A fungal microRNA-like RNA regulated effector promotes pathogen infection by targeting a host defense-related transcription factor.

Effectors play important roles in facilitating the infection of plant pathogenic fungi. However, the gene expression regulatory mechanism of effector genes, in particular at the post-transcriptional level, is largely unknown. In this study, we uncovered the post-transcriptional regulation of an effector gene VmSP1 by a miRNA-like RNA (Vm-milR16) facilitating the infection of the apple tree Valsa canker pathogen Valsa mali. Genetic and molecular biological assays indicated that the expression of VmSP1 could be suppressed by Vm-milR16-mediated mRNA cleavage in a sequence-specific manner. During V. mali infection, Vm-milR16 was downregulated, whereas VmSP1 was upregulated, which further indicated the regulation relationship. VmSP1 was further demonstrated to be a secreted protein and could suppress plant immunity. Deletion of VmSP1 did not affect the vegetative growth but significantly reduced the virulence of V. mali. Further study indicated that VmSP1 could interact with the transcription factor MdbHLH189 of apple. Transiently overexpression of MdbHLH189 enhanced host resistance to V. mali by enhancing the expression of apple defense-related genes, together with the increased callose deposition. Silencing of MdbHLH189 compromised host resistance to V. mali. Our findings uncovered the novel epigenetic regulation mechanism of a virulence-associated effector gene mediated by a fungal milRNA at the post-transcriptional level, and the results enriched the understanding of the function and action mechanism of effectors in tree pathogenic fungi.

Abietane-Type Diterpenoids from the Arils of Torreya grandis.

A chemical investigation of the arils of Torreya grandis led to the isolation of seven abietane-type diterpenoids (compounds 1-7) including three previously undescribed compounds, one unreported natural product, and three known analogs. The structures of these compounds were determined by means of spectroscopy, single-crystal X-ray diffraction, and ECD spectra. An antibacterial activity assay showed that compounds 5 and 6 had significant inhibitory effects on methicillin-resistant Staphylococcus aureus, with MIC values of 100 µM. Moreover, compounds 1, 3, 4, and 7 exhibited anti-neuroinflammatory activity in LPS-stimulated BV-2 microglia cells, with the IC50 values ranging from 38.4 to 67.9 µM.

Hhex and Prox1a synergistically dictate the hepatoblast to hepatocyte differentiation in zebrafish.

The specification of endoderm cells to prospective hepatoblasts is the starting point for hepatogenesis. However, how a prospective hepatoblast gains the hepatic fate remains elusive. Previous studies have shown that loss-of-function of either hhex or prox1a alone causes a small liver phenotype but without abolishing the hepatocyte differentiation, suggesting that absence of either Hhex or Prox1a alone is not sufficient to block the hepatoblast differentiation. Here, via genetic studies of the zebrafish two single (hhex-/- and prox1a-/-) and one double (hhex-/-prox1a-/-) mutants, we show that simultaneous loss-of-function of the hhex and prox1a two genes does not block the endoderm cells to gain the hepatoblast potency but abolishes the hepatic differentiation from the prospective hepatoblast. Consequently, the hhex-/-prox1a-/- double mutant displays a liverless phenotype that cannot be rescued by the injection of bmp2a mRNA. Taken together, we provide strong evidences showing that Hhex teams with Prox1a to act as a master control of the differentiation of the prospective hepatoblasts towards hepatocytes.

Optimized screening of DNA methylation sites combined with gene expression analysis to identify diagnostic markers of colorectal cancer.

