The transcriptomic responses of blunt snout bream (Megalobrama amblycephala) to acute hypoxia stress alone, and in combination with bortezomib.

BACKGROUND: Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia. A new blunt snout bream strain, "Pujiang No.2", was developed to overcome this shortcoming. As a proteasome inhibitor, bortezomib (PS-341) has been shown to affect the adaptation of cells to a hypoxic environment. In the present study, bortezomib was used to explore the hypoxia adaptation mechanism of "Pujiang No.2". We examined how acute hypoxia alone (hypoxia-treated, HN: 1.0 mg·L- 1), and in combination with bortezomib (hypoxia-bortezomib-treated, HB: Use 1 mg bortezomib for 1 kg fish), impacted the hepatic ultrastructure and transcriptome expression compared to control fish (normoxia-treated, NN). RESULTS: Hypoxia tolerance was significantly decreased in the bortezomib-treated group (LOEcrit, loss of equilibrium, 1.11 mg·L- 1 and 1.32 mg·L- 1) compared to the control group (LOEcrit, 0.73 mg·L- 1 and 0.85 mg·L- 1). The HB group had more severe liver injury than the HN group. Specifically, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the HB group (52.16 U/gprot, 32 U/gprot) were significantly (p < 0.01) higher than those in the HN group (32.85 U/gprot, 21. 68 U/gprot). In addition, more severe liver damage such as vacuoles, nuclear atrophy, and nuclear lysis were observed in the HB group. RNA-seq was performed on livers from the HN, HB and NN groups. KEGG pathway analysis disclosed that many DEGs (differently expressed genes) were enriched in the HIF-1, FOXO, MAPK, PI3K-Akt and AMPK signaling pathway and their downstream. CONCLUSION: We explored the adaptation mechanism of "Pujiang No.2" to hypoxia stress by using bortezomib, and combined with transcriptome analysis, accurately captured the genes related to hypoxia tolerance advantage.

Targeting the pathways of regulated necrosis: a potential strategy for alleviation of cardio-cerebrovascular injury.

Apoptosis, necrosis and autophagy-dependent cell death are the three major types of cell death. Traditionally, necrosis is thought as a passive and unregulated form of cell death. However, certain necrosis can also occur in a highly regulated manner, referring to regulated necrosis. Depending on the signaling pathways, regulated necrosis can be further classified as necroptosis, pyroptosis, ferroptosis, parthanatos and CypD-mediated necrosis. Numerous studies have reported that regulated necrosis contributes to the progression of multiple injury-relevant diseases. For example, necroptosis contributes to the development of myocardial infarction, atherosclerosis, heart failure and stroke; pyroptosis is involved in the progression of myocardial or cerebral infarction, atherosclerosis and diabetic cardiomyopathy; while ferroptosis, parthanatos and CypD-mediated necrosis participate in the pathological process of myocardial and/or cerebral ischemia/reperfusion injury. Thereby, targeting the pathways of regulated necrosis pharmacologically or genetically could be an efficient strategy for reducing cardio-cerebrovascular injury. Further study needs to focus on the crosstalk and interplay among different types of regulated necrosis. Pharmacological intervention of two or more types of regulated necrosis simultaneously may have advantages in clinic to treat injury-relevant diseases.

Determination of reference microRNAs for relative quantification in grass carp (Ctenopharyngodon idella).

