activity, thus increasing reactive oxygen species (ROS) generation and subsequent cell death. However, we observed significant increases in neuronal survival in neuron-astrocyte co-cultures that were subjected to IP prior to OGD/R. Moreover, the addition of CBX enhanced the protective effects of IP during the re-oxygenation period following OGD, by means of blocking the release of glutamate, increasing the level of the excitatory amino acid transporter 1, and downregulating glutamine expression. Conclusions Our results suggest that combined use of IP and CBX represents a novel therapeutic strategy to attenuate damage from cerebral ischemia with minimal adverse side effects

Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone

Abstract Background Glia-mediated neuroinflammation is related to brain injury exacerbation after cerebral ischemia/reperfusion (I/R) injury. Astrocytic hemichannels or gap junctions, which were mainly formed by connexin-43, have been implicated in I/R damage. However, the exact roles of astrocytic hemichannels and gap junction in neuroinflammatory responses induced by I/R injury remain unknown. Methods Primary cultured astrocytes were subjected to OGD/R injury, an in vitro model of I/R injury. Salvianolic acid B (SalB) or carbenoxolone (CBX) were applied for those astrocytes. Besides, Cx43 mimetic peptides Gap19 or Gap26 were also applied during OGD/R injury; Cx43 protein levels were determined by western blot and cytoimmunofluorescene staining, hemichannel activities by Ethidium bromide uptake and ATP concentration detection, and gap junction intercellular communication (GJIC) permeability by parachute assay. Further, astrocyte-conditioned medium (ACM) was collected and incubated with microglia. Meanwhile, ATP or apyrase were applied to explore the role of ATP during OGD/R injury. Microglial activation, M1/M2 phenotypes, and M1/M2-related cytokines were detected. Also, microglia-conditioned medium (MEM) was collected and incubated with astrocytes to further investigate its influence on astrocytic hemichannel activity and GJIC permeability. Lastly, effects of ACM and MCM on neuronal viability were detected by flow cytometry. Results We found that OGD/R induced abnormally opened hemichannels with increased ATP release and EtBr uptake but reduced GJIC permeability. WB tests showed decreased astrocytic plasma membrane’s Cx43, while showing an increase in cytoplasma. Treating OGD/R-injured microglia with ATP or OGD/R-ACM induced further microglial activation and secondary pro-inflammatory cytokine release, with the M1 phenotype predominating. Conversely, astrocytes incubated with OGD/R-MCM exhibited increased hemichannel opening but reduced GJIC coupling. Both SalB and CBX inhibited abnormal astrocytic hemichannel opening and ATP release and switched the activated microglial phenotype from M1 to M2, thus providing effective neuroprotection. Application of Gap19 or Gap26 showed similar results with CBX. We also found that OGD/R injury caused both plasma membrane p-Cx43(Ser265) and p-Src(Tyr416) significantly upregulated; application of SalB may be inhibiting Src kinase and attenuating Cx43 internalization. Meanwhile, CBX treatment induced obviously downregulation of p-Cx43(Ser368) and p-PKC(Ser729) protein levels in plasma membrane. Conclusions We propose a vicious cycle exists between astrocytic hemichannel and microglial activation after OGD/R injury, which would aggravate neuroinflammatory responses and neuronal damage. Astrocytic Cx43, hemichannels, and GJIC play critical roles in OGD/R injury-induced neuroinflammatory responses; treatment differentially targeting astrocytic Cx43, hemichannels, and GJIC may provide novel avenues for therapeutics during cerebral I/R injury

