GluA1 Degradation by Autophagy Contributes to Circadian Rhythm Effects on Cerebral Ischemia Injury.

The mechanisms of many diseases, including central nervous system disorders, are regulated by circadian rhythms. The development of brain disorders such as depression, autism, and stroke is strongly associated with circadian cycles. Previous studies have shown that cerebral infarct volume is smaller at night (active phase) than during the day (inactive phase) in ischemic stroke rodent models. However, the underlying mechanisms remain unclear. Increasing evidence suggests that glutamate systems and autophagy play important roles in the pathogenesis of stroke. Here, we report that GluA1 expression was decreased and autophagic activity was increased in active-phase male mouse models of stroke compared with the inactive-phase models. In the active-phase model, induction of autophagy decreased the infarct volume, whereas inhibition of autophagy increased the infarct volume. Meanwhile, GluA1 expression was decreased following activation of autophagy and increased following inhibition of autophagy. We used Tat-GluA1 to uncouple p62, an autophagic adapter, from GluA1 and found that this blocked the degradation of GluA1, an effect similar to that of inhibition of autophagy in the active-phase model. We also demonstrated that knock-out of the circadian rhythm gene Per1 abolished the circadian rhythmicity of the volume of infarction and also abolished GluA1 expression and autophagic activity in wild-type (WT) mice. Our results suggest an underlying mechanism by which the circadian rhythm participates in the autophagy-dependent regulation of GluA1 expression, which influences the volume of infarction in stroke.SIGNIFICANCE STATEMENT Circadian rhythms affect the pathophysiological mechanisms of disease. Previous studies suggested that circadian rhythms affect the infarct volume in stroke, but the underlying mechanisms remain largely unknown. Here, we demonstrate that the smaller infarct volume after middle cerebral artery occlusion/reperfusion (MCAO/R) during the active phase is related to lower GluA1 expression and activation of autophagy. The decrease in GluA1 expression during the active phase is mediated by the p62-GluA1 interaction, followed by direct autophagic degradation. In short, GluA1 is the substrate of autophagic degradation, which mainly occurs after MCAO/R during the active phase but not the inactive phase.

Upregulation of HLA-II related to LAG-3+CD4+ T cell infiltration is associated with patient outcome in human glioblastoma.

Glioblastoma (GBM) is the most common malignant diffuse glioma of the brain. Although immunotherapy with immune checkpoint inhibitors (ICIs), such as programmed cell death protein (PD)-1/PD ligand-1 inhibitors, has revolutionized the treatment of several cancers, the clinical benefit in GBM patients has been limited. Lymphocyte-activation gene 3 (LAG-3) binding to human leukocyte antigen-II (HLA-II) plays an essential role in triggering CD4+ T cell exhaustion and could interfere with the efficiency of anti-PD-1 treatment; however, the value of LAG-3-HLA-II interactions in ICI immunotherapy for GBM patients has not yet been analyzed. Therefore, we aimed to investigate the expression and regulation of HLA-II in human GBM samples and the correlation with LAG-3+CD4+ T cell infiltration. Human leukocyte antigen-II was highly expressed in GBM and correlated with increased LAG-3+CD4+ T cell infiltration in the stroma. Additionally, HLA-IIHighLAG-3High was associated with worse patient survival. Increased interleukin-10 (IL-10) expression was observed in GBM, which was correlated with high levels of HLA-II and LAG-3+ T cell infiltration in stroma. HLA-IIHighIL-10High GBM associated with LAG-3+ T cells infiltration synergistically showed shorter overall survival in patients. Combined anti-LAG-3 and anti-IL-10 treatment inhibited tumor growth in a mouse brain GL261 tumor model. In vitro, CD68+ macrophages upregulated HLA-II expression in GBM cells through tumor necrosis factor-α (TNF-α). Blocking TNF-α-dependent inflammation inhibited tumor growth in a mouse GBM model. In summary, T cell-tumor cell interactions, such as LAG-3-HLA-II, could confer an immunosuppressive environment in human GBM, leading to poor prognosis in patients. Therefore, targeting the LAG-3-HLA-II interaction could be beneficial in ICI immunotherapy to improve the clinical outcome of GBM patients.

Quercetin promotes the proportion and maturation of NK cells by binding to MYH9 and improves cognitive functions in aged mice.

