Critical role of cystic fibrosis transmembrane conductance regulator in hypoxia-induced apoptosis of H9c2 cardiomyocytes

AIM:To explore the roles of cystic fibrosis transmembrane conductance regulator (CFTR) in hypoxia-induced apoptosis of H9c2 cardiomyocytes and the underlying mechanisms. METHODS:The rat H9c2 cardiomyocytes were exposed to a 1% hypoxic environment in a hypoxic chamber. After CFTR overexpression, H9c2 cardiomyocytes were cultured in a hypoxic environment. The mRNA and protein levels of CFTR were examined by RT-qPCR and Western blot, respectively. The cell viability was measured by MTT assay. The apoptotic rate was determined by Hoechst 33342 and Annexin V-FITC/PI staining, and the production of reactive oxygen species (ROS) was examined by dichloro-dihydro-fluorescein diacetate (DCF-DA) staining. RESULTS:Hypoxic exposure caused the apoptosis of H9c2 cardiomyocytes, which was accompanied by the down-regulation of CFTR at mRNA and protein levels and over-production of ROS (P<0.05). After CFTR overexpression, the apoptotic rate of the H9c2 cardiomyocytes induced by hypoxia was significantly reduced, with a prominent inhibition of ROS production (P<0.05). However, pretreatment with CFTRinh-172, a specific inhibitor of CFTR, reversed the protective effect of CFTR overexpression in H9c2 cardiomyocytes. CONCLUSION:CFTR has a critical role in protecting against hypoxia-induced apoptosis of H9c2 cells, which may be through inhibiting the generation of ROS.     

Interannual variation in quantitative relationships among egg production and densities of larvae and juveniles of the Japanese mantis shrimp <Emphasis Type="Italic">Oratosquilla oratoria</Emphasis> in Tokyo Bay,Japan

To explore which lifestages affect the stock size of young-of-the-year mantis shrimp Oratosquilla oratoria in Tokyo Bay, Japan, we investigated interannual variations in the quantitative relationships among egg production, larval density, and juvenile density. We collected adult females, larvae, and juveniles during monthly field surveys from 2004 to 2007. The interannual trend for the juvenile density index differed from those for egg production and larval density; although indices of both egg production and larval density were high in 2004 and 2007, the juvenile density index was high only in 2007, suggesting high mortality during the pelagic larval stage or the early phase of the postsettlement juvenile stage in 2004. We found that larval settlement started at the end of August and peaked in October, although larvae from the early spawning season (May–June) should have settled in August or earlier. Juveniles were found throughout the bay except in areas where bottom hypoxia occurred, suggesting that hypoxia restricts the spatial distribution of juveniles. Our results suggest that mortality during the early life history fluctuates among years, probably because of changes in environmental conditions in the bay, resulting in interannual variation in the stock size of young-of-the-year juvenile O. oratoria.     

Expression relevance of GATA-1 and miR-451a in erythroid differentiation of K562 cells under hypoxia

AIM To investigate the expression relevance of GATA binding protein-1 (GATA-1) and microR?NA-451a (miR-451a) in erythroid differentiation of human chronic myeloid leukemia K562 cells under hypoxia. METHODS The K562 cells were divided into 2 groups: normoxia group and hypoxia (1% O₂) group, and 40 μmol/L hemin chloride was used to induce K562 cell differentiation for 48 and 72 h. The mRNA expression of γ-globin was detected by RT-qPCR, hemoglobin production was observed by benzidine staining, and flow cytometry was used to detect CD235a expression for verifying erythroid differentiation model. The protein expression of GATA-1 during K562 cell differentiation under normoxia and hypoxia was determined by Western blot. RT-qPCR was used to detect the mRNA expression of GATA-1 and the expression level of miR-451a, and their correlation was analysis. The K562 cells were infected by lentivirus for over-expression or knock-down of GATA-1. Meanwhile, the morphological changes of the cells in the above groups were analyzed by Wright-Giemsa staining method to clarify the erythroid differentiation of K562 cells. The expression miR-451a was detected by RT-qPCR after GATA-1 over-expression or knock-down. REULTS: Under normoxia and hypoxia conditions, the expression levels of γ?-globin and CD235a and the positive rate of benzidine staining at 48 and 72 h were significantly higher than those at 0 h (P<0.05).At 72 h, the expression levels of γ?-globin and CD235a and the benzidine staining positive rate in hypoxia group were significantly higher than normoxia group (P<0.05). The expression of GATA-1 mRNA and miR-451a under hypoxia showed an upward trend during the erythroid differentiation of K562 cells, and was significantly higher than that in normoxia group at 72 h (P<0.05). Correlation analysis showed that the mRNA expression of GATA-1 was positively correlated with miR-451a expression under hypoxia (P<0.01). After over-expression of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the positive rate of benzidine staining, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly higher than those in negative control group (P<0.05). After knock-down of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the benzidine staining positive rate, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly lower than those in negative control group (P<0.05). Compared with negative control group under hypoxia, the expression of miR-451a was significantly increased after GATA-1 over-expression (P<0.05), while the expression of miR-451a was significantly decreased after GATA-1 knock-down (P<0.05). CONCLUSION Hypoxia increases the expression of GATA-1 and then up-regulates miR-451a to promote erythroid differentiation of K562 cells.     

