[The Effect of SIRT5 Deletion on Recovery of Hematopoietic Stem Cells after Injury in Mouse].

OBJECTIVE: To investigate the effect of deacylase Sirtuin 5 in the recovery of hematopoietic stem cells (HSCs) after treated by 5-FU in mouse. METHODS: Flow cytometry was used to analyze the effect of SIRT5 deletion on the proportion of hematopoietic stem/progenitor cells (HSPCs) in bone marrow (BM), the proportion of T cells, B cells and myeloid cells (TBM) in peripheral blood (PB) and spleen, and the development of T cells in thymus. Mouse were treated with 5-FU to study the effect of SIRT5 deletion on the cell cycle, apoptosis and the proportion of HSPCs in BM. The effect of SIRT5 deletion on the proliferation of HSCs was analyzed by flow sorting in vitro. RESULTS: SIRT5 deletion did not affect the development of T cells in thymus and the proportion of TBM cells in PB and spleen compared with wild type mice. SIRT5 deletion increased proportion of HSPCs in BM. After 5-FU treatment, the proportion of HSCs in SIRT5 deletion mice was significant decreased (P < 0.05), the HSPC in SIRT5 deletion mice was activated from G0 to G1 phase (P < 0.05), and the proportion of early apoptosis increased (P < 0.05). By monoclonal culture in vitro, the ability of HSCs to form clones in SIRT5 deletion mice was decreased significantly (P < 0.05). CONCLUSION: SIRT5 deletion lead to a decreased the ability of HSCs to clone in vitro. SIRT5 deletion is not conducive to the recovery of HSPCs injury in mice under hematopoietic stress.

AI-assisted mass spectrometry imaging with in situ image segmentation for subcellular metabolomics analysis.

Subcellular metabolomics analysis is crucial for understanding intracellular heterogeneity and accurate drug-cell interactions. Unfortunately, the ultra-small size and complex microenvironment inside the cell pose a great challenge to achieving this goal. To address this challenge, we propose an artificial intelligence-assisted subcellular mass spectrometry imaging (AI-SMSI) strategy with in situ image segmentation. Based on the nanometer-resolution MSI technique, the protonated guanine and threonine ions were respectively employed as the nucleus and cytoplasmic markers to complete image segmentation at the subcellular level, avoiding mutual interference of signals from various compartments in the cell. With advanced AI models, the metabolites within the different regions could be further integrated and profiled. Through this method, we decrypted the distinct action mechanism of isomeric drugs, doxorubicin (DOX) and epirubicin (EPI), only with a stereochemical inversion at C-4'. Within the cytoplasmic region, fifteen specific metabolites were discovered as biomarkers for distinguishing the drug action difference between DOX and EPI. Moreover, we identified that the downregulations of glutamate and aspartate in the malate-aspartate shuttle pathway may contribute to the higher paratoxicity of DOX. Our current AI-SMSI approach has promising applications for subcellular metabolomics analysis and thus opens new opportunities to further explore drug-cell specific interactions for the long-term pursuit of precision medicine.

Ambient Temperature Affects Protein Self-Assembly by Interfering with the Interfacial Aggregation Behavior.

Amyloid fibrillation is known to be associated with degenerative diseases, and mature fibrils are also considered as valuable biomedical materials. Thus, the mechanism and influencing factors of fibrillation have always been the focus of research. However, in vitro studies are always plagued by low reproducibility of kinetics and the molecular mechanism of amyloid fibrillation is under debate until now. Here, we identified the ambient temperature (AT) as a non-negligible interfering factor in in vitro self-assembly of globular protein hen egg-white lysozyme for the first time. By multimodal molecular spectroscopy methods, not only the effect of ATs on the kinetics of protein aggregation was described but also the conformational changes of the molecular structure with different ATs were captured. Through investigating the dependence of interfacial area and catalysis, the reason for this influence was construed by the various aggregation behaviors of protein molecules in the two-phase interface. The results suggest that in vitro mechanism research on protein fibrillation needs to first clarify the AT for a more accurate comparative analysis. The proposal of this concept will provide a new clue for a deeper understanding of the mechanism of protein self-assembly and may have an impact on evaluating the efficiency of amyloid accelerators or inhibitors based on the comparative analysis of protein self-assembly.