BACKGROUND: Quercetin is a flavonol compound widely distributed in plants that possesses diverse biological properties, including antioxidative, anti-inflammatory, anticancer, neuroprotective and senescent cell-clearing activities. It has been shown to effectively alleviate neurodegenerative diseases and enhance cognitive functions in various models. The immune system has been implicated in the regulation of brain function and cognitive abilities. However, it remains unclear whether quercetin enhances cognitive functions by interacting with the immune system. RESULTS: In this study, middle-aged female mice were administered quercetin via tail vein injection. Quercetin increased the proportion of NK cells, without affecting T or B cells, and improved cognitive performance. Depletion of NK cells significantly reduces cognitive ability in mice. RNA-seq analysis revealed that quercetin modulated the RNA profile of hippocampal tissues in aging animals towards a more youthful state. In vitro, quercetin significantly inhibited the differentiation of Lin-CD117+ hematopoietic stem cells into NK cells. Furthermore, quercetin promoted the proportion and maturation of NK cells by binding to the MYH9 protein. CONCLUSIONS: In summary, our findings suggest that quercetin promotes the proportion and maturation of NK cells by binding to the MYH9 protein, thereby improving cognitive performance in middle-aged mice.

Ochratoxin A-enhanced glycolysis induces inflammatory responses in human gastric epithelium cells through mTOR/HIF-1α signaling pathway.

Ochratoxin A (OTA) is a mycotoxin commonly found in several food commodities worldwide with potential nephrotoxic, hepatotoxic and carcinogenic effects. We previously showed for the first time that OTA treatment enhanced glycolysis in human gastric epithelium (GES-1) cells in vitro. Here, we found that OTA exposure activated inflammatory responses, evidenced by increasing of NF-κB signaling pathway-related protein (p-p65 and p-IκBα) expressions and elevating of inflammatory cytokine (IL-1ß and IL-6) mRNA expressions in GES-1 cells. To elucidate the role of glycolysis in inflammatory effects triggered by OTA, we pretreated GES-1 cells with glycolysis inhibitor (2-deoxy-D-glucose, 2-DG) before OTA exposure. The result showed that 2-DG reduced the protein expressions of p-p65 and p-IκBα and alleviated the mRNA expressions of inflammatory cytokines in OTA-treated GES-1 cells. Furthermore, OTA activated the mTOR/HIF-1α pathway by increasing the protein expressions of p-mTOR, p-eIF4E and HIF-1α, and inhibition of mTOR with rapamycin or silencing HIF-1α with siRNA significantly attenuated OTA-enhanced glycolysis by reducing glycolysis related genes and thereby decreasing inflammatory effects of GES-1 cells. These results demonstrate that OTA activates inflammatory responses in GES-1 cells and this is controlled by mTOR/HIF-1α pathway-mediated glycolysis enhancement. Our findings provide a novel mechanistic view into OTA-induced gastric cytotoxicity.

Unbalanced regulation of Sec22b and Ykt6 blocks autophagosome axonal retrograde flux in neuronal ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury in ischemic penumbra is accountable for poor outcome of ischemic stroke patients receiving recanalization therapy. Compelling evidence previously demonstrated a dual role of autophagy in stroke. This study aimed to understand the traits of autophagy in the ischemic penumbra and the potential mechanism that switches the dual role of autophagy. We found that autophagy induction by rapamycin and lithium carbonate performed before ischemia reduced neurological deficits and infarction, while autophagy induction after reperfusion had the opposite effect in the male murine middle cerebral artery occlusion/reperfusion model, both of which were eliminated in mice lacking autophagy (Atg7flox/flox; Nestin-Cre). Autophagic flux determination showed that reperfusion led to a blockage of axonal autophagosome retrograde transport in neurons, which then led to autophagic flux damage. Then, we found that ischemia-reperfusion induced changes in the protein levels of Sec22b and Ykt6 in neurons, two autophagosome transport-related factors, in which Sec22b significantly increased and Ykt6 significantly decreased. In the absence of exogenous autophagy induction, Sec22b knockdown and Ykt6 overexpression significantly alleviated autophagic flux damage, infarction, and neurological deficits in neurons or murine exposed to cerebral ischemia-reperfusion in an autophagy-dependent manner. Furthermore, Sec22b knockdown and Ykt6 overexpression switched the outcome of rapamycin post-treatment from deterioration to neuroprotection. Thus, Sec22b and Ykt6 play key roles in neuronal autophagic flux, and modest regulation of Sec22b and Ykt6 may help to reverse the failure of targeting autophagy induction to improve the prognosis of ischemic stroke.Significance Statement:The highly polarized architecture of neurons with neurites presents challenges for material transport, such as autophagosomes, which form at the neurite tip and need to be transported to the cell soma for degradation. Here, we demonstrate that Sec22b and Ykt6 act as autophagosome porters and play an important role in maintaining the integrity of neuronal autophagic flux. Ischemia-reperfusion-induced excess Sec22b and loss of Ykt6 in neurons lead to axonal autophagosome retrograde trafficking failure, autophagic flux damage, and finally neuronal injury. Facilitated axonal autophagosome retrograde transport by Sec22b knockdown and Ykt6 overexpression may reduce ischemia-reperfusion-induced neuron injury and extend the therapeutic window of pharmacological autophagy induction for neuroprotection.

