Hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen–glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy

In this study, we investigated the protective effect of hyperbaric oxygen (HBO) on PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion and its possible mechanism. PC12 and H9C2 cell oxygen-glucose deprivation/reperfusion model were established. Cells were divided into a control group, model group, hyperbaric air (HBA) group and HBO group. The cell viability was detected by the CCK8 assay. Hoechst 33342 and PI staining assays and mitochondrial membrane potential (MMP) assays were used to detect cell apoptosis. The ultrastructure of cells, including autophagosomes, lysosomes, and apoptosis, were examined using a transmission electron microscope. The expression of autophagy-related proteins was detected by cellular immunofluorescence and immunocytochemistry. Our results showed that HBO can significantly improve the vitality of damaged PC12 and H9C2 cells caused by oxygen–glucose deprivation/reperfusion. HBO can significantly inhibit apoptosis of PC12 and H9C2 cells caused by oxygen-glucose deprivation/reperfusion. Importantly, we found that the protective mechanism of PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion may be related to the inhibition of the autophagy pathway. In this study, the results of cellular immunofluorescence and immunocytochemistry experiments showed that the 4E-BP1, p-AKt and mTOR levels of PC12 and H9C2 cells in the model group decreased, while the levels of LC3B, Atg5 and p53 increased. However, after HBO treatment, these autophagy-related indexes were reversed. In addition, observation of the cell ultrastructure with transmission electron microscopy found that in the model group, a significant increase in the number of autophagic vesicles was observed. In the HBO group, a decrease in autophagic vesicles was observed. The study demonstrated that hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy.     

Hypoxia induced apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway

The pathogenesis of high altitude-related gastric mucosal injury remains poorly understood, this study aimed to investigate the role of autophagy in hypoxia-induced apoptosis of rat gastric mucosal cells. Rats were randomized into four groups which were maintained at an altitude of 400 m (P) or received no treatment (H), autophagy inducer rapamycin (H+AI) or autophagy inhibitor 3-MA (H+AB) at an altitude of 4,300 m for 1, 7, 14 and 21 days, respectively, and the morphology, ultrastructure, autophagy, and apoptosis of gastric mucosal tissues were examined. Gastric mucosal epithelial cells CC-R039 were cultured under conditions of normoxia, 2% O2 (hypoxia), or 2% O2+anti-mTORC1 for 0, 24, 48, and 72 h, respectively, and the autophagy and apoptosis were analyzed. CC-R039 cells were transfected with siHIF-1α, siTERT, or siRNA and the autophagy was examined. The results showed that the exposure to hypoxia increased the autophagy and apoptosis of gastric mucosal cells in rats, and apoptosis was aggravated by rapamycin treatment but alleviated by 3-MA treatment. Increased duration of hypoxia from 0 to 72 h could increase the autophagy and apoptosis but decrease the proliferation of gastric mucosal cells. Inhibition of mTORC1 with rapamycin led to further increase in apoptosis and even substantial cell death, and inhibition of HIF- 1α and TERT increased mTORC1 expression and reduced autophagy. Moreover, the inhibition of HIF-1α reduced TERT expression. In conclusion, hypoxia could induce apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway     

Effect of Peroxiredoxin 1 on the biological function of airway epithelial cells and epithelial-mesenchymal transition

Peroxiredoxin 1 (PRDX1) participates in tumor cell proliferation, apoptosis, migration, invasion, and the epithelial-to-mesenchymal transition (EMT). This study aimed to investigate the effect of PRDX1 on the EMT of airway epithelial cells stimulated with lipopolysaccharide (LPS) and transforming growth factor-beta 1 (TGF-β1). PRDX1 overexpression significantly increased the proliferation and migration of human bronchial epithelial (BEAS-2B) cells, reduced cell apoptosis (p < 0.01), and induced EMT and collagen deposition by upregulating the expression of the matrix metallopeptidase (MMP)2, MMP9, α-smooth muscle actin (α-SMA), N-cadherin, vimentin and twist proteins and inhibiting E-cadherin expression (p < 0.05). PRDX1 overexpression promoted TGF-β1-mediated inhibition of cell proliferation and migration and significantly enhanced the TGF-β1-induced EMT and collagen synthesis (p < 0.05). Knockdown of PRDX1 inhibited cell proliferation, migration, EMT, and collagen synthesis (p < 0.01), reversed LPS-mediated inhibition of cell proliferation and migration, and significantly suppressed LPS-induced EMT and collagen synthesis (p < 0.01). The result indicating that PRDX1 may be involved in LPS/TGF-1-induced EMT and collagen synthesis in human bronchial epithelial cells.     