Establishment and comparative analysis of 2 hypoxia models of human breast cancer MDA-MB-231 cells

AIM: To explore the differences in the effects of 2 representative hypoxia models on human breast cancer MDA-MB-231 cells. METHODS: To establish a chemical hypoxia model, MDA-MB-231 cells were treated with CoCl₂ at different concentrations (0~300 μmol/L) for different time (24 h, 48 h and 72 h). On the other hand, MDA-MB-231 cells were exposed to hypoxia with different volume fractions (1%, 2% and 5%) of oxygen for different time (4 h, 16 h and 24 h) to establish a physical hypoxia model. The viability of the cells in the 2 hypoxia models was measured by CCK-8 assay. The protein levels of hypoxia-inducible factor-1α (HIF-1α), Bcl-2 and matrix metalloproteinase-2 (MMP-2) were determined by Western blot. The apoptosis of the cells was analyzed by flow cytometry. The cell invasion and migration were examined by Transwell assay and wound-healing assay, respectively. RESULTS: The HIF-1α was expressed in time- and dose-dependent manners. These results indicated that treatment of MDA-MB-231 cells with CoCl₂ at 100 μmol/L for 24 h served as the final chemical hypoxia model, and exposure of MDA-MB-231 cells to hypoxia with 2% oxygen for 24 h served as the final physical hypoxia model. Compared with the cells with chemical hypoxia, the protein le-vel of HIF-1α was significantly increased in the cells with physical hypoxia. In addition, reoxygenation from hypoxia caused a rapidly degraded HIF-1α expression level in physical hypoxia model. Flow cytometry results showed that apoptosis was increased and the protein expression level of anti-apoptotic Bcl-2 was decreased in the cells with physical hypoxia, while no significant change was observed in the cells with chemical hypoxia. The protein expression level of MMP-2 in both hypoxia models was increased, and the invasion and migration capabilities of the cells with chemical hypoxia were enhanced. CONCLUSION: Two hypoxia models were successfully constructed. There were differences in the expression and stability of HIF-1α protein, and the viability, apoptosis, invasion and migration of the cells in the 2 different hypoxia models, which provide references for the selection of hypoxia models and the further study of characteristics of tumor cells in hypoxia environment.     