Identification, evolution, and expression of GDSL-type Esterase/Lipase (GELP) gene family in three cotton species: a bioinformatic analysis.

BACKGROUND: GDSL esterase/lipases (GELPs) play important roles in plant growth, development, and response to biotic and abiotic stresses. Presently, an extensive and in-depth analysis of GELP family genes in cotton is still not clear enough, which greatly limits the further understanding of cotton GELP function and regulatory mechanism. RESULTS: A total of 389 GELP family genes were identified in three cotton species of Gossypium hirsutum (193), G. arboreum (97), and G. raimondii (99). These GELPs could be classified into three groups and eight subgroups, with the GELPs in same group to have similar gene structures and conserved motifs. Evolutionary event analysis showed that the GELP family genes tend to be diversified at the spatial dimension and certain conservative at the time dimension, with a trend of potential continuous expansion in the future. The orthologous or paralogous GELPs among different genomes/subgenomes indicated the inheritance from genome-wide duplication during polyploidization, and the paralogous GELPs were derived from chromosomal segment duplication or tandem replication. GELP genes in the A/D subgenome underwent at least three large-scale replication events in the evolutionary process during the period of 0.6-3.2 MYA, with two large-scale evolutionary events between 0.6-1.8 MYA that were associated with tetraploidization, and the large-scale duplication between 2.6-9.1 MYA that occurred during diploidization. The cotton GELPs indicated diverse expression patterns in tissue development, ovule and fiber growth, and in response to biotic and abiotic stresses, combining the existing cis-elements in the promoter regions, suggesting the GELPs involvements of functions to be diversification and of the mechanisms to be a hormone-mediated manner. CONCLUSIONS: Our results provide a systematic and comprehensive understanding the function and regulatory mechanism of cotton GELP family, and offer an effective reference for in-depth genetic improvement utilization of cotton GELPs.

Tumor Necrosis Factor α-Dependent Lung Inflammation Promotes the Progression of Lung Adenocarcinoma Originating From Alveolar Type II Cells by Upregulating MIF-CD74.

Lung adenocarcinoma is the most common type of lung cancer. We recently reported that inflammation-driven lung adenocarcinoma (IDLA) originates from alveolar type (AT)-II cells, which depend on major histocompatibility complex (MHC) class II to promote the expansion of regulatory T cells. The MHC class II-associated invariant chain (CD74) binds to the macrophage migration inhibitory factor (MIF), which is associated with promoting tumor growth and invasion. However, the role of MIF-CD74 in the progression of lung adenocarcinoma and the underlying mechanisms remain unclear. We aimed to explore the role of MIF-CD74 in the progression of lung adenocarcinoma and elucidate the mechanisms by which tumor necrosis (TNF)-α-mediated inflammation regulates CD74 and MIF expression in IDLA. In human lung adenocarcinoma, CD74 was upregulated on the surface of tumor cells originating from AT-II cells, which correlated positively with lymph node metastasis, tumor origin/nodal involvement/metastasis stage, and TNF-α expression. MIF interaction with CD74 promoted the proliferation and migration of A549 and H1299 cells in vitro. Using a urethane-induced IDLA mouse model, we observed that CD74 was upregulated in tumor cells and macrophages. MIF expression was upregulated in macrophages in IDLA. Blocking TNF-α-dependent inflammation downregulated CD74 expression in tumor cells and CD74 and MIF expression in macrophages in IDLA. Conditioned medium from A549 cells or activated mouse AT-II cells upregulated MIF in macrophages by secreting TNF-α. TNF-α-dependent lung inflammation contributes to the progression of lung adenocarcinoma by upregulating CD74 and MIF expression, and AT-II cells upregulate MIF expression in macrophages by secreting TNF-α. This study provides novel insights into the function of CD74 in the progression of IDLA.