Morphological changes in mouse embryos cryopreserved by different techniques

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnologies. Subjective evaluation to determine embryo viability is often used. The determination of the best cryopreservation protocol depends on morphological and molecular analysis of cellular injuries. The main objective of this study was to compare two methods of cryopreservation by assessing morphological alterations of frozen embryos using light, fluorescence, and transmission electron microscope. Fresh (control), slow frozen, and vitrified mouse embryos were composed. To evaluate the viability of the embryos, the cell membrane integrity was assessed using Hoechst33342 and propidium iodide (H/PI) staining. Morphological analyses using hematoxylin and eosin (HE) staining were performed to test different techniques (in situ, paraffin, and historesin) by both light and fluorescence microscopy. Transmission electron microscope was used to detect ultrastructural alterations in Spurr- and Araldite-embedded samples. H/PI staining detected more membrane permeability in the vitrification (69.8%) than in the slow freezing (48.4%) or control (13.8%) groups (P < 0.001). Historesin-embedded samples showed to be more suitable for morphological analyses because cellular structures were better identified. Nuclear evaluation in historesin sections showed the induction of pycnosis in slow freezing and vitrification groups. Cytoplasm evaluation revealed a condensation and an increase in eosinophilic intensity (indicating apoptosis) in the slow freezing group, and weakly eosinophilic structures and degenerated cells (indicating oncosis) in the vitrification group (P < 0.05). Ultrastructural analyses confirmed HE morphological findings. It was concluded that both cryopreservation techniques resulted in oncosis and apoptosis injuries. However, vitrification caused more severe cellular alterations and reduced embryonic viability compared to slow freezing.     

MicroRNA expression profile and lipid metabolism characteristics in liver of rat undergoing high-fat diet

This study aimed to investigate the microRNA expression profile and the characteristics of lipid metabolism in the livers of rats undergoing a high-fat diet. Fifty male Sprague-Dawley (SD) rats were divided into a standard chow group (C group, N = 10) and a high-fat diet group (H group, N = 40). After 12 weeks, the rat body weight, body length, fat mass, and serum lipid concentration were measured. The expression profile of microRNAs and the gene and protein expression levels involved in lipid metabolism in rat liver were detected. Body fat and serum lipid concentrations were all significantly higher in the H group than those in the C group (p < 0.05 or p < 0.01). The expression of 10 microRNAs showed significant differences in the liver (p < 0.05). In particular, the let-7 family expression levels significantly increased (p < 0.05) in the H group compared with those in the C group. Compared with the C group, the high-fat diet resulted in low FAS, CPT1A, and ApoAI mRNA expression levels (p < 0.05 or p < 0.01) and high PPARα and FAT/CD36 mRNA expression levels in the H group rat liver (p < 0.01). Meanwhile, the protein PPARα, FAS, CPT1A, FAT/CD36, and ApoAI expression levels were all significantly lower in the H group than those in the C group (p < 0.05 or p < 0.01). In conclusion, the high-fat diet increased the body fat and serum lipid levels and altered the 10 microRNA expression levels in the liver. The high-fat diet may affect hepatic carbohydrate metabolism and increase ectopic fat accumulation through let-7 family overexpression. The high-fat diet for 12 weeks decreased lipid metabolism level in the liver, thereby decreasing fatty acid synthesis, oxidation, and transport by down-regulating the PPARα, FAS, CPT1A, FAT/CD36, and ApoAI protein levels.     

Development of electrically conductive hybrid nanofibers based on CNT‐polyurethane nanocomposite for cardiac tissue engineering

Conductive nanofibers have been considered as one of the most interesting and promising candidate scaffolds for cardiac patch applications with capability to improve cell–cell communication. Here, we successfully fabricated electroconductive nanofibrous patches by simultaneous electrospray of multiwalled carbon nanotubes (MWCNTs) on polyurethane nanofibers. A series of CNT/PU nanocomposites with different weight ratios (2:10, 3:10, and 6:10wt%) were obtained. Scanning electron microscopy, conductivity analysis, water contact angle measurements, and tensile tests were used to characterize the scaffolds. FESEM showed that CNTs were adhered on PU nanofibers and created an interconnected web‐like structures. The SEM images also revealed that the diameters of nanofibers were decreased by increasing CNTs. The electrical conductivity, tensile strength, Young's modulus, and hydrophilicity of CNT/PU nanocomposites also enhanced after adding CNTs. The scaffolds revealed suitable cytocompatibility for H9c2 cells and human umbilical vein endothelial cells (HUVECs). This study indicated that simultaneous electrospinning and electrospray can be used to fabricate conductive CNT/PUnanofibers, resulting in better cytocompatibility and improved interactions between the scaffold and cardiomyoblasts.     