Nanometer Resolution Mass Spectro-Microtomography for In-Depth Anatomical Profiling of Single Cells.

Visually identifying the molecular changes in single cells is of great importance for unraveling fundamental cellular functions as well as disease mechanisms. Herein, we demonstrated a mass spectro-microtomography with an optimal voxel resolution of ∼300 × 300 × 25 nm3, which enables three-dimensional tomography of chemical substances in single cells. This mass imaging method allows for the distinguishment of abundant endogenous and exogenous molecules in subcellular structures. Combined with statistical analysis, we demonstrated this method for spatial metabolomics analysis of drug distribution and subsequent molecular damages caused by intracellular drug action. More interestingly, thanks to the nanoprecision ablation depth (∼12 nm), we realized metabolomics profiling of cell membrane without the interference of cytoplasm and improved the distinction of cancer cells from normal cells. Our current method holds great potential to be a powerful tool for spatially resolved single-cell metabolomics analysis of chemical components during complex biological processes.

The Effect of GPX2 on the Prognosis of Lung Adenocarcinoma Diagnosis and Proliferation, Migration, and Epithelial Mesenchymal Transition.

Objective: To investigate the expression of glutathione peroxidase 2 (GPX2) in human lung adenocarcinoma tissues and its effect on the biological function of lung adenocarcinoma A549 cells. Methods: The expression of GPX2 in lung adenocarcinoma and its effect on survival were analyzed by the TCGA database and the GEPIA 2 database. A total of 45 cases of primary lung adenocarcinoma tissue specimens and 45 cases of their paracancerous tissue specimens were collected, and the expression of GPX2 in the two types of tissues was detected by immunohistochemistry. Lung adenocarcinoma A549 cells were divided into the GPX2 overexpression group (GPX2), the GPX2 knockdown group (si-GPX2), the empty vector group (Vector), the siRNA negative control group (si-NC), and the WT group; the mRNA level and protein expression of GPX2 in each group of A549 cells were detected by real-time fluorescence quantitative PCR and Western blotting; the proliferation activity of each group of cells was detected by the CCK-8 assay; the effect of GPX2 on cell migration and invasion ability was detected by the scratch assay and the Transwell invasion assay; the apoptosis of each group of cells was detected by flow cytometry; Western blotting was performed to detect the expression levels of Bax, Bcl-2, E-cadherin, vimentin, and MMP2 and MMP9 proteins in each group of cells. Results: Bioinformatics analysis showed that the expression of GPX2 was strongly correlated with the prognosis of lung adenocarcinoma patients (P < 0.01). The positive expression rates of GPX2 in lung adenocarcinoma and its paracancerous tissues were 66.0% and 15.7%, respectively (P < 0.05). The results of RT-qPCR and Western blotting showed that the expression level of GPX2 mRNA and protein in A549 cells in the GPX2 group increased, which was significantly higher than that in the WT group (P < 0.05); the expression levels of GPX2 mRNA and protein in A549 cells in the si-GPX2 group were the same, that is, significantly lower than the WT group (P < 0.05). GPX2 overexpression promoted the proliferation, migration, and invasion of A549 cells and inhibited their apoptosis; the results in the si-GPX2 group were opposite to those in the GPX2 group. Compared with the WT group, the expression of Bcl-2, vimentin, and MMP2 and MMP9 protein in the GPX2 group increased (P < 0.05), while the expression of Bax and E-cadherin protein decreased in the GPX2 group (P < 0.05); the results in the si-GPX2 group were opposite to those in the GPX2 group. Conclusion: The expression of GPX2 in lung adenocarcinoma is related to the prognosis of patients. It is proved that GPX2 can promote the migration and invasion of lung adenocarcinoma cells and is related to the EMT/ß-catenin pathway. Thus, GPX2 is expected to be an important target for the diagnosis and treatment of lung adenocarcinoma.

Living-DNA Nanogel Appendant Enables In Situ Modulation and Quantification of Regulation Effects on Membrane Proteins.