Relative quantification is the strategy of choice for processing real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) data in microRNA (miRNA) expression studies. Normalization of relative quantification data is performed by comparison to reference genes. In teleost species, such as grass carp (Ctenopharyngodon idella), the determination of reference miRNAs and the optimal numbers of these that should be used has not been widely studied. In the present study, the stability of seven miRNAs (miR-126-3p, miR-101a, miR-451, miR-22a, miR-146, miR-142a-5p and miR-192) was investigated by RT-qPCR in different tissues and in different development stages of grass carp. Stability values were calculated with geNorm, NormFinder, BestKeeper and Delta CT algorithms. The results showed that tissue type is an important variability factor for miRNA expression stability. All seven miRNAs had good stability values and, therefore, could be used as reference miRNAs. When all tissues and developmental stages were considered, miR-101a was the most stable miRNA. When each tissue type was considered separately, the most stable miRNAs were 126-3p in blood and liver, 101a in the gills, 192 in the kidney, 451 in the intestine and 22a in the brain, head kidney, spleen, heart, muscle, skin and fin. 126-3p was the most stable reference miRNA gene during developmental stages 1-5, while 22a was the most stable during developmental stages 6-18. Overall, this study provides valuable information about the reference miRNAs that can be used to perform appropriate normalizations when undertaking relative quantification in RT-qPCR studies of grass carp.

CARD11-BCL10-MALT1 complex-dependent MALT1 activation facilitates myocardial oxidative stress in doxorubicin-treated mice via enhancing k48-linked ubiquitination of Nrf2.

AIMS: Down-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) contributes to doxorubicin (DOX)-induced myocardial oxidative stress, and inhibition of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) increased Nrf2 protein level in rat heart suffered ischemia/reperfusion, indicating a connection between MALT1 and Nrf2. This study aims to explore the role of MALT1 in DOX-induced myocardial oxidative stress and the underlying mechanisms. RESULTS: The mice received a single injection of DOX (15 mg/kg, i.p.) to induce myocardial oxidative stress, evidenced by increases in the levels of reactive oxidative species while decreases in the activities of anti-oxidative enzymes, concomitant with a down-regulation of Nrf2; these phenomena were reversed by MALT1 inhibitor. Similar phenomena were observed in DOX-induced oxidative stress in cardiomyocytes. Mechanistically, knockdown or inhibition of MALT1 notably attenuated the interaction between Nrf2 and MALT1, and decreased the k48-linked ubiquitination of Nrf2. Furthermore, inhibition or knockdown of calcium/calmodulin-dependent protein kinase II (CaMKII-δ) reduced the phosphorylation of caspase recruitment domain-containing protein 11 (CARD11), and subsequently disrupted the assembly of CARD11, B-cell lymphoma 10 (BCL10) and MALT1 (CBM) complex, and reduced the MALT1-dependent k48-linked ubiquitination of Nrf2 in DOX-treated mice or cardiomyocytes. INNOVATION AND CONCLUSION: The E3 ubiquitin ligase function of MALT1 accounts for the down-regulation of Nrf2 and aggravation of myocardial oxidative stress in DOX-treated mice, and CaMKII-δ-dependent phosphorylation of CARD11 triggered the assembly of CBM complex and subsequent activation of MALT1.

DL-3-n-butylphthalide improves the endothelium-dependent vasodilation in high-fat diet-fed ApoE-/- mice via suppressing inflammation, endothelial necroptosis and apoptosis.

Impaired endothelium-dependent vasodilation in atherosclerosis is a high-risk factor for myocardial infarction and ischemic stroke, and inflammation, necroptosis and apoptosis contribute to endothelial dysfunction in atherosclerosis. Although DL-3-n-butylphthalide (NBP) has been widely used in treating ischemic stroke, its effect on endothelium-dependent vasodilation remains unknown. This study aims to explore whether NBP is able to improve endothelium-dependent vasodilation in atherosclerosis and the underlying mechanisms. Male ApoE-/- mice were fed with a high-fat diet (HFD) for 9-16 weeks to establish a model of atherosclerosis. NBP were given to the mice after eating HFD for 6 weeks and atorvastatin served as a positive control. The endothelium-dependent vasodilation, the blood flow velocity, the atherosclerotic lesion area, the serum levels of lipids, inflammatory cytokines and necroptosis-relevant proteins (RIPK1, RIPK3 and MLKL), and the endothelial necroptosis and apoptosis within the aorta were measured. Human umbilical vein endothelial cells (HUVECs) were incubated with oxidized low-density lipoprotein (ox-LDL) for 48 h to mimic endothelial injury in atherosclerosis, lactate dehydrogenase release, the ratio of necroptosis and apoptosis and the expression of necroptosis-relevant proteins were examined. Similar to atorvastatin, NBP improves endothelium-dependent vasodilation, decreases aortic flow velocity and reduces atherosclerotic lesion area in HFD-fed ApoE-/- mice, concomitant with a reduction in serum lipids, inflammatory cytokines and necroptosis-relevant proteins, and endothelial necroptosis and apoptosis. Consistently, NBP inhibited necroptosis and apoptosis in ox-LDL-treated HUVECs. Based on these observations, we conclude that NBP exerts beneficial effects on improving the endothelium-dependent vasodilation in atherosclerosis via suppressing inflammation, endothelial necroptosis and apoptosis.