Cobalt chloride stimulates phosphoinositide 3-kinase/Akt signaling through the epidermal growth factor receptor in oral squamous cell carcinoma

Tumor cells are often found under hypoxic conditions due to the rapid outgrowth of their vascular supply, and, in order to survive hypoxia, these cells induce numerous signaling factors. Akt is an important kinase in cell survival, and its activity is regulated by the upstream phosphoinositide 3-kinase (PI3K) and receptor tyrosine kinases (RTKs). In this study, we examined Akt activation and RTKs/PI3K/Akt signaling using the hypoxia-mimetic cobalt chloride in oral squamous carcinoma cells. Cobalt chloride increases Akt phosphorylation in both a dose- and time-dependent manner. Blocking the activation of the PI3K/Akt pathway using LY294002 abolished Akt activation in response to cobalt chloride, suggesting that Akt phosphorylation by cobalt chloride is dependent on PI3K. In addition, activation of the PI3K/Akt pathway seems to rely on the epidermal growth factor receptor (EGFR), since the inhibition of EGFR attenuated cobalt chloride-induced Akt activation. The results in this study also demonstrate that cobalt chloride increases EGFR protein levels and induces oral squamous cell carcinoma cells to enter S phase.     

Imaging of reactive oxygen species burst from mitochondria using laser scanning confocal microscopy

Objective: Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM). Methods: Cardiac H9c2 cells were exposed to H₂O₂ (1200μM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H₂DCFDA). Results: A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H₂O₂ compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H₂DCFDA. Conclusions: Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to. Microsc. Res. Tech. 76:612–617, 2013. © 2013 Wiley Periodicals, Inc.     

Expression of caspase-8 and caspase-9 in rat hippocampus during postnatal development

The role of caspases in the regulation of apoptosis of neurons during development is well established. An emerging body of evidence indicates that caspases may also play significant roles which are nonapoptotic. We have demonstrated previously that the executor caspase-3 exhibited a unique pattern of spatiotemporal expression in the postnatal rat hippocampal subregions, and the activation of caspase-3 in different hippocampal neurons appeared to have distinct roles during postnatal development. In the present study, we examined the expressions of initiator caspases in the hippocampus, using immunofluorescent staining for caspase-8 and caspase-9, and Hoechst 33342 staining for nuclear chromatin to assess caspase-8 and -9 expression in the CA1, CA3, and the dentate gyrus (DG) on postnatal days (P) 0, P2, P4, P7, P14, P21, P28, P56. The results indicate that caspase-8 and caspase-9 were expressed in pyramidal neurons of CA1 and CA3 fields, and granular neurons of the DG during development. Caspase-8 was expressed in a general upward trend while caspase-9 showed a slight downward pattern, but still remained at high levels in the adult hippocampus. The expression profiles of caspases-8 and -9 are distinct from that of the apoptotic cells. These data indicate that caspase-8 may be involved not only in the classical apoptotic function, but also in the cell death of necrosis, and in response to different insults and other nonapoptotic functions. Caspase-9 plays a role in apoptosis during postnatal development, but it may have other functions as well.     

Kidney injury and cell therapy: Preclinical study

The aim of this study is to show histological and immunofluorescence analysis of renal parenchyma of agoutis affected by gentamicin‐induced renal disease after the infusion of bone marrow mononuclear cells (BMMC) stained with Hoechst®. Nine agouti's males were divided into three groups: Test group (TG): renal disease by gentamicin induced (n = 3), cell therapy group (CTG): renal disease by gentamicin induced and BMMC infusion (n = 3), and control group (CG): nonrenal disease and BMMC infusion (n = 3). TG and CTG were submitted to the protocol of renal disease induction using weekly application of gentamicin sulfate for 4 months. CG and CTG received a 1 × 108 BMMC stained with Hoechst and were euthanized for kidney examination 21 days after BMMC injection and samples were collected for histology and immunofluorescence analysis. Histological analysis demonstrated typical interstitial lesions in kidney similarly to human disease, as tubular necrosis, glomerular destruction, atrophy tubular, fibrotic areas, and collagen deposition. We conclude that histological analysis suggest a positive application of agouti's as a model for a gentamicin inducing of kidney disease, beyond the immunofluorescence analysis suggest a significant migration of BMMC to sites of renal injury in CTG. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.     