Screening appendants on membrane proteins to understand their varied regulation effects is desirable for finding the potential candidates of the membrane-protein-targeted drugs. However, most artificial appendants can hardly support in situ condition screening because they cannot evolve in situ, neither can they send out signals to reflect the modulation. Here, we designed living-DNA appendants to enable such screening. First, the living-cell rolling-circle amplification (LCRCA) strategy was developed to elongate the DNA appendants for self-tangled physical nanogels. The nanogels unify both the functions of membrane-protein modulation and quantification: their sizes increase with the increased time length of LCRCA, which change the regulation effect on the membrane proteins; their large number of repeating short sequences allow quantification of their sizes in the presence of the complementary fluorophore-tagged short DNA. Then, the performance of the living-DNA appendants was examined taking α6ß4 integrins as the target, where effective regulation over the distribution of actin filaments, cell viability, and chances of anoikis are all validated. The screening also clearly elucidates the interesting nonlinear relationships between the regulations and the effects. We hope this screening strategy based on living-DNA appendants can stand for a prototype for deeper understanding of natural behaviors of membrane proteins and help in the accurate designing of the membrane-protein-targeted drugs.

Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis.

BACKGROUND: P53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells. PURPOSE: To explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation. STUDY DESIGN AND METHODS: Different mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53. RESULTS: The triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation. CONCLUSION: Our study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.

Downregulation of LINC00515 in high-grade serous ovarian cancer and its relationship with platinum resistance.

Aim: To investigate the expression of long intergenic noncoding RNA 00515 (LINC00515) in high-grade serous ovarian cancer (HGSOC) and its potential correlation with platinum resistance. Patients & methods: Expression of LINC00515 in HGSOC (n = 115) and normal (n = 19) tissues was detected via quantitative real-time PCR (qRT-PCR). We further explored the statistical significance of the relationship between LINC00515 expression and platinum resistance in HGSOC. Results: LINC00515 was gradually downregulated in the order of normal > platinum-sensitive > platinum-resistant tissue (p < 0.05). Results demonstrated that LINC00515 downregulation was correlated with platinum resistance and relapse-free survival (RFS) of HGSOC (p < 0.05). Conclusion: LINC00515 downregulation is correlated with HGSOC development, platinum resistance and RFS, supporting its utility as a potential biomarker to predict platinum resistance and prognosis of RFS.

Phosphatase CDC25B Inhibitors Produced by Basic Alumina-Supported One-Pot Gram-Scale Synthesis of Fluorinated 2-Alkylthio-4-aminoquinazolines Using Microwave Irradiation.

An efficient, environmentally benign, and inexpensive procedure has been developed for the synthesis of fluorinated 2-alkylthio-4-aminoquinazolines by microwave irradiation using basic alumina as a solid-support agent as well as a solid base. Notably, this protocol features improved energy efficiency, broad isothiourea substrate scope, easily available starting materials, and high atom efficiency and applicability toward gram-scale synthesis. Additionally, the target compounds were evaluated for the cytotoxic effect against human colon adenocarcinoma (HCT116 and HT29), human gastric cancer (SGC-7901), human lung adenocarcinoma (A549), and human hepatocyte carcinoma (HepG2) cells, and it was found that these compounds have excellent antitumor activities. Among them, compound 3e was found to be one of the most potent derivatives with IC50 values lower than 9.44 µM against five human tumor cell lines, making it more active than cisplatin (DDP). Furthermore, for the first time, the fluorinated 2-alkylthio-substituted 4-aminoquinazolines were identified as phosphatase CDC25B inhibitors.

Simultaneous determination of six tyrosine kinase inhibitors in human plasma using HPLC-Q-Orbitrap mass spectrometry.