低氧预适应对魁蚶在低氧胁迫下生理生化指标的影响

本研究以未进行低氧预适应的魁蚶(Scapharca broughtonii)为对照组(C组),分析了2次低氧预适应(H2组)和4次低氧预适应(H4组)的魁蚶在溶解氧(DO)约为2.0 mg/L低氧胁迫48 h内的摄食、呼吸代谢和酶活力的变化规律。结果显示,3组魁蚶的摄食率(IR)在胁迫初期急剧下降,后期均随时间的延长逐渐恢复,至48 h时,H组恢复程度显著高于C组(P＜0.05＝;C组、H2组和H4组魁蚶的耗氧率(OR)随时间变化呈逐渐升高的趋势,48 h比0 h分别提高了1.15、1.08、0.73倍;3组排氨率(NR)表现出不同的变化趋势,至48 h时,C组、H2组和H4组分别为0 h的1.67、1.30、0.97倍;C组的氧氮比(O/N)相对平稳,H组的变化范围相对较大。3组的细胞色素C氧化酶(COX)随着低氧胁迫时间的延长呈逐渐降低的趋势,乳酸脱氢酶(LDH)活力和还原型谷胱甘肽酶(GSH)含量整体呈上升趋势,与对照组相比,预低氧组的酶活力在低氧胁迫期间变化相对平稳,应激反应小。研究表明,魁蚶经低氧预适应后再次受到低氧胁迫时,IR升高,OR降低,酶活性相对稳定,低氧预适应能提高魁蚶的耐低氧能力。本研究丰富了魁蚶低氧耐受相关研究的基础数据,为进一步探讨魁蚶低氧耐受机制和创制耐低氧新种质提供了参考资料。     

低氧胁迫下中华圆田螺的肝脏转录组学分析

为了探究低氧胁迫下中华圆田螺(Cipangopaludina cathayensis)肝脏组织基因的差异表达,本研究通过高通量测序技术,分析中华园田螺低氧胁迫组(2.5 mg/L)和常氧组(6.9 mg/L)某些基因的差异表达,并对差异基因进行生物信息学分析,进一步采用实时荧光定量PCR (RT-qPCR)对关键差异表达基因进行验证。结果显示,测序共获得232 379条基因(unigenes),与对照组比较,低氧胁迫组筛到176个差异基因,包含64个上调基因和112个下调基因。GO功能注释分析显示,差异基因主要富集在生物学过程中的几丁质代谢过程和含氨基葡萄糖的复合代谢过程,细胞组分中的胶原三聚体成分,分子功能中的几丁质结合功能和糖衍生物结合功能。KEGG通路富集分析显示,差异基因主要集中于环境信息处理、遗传信息处理、代谢和生物系统这4大类通路。6个关键差异基因的RT-qPCR结果显示,热休克蛋白70B2和热休克蛋白β-6基因表达量上调,几丁质酶蛋白4、α-1胶原蛋白(XIV)、α-4胶原蛋白(XIV)、5-磷酸酶蛋白基因表达量下调,证实了转录组测序结果的可靠性。本研究发现,低氧胁迫激活了中华圆田螺适应缺氧的生理活动,并获得了低氧胁迫下中华圆田螺肝脏组织中相关功能基因的表达信息,为深入研究中华圆田螺响应低氧胁迫的调控机制提供了基础数据和理论依据。     

Role of retinoic acid X receptor in regulation of autophagy in rat alveolar type Ⅱ epithelial cells induced by hypoxia/reoxygenation

AIM To investigate the regulatory effect of retinoic acid X receptor （RXR） on autophagy induced by hypoxia/reoxygenation （H/R） in rat alveolar type Ⅱ epithelial cells （AEC Ⅱ） and its molecular mechanism. METHODS AEC Ⅱ were cultured in normoxia. The cells growing to logarithmic growth phase were randomly divided into 5 groups： （1） control （Con） group： cells were cultured for 30 h under normal operation; （2） H/R group： cells were cultured in hypoxia condition for 6 h and then in reoxygenation condition for 24 h; （3） DMSO group： cells were pretreated 1.5 h with medium containing less than 0.1% DMSO before modeling, and the rest were treated the same as the H/R group; （4） 9-cis-retinoic acid （9-RA） group： cells were pretreated for 1 h with 9-RA （100 nmol/L） before hypoxia; （5） HX531 group： cells were treated with 9-RA （100 nmol/L） for 0.5 h, then treatment with HX531 （2.5 μmol/L） for 1 h. CCK-8 assay was used to detect the cell viability. Immunofluorescence staining was used to observe the expression of RXRα. Transmission electron microscope was used to observe the changes of intracellular ultrastructure, and the mRNA expression of adenosine AMP-activated protein kinase （AMPK）, beclin 1, LC3, mammalian target of rapamycin （mTOR） and P62 was detected by RT-PCR. Western blot was used to detected the protein levels of p-AMPK, beclin 1, LC3-Ⅱ, p-mTOR and P62. RESULTS Compared with Con group, the cell viability in H/R, DMSO, 9-RA and HX531 groups were significantly decreased. The mRNA expression of AMPK, beclin 1 and LC3 was significantly increased, and the protein levels of p-AMPK, beclin 1 and LC3-Ⅱ were also increased. The mRNA expression of mTOR and P62 was decreased, and the protein levels of p-mTOR and P62 were also decreased （P<0.05）. The cell injury in 9-RA group was alleviated and autophagy level was significantly lower than that in H/R, DMSO and HX531 groups （P<0.05）, and no significant difference among H/R, DMSO and HX531 groups was observed （P>0.05）. CONCLUSION H/R induces autophagy of AEC Ⅱ. Activating RXR reduce the damage of AEC Ⅱ cells induced by H/R, and its mechanism may be related to the inhibition of autophagy.     