BACKGROUND: The prognosis of patients with colorectal cancer is related to early detection. However, commonly used screening markers lack sensitivity and specificity. In this study, we identified diagnostic methylation sites for colorectal cancer. METHODS: After screening the colorectal cancer methylation dataset, diagnostic sites were identified via survival analysis, difference analysis, and ridge regression dimensionality reduction. The correlation between the selected methylation sites and the estimation of immune cell infiltration was analyzed. The accuracy of the diagnosis was verified using different datasets and the 10-fold crossover method. RESULTS: According to Gene Ontology, the main enrichment pathways of genes with hypermethylation sites are axon development, axonogenesis, and pattern specification processes. However, the Kyoto Encyclopedia of Genes and Genomes (KEGG) suggests the following main enrichment pathways: neuroactive ligand-receptor interaction, calcium signaling, and cAMP signaling. In The Cancer Genome Atlas (TCGA) and GSE131013 datasets, the area under the curve of cg07628404 was > 0.95. For the NaiveBayes machine model of cg02604524, cg07628404, and cg27364741, the accuracies of 10-fold cross-validation in the GSE131013 and TCGA datasets were 95% and 99.4%, respectively. The survival prognosis of the hypomethylated group (cg02604524, cg07628404, and cg27364741) was better than that of the hypermethylated group. The mutation risk did not differ between the hypermethylated and hypomethylated groups. The correlation coefficient between the three loci and CD4 central memory T cells, hematological stem cells, and other immune cells was not high (p < 0.05). CONCLUSION: In cases of colorectal cancer, the main enrichment pathway of genes with hypermethylated sites was axon and nerve development. In the biopsy tissues, the hypermethylation sites were diagnostic for colorectal cancer, and the NaiveBayes machine model of the three loci showed good diagnostic performance. Site (cg02604524, cg07628404, and cg27364741) hypermethylation predicts poor survival for colorectal cancer. Three methylation sites were weakly correlated with individual immune cell infiltration. Hypermethylation sites may be a useful repository for diagnosing colorectal cancer.

Spatiotemporal optical properties of dissolved organic matter in a sluice-controlled coastal plain river with both salinity and trophic gradients.

Due to the combined effect of sluices and sea tide, the sluice-controlled coastal plain river would be characterized by both trophic state and salinity gradients, affecting the spatiotemporal optical properties of dissolved organic matter (DOM). In this study, we investigated the spatiotemporal variation of water quality parameters and optical properties of DOM in the Haihe River, a representative sluice-controlled coastal plain river in Tianjin, China. A significant salinity gradient and four trophic states were observed in the water body of the Haihe River. Two humic- and one protein-like substances were identified from the DOM by the three-dimensional fluorescence spectra combined with the parallel factor (PARAFAC) analysis. Pearson's correlation analysis and redundancy analysis (RDA) showed that the salinity significantly affected the abundance of chromophoric DOM (CDOM) but did not cause significant changes in the fluorescence optical characteristics. In addition, the effect of Trophic state index (TSI) on the CDOM abundance was greater than that on the fluorescence intensity of fluorescent dissolved organic matter (FDOM). In the water body with both salinity and trophic state gradients, TSI posed a greater influence than salinity on the CDOM abundance. Our results fill the research gap in spatiotemporal DOM characteristics and water quality variation in water bodies with both salinity and trophic state gradients. These results are beneficial for clarifying the joint influence of saline intrusion and sluices on the DOM characteristics and water quality in sluice-controlled coastal plain rivers.

A fungal microRNA-like RNA subverts host immunity and facilitates pathogen infection by silencing two host receptor-like kinase genes.

Small RNAs (sRNAs) play important roles in various biological processes by silencing their corresponding target genes in most eukaryotes. However, cross-kingdom regulation mediated by fungal microRNA-like RNAs (milRNAs) in plant-pathogen interactions is still largely unknown. Using molecular, genetic, histological, and biochemical approaches, we found that the apple tree Valsa canker pathogen Valsa mali milRNA Vm-milR1 could suppress the host immunity by silencing two host receptor-like kinase genes, MdRLKT1 and MdRLKT2. Vm-milR1 was highly induced during V. mali infection. Deletion of Vm-milR1 precursor abolished the generation of Vm-milR1 and reduced the virulence of V. mali. Inoculation of Vm-milR1 deletion mutants induced the host defence responses, including reactive oxygen species (ROS) accumulation, callose deposition, and high expression of defence-related genes. Furthermore, Vm-milR1 was confirmed to be able to suppress the expression of MdRLKT1 and MdRLKT2 in a sequence-specific manner. Moreover, overexpression of either MdRLKT1 or MdRLKT2 enhanced apple resistance to V. mali by activating the host defence responses. Furthermore, knockdown of MdRLKT1 or MdRLKT2 compromised the host resistance to V. mali. Our study revealed that V. mali was equipped with Vm-milR1 as an sRNA effector to silence host receptor-like kinase genes, suppress the host defence responses, and facilitate pathogen infection.