AIM: Gefitinib, erlotinib, icotinib, crizotinib, lapatinib and apatinib are targeted cancer therapy agents acting through inhibition of tyrosine kinase. Method for quantifying these six drugs in human plasma of patients was required. MATERIALS & METHODS: An HPLC-Q-Orbitrap method (based on HPLC-MS/MS) was developed and validated for the simultaneous detection and quantitation of six tyrosine kinase inhibitors in human plasma. Sample was extracted by liquid-liquid extraction (ethyl acetate: tert-Butyl methyl ether, 1:1 v/v). The method shows a high level of accuracy and reproducibility. The lower limit of quantification was 0.02 ng/ml for apatinib, 0.1 ng/ml for crizotinib, 2.0 ng/ml for lapatinib and 0.05 ng/ml for erlotinib, gefitinib and icotinib. This method was successfully used for apatinib monitoring in plasma of patients with NSCLC. CONCLUSION: This simple and reproducible method has potential for monitoring of tyrosine kinase inhibitors in patients' plasma.

13C-assisted metabolomics analysis reveals the positive correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size in Saccharopolyspora erythraea.

Metabolomics analysis is extremely essential to explore the metabolism characteristics of Saccharopolyspora erythraea. The lack of suitable methods for the determination of intracellular metabolites, however, hinders the application of metabolomics analysis for S. erythraea. Acyl-CoAs are important precursors of erythromycin; phosphorylated sugars are intermediate metabolites in EMP pathway or PPP pathway; organic acids are intermediate metabolites in TCA cycle. Reliable determination methods for intracellular acyl-CoAs, phosphorylated sugars, and organic acids of S. erythraea were designed and validated in this study. Using the optimized determination methods, the pool sizes of intracellular metabolites during an erythromycin fermentation process were precisely quantified by isotope dilution mass spectroscopy method. The quantification results showed that the specific erythromycin production rate was positively correlated with the pool sizes of propionyl-CoA as well as many other intracellular metabolites. The experiment under the condition without propanol, which is a precursor of propionyl-CoA and an important substrate in industrial erythromycin production process, also corroborated the correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size. As far as we know, this is the first paper to conduct the metabolomics analysis of S. erythraea, which makes the metabolomics analysis of S. erythraea in the industrial erythromycin production process possible.

Immune recovery after fluid resuscitation in rats with severe hemorrhagic shock.

OBJECTIVE: To investigate the effects of resuscitation with normal saline (NS), hypertonic saline (HTS), and hydroxyethyl starch (HES) on regulatory T cells (Tregs), helper T 1 (Th1)/Th2 and cytotoxic T 1 (Tc1)/Tc2 profiles in the treatment of hemorrhagic shock. METHODS: Rats subjected to severe hemorrhagic shock were resuscitated for 30 min with NS (n=8), HTS (n=8), or HES (n=8); sham (n=8) and naive control (n=8) groups were used for comparison. Following fluid resuscitation, the whole shed blood was reinfused for 30 min, and the rats were observed with continuous hemodynamic monitoring for 120 min. CD4+CD25+Foxp3+ Treg proportions, Th1/Th2 and Tc1/Tc2 profiles in spleen were analyzed by three-color flow cytometry. RESULTS: The proportion of CD4+CD25+Foxp3+ Tregs and ratios of Th1/Th2 and Tc1/Tc2 did not differ among control, sham, and HTS groups, but were significantly lower in NS and HES groups (both P<0.05 vs. sham); NS and HES levels were similar. The level of Tc1 was significantly increased in HTS (P<0.05 vs. sham), and levels of Tc2 were increased in NS, HES, and HTS groups compared to sham (all P<0.05), but did not differ from each other. CONCLUSIONS: HTS resuscitation has a greater impact on immune system recovery than NS or HES by preserving the proportion of Tregs and maintaining the balance between Th1/Th2 and Tc1/Tc2 cells in the spleen. Thus, HTS resuscitation provides potential immunomodulatory activity in the early stage after hemorrhagic shock.

Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics.