Receptor-interacting protein 3-phosphorylated Ca2+ /calmodulin-dependent protein kinase II and mixed lineage kinase domain-like protein mediate intracerebral hemorrhage-induced neuronal necroptosis.

Necroptosis-mediated cell death is an important mechanism in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI). Our previous study has demonstrated that receptor-interacting protein 1 (RIP1) mediated necroptosis in SBI after ICH. However, further mechanisms, such as the roles of receptor-interacting protein 3 (RIP3), mixed lineage kinase domain-like protein (MLKL), and Ca2+ /calmodulin-dependent protein kinase II (CaMK II), remain unclear. We hypothesized that RIP3, MLKL, and CaMK II might participate in necroptosis after ICH, including their phosphorylation. The ICH model was induced by autologous blood injection. First, we found the activation of necroptosis after ICH in brain tissues surrounding the hematoma (propidium iodide staining). Meanwhile, the phosphorylation and expression of RIP3, MLKL, and CaMK II were differently up-regulated (western blotting and immunofluorescent staining). The specific inhibitors could suppress RIP3, MLKL, and CaMK II (GSK'872 for RIP3, necrosulfonamide for MLKL, and KN-93 for CaMK II). We found the necroptosis surrounding the hematoma and the concrete interactions in RIP3-MLKL/RIP3-CaMK II also both decreased after the specific intervention (co-immunoprecipitation). Then we conducted the short-/long-term neurobehavioral tests, and the rats with specific inhibition mostly had better performance. We also found less blood-brain barrier (BBB) injury, and less neuron loss (Nissl staining) in intervention groups, which supported the neurobehavioral tests. Besides, oxidative stress and inflammation were also alleviated with intervention, which had significant less reactive oxygen species (ROS), tumor necrosis factor (TNF)-α, lactate dehydrogenase (LDH), Iba1, and GFAP surrounding the hematoma. These results confirmed that RIP3-phosphorylated MLKL and CaMK II participate in ICH-induced necroptosis and could provide potential targets for the treatment of ICH patients.

Limb expression 1-like protein promotes epithelial-mesenchymal transition and epidermal growth factor receptor-tyrosine kinase inhibitor resistance via nucleolin-mediated ribosomal RNA synthesis in non-small cell lung cancer.

Limb expression 1-like protein (LIX1L) might be an RNA-binding protein involved in post-transcriptional regulation. However, little is known regarding the biological function and mechanism of LIX1L in cancer cells. Here we demonstrate a clear correlation between LIX1L expression and epithelial-mesenchymal transition (EMT) markers in 81 non-small cell lung cancer (NSCLC) tissues and The Cancer Genome Atlas database, suggesting that LIX1L is a mesenchymal marker. Besides, LIX1L expression is obviously elevated in TGFß1-induced EMT NSCLC cells and enhances cell migration, invasion, anoikis resistance, epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance, and proliferation. Interestingly, the increased LIX1L expression prominently localizes to the nucleoli, where it physically interacts with the key ribosome biogenesis regulator NCL protein, inducing ribosomal RNA (rRNA) synthesis in EMT NSCLC cells. NCL knockdown or inhibition of rRNA synthesis reverses the enhanced EMT functions and proliferation ability caused by LIX1L overexpression in NSCLC cells, indicating that NCL expression and rRNA synthesis participates in LIX1L-mediated biological functions during EMT. Collectively, our findings suggest that the LIX1L-NCL-rRNA synthesis axis is a novel EMT-activated mechanism. Targeting the pathway might be a therapeutic option for EMT and EGFR-TKI resistance in NSCLC.

TNF-α-dependent lung inflammation upregulates PD-L1 in monocyte-derived macrophages to contribute to lung tumorigenesis.