A multifaceted module of BRI1 ETHYLMETHANE SULFONATE SUPRESSOR1 (BES1)-MYB88 in growth and stress tolerance of apple.

The R2R3 transcription factor MdMYB88 has previously been reported to function in biotic and abiotic stress responses. Here, we identify BRI1 ETHYLMETHANE SULFONATE SUPRESSOR1 (MdBES1), a vital component of brassinosteroid (BR) signaling in apple (Malus × domestica) that directly binds to the MdMYB88 promoter, regulating the expression of MdMYB88 in a dynamic and multifaceted mode. MdBES1 positively regulated expression of MdMYB88 under cold stress and pathogen attack, but negatively regulated its expression under control and drought conditions. Consistently, MdBES1 was a positive regulator for cold tolerance and disease resistance in apple, but a negative regulator for drought tolerance. In addition, MdMYB88 participated in BR biosynthesis by directly regulating the BR biosynthetic genes DE ETIOLATED 2 (MdDET2), DWARF 4 (MdDWF4), and BRASSINOSTEROID 6 OXIDASE 2 (MdBR6OX2). Applying exogenous BR partially rescued the erect leaf and dwarf phenotypes, as well as defects in stress tolerance in MdMYB88/124 RNAi plants. Moreover, knockdown of MdMYB88 in MdBES1 overexpression (OE) plants decreased resistance to a pathogen and C-REPEAT BINDING FACTOR1 expression, whereas overexpressing MdMYB88 in MdBES1 OE plants increased expression of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 （MdSPL3） and BR biosynthetic genes, suggesting that MdMYB88 contributes to MdBES1 function during BR biosynthesis and the stress response. Taken together, our results reveal multifaceted regulation of MdBES1 on MdMYB88 in BR biosynthesis and stress tolerance.

Structurally Diverse Sesquiterpenoid Glycoside Esters from Pittosporum qinlingense with Anti-neuroinflammatory Activity.

Thirteen new sesquiterpenoid glycoside esters, including 11 aromadendrane-type compounds, pitqinlingosides A-K (1-11), one cadinane-type compound, pitqinlingoside L (12), and one eudesmane-type compound, pitqinlingoside M (13), together with seven known analogues (14-20) were isolated from the twigs, fruits, and leaves of Pittosporum qinlingense. Structures were elucidated by analysis of spectroscopic data, gas chromatography mass spectrometry (GC-MS), and chemical methods. The absolute configuration was confirmed by single-crystal X-ray crystallography analysis or electronic circular dichroism spectra. Unusual glycoside esters are characterized by the presence of polyacylated ß-d-fucopyranosyl, ß-d-glucopyranosyl, and ß-d-xylopyranosyl units. Pitqinlingosides A (1), B (2), D (4), and F (6), pittosporanoside A1 acetate (14), and pittosporanoside A1 (16) showed significant nitric oxide production inhibition in lipopolysaccharide (LPS)-induced BV-2 microglial cells with IC50 values ranging from 0.95 to 24.12 µM. Structure-activity relationships of the isolated compounds are discussed.

Triterpenoids and meroterpenoids from the edible Ganoderma resinaceum and their potential anti-inflammatory, antioxidant and anti-apoptosis activities.