Docosahexaenoic acid reduces hypoglycemia-induced neuronal necroptosis via the peroxisome proliferator-activated receptor γ/nuclear factor-κB pathway.

DHA has been shown to be neuroprotective and important to neurogenesis, but its role in HG-induced brain injury and the underlying mechanisms remain unknown. To elucidate the therapeutic effect of DHA, we established a mouse model with insulin-induced hypoglycemic brain injury and an in vitro model of HT-22 cells using a sugar-free medium. DHA treatment significantly reduced neuronal death and improved HG-induced learning and memory deficits. Moreover, DHA inhibited neuronal necroptosis and decreased the concentrations of TNF-α, IL-1ß and TNFR1. DHA also activated PPAR-γ and suppressed the NF-κB pathway in mouse brain tissues. In vitro, DHA treatment restored the viability and decreased necroptosis of HT-22 cells treated with glucose deprivation. However, the inhibition of PPAR-γ with T0070907 reversed neuroprotective and anti-necroptosis effects of DHA in HG-induced brain injury, which is associated with the activation of the downstream NF-κB pathway. We conclude that DHA displays a protective effect against HG-induced brain injury through the PPAR-γ/NF-κB pathway and represents a promising method to prevent HG-induced brain injury.

The SPATA2/CYLD pathway contributes to doxorubicin-induced cardiomyocyte ferroptosis via enhancing ferritinophagy.

Ferroptosis is an iron-dependent cell death and contributes to doxorubicin-induced cardiotoxicity, but the mechanisms behind intracellular iron overload in cardiomyocyte after administration of doxorubicin remain largely unknown. Ferritinophagy is a selective type of autophagy and could be a novel source for intracellular free iron. Spermatogenesis-associated protein 2 (SPATA2), a member of the TNF signaling pathway, can recruit cylindromatosis (CYLD, a deubiquitinating enzyme) to regulate cell death. This study aims to explore whether ferritinophagy is the source for intracellular iron overload in cardiomyocyte upon doxorubicin treatment and whether the SPATA2/CYLD pathway is involved in regulation of nuclear receptor coactivator 4 (NCOA4) level, the selective cargo receptor for ferritinophagy. The C57BL/6J mice were subjected to a single injection of doxorubicin, which showed the compromised cardiac functions, accompanied by the upregulation of SPATA2 and CYLD and the enhanced interaction between them, the increases in ferritinophagy (reflecting by increases in NCOA4 and ratio of LC3Ⅱ/LC3Ⅰ while decreases in NCOA4 ubiquitination and ferritin) and ferroptosis (reflecting by intracellular iron overload and increase of acyl-CoA synthetase long chain family member 4). Consistently, similar results were achieved in the cultured cardiomyocytes after incubation with doxorubicin. Knocked down of SPATA2 notably reduced doxorubicin-induced cardiomyocyte injury concomitant with the attenuated ferritinophagy and the decreased ferroptosis. Based on these observations, we conclude that a novel pathway of SPATA2/CYLD has been identified, which contributes to doxorubicin-induced cardiomyocyte ferroptosis via enhancing ferritinophagy through a mechanism involving the deubiquitination of NCOA4.