Chronic inflammation, which is dominated by macrophage-involved inflammatory responses, is an instigator of cancer initiation. Macrophages are the most abundant immune cells in healthy lungs, and associated with lung tumor development and promotion. PD-L1 is a negative molecule in macrophages and correlated with an immunosuppressive function in tumor environment. Macrophages expressing PD-L1, rather than tumor cells, exhibits a critical role in tumor growth and progression. However, whether and how PD-L1 in macrophages contributes to inflammation-induced lung tumorigenesis requires further elucidation. Here, we found that higher expression of PD-L1 in CD11b+ CD206+ macrophages was positively correlated with tumor progression and PD-1+ CD8+ T cells population in human adenocarcinoma patients. In the urethane-induced inflammation-driven lung adenocarcinoma (IDLA) mouse model, the infiltration of circulating CD11bhigh F4/80+ monocyte-derived macrophages (MoMs) was increased in pro-tumor inflamed lung tissues and lung adenocarcinoma. PD-L1 was mainly upregulated in MoMs associated with enhanced T cells exhaustion in lung tissues. Anti-PD-L1 treatment can reduce T cells exhaustion at pro-tumor inflammatory stage, and then inhibit tumorigenesis in IDLA. The pro-tumor lung inflammation depended on TNF-α to upregulate PD-L1 and CSN6 expression in MoMs, and induced cytokines production by alveolar type-II cells (AT-II). Furthermore, inflammatory AT-II cells could secret TNF-α to upregulate PD-L1 expression in bone-marrow driven macrophages (BM-M0). Inhibition of CSN6 decreased PD-L1 expression in TNF-α-activated macrophage in vitro, suggesting a critical role of CSN6 in PD-L1 upregulation. Thus, pro-tumor inflammation can depend on TNF-α to upregulate PD-L1 in recruited MoMs, which may be essential for lung tumorigenesis.

Circadian Rhythm Regulator REV-ERBα Attenuates Neuroapoptosis in Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats.

Subarachnoid hemorrhage (SAH) is associated with circadian rhythm abnormalities, in which REV-ERBα plays a major regulatory role. Our ambition was to investigate the capacity of REV-ERBα to inhibit neuronal neuroapoptosis induced by early brain injury (EBI) after SAH. The endovascular perforation model was used to produce experimental SAH in Sprague-Dawley rats. Specific small-interfering RNA was used to downregulate the expression REV-ERBα while SR9009 was used to upregulate the expression before assessments. Short- and long-term neurobehavior assessments, immunofluorescence staining, TUNEL staining, Nissl staining, brain water content, and Western blot were performed. The expression level of endogenous REVERBα tended to increase and then decrease after SAH and peaked at 48 h. REV-ERBα upregulation diminished neuronal apoptosis and enhanced neurological function deficits. Meanwhile, REV-ERBα downregulation aggravated the damage. Furthermore, the levels of downstream proteins of REV-ERBα (i.e., brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK)) changed accordingly with REV-ERBα regulation. REV-ERBα may attenuate neuronal apoptosis in EBI after SAH through the BMAL1/CLOCK pathway.

Glycyrrhizic acid inhibits myeloid differentiation of hematopoietic stem cells by binding S100 calcium binding protein A8 to improve cognition in aged mice.

BACKGROUND: Glycyrrhizic acid (GA), a saponin compound often used as a flavoring agent, can elicit anti-inflammatory and anti-tumor effects, and alleviate aging. However, the specific mechanism by which GA alters immune cell populations to produce these beneficial effects is currently unclear. RESULTS: In this study, we systematically analyzed single-cell sequencing data of peripheral blood mononuclear cells from young mice, aged mice, and GA-treated aged mice. Our in vivo results show that GA reduced senescence-induced increases in macrophages and neutrophils, and increased numbers of lymphoid lineage subpopulations specifically reduced by senescence. In vitro, GA significantly promoted differentiation of Lin-CD117+ hematopoietic stem cells toward lymphoid lineages, especially CD8+ T cells. Moreover, GA inhibited differentiation of CD4+ T cells and myeloid (CD11b+) cells by binding to S100 calcium-binding protein 8 (S100A8) protein. Overexpression of S100A8 in Lin- CD117+ hematopoietic stem cells enhanced cognition in aged mice and the immune reconstitution of severely immunodeficient B-NDG (NOD.CB17-Prkdcscid/l2rgtm1/Bcgen) mice. CONCLUSIONS: Collectively, GA exerts anti-aging effects by binding to S100A8 to remodel the immune system of aged mice.

MIC19 Exerts Neuroprotective Role via Maintaining the Mitochondrial Structure in a Rat Model of Intracerebral Hemorrhage.