Ganoderma resinaceum, as a traditional edible mushroom, has been widely reported to improve neurodegenerative diseases characterized by oxidative stress and inflammation. In this study, five new terpenoids, including four lanostane triterpenoids, named ganoresinoid A-D (1-4) and one meroterpenoid, named ganoresinoid E (5), along with 27 known compounds (6-32), were isolated from the fruiting bodies of edible mushroom G. resinaceum. These structures were identified by NMR, HRESIMS data analysis. All metabolites were evaluated for anti-inflammatory, antioxidative and anti-apoptosis activities. Among them, ganoresinoid A showed notably restrained nitric oxide (NO), IL-1ß, IL-6 and TNF-α levels in LPS-activated BV-2 microglial cells via suppressing TLR-4/ NF-κB and MAPK signaling pathway. Simultaneously, ganoresinoid A remarkably alleviated LPS-induced apoptosis by means of the decrease of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). In addition, ganoresinoid A demonstrated antioxidant effects in H2O2-induced SH-SY5Y cells by activating the Akt/GSK-3ß/Nrf2 signaling pathway. Taken together, these results may provide a stronger theoretical basis for ganoresinoid A from G. resinaceum as nutrition intervention to alleviate neurodegenerative diseases.

Carbohydrates deteriorate fatty liver by activating the inflammatory response.

Non-alcoholic fatty liver disease (NAFLD) was defined in 1980 and has the same histological characteristics as alcoholic liver disease except for alcohol consumption. After 40 years, the understanding of this disease is still imperfect. Without specific drugs available for treatment, the number of patients with NAFLD is increasing rapidly, and NAFLD currently affects more than one-quarter of the global population. NAFLD is mostly caused by a sedentary lifestyle and excessive energy intake of fat and sugar. To ameliorate or avoid NAFLD, people commonly replace high-fat foods with high-carbohydrate foods (especially starchy carbohydrates) as a way to reduce caloric intake and reach satiety. However, there are few studies that concentrate on the effect of carbohydrate intake on liver metabolism in patients with NAFLD, much fewer than the studies on fat intake. Besides, most of these studies are not systematic, which has made identification of the mechanism difficult. In this review, we collected and analysed data from studies on human and animal models and, surprisingly, found that carbohydrates and liver steatosis could be linked by inflammation. This review not only describes the effects of carbohydrates on NAFLD and body lipid metabolism but also analyses and predicts possible molecular pathways of carbohydrates in liver lipid synthesis that involve inflammation. Furthermore, the limitations of recent research and possible targets for regulating inflammation and lipogenesis are discussed. This review describes the effects of starchy carbohydrates, a nutrient signal, on NAFLD from the perspective of inflammation.

Stanniocalcin-1 Protected Astrocytes from Hypoxic Damage Through the AMPK Pathway.

Our previous studies revealed that the expression of stanniocalcin-1 (STC1) in astrocytes increased under hypoxic conditions. However, the role of STC1 in hypoxic astrocytes is not well understood. In this work, we first showed the increased expression of STC1 in astrocyte cell line and astrocytes in the brain tissues of mice after exposure to hypoxia. Then, we found that knockdown of STC1 inhibited cell viability and increased apoptosis. These effects were mediated by decreasing the levels of SIRT3, UCP2, and glycolytic genes and increasing the levels of ROS. Further studies suggested that STC1 silencing promoted oxidative stress and suppressed glycolysis by downregulating AMPKα1. Moreover, HIF-1α knockdown in hypoxic astrocytes led to decreased expression of STC1 and AMPKα1, indicating that the expression of STC1 was regulated by HIF-1α. In conclusion, our study showed that HIF-1α-induced STC1 could protect astrocytes from hypoxic damage by regulating glycolysis and redox homeostasis in an AMPKα1-dependent manner.

Bright chemiluminescent dioxetane probes for the detection of gaseous transmitter H2S.