SPAG9 promotes prostate cancer growth and metastasis

Sperm-associated antigen 9 (SPAG9) expression is increased in prostate tissues of prostate cancer patients. This experimental study aimed to investigate the role of SPAG9 in bone metastasis of prostate cancer. Immunohistochemical analysis showed that SPAG9 staining was positive in 81.67% of 240 cases of prostatic carcinoma but only in 6.67% of 120 cases of benign prostate hyperplasia. Strong PAG9 staining was positively correlated with Gleason score and bone metastasis in 240 prostate cancer patients (p < 0.05), but not with the age or serum prostatespecific antigen level (p > 0.05). PC-3 cells were transfected with shRNA against SPAG9, and CCK-8 assay in triplicate showed that PC-3 cell viability was inhibited by SPAG9 knockdown. In addition, transwell assay in triplicate showed that PC-3 cell invasion was inhibited by SPAG9 knockdown. Furthermore, total 2 × 106 PC-3 cells were injected subcutaneously into the right flank of nude mice which were randomly divided into three groups (N = 8) and treated by intratumoral injection of SPAG9 shRNA, control shRNA or PBS, respectively. SPAG9 shRNA inhibited the growth, invasion and angiogenesis while promoted apoptosis of xenografted PC-3 cells. SPAG9 knockdown led to the upregulation of E-cadherin and the downregulation of MMP2 and vimentin in xenografted tumors. In conclusion, this is the first study to provide evidence that SPAG9 promotes bone metastasis of prostate cancer, and SPAG9 is a promising target to prevent or treat bone metastasis of prostate cancer.     

荧光显微镜观察银杏内酯B对大鼠心肌细胞凋亡的影响

目的：建立一种简便、快速的细胞凋亡检测方法，探讨银杏内酯B（GB）对心肌细胞凋亡的影响。方法：体外培养大鼠心肌细胞，利用荧光显微镜观察经Hoeehst 33258染色的心肌细胞，记录凋亡细胞核形态学改变并计算凋亡百分率。结果：H2O2可诱导心肌细胞发生凋亡，与H2O2组相比，GB组大鼠心肌细胞凋亡率明显降低，并有显著性差异（P〈0．05）。结论：银杏内酯B对大鼠心肌细胞凋亡具有保护作用。     

Brief Note: Isolation,culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85± 4.6%), and a correspondingly low proportion K3 positive cells (15 ± 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean ± SE, n=10). Cultures reached full confluency after 17.3 ± 1.2 days when the medium was supplemented with human EGF, while 21.7 ± 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.     

<i>miR-103-3p</i> regulates the differentiation of bone marrow mesenchymal stem cells in myelodysplastic syndrome

The pathogenesis of myelodysplastic syndrome (MDS) may be related to the abnormal expression of microRNAs (miRNAs), which could influence the differentiation capacity of mesenchymal stem cells (MSCs) towards adipogenic and osteogenic lineages. In this study, exosomes from bone marrow plasma were successfully extracted and identified. Assessment of miR-103-3p expression in exosomes isolated from BM in 34 MDS patients and 10 controls revealed its 0.52-fold downregulation in patients with MDS compared with controls (NOR) and was downregulated 0.55-fold in MDS-MSCs compared with NOR-MSCs. Transfection of MDS-MSCs with the miR-103-3p mimic improved osteogenic differentiation and decreased adipogenic differentiation in vitro, while inhibition of miR-103-3p showed the opposite results in NOR-MSCs. Thus, the expression of miR-103-3p decreases in MDS BM plasma and MDS-MSCs, significantly impacting MDS-MSCs differentiation. The miR-103-3p mimics may boost MDS-MSCs osteogenic differentiation while weakening lipid differentiation, thereby providing possible target for the treatment of MDS pathogenesis.     

Effect of VirD4 on gastric epithelial-1 cells and its mechanism

The gene of Helicobacter pylori can encode three to four type IV secretory systems, of which a new gene region has been found in the H. pylori plasticity region. The coding products of this region can form a new T4SS named tfs3, but its function is unclear. This study investigated the effect of VirD4 recombinant protein in the tfs3 secretory system of the H. pylori clinical strain SBK on GES-1 cells. We observed changes in cell morphology after VirD4 treatment. Further analysis indicated that VirD4 increased inflammation by increasing the activation of NF-κB. VirD4 can also inhibited proliferation, and induced migration of cells. Moreover, VirD4 caused apoptosis in GES-1 cells in caspase and ERK1/2/Ras dependent signaling events. Our study laid a foundation for further research on the biological function of VirD4 and the detection and treatment of H. pylori-related diseases.     