As an essential constituent of the mitochondrial contact site and cristae organization system (MICOS), MIC19 plays a crucial role in maintaining the stability of mitochondrial function and microstructure. However, the mechanisms and functions of MIC19 in intracerebral hemorrhage (ICH) remain unknown and need to be investigated. Sprague Dawley (SD) rats injected with autologous blood obtained from the caudal artery, and cultured neurons exposed to oxygen hemoglobin (OxyHb) were used to establish and emulate the ICH model in vivo and in vitro. Lentiviral vector encoding MIC19 or MIC19 short hairpin ribonucleic acid (shRNA) was constructed and administered to rats by intracerebroventricular injection to overexpress or knock down MIC19, respectively. First, MIC19 protein levels were increased after ICH modeling. After virus transfection and subsequent ICH modeling, we observed that overexpression of MIC19 could mitigate cell apoptosis and neuronal death, as well as abnormalities in mitochondrial structure and function, oxidative stress within mitochondria, and neurobehavioral deficits in rats following ICH. Conversely, knockdown of MIC19 had the opposite effect. Moreover, we found that the connection between MIC19 and SAM50 was disrupted after ICH, which may be a reason for the impairment of the mitochondrial structure after ICH. In conclusion, MIC19 exerts a protective role in the subsequent injury induced by ICH. The investigation of MIC19 may offer clinicians novel therapeutic insights for patients afflicted with ICH.

TREM2 modulates neuroinflammation with elevated IRAK3 expression and plays a neuroprotective role after experimental SAH in rats.

BACKGROUND: The modulation of neuroinflammation is a new direction that may alleviate the early brain injury after subarachnoid hemorrhage (SAH). Brain resident microglia/macrophages (Mi/MΦ) are the key drivers of neuroinflammation. Triggering receptor expressed on myeloid cells 2 (TREM2) has been reported to play a neuroprotective role by activating phagocytosis and suspending inflammatory response in experimental ischemic stroke and intracerebral hemorrhage. This study was designed to investigate the role of TREM2 on neuroinflammation and neuroprotective effects in a rat SAH model. METHODS: Adult male Sprague-Dawley rats were induced SAH through endovascular perforation. Lentivirus vectors were administered by i.c.v. to induce TREM2 overexpression or knockdown 7 days before SAH induction. Short- and long-term neurobehavioral tests, western blotting, immunofluorescence, enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase dUTP nick end labeling and Nissl staining were performed to explore the neuroprotective role of TREM2 after SAH. RESULTS: The expression of TREM2 elevated in a rat SAH model with a peak at 48 h after SAH and mainly expressed in Mi/MΦ in brain. TREM2 overexpression improved short- and long-term neurological deficits induced by SAH in rats, while TREM2 knockdown worsened neurological dysfunction. The rats with TREM2 overexpressed presented less neuronal apoptosis and more neuronal survival at 48 h after SAH, while the rats with TREM2 knockdown presented on the contrary. TREM2 overexpression manifested activated phagocytosis and suppressed inflammatory response, with the increase of CD206+/CD11b+ cells and IL-10 expression as well as the decrease of the infiltration of MPO+ cells and the expression of TNF-α, IL-1ß. While TREM2 knockdown abolished these effects. The protein level of IRAK3, a negative regulatory factor of inflammation, was significantly elevated after TREM2 overexpression and declined after TREM2 knockdown. CONCLUSIONS: Our research suggested TREM2 played a neuroprotective role and improved the short- and long-term neurological deficits by modulating neuroinflammation after SAH. The modulation on neuroinflammation of TREM2 after SAH was related with the elevated protein level of IRAK3.

Reconstruction of karyotypic evolution in Saccharum spontaneum species by comparative oligo-FISH mapping.

BACKGROUND: Karyotype dynamics driven by chromosomal rearrangements has long been considered as a fundamental question in the evolutionary genetics. Saccharum spontaneum, the most primitive and complex species in the genus Saccharum, has reportedly undergone at least two major chromosomal rearrangements, however, its karyotypic evolution remains unclear. RESULTS: In this study, four representative accessions, i.e., hypothetical diploid sugarcane ancestor (sorghum, x = 10), Sa. spontaneum Np-X (x = 10, tetraploid), 2012-46 (x = 9, hexaploid) and AP85-441 (x = 8, tetraploid), were selected for karyotype evolution studies. A set of oligonucleotide (oligo)-based barcode probes was developed based on the sorghum genome, which allowed universal identification of all chromosomes from sorghum and Sa. spontaneum. By comparative FISH assays, we reconstructed the karyotype evolutionary history and discovered that although chromosomal rearrangements resulted in greater variation in relative lengths of some chromosomes, all chromosomes maintained a conserved metacentric structure. Additionally, we found that the barcode oligo probe was not applicable for chromosome identification in both Sa. robustum and Sa. officinarum species, suggesting that sorghum is more distantly related to Sa. robustum and Sa. officinarum compared with Sa. spontaneum species. CONCLUSIONS: Our study demonstrated that the barcode oligo-FISH is an efficient tool for chromosome identification and karyotyping research, and expanded our understanding of the karyotypic and chromosomal evolution in the genus Saccharum.