Hydrogen sulfide (H2S), the third gaseous transmitter after CO and NO, is a double-edged sword in the human body. A specific concentration of H2S can attenuate myocardial ischemia-reperfusion injury by preserving mitochondrial function, in contrast, cause illness, including inflammation and stroke. There are already some probes for the real-time monitoring of the level of H2S in the biological environment. However, they have some disadvantages, such as phototoxicity, low sensitivity, and low quantum yield. In this research, by linking 4-dinitrophenyl-ether (DNP), a specific recognition group for H2S, with a chemiluminophore 1,2-dioxetane, we designed and synthesized the probe SCL-1. To tackle the barrier that the traditional chemiluminescent group has a short emission wavelength and is not easy to penetrate deep tissues, an acrylonitrile electron-withdrawing substituent was installed to the ortho-position of the 1,2-dioxanol hydroxy group. According to the same design strategy as SCL-1, the probe SCL-2 was designed with the modified chemiluminescent group. Studies have shown that SCL-2 with electron-withdrawing acrylonitrile has higher luminescence quantum yield and high sensitivity than SCL-1, realizing real-time detection of H2S in vitro and in vivo. The LOD of SCL-2 was 0.185 µM, which was the best among the currently available luminescent probes for detecting H2S. We envisage that SCL-2 may be a practical toolbox for studying the biological functions of H2S and H2S-related diseases.

Asymmetric synthesis of 9-alkyl tetrahydroxanthenones via tandem asymmetric Michael/cyclization promoted by chiral phosphoric acid.

A tandem asymmetric Michael-addition/cyclization of cyclic 1,3-dicarbonyl compounds to ß,γ-unsaturated α-ketoesters catalyzed by chiral phosphoric acid is presented. This protocol provides a facile approach for the construction of enantioenriched 9-alkyl tetrahydroxanthenones, an ubiquitous framework found in a number of natural products and pharmaceutical molecules, in high yields with good to high enantioselectivities.

Acoustic scattering by a finite-length elastic elliptical cylinder in a plane wave.

In this paper, based on the deformed cylinder method, the expression of the far-field acoustic scattering form function of a finite-length elastic elliptical cylinder is obtained. The target strength of elastic aluminum and polymethylmethacrylate (PMMA) elliptical cylinders, with different lengths varying with dimensionless frequency ka, is studied for different incident angles. In addition, the change of the target strength of a PMMA elliptical cylinder with incident angle is explored theoretically and experimentally when the frequency range is 20-40 kHz. Simulation results show that there is almost no change in the peak of the curve of the backscattering target strength versus frequency for a finite-length elastic elliptical cylinder with different lengths. As the length decreases, the target strength gradually decreases. When the theoretical simulation results are compared with the experimental results, it is found that the theoretical predictions are in good agreement with the experimental results. Furthermore, the study will provide theoretical and experimental basis for the prediction and recognition of underwater targets.

Water quality characteristics and assessment of Yongding New River by improved comprehensive water quality identification index based on game theory.

The Yongding New River is essential for the water supplies of Tianjin. To date, there is no comprehensive report that assesses the year-round water quality of the Yongding New River Main stream. Moreover, little attention has been given to determining a combined weight for improving the traditional comprehensive water quality identification index (ICWQII) by the game theory. Seven water quality parameters were investigated monthly along the main stream of the Yongding New River from May 2018 to April 2019. Organic contaminants and nitrogen pollution were mainly caused by point sources pollution, and the total phosphorus mainly by non-point source pollution. Dramatic spatio-temporal variations of water quality parameters were jointly caused by different pollutant sources and hydrometeorological factors. In terms of this study, an improved comprehensive water quality identification index (ICWQII) based on entropy weight or variation coefficient and traditional CWQII underestimated the water qualities, and an ICWQII based on the superstandard multiple method overvalued the assessments. By contrast, water qualities assessments done with an ICWQII based on the game theory matched perfectly with the practical situation. The ICWQII based on game theory proposed in this study takes into account not only the degree of disorder and variation of water quality data, but also the influence of standard-exceeded pollution indicators, whose results are relatively reasonable. All findings and the ICWQII based on game theory can provide scientific support for decisions related to the water environment management of the Yongding New River and other waters.