Effects of adenosine A1 receptor antagonist DPCPX on cerebral neurons after hypoxia and reoxygenation

AIM: To investigate the effects of adenosine A₁ receptor antagonist DPCPX on the release of cerebral neuronal lactate dehydrogenase (LDH), the activity of calcineurin (CaN) and acetylcholinesterase (AChE), and the level of extracellular amino acid after hypoxia and reoxygenation (H/R).METHODS: Primary cultured rat cerebral cortical neurons were used to establish an H/R injury model. Different concentrations of DPCPX (the final concentrations were as follows: 0, 25, 50 and 100 nmol/L) were added at the same time of hypoxia treatment for 8 h, 12 h or 24 h,followed by reoxygenation for 24 h. The LDH release from the neurons was measured. The effects of DPCPX (100 nmol/L) on the activity of CaN and AChE, and the level of extracellular amino acid in neurons treated with hypoxia for 12 h followed by reoxygenation were observed. RESULTS: Compared to the cells in control groups, the neurons treated with 100 nmol/L DPCPX and exposed to hypoxia for 12 h followed by reoxygenation, showed significantly higher LDH release, higher activity of CaN and AChE, and lower level of extracellular γ-aminobutyric acid.CONCLUSION: These results suggest that DPCPX increases the LDH release and the activity of CaN and AChE, decreases the level of extracellular γ-aminobutyric acid in neurons with H/R.     

Expression of STC1 in colorectal cancer and its significance in tumor microenvironment

AIM:To explore the expression of stanniocalcin 1 (STC1) in colorectal cancer tissues and to analyze its relationship with prognosis, further to investigate the correlation between STC1 and tumor microenvironment. METHODS:The data of the STC1 mRNA expression were accessed by the UALCAN database (http://ualcan.path.uab.edu/index.html) and Oncomine database (https://www.oncomine.org). RT-qPCR and Western blot were used to detect the expression of STC1 in the clinical samples. OncoLnc (http://www.oncolnc.org/) analytical tool was adopted to evaluate the correlation of STC1 level and the prognosis of colorectal cancer. CoCl₂ was used to establish the hypoxia status and lipopolysaccharide (LPS) was utilized to establish inflammatory condition in the colorectal cancer HT-29 cells, respectively, and then STC1 expression was examined by RT-qPCR and Western blot. RESULTS:STC1 was over-expressed in the colorectal cancer tissues, and its high expression was positively correlated with poor prognosis of the colorectal cancer patients (P<0.01). In colorectal cancer HT-29 cells, treatment of CoCl₂ up-regulated the expression of STC1. Under the condition of inflammation by stimulating with LPS, the expression of STC1 was significantly increased. In the colorectal cancer, STC1 level was positively correlated with hypoxia-inducible factor-1α or inflammatory molecules expression, and the levels of tumor-infiltrating immune cells in colorectal cancer tissue. CONCLUSION:STC1 is over-expressed in the colorectal tumor tissue, and the high level of STC1 is an important prognosis indicator for colorectal cancer. Furthermore, STC1 is closely correlated with tumor microenvironment, especially with the tumor hypoxia and tumor immune regulation of colorectal cancer.