Integrative analysis of the pharmaceutical active ingredient and transcriptome of the aerial parts of Glycyrrhiza uralensis under salt stress reveals liquiritin accumulation via ABA-mediated signaling.

The aerial parts of Glycyrrhiza uralensis supply substantial raw material for the extraction of active pharmaceutical ingredients comprehensively utilized in many industries. Our previous study indicated that salt stress increased the content of active ingredients. However, the regulatory mechanism remains unclear. In this study, RNA-sequencing (RNA-seq) of the aerial parts of G. uralensis treated with 150 mM NaCl for 0, 2, 6, and 12 h was performed to identify the key genes and metabolic pathways regulating pharmacological active component accumulation. The main active component detection showed that liquiritin was the major ingredient and exhibited more than a ten-fold significant increase in the 6 h NaCl treatment. Temporal expression analysis of the obtained 4245 differentially expressed genes (DEGs) obtained by RNA-seq revealed two screened profiles that included the significant up-regulated DEGs (UDEGs) at different treatment points. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these UDEGs identified phenylpropanoid metabolism and flavonoid biosynthesis as the most significantly enriched pathways in 2 h treated materials. Interestingly, the carotenoid biosynthesis pathway that is related to ABA synthesis was also discovered, and the ABA content was significantly promoted after 6 h NaCl treatment. Following ABA stimulation, the content of liquiritin demonstrated a significant and immediate increase after 2 h treatment, with the corresponding consistent expression of genes involved in the pathways of ABA signal transduction and flavonoid biosynthesis, but not in the pathway of glycyrrhizic acid biosynthesis. Our study concludes that salt stress might promote liquiritin accumulation through the ABA-mediated signaling pathway, and provides effective reference for genetic improvement and comprehensive utilization of G. uralensis.

Apelin-13 attenuates early brain injury through inhibiting inflammation and apoptosis in rats after experimental subarachnoid hemorrhage.

BACKGROUND: Early brain injury (EBI) has been considered as the major contributor to the neurological dysfunction and poor clinical outcomes of subarachnoid hemorrhage (SAH). Studies showed that apelin-13 exhibits a neuroprotective effect in brain damage induced by cerebral ischemia. However, it remains unclear whether apelin-13 could exhibit the protective functions following SAH. The present study aimed to validate the neuroprotective role of apelin-13 in SAH, and further investigated the underlying mechanisms. METHODS AND RESULTS: We constructed SAH rat model and we found that apelin-13 significantly alleviated neurological disorder and brain edema, improved memory deficits in SAH rats. Apelin-13 treatment decreased contents of TNF-α and IL-1ß in cerebral spinal fluid of SAH rat by using ELISA. Apelin-13 treatment promoted the expression of APJ and Bcl-2, and decreased the level of active caspase-3 and Bax in the temporal cortex after SAH by using western blot. Also, apelin-13 attenuated the cortical cell death and neuronal degeneration as shown by TUNEL, FJB and Nissl staining. However, ML221, an inhibitor of APJ, significantly reversed all the above neuroprotective effects of apelin-13. Moreover, a neuron-microglia co-culture system, which mimic SAH in vitro, confirmed the protective effect of apelin-13 on neurons and the inhibitory effect on inflammation through apoptosis-related proteins. CONCLUSIONS: These data demonstrated that apelin-13 exhibit a neuroprotective role after SAH through inhibition of apoptosis in an APJ dependent manner.

Multistage pH-responsive codelivery liposomal platform for synergistic cancer therapy.