Allopurinol attenuates oxidative injury in rat hearts suffered ischemia/reperfusion via suppressing the xanthine oxidase/vascular peroxidase 1 pathway.

Allopurinol, a xanthine oxidase (XO) inhibitor, is reported to alleviate myocardial ischemia/reperfusion (I/R) injury by reducing the production of reactive oxygen species (ROS). As an XO-derived product, H2O2 can act as a substrate of vascular peroxidase 1 (VPO1) to induce the generation of hypochlorous acid (HOCl), a potent oxidant. This study aims to explore whether the XO/VPO1 pathway is involved in the anti-oxidative effects of allopurinol on the myocardial I/R injury. In a rat heart model of I/R, allopurinol alleviated I/R oxidative injury accompanied by decreased XO activity, XO-derived products (H2O2 and uric acid), and VPO1 expression (mRNA and protein). In a cardiac cell model of hypoxia/reoxygenation (H/R), allopurinol or XO siRNA reduced H/R injury concomitant with decreased XO activity, VPO1 expression as well as the XO and VPO1-derived products (H2O2, uric acid, and HOCl). Although knockdown of VPO1 could also exert a beneficial effect on H/R injury, it did not affect XO activity, XO expression, and XO-derived products. Based on these observations, we conclude that the novel pathway of XO/VPO1 is responsible for, at least partly, myocardial I/R-induced oxidative injury, and allopurinol exerted the cardioprotective effects on myocardial I/R injury via inhibiting the XO/VPO1 pathway.

Regulation of the Golgi apparatus via GOLPH3-mediated new selective autophagy.

AIMS: Although previous studies elaborated that selective autophagy was involved in quality control of some organelles, including nucleus, mitochondria, the endoplasmic reticulum and peroxisomes, it remained unclear whether the selective autophagy of the Golgi apparatus (Golgiphagy) existed or not. MAIN METHODS: In this study, H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells were treated with autophagy inducers, Golgi stress inducers and cardiomyocytes hypertrophy stimulators. The Golgiphagy was evaluated by analysing the co-localization of Golgi markers and LC3B. Furthermore, the transmission electron microscope was used to observe the occurrence of Golgiphagy. The co-immunoprecipitation assay was used to evaluate the interaction of GOLPH3 and LC3B. KEY FINDINGS: Results showed that starvation promoted the co-localization of both GM130-positive and TGN46-positive Golgi fragments with LC3B-positive autophagosomes in H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells. Transmission electron microscopy images showed that Golgi apparatus was sequestered into the autophagosomes in the starvation group. Moreover, Golgi stress inducers also facilitated the co-localization of Golgi markers and LC3B in H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells. Furthermore, cardiomyocyte hypertrophy stimulators also triggered the appearance of Golgiphagy in H9c2 cells. Importantly, the co-immunoprecipitation assay indicated endogenous GOLPH3 interacted with LC3B in H9c2 cells, HUVECs, HA-VSMCs. However, knocking down GOLPH3 inhibited the Golgiphagy. SIGNIFICANCE: This study unveiled a new selective autophagy of the Golgi apparatus (Golgiphagy). In addition, GOLPH3 might act as a novel cargo receptor to regulate Golgiphagy. Maintaining homeostasis of the Golgi apparatus via GOLPH3-mediated autophagy was indispensable for cell survival.

Down-regulated miR-21 promotes learning-memory recovery after brain injury.