RATIONALE: Mass spectrometry (MS)-based protein identification depends mainly on protein extraction and digestion. Although sodium dodecyl sulfate (SDS) can preclude enzymatic digestion and interfere with MS analysis, it is still the most widely used surfactant in these steps. To overcome these disadvantages, a SDS-compatible proteomic technique for SDS removal prior to MS-based analyses was developed, namely filter-aided sample preparation (FASP). METHODS: Herein, based on the effectiveness of sodium deoxycholate and a detergent removal spin column, we developed a modified FASP (mFASP) method and compared its overall performance, total number of peptides and proteins identified for shotgun proteomic experiments with that of the FASP method. RESULTS: Identification of 4570 ± 392 and 9139 ± 317 peptides and description of 862 ± 46 and 1377 ± 33 protein groups with two or more peptides from the ovarian cancer cell line A2780 was accomplished by FASP and mFASP methods, respectively. The mFASP method (21.2 ± 0.2%) had higher average peptide to protein coverage than FASP method (13.2 ± 0.5%). More hydrophobic peptides were identified by mFASP than by FASP, as indicated by the GRAVY score distribution. CONCLUSIONS: The reported method enables reliable and efficient identification of proteins and peptides in whole-cell extracts containing SDS. The new approach allows for higher throughput (the simultaneous identification of more proteins), a more comprehensive investigation of proteins, and potentially the discovery of new biomarkers. Copyright © 2016 John Wiley & Sons, Ltd.

Effect of hypertonic versus isotonic saline resuscitation on heme oxygenase-1 expression in visceral organs following hemorrhagic shock in rats.

To compare the early effects of hypertonic and isotonic saline resuscitation on heme oxygenase-1 (HO-1) expression in organs of rats with hemorrhagic shock. Rats were randomly divided into hypertonic saline resuscitation (HTS), normal saline resuscitation (NS) and sham groups. HO-1 mRNA, protein expression and apoptosis were evaluated in organs. In the HTS group, significant difference was noted in HO-1 protein in small intestinal mucosa and liver compared with the NS and sham groups, and in HO-1 mRNA in liver and kidney compared with the sham group. The apoptosis of small intestinal mucosa, liver, heart, and lung was significantly lower in the HTS group than that in the NS group. In this study, small volume resuscitation with HTS can efficiently up-regulate the expression level of HO-1 in small intestinal mucosa and liver, which may be one of the mechanisms alleviating organ damage.

Hypertonic saline resuscitation contributes to early accumulation of circulating myeloid-derived suppressor cells in a rat model of hemorrhagic shock.

BACKGROUND: Hemorrhagic shock is usually associated with complicated immune and inflammatory responses, which are sometimes crucial for the prognosis. As regulators of the immune and inflammatory system; proliferation, migration, distribution and activation of myeloid-derived suppressor cells (MDSCs) are intimately linked to the inflammation cascade. METHODS: In a model of severe hemorrhagic shock, thirty-five rats were randomly divided into control, sham, normal saline resuscitation (NS), hypertonic saline resuscitation (HTS), and hydroxyethyl starch resuscitation (HES), with seven in each group. MDSCs were analyzed by flow cytometric staining of CD11b/c(+)Gra(+) in peripheral blood mononuclear cells (PBMC), spleen cell suspensions, and bone marrow nucleated cells (BMNC). Simultaneously, the expressions of arginase-1 (ARG-1) and inducible nitric oxide synthase (iNOS) mRNA in MDSCs were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: In the early stage after hemorrhagic shock, fluid resuscitation and emergency treatment, the MDSCs in the PBMC of NS, HTS and HES groups markedly increased, and MDSCs in BMNC of these groups decreased accordingly, significantly different to the control group. In hemorrhagic shock rats infused with HTS at the early resuscitation stage, MDSCs in PBMC increased about 2 and 4 folds, and MDSCs in BMNC decreased about 1.3 and 1.6 folds, as compared to the sham group respectively, with statistically significant difference. Furthermore, compared to the NS and HES groups, the MDSCs in PBMC of HTS group increased 1.6 and 1.8 folds with statistically significant differences; the MDSCs decrease in BMNC was not significant. However, there was no statistically significant difference in MDSCs of spleen among the five groups. In addition, compared to the control, sham, NS and HES groups, the ARG-1 and iNOS mRNA of MDSCs in PBMC, spleen and BMNC in the HTS group had the highest level of expression, but no statistically significant differences were noted. CONCLUSIONS: In this model of rat with severe and controlled hemorrhagic shock, small volume resuscitation with HTS contributes to dramatically early migration and redistribution of MDSCs from bone marrow to peripheral circulation, compared to resuscitation with NS or HES.