BACKGROUND: Small interfering RNA (siRNA) is utilized as a potent agent for cancer therapy through regulating the expression of genes associated with tumors. While the widely application of siRNAs in cancer treatment is severely limited by their insufficient biological stability and its poor ability to penetrate cell membranes. Targeted delivery systems hold great promise to selectively deliver loaded drug to tumor site and reduce toxic side effect. However, the elevated tumor interstitial fluid pressure and efficient cytoplasmic release are still two significant obstacles to siRNA delivery. Co-delivery of chemotherapeutic drugs and siRNA represents a potential strategy which may achieve synergistic anticancer effect. Herein, we designed and synthesized a dual pH-responsive peptide (DPRP), which includes three units, a cell-penetrating domain (polyarginine), a polyanionic shielding domain (ehG)n, and an imine linkage between them. Based on the DPRP surface modification, we developed a pH-responsive liposomal system for co-delivering polo-like kinase-1 (PLK-1) specific siRNA and anticancer agent docetaxel (DTX), D-Lsi/DTX, to synergistically exhibit anti-tumor effect. RESULTS: In contrast to the results at the physiological pH (7.4), D-Lsi/DTX lead to the enhanced penetration into tumor spheroid, the facilitated cellular uptake, the promoted escape from endosomes/lysosomes, the improved distribution into cytoplasm, and the increased cellular apoptosis under mildly acidic condition (pH 6.5). Moreover, both in vitro and in vivo study indicated that D-Lsi/DTX had a therapeutic advantage over other control liposomes. We provided clear evidence that liposomal system co-delivering siPLK-1 and DTX could significantly downregulate expression of PLK-1 and inhibit tumor growth without detectable toxic side effect, compared with siPLK-1-loaded liposomes, DTX-loaded liposomes, and the combinatorial administration. CONCLUSION: These results demonstrate great potential of the combined chemo/gene therapy based on the multistage pH-responsive codelivery liposomal platform for synergistic tumor treatment.

The STING-IRF3 pathway contributes to paraquat-induced acute lung injury.

Immune and inflammatory responses play significant roles in paraquat (PQ)-induced acute lung injury (ALI), but the specific mechanisms remain unclear. Our study aimed to investigate the action of STING-IRF3 signaling on PQ-induced ALI in mice. Following PQ administration, samples were collected at 2, 12, 24, and 48 h for in vivo studies, and 24 h for in vitro studies. Following PQ administration (30 mg/kg, i.p.), injury to mouse lungs was evaluated by H&E staining and wet/dry ratios, and lung oxidative damage was evaluated by MDA and SOD assays. The mRNA levels of Sting, Irf3, and Ifnß were detected by RT-PCR, the expression of STING and IRF3 were assessed by western blotting and IHC/IF, and the secretion of IFNß was detected by ELISA. In vivo, PQ administration induced pathological changes and increased wet/dry ratios in lungs after 48 h. Sting, Irf3, and Ifnß mRNA levels in lung tissues, STING and pIRF3 protein levels in lung tissues, and IFNß secretion in serum, were upregulated by PQ in a time-dependent manner. PQ administration promoted IRF3 nuclear translocation in lung tissues after 48 h. The above changes were all attenuated by dexamethasone treatment (5 mg/kg, i.p., qd). In vitro, PQ induced STING and IRF3 translocation. Irf3 or Sting silencing decreased the mRNA levels and supernatant secretion of IFNß in PQ-treated RAW264.7 mouse macrophages. Sting silencing also inhibited the protein and mRNA levels of IRF3 in vitro. Our study suggests that STING-IRF3 signaling contributes to PQ-induced ALI, providing new information for future treatment strategies.

Isorhynchophylline ameliorates paraquat-induced acute kidney injury by attenuating oxidative stress and mitochondrial damage via regulating toll-interacting expression.

Isorhynchophylline (IRN) is an alkaloid with anti-inflammatory and anti-oxidative activities in cardiovascular and brain diseases, but its role in paraquat (PQ)-induced acute kidney injury (AKI) is yet unknown. The model of PQ-induced AKI in rats was established by intraperitoneal injection of PQ (25 mg/kg). We found that the tail vein injection of IRN (4 mg/kg) significantly increased the survival rate of PQ-intoxicated rats. IRN administration alleviated PQ-induced renal injury and renal dysfunction in rats, as evidenced by decreased apoptosis in renal cortex and reduced serum creatinine, serum BUN, and urine NGAL levels. Furthermore, IRN treatment improved the PQ-triggered oxidative stress in renal cortex by increasing the levels of anti-oxidant indicators (SOD activity, GSH/GSSG ratio, levels of Nrf-2, NQO-1, and HO-1 in renal cortex) and decreasing the levels of oxidative stress indexes (ROS and MDA levels in renal cortex). Interestingly, toll-interacting protein (Tollip), a negative regulator of interleukin 1 receptor associated kinase 1 (IRAK1) phosphorylation, was demonstrated to be increased by IRN injection in the renal cortex of PQ-intoxicated rats. In vitro experiments revealed that IRN protected renal tubular epithelial cells against PQ toxicity through suppressing oxidative stress and mitochondrial damage, and these protective effects were reversed by Tollip shRNA. Collectively, the present study demonstrated that IRN ameliorated PQ-induced AKI by attenuating oxidative stress and mitochondrial damage through upregulating Tollip, which provides new insights into the pathogenesis and treatment of PQ poisoning.