Integrated analysis identified core signal pathways and hypoxic characteristics of human glioblastoma.

As a hallmark for glioblastoma (GBM), high heterogeneity causes a variety of phenotypes and therapeutic responses among GBM patients, and it contributes to treatment failure. Moreover, hypoxia is a predominant feature of GBM and contributes greatly to its phenotype. To analyse the landscape of gene expression and hypoxic characteristics of GBM cells and their clinical significance in GBM patients, we performed transcriptome analysis of the GBM cell line U87-MG and the normal glial cell line HEB under normoxia and hypoxia conditions, with the results of which were analysed using established gene ontology databases as well as The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia. We revealed core signal pathways, including inflammation, angiogenesis and migration, and for the first time mapped the components of the toll-like receptor 6 pathway in GBM cells. Moreover, by investigating the signal pathways involved in homoeostasis, proliferation and adenosine triphosphate metabolism, the critical response of GBM to hypoxia was clarified. Experiments with cell lines, patient serum and tissue identified IL1B, CSF3 and TIMP1 as potential plasma markers and VIM, STC1, TGFB1 and HMOX1 as potential biopsy markers for GBM. In conclusion, our study provided a comprehensive understanding for signal pathways and hypoxic characteristics of GBM and identified new biomarkers for GBM patients.

Targeting mitochondria-associated membranes as a potential therapy against endothelial injury induced by hypoxia.

Mitochondrial dysfunction plays a principal role in hypoxia-induced endothelial injury, which is involved in hypoxic pulmonary hypertension and ischemic cardiovascular diseases. Recent studies have identified mitochondria-associated membranes (MAMs) that modulate mitochondrial function under a variety of pathophysiological conditions such as high-fat diet-mediated insulin resistance, hypoxia reoxygenation-induced myocardial death, and hypoxia-evoked vascular smooth muscle cell proliferation. However, the role of MAMs in hypoxia-induced endothelial injury remains unclear. To explore this further, human umbilical vein endothelial cells and human pulmonary artery endothelial cells were exposed to hypoxia (1% O2 ) for 24 hours. An increase in MAM formation was uncovered by immunoblotting and immunofluorescence. Then, we performed small interfering RNA transfection targeted to MAM constitutive proteins and explored the biological effects. Knockdown of MAM constitutive proteins attenuated hypoxia-induced elevation of mitochondrial Ca2+ and repressed mitochondrial impairment, leading to an increase in mitochondrial membrane potential and ATP production and a decline in reactive oxygen species. Then, we found that MAM disruption mitigated cell apoptosis and promoted cell survival. Next, other protective effects, such as those pertaining to the repression of inflammatory response and the promotion of NO synthesis, were investigated. With the disruption of MAMs under hypoxia, inflammatory molecule expression was repressed, and the eNOS-NO pathway was enhanced. This study demonstrates that the disruption of MAMs might be of therapeutic value for treating endothelial injury under hypoxia, suggesting a novel strategy for preventing hypoxic pulmonary hypertension and ischemic injuries.

Bioluminescent Probe for Monitoring Endogenous Fibroblast Activation Protein-Alpha.

Fibroblast activation protein-α (FAP), as a crucial member of cell surface glycoprotein, highly expresses in reactive fibroblasts of tumors and several fibrosis diseases. It is a potential target for drug design and also reported as a prodrug strategy to increase the therapeutic window of some anticancer agents. In this work, we developed the first bioluminogenic probe for FAP with a limit-of-detection of 0.254 ng/mL, which could be applied to evaluate the FAP inhibitors in vitro. The experiments of transgenic mice and tumor-bearing nude mice validated our probe 1 could reflect the endogenous FAP level in vivo. Furthermore, this probe was successfully used to reflect FAP up-regulation in the lung homogenates of the bleomycin-induced idiopathic pulmonary fibrosis mice.