20(R)-ginsenoside Rg3, a product of high-efficiency thermal deglycosylation of ginsenoside Rd,exerts protective effects against scrotal heat-induced spermatogenic damage in mice

Heat stress (HS) reaction can lead to serious physiological dysfunction associated with cardiovascular and various organ diseases. Ginsenoside Rg3 (G-Rg3) is a representative component of ginseng rare saponin and can protect against multiple organs, also used as functional food to adjust the balance of the human body, but the therapeutic effect and molecular mechanism of G-Rg3 on male diseases under HS are underexplored. The aim of the present study, G-Rg3 was prepared through the efficient conversion of ginsenoside Rd and investigate the contribution of G-Rg3 to testicular injury induced exposure to HS. All mice were divided into four groups as follows: normal group, HS group, and HS+G-Rg3 (5 and 10 mg/kg) groups. G-Rg3 was administered orally for 14 days, then exposed to a single scrotal heat treatment (43°C, 18min) on the 7th day. After HS treatment, the morphology of testis and epididymis changes, and caused a significant loss of multinucleated giant cells, desquamation of germ cells in destructive seminiferous tubules, and degenerative Leydig cells, further destroying the production of sperm. After administration G-Rg3 (5 and 10 mg/kg/day) for 2 weeks, the spermatogenic-related indexes of testosterone levels and superoxide dismutase (SOD) activity, glutathione (GSH) content significantly (p < 0.01) increase compared with the HS group. Moreover, G-Rg3 treatment effectively ameliorated the production of malondialdehyde (MDA) (p < 0.05 or p < 0.01). Importantly, G-Rg3 exhibited the protective potential against HS-induced injury not only suppressing the protein levels of heme oxygenase-1 (HO-1), hypoxia-inducible factor-1α (HIF-1α), and heat shock protein 70 (HSP70) but also modulating the Bcl-2 family (p < 0.01 or p < 0.001) and activation of mitogen-activated protein kinase (MAPK) signaling pathways (p < 0.01). For most of the parameters tested, the HS+G-Rg3 (10 mg/kg) group exhibited potent effects compared with those exhibited by the low dose (5 mg/kg) group. In conclusion, the present study demonstrated that G-Rg3 exerted protective effects against HS-induced testicular dysfunction via inhibiting the MAPK-mediated oxidative stress and apoptosis in mice.     

Effects of human umbilical cord mesenchymal stem cells on co‐cultured ovarian carcinoma cells

Ovarian carcinoma is mainly treated by surgery aided by chemotherapy. If supplemented by stem cells treatment, its recurrence rate and mortality rate will be decreased. This is a new therapy. In this study, ovarian cancer cells were cultured together with umbilical cord mesenchymal stem cells (UCMSCs), and the interactions between them were observed. The results showed that the survival rates of UCMSCs increased to 83.8 ± 2.2% from 56.5 ± 5.5%, and the survival rates of ovarian cancer cells decreased to 16.2 ± 2.2% from 43.5 ± 5.5% with the progression of the cultural time from 24 to 96 hr. There was a significant difference between them (p < .05). It revealed that UCMSCs could inhibit the proliferation of ovarian cancer cells.     

Three‐dimensional morphometric comparison of normal and apoptotic endothelial cells based on laser scanning confocal microscopy observation

Three‐dimensional (3D) morphometric analysis of cellular and subcellular structures provides an effective method for spatial cell biology. Here, 3D cellular and nuclear morphologies are reconstructed to quantify and compare morphometric differences between normal and apoptotic endothelial cells. Human umbilical vein endothelial cells (HUVECs) are treated with 60 μM H₂O₂ to get apoptotic cell model and then a series of sectional images are acquired from laser scanning confocal microscopy. The 3D cell model containing plasma membrane and cell nucleus is reconstructed and fused utilizing three sequential softwares or packages (Mimics, Geomagic, and VTK). The results reveal that H₂O₂ can induce apoptosis effectively by regulating the activity of apoptosis‐related biomolecules, including pro‐apoptotic factors p53 and Bax, and anti‐apoptotic factor Bcl‐2. Compared with the normal HUVECs, the apoptotic cells exhibit significant 3D morphometric parameters (height, volume and nucleus‐to‐cytoplasm ratio) variation. The present research provides a new perspective on comparative quantitative analysis associated with cell apoptosis and points to the value of LSCM as an objective tool for 3D cell reconstruction. Microsc. Res. Tech. 76:1154–1162, 2013. © 2013 Wiley Periodicals, Inc.