Early changes of CD4⁺CD25⁺Foxp3⁺ regulatory T cells and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of rats with controlled hemorrhagic shock and no fluid resuscitation.

BACKGROUND: Hemorrhagic shock induces immune dysfunction. Regulatory T cells (Tregs), T-helper (Th) cells, and cytotoxic T-lymphocytes (CTLs) can execute many crucial actions in immune and inflammatory responses. This study was conducted to investigate the early pathophysiological changes of CD4(+)CD25(+)Foxp3(+) Treg and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of rats with controlled hemorrhagic shock and no fluid resuscitation. METHODS: A rat model of controlled hemorrhagic shock with no fluid resuscitation was established. Peripheral blood samples were taken before and four hours after hemorrhagic shock with no fluid resuscitation. Three color flow cytometry was used to detect Tregs, Th1, Th2, Tc1 and Tc2 cells in the samples. RESULTS: In the peripheral blood of rats, the percentage of Tregs four hours after hemorrhagic shock was significantly lower than before hemorrhagic shock (P = 0.001). The ratios of Th1/Th2 and Tc1/Tc2 were changed from (23.08 ± 8.98)% to (23.91 ± 15.36)%, and from (40.40 ± 21.56)% to (65.48 ± 23.88)%, respectively. CONCLUSIONS: At an early stage, the advent of hemorrhagic shock is related to an early decrease of Tregs, and a mild shift in the Th1/Th2, Tc1/Tc2 balance toward Th1 and Tc1 dominance. These changes are part of a hyper-inflammatory state of the host, and will deteriorate the maintenance of immune balance. Further influences and detailed mechanisms need to be investigated.

Comprehensive profiling of the low molecular weight proteins and peptides in weak cation exchange beads human serum retentate.

Mass spectrometric profiling using ProteinChip and magnetic beads has rapidly grown over the past years, particularly to generate serum profiles for cancer diagnosis. The molecular weights of these distinguishing peaks are usually under 30 kDa. To identify those low molecular weight proteins and peptides is important for specific assays to be developed and increases biological insight. In this study, low molecular weight proteins and peptides from serum were purified by a combination of weak cation exchange magnetic beads and high performance liquid chromatography. The purified proteins and peptides were analyzed by 1D SDS PAGE, SELDI and LC-MS/MS. 246 proteins were identified from the HPLC fractions by LC-MS/MS. 95(38.62%) proteins were first identified in serum compare with Sys-BodyFluid database. 11(11/96) proteins were documented cancer associated proteins. We also observed about 109 proteins/peptides in SELDI mass spectrum, and 13 of the SELDI features were identified.

Effect of hypertonic saline resuscitation on heme oxygenase-1 mRNA expression and apoptosis of the intestinal mucosa in a rat model of hemorrhagic shock.

BACKGROUND: Massive blood loss due to trauma is the leading cause of death in trauma patients and military combatants. The fluid category of resuscitation for hypotensive trauma patients is open to debate. This study was conducted to investigate the early effects of hypertonic and isotonic saline solutions on heme oxygenase-1 (HO-1) mRNA expression and apoptosis in the intestinal mucosa of rats with hemorrhagic shock. METHODS: A model of severe hemorrhagic shock was established in 21 Sprague-Dawley rats. The rats were randomly divided into sham, normal saline resuscitation (NS), and hypertonic saline resuscitation (HTS) groups, with 7 in each group. We assessed and compared the HO-1 mRNA expression and apoptosis in the small intestinal mucosa of rats after hemorrhagic shock and resuscitation using the SYBR Green I fluorescence quantitative reverse transcriptase polymerase chain reaction, fluorescein-iso-thiocyanate-annexin V/propidium iodide double staining, and flow cytometry. RESULTS: In the early stage of hemorrhagic shock and resuscitation, marked apoptosis occurred in the small intestinal mucosa from both the NS and HTS groups. The apoptotic rate in the NS group was higher than that in the HTS group (P < 0.01). Among the three groups, HO-1 mRNA mucosa from the HTS group had the highest level of expression; however, the differences were not significant. There was a significant negative correlation between HO-1 mRNA expression and apoptosis in the small intestinal mucosa from the NS and HTS groups after hemorrhagic shock and resuscitation. CONCLUSIONS: In this rat model of severe hemorrhagic shock, HTS resuscitation with a small volume is more effective than NS resuscitation in reducing apoptosis of the intestinal mucosa. Further, HO-1 mRNA over-expression in the intestinal mucosa may be one of the molecular mechanisms of HTS in the resuscitation of hemorrhagic shock.