BACKGROUND: MicroRNA is anendogenous non-coding single strand RNA which consists of 22 nt. It post-transcriptionally regulates gene expression and development. MicroRNA 21 plays an important role in repairing injured brain tissues. Thus, in this research, we explored the function of miR-21 in learning-memory recovery after brain injury. METHOD: 3 days old newborn SD rats were separated into three groups: Sham operation group (Sham), inflammation-sensitized hypoxic-ischemic brain injury (LPS+HI) group and miR-21 inhibitor group. 28 days later, the learning and memory capability was assayed by water maze. H&E staining and Nissl's staining were used to assay pathologic changes and TUNEL was used to assay neuron apoptosis in brain tissue. RESULTS: Water maze assay showed that the capability of positioning navigation in the IH-HI group was worse than in the Sham group and miR-21 inhibition group, and the Sham group wass better than miR-21 group. Both of the comparisons had statistical significance (P < 0.05). H&E staining in the sham group showed that the neurons were arranged well in hippocampus. In LPS+HI group, some neurons in hippocampus had vacuolar degeneration and the neurons were not well arranged well. In the hippocampus of miR-21 inhibitor group, the neuron cell layers were decreased but the neurons were arranged better than in the LPS+HI group. Nissl's staining in LPS+HI group showed neuronal edema, neurons decreased, and Nissl's bodies decreased in the cytoplasm compared with the sham group. However, compared with the LPS+HI group, Nissl's staining in miR-21 inhibitor group showed that the neuronal edema was alleviated and neurons were better arranged. TUNEL assay showed that the apoptosis rate of LPS+HI group was higher than in the miR-21 inhibitor group and miR-21 inhibitor group was higher than the sham group. CONCLUSION: Down-regulated miR-21 can alleviate LPS+HI injury in the brain.

MicroRNA-induced negative regulation of TLR-5 in grass carp, Ctenopharyngodon idella.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in antibacterial defence in fish has not been fully determined. Here, we identified that nine miRNAs are differentially expressed in kidney between susceptible and resistant grass carp strains. Analysis of spatial and temporal miRNA expression patterns suggests that cid-miRn-115 and miR-142a-3p are potential regulators of anti-bacterial activity. Overexpressing of cid-miRn-115 and miR-142a-3p results in a visible change in Ctenopharyngodon idella kidney (CIK) cells immune effector activity. Bioinformatics analysis and overexpressing assay shows that cid-miRn-115 and miR-142a-3p directly regulate tlr5 expression. cid-miRn-115 and miR-142a-3p overexpressing leads to a significant decrease in tlr5 expression in CIK, thereby repressing its downstream genes, such as il-1ß, il-8 and tnf-α. These findings provide a novel insight into the determination of anti-bacterial compounds in grass carp.

Screening and functional pathway analysis of genes associated with pediatric allergic asthma using a DNA microarray.

The present study aimed to identify differentially expressed genes (DEGs) associated with pediatric allergic asthma, and to analyze the functional pathways of the selected target genes, in order to explore the pathogenesis of the disease. The GSE18965 gene expression profile was downloaded from the Gene Expression Omnibus database and was preprocessed. This gene expression profile consisted of seven normal samples and nine samples from patients with pediatric allergic asthma. The DEGs between the normal and pediatric allergic asthma samples were screened using limma package in R, and the cut­off value was set at false discovery rate <0.05 and log fold change >1. Following hierarchical clustering of the DEGs based on the expression profiles, the up­ and downregulated genes underwent a functional enrichment analysis by topological approach (P<0.05), using the Database for Annotation, Visualization and Integrated Discovery. A total of 127 DEGs were identified between the normal and pediatric allergic asthma samples. The up­ and downregulated genes were significantly enriched in the actin filament­based process and the monosaccharide metabolic process, respectively. Seven downregulated DEGs (M6PR, TPP1, GLB1, NEU1, ACP2, LAMP1 and HGSNAT) were identified in the lysosomal pathway, with P=6.4x10(­9). These results suggested that variation in lysosomal function, triggered by the seven downregulated genes, may lead to aberrant functioning of the T lymphocytes, resulting in asthma. Further research regarding the treatment of pediatric allergic asthma through targeting lysosomal function is required.

Suppression of RNA interference pathway in vitro by Grass carp reovirus.

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.