Additional file 1: of Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone

Figure S1. Analysis of purity of primary cultured astrocytes or microglia. Primary glial cells were prepared, astrocytes and microglial cells were prepared and purified. (A1) Cells were stained with anti-CD11b-FITC antibody and detected with flow cytometry. The staining showed that < 0.1% of the cultured cells were microglia; (A2) Immunofluorescent staining revealed astrocytes stained with GFAP (green). Nuclei were stained with DAPI (blue). The GFAP staining showed that > 98% of the cultured cells were astrocytes. For microglial cells separated from the mix culture, both flow cytometry analysis and immunofluorescent staining showed that > 99% of the cultured cells were microglia in (B1-B2). Scale bar = 50 μm. Figure S2. MTT assay for cell viability of astrocytes undergone OGD/R injury. Primary astrocytes were prepared from newborn mice and subjected to OGD/R injury. (A) MTT assay to measure cell viability in astrocytes after treatment with SalB at 5 to 100 μg/mL concentrations. Con: control; (B) MTT assay to measure cell viability in astrocytes after treatment with CBX at 10 to 5000 μM concentrations. Con: control; (C) MTT assay to measure cell viability in astrocytes after treatment with CBX at 10 μM, SalB at 20 μg/mL, Gap19 at 100 μM, Gap26 at 100 μM; Also, Gap19, Gap26 or CBX pretreatment followed by SalB incubation and SalB pretreatment for 30 min followed by Gap19, Gap26 or CBX incubation with the above indicated concentrations; All error bars:±SEM. We evaluated the statistical significance with ANOVA and Duncan’s multiple comparisons test. *p < 0.05, **p < 0.01, and ***p < 0.001. Figure S3. Standard curve for ATP detection. ATP levels in conditioned medium were determined. The fluorescence levels from five serial ATP dilutions—0, 10, 30, 60, 100, 300, and 1000 nM are shown. Figure S4 (A-B) Western blotting were performed to evaluate the M2 marker arginase-1. Arginase-1 protein expression was decreased in the OGD/R group’s activated microglia, but SalB reversed this effect; (C-D) Arginase-1 expression was decreased in OGD/R-ACM-treated microglia while increased in microglia treated with OGD/R-SalB-ACM or OGD/R-CBX-ACM. We evaluated the statistical significance with ANOVA and Duncan’s multiple comparisons test. *p < 0.05, **p < 0.01, and ***p < 0.001. (PPTX 11400 kb

The <i>MKK7</i> p.Glu116Lys Rare Variant Serves as a Predictor for Lung Cancer Risk and Prognosis in Chinese

<div><p>Accumulated evidence indicates that rare variants exert a vital role on predisposition and progression of human diseases, which provides neoteric insights into disease etiology. In the current study, based on three independently retrospective studies of 5,016 lung cancer patients and 5,181 controls, we analyzed the associations between five rare polymorphisms (i.e., p.Glu116Lys, p.Asn118Ser, p.Arg138Cys, p.Ala195Thr and p.Leu259Phe) in <i>MKK7</i> and lung cancer risk and prognosis. To decipher the precise mechanisms of <i>MKK7</i> rare variants on lung cancer, a series of biological experiments was further performed. We found that the <i>MKK7</i> p.Glu116Lys rare polymorphism was significantly associated with lung cancer risk, progression and prognosis. Compared with Glu/Glu common genotype, the 116Lys rare variants (Lys/Glu/+ Lys/Lys) presented an adverse effect on lung cancer susceptibility (odds ratio [OR] = 3.29, 95% confidence interval [CI] = 2.70–4.01). These rare variants strengthened patients’ clinical progression that patients with 116Lys variants had a significantly higher metastasis rate and advanced N, M stages at diagnosis. In addition, the patients with 116Lys variants also contributed to worse cancer prognosis than those carriers with Glu/Glu genotype (hazard ratio [HR] = 1.53, 95% CI = 1.32–1.78). Functional experiments further verified that the <i>MKK7</i> p.116Lys variants altered the expression of several cancer-related genes and thus affected lung cancer cells proliferation, tumor growth and metastasis <i>in vivo</i> and <i>in vitro</i>. Taken together, our findings proposed that the <i>MKK7</i> p.Glu116Lys rare polymorphism incurred a pernicious impact on lung cancer risk and prognosis through modulating expressions of a serial of cancer-related genes.</p></div

The effects of <i>MKK7</i> p.Glu116Lys on tumor growth and metastasis.

<p>(A, B). Subcutaneously implanted <i>MKK7</i>-116Glu (A549<i>-MKK7</i>-116Glu and L78<i>-MKK7</i>-116Glu cells) and <i>MKK7</i>-116Lys (A549<i>-MKK7</i>-116Lys and L78<i>-MKK7</i>-116Lys cells) cells xenografted tumors were established and were observed for a total of 4 weeks. Tumor volumes represented the mean ± SD of 6 mice per group. Columns, mean; bars, SD. The symbol “^★” indicated a statistical significance with <i>P</i> < 0.05 between the cells transfected with the two different transfectants. (C-E). A549<i>-MKK7</i>-116Glu or A549<i>-MKK7</i>-116Lys cells were separately injected into the tail vein of each mouse. After proximately 10 weeks, lung metastases were evaluated using magnetic resonance imaging, macroscopic observation and histomorphology under microscopy. The red loops and arrows indicate the metastases.</p