Nogo-B Receptor Directs Mitochondria-Associated Membranes to Regulate Vascular Smooth Muscle Cell Proliferation.

Mitochondria-associated membranes (MAM) are a well-recognized contact link between the mitochondria and endoplasmic reticulum that affects mitochondrial biology and vascular smooth muscle cells (VSMCs) proliferation via the regulation of mitochondrial Ca2+(Ca2+m) influx. Nogo-B receptor (NgBR) plays a vital role in proliferation, epithelial-mesenchymal transition, and chemoresistance of some tumors. Recent studies have revealed that downregulation of NgBR, which stimulates the proliferation of VSMCs, but the underlying mechanism remains unclear. Here, we investigated the role of NgBR in MAM and VSMC proliferation. We analyzed the expression of NgBR in pulmonary arteries using a rat model of hypoxic pulmonary hypertension (HPH), in which rats were subjected to normoxic recovery after hypoxia. VSMCs exposed to hypoxia and renormoxia were used to assess the alterations in NgBR expression in vitro. The effect of NgBR downregulation and overexpression on VSMC proliferation was explored. The results revealed that NgBR expression was negatively related with VSMCs proliferation. Then, MAM formation and the phosphorylation of inositol 1,4,5-trisphosphate receptor type 3 (IP3R3) was detected. We found that knockdown of NgBR resulted in MAM disruption and augmented the phosphorylation of IP3R3 through pAkt, accompanied by mitochondrial dysfunction including decreased Ca2+m, respiration and mitochondrial superoxide, increased mitochondrial membrane potential and HIF-1α nuclear localization, which were determined by confocal microscopy and Seahorse XF-96 analyzer. By contrast, NgBR overexpression attenuated IP3R3 phosphorylation and HIF-1α nuclear localization under hypoxia. These results reveal that dysregulation of NgBR promotes VSMC proliferation via MAM disruption and increased IP3R3 phosphorylation, which contribute to the decrease of Ca2+m and mitochondrial impairment.

Hypoxia-induced apoptosis of mouse spermatocytes is mediated by HIF-1α through a death receptor pathway and a mitochondrial pathway.

Hypoxia in vivo induces oligozoospermia, azoospermia, and degeneration of the germinal epithelium, but the underlying molecular mechanism of this induction is not fully clarified. The aim of this study was to investigate the role of the death receptor pathway and the mitochondrial pathway in hypoxia-induced apoptosis of mouse GC-2spd (GC-2) cells and the relationship between HIF-1α and apoptosis of GC-2 cells induced by hypoxia. GC-2 cells were subjected to 1% oxygen for 48 hr. Apoptosis was detected by flow cytometry, TUNEL staining, LDH, caspase-3/8/9 in the absence and presence of HIF-1α siRNA. The protein levels of apoptosis-related markers were determined by Western blot in the presence and absence of HIF-1α siRNA. Mitochondrial transmembrane potential change was observed by in situ JC-1 staining. Cell viability was assessed upon treatment of caspase-8 and 9 inhibitors. The results indicated that hypoxia at 1% oxygen for 48 hr induced apoptosis of GC-2 cells. A prolonged exposure of GC-2 cells to hypoxic conditions caused downregulation of c-FLIP, Dc R2 and Bcl-2 and upregulation of DR5 , TRAIL, Fas, p53, and Bax, with an overproduction of caspase-3/8/9. Moreover, hypoxia at this level had an effect on mitochondrial depolarization. In addition, specific inhibitors of caspase-8/9 partially suppressed hypoxia-induced GC-2 cell apoptosis, and the anti-apoptotic effects of the caspase inhibitors were additive. Of note, HIF-1α knockdown attenuated hypoxia and induced apoptosis of GC-2 cells. In conclusion, our data suggest that the death receptor pathway and mitochondrial pathway, which are likely mediated by HIF-1α, contribute to hypoxia-induced GC-2 cell apoptosis.