[Study of the metastasis-associated genes and its copy numbers variation in highly metastatic epithelial ovarian cancer].

OBJECTIVE: To investigate the relationship of the metastasis-associated genes and its copy numbers variation in the highly metastatic human epithelial ovarian cancer cell line HO-8910PM. METHODS: The differentially expressed genes and its copy number variation between HO-8910PM cell line and normal ovarian tissues was detected by human genome U133A 2.0 gene chip and human mapping 10K array 2.0 gene chip, and the data was analyzed by bioinformatics. Some of metastasis-associated genes were validated the results of single nucleotide polymorphism (SNP) and cDNA chips by fluorescence in situ hybridization (FISH) and real-time quantitative PCR. RESULTS: Integrate analysis of two gene chips data showed that there were 385 differentially expressed genes in the same and 379 SNP positional point (6 of them, included 2 genes) between HO-8910PM cell line and normal ovarian tissues, these copy number amplification of 379 SNP positional point of chromosome were > or = 3, which had 240, deletion < or = 1 had 139. Chromosome location analysis showed that there were 385 differentially expressed genes located at all chromosomes, and 261 of them (67.8%, 261/385) located at 10 chromosomes, included that 34 (8.8%), 33 (8.6%), 28 (7.3%), 27 (7.0%), 25 (6.5%), 24 (6.2%) of them located at chromosome 3, 2, 9, 10, 1 and 11 respectively, and 23 (6.0%) of them at chromosome 6 and 12 each, 22 (5.7%) of them at chromosome 4 and 5 each. For the function of differentially expressed genes, the results showed that 99 (25.7%) genes belonged to the family of enzymes and their regulators, 54 (14.0%) genes associated with signal transduction, 50 (13.0%) genes associated with nucleic acid binding, and 36 (9.4%) genes associated with protein binding. CONCLUSION: We have demonstrated that there are 4 kinds of differentially expressed genes related to metastasis of ovarian cancer, which belonged to the families enzyme and its regulator, nucleic acid binding, signal transduction and protein binding, and located at chromosome 1, 2, 3, 4, 5, 6, 9, 10, 11 and 12.

Hypertonic saline resuscitation reduces apoptosis of intestinal mucosa in a rat model of hemorrhagic shock.

OBJECTIVE: To investigate the early effects of hypertonic and isotonic saline solutions on apoptosis of intestinal mucosa in rats with hemorrhagic shock. METHODS: A model of rat with severe hemorrhagic shock was established in 21 Sprague-Dawley (SD) rats. The rats were randomly divided into the sham group, normal saline resuscitation (NS) group, and hypertonic saline resuscitation (HTS) group, with 7 in each group. We detected and compared the apoptosis in small intestinal mucosa of rats after hemorrhagic shock and resuscitation by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), FITC (fluorescein-iso-thiocyanate)-Annexin V/PI (propidium iodide) double staining method, and flow cytometry. RESULTS: In the early stage of hemorrhagic shock and resuscitation, marked apoptosis of small intestinal mucosa in the rats of both NS and HTS groups was observed. The numbers of apoptotic cells in these two groups were significantly greater than that in the sham group (P<0.01). In the HTS group, the apoptic cells significantly decreased, compared with the NS group (P<0.01). CONCLUSION: In this rat model of severe hemorrhagic shock, the HTS resuscitation of small volume is more effective than the NS resuscitation in reducing apoptosis of intestinal mucosa in rats, which may improve the prognosis of trauma.