Differential mRNA Expressions in HCMV infected HUVECs.

Objective: The aim was to identify the gene expressions of human cytomegalovirus (HCMV)-infected human umbilical vein endothelial cells (HUVECs) and to study its possible pathogenic mechanism on atherosclerosis using microarray technology. Methods: The gene expression differences in HCMV AD169 strain-infected HUVECs were studied by the microarray technology to explore the potential molecular mechanism of HCMV infection. The qPCRs were performed to verify the transcriptome results. Results: A total of 2,583 differentially expressed genes, including 407 down-regulated genes and 2,176 up-regulated genes, were detected by the systematic bioinformatics analysis. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the significantly differentially expressed genes were mainly involved in regulating protein kinase activity, inflammatory response, ubiquitination, protein phosphorylation, cell metabolism, and exosomes, among which 12 genes had significant changes and were screened by protein-protein interaction (PPI) analysis and verified by qPCR. The experimental qPCR results were consistent with the microarray results. Conclusion: The GO and KEGG analyses revealed that the regulation of protein kinase activity, inflammatory response, ubiquitination, protein phosphorylation, and cell metabolism played important roles in the process of endothelial cell infection. Furthermore, 12 genes were involved in the process of HCMV infection of endothelial cells and contributed to the current understanding of the infection and pathogenic mechanisms of atherosclerosis.

[Effect of the focal adhesion kinase inhibitor TAE226 on the epithelial-mesenchymal transition in human oral squamous cell carcinoma cell line].

OBJECTIVE: To study the effect of the focal adhesion kinase inhibitor TAE226 on epithelial-mesenchymal transition (EMT) in human oral squamous cell carcinoma (OSCC) cell line. METHODS: HSC-3 and HSC-4 cells were cultured with TAE226 under different concentrations (0, 1, 5, and 10 µmol·L⁻¹) for 24, 48, and 72 h. Real-time quantitative polymerase chain reaction was performed to detect the mRNA expressions of E-cadherin and Vimentin. The protein expressions of E-cadherin and Vimentin were determined by Western blot assay after 48 h of TAE226 treatment. RESULTS: Real-time quantitative polymerase chain reaction showed that increasing the TAE226 dose and reaction time resulted in increased and decreased E-cadherin and Vimentin mRNA expressions, respectively (P<0.05). Western blot assays showed that increasing the TAE226 dose resulted in increased and decreased E-cadherin and Vimentin protein expressions, respectively (P<0.05). CONCLUSIONS: TAE226, which is expected to be an effective drug for OSCC treatment, can effectively inhibit the EMT of the OSCC cell line.

Oct-4 Enhanced the Therapeutic Effects of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Acute Kidney Injury.

BACKGROUND/AIMS: Acute kidney injury (AKI) is a common clinical condition that can lead to chronic kidney failure. Although mesenchymal stem cell-derived extracellular vesicles (MSC EVs) are regarded as a potent AKI treatment, the mechanisms underlying their beneficial effects remain unclear. Oct-4 may play an important role in tissue injury repair. We thus hypothesized that oct-4 overexpression might enhance the therapeutic effects of MSC EVs in AKI treatment. METHODS: Renal tubular epithelial cells were cultured in a low oxygen environment, then cocultured with MSC EVs or control medium for 48 h. BrdU and transferase-mediated dUTP nick-end labeling (TUNEL) staining were used to assess cell proliferation and apoptosis. Mice subjected to ischemia reperfusion were randomly divided into 4 groups, then injected with either phosphate-buffered saline (vehicle), EVs, EVs overexpressing oct-4 (EVs+Oct-4), and EVs not expressing Oct-4 (EVs-Oct-4). Blood creatinine (CREA) and urine nitrone levels were assessed 48 h and 2 weeks after injection. After ischemia reperfusion, renal tissues from each group were stained with TUNEL and proliferating cell nuclear antigen (PCNA) to determine the degree of apoptosis and proliferation. Masson trichrome staining was used to evaluate renal fibrosis progression. Snail gene expression was assessed using polymerase chain reaction (PCR). RESULTS: At 48 h after hypoxic treatment, TUNEL and BrdU staining indicated that the EVs+Oct-4 group had the least apoptosis and the most proliferation, respectively. Treatment with EVs overexpressing Oct-4 significantly decreased serum Crea and blood urea nitrogen levels and rescued kidney fibrosis, as indicated by the low proportion of Masson staining, high number of PCNA-positive cells, and low number of TUNEL-positive cells. PCR analysis indicated that Snail was most upregulated in the vehicle group and least upregulated in the EVs+Oct-4 group. CONCLUSIONS: MSC EVs had a pronounced therapeutic effect on ischemic reperfusion injury-related AKI, and Oct-4 overexpression enhanced these therapeutic effects. Our results may inspire a new direction for AKI treatment with MSC EVs.

Comprehensive miRNA Analysis of Human Umbilical Cord-Derived Mesenchymal Stromal Cells and Extracellular Vesicles.

BACKGROUND/AIMS: Mesenchymal stromal cells (MSCs) participate in the tissue-specific repair of many different organs, especially the kidney. Their effects are primarily mediated by the paracrine release of factors including extracellular vesicles (EVs), which are composed of micro-vesicles and exosomes. The corresponding microRNAs (miRNAs) of EVs are considered important for their biological functions. METHODS: MSCs were cultured from the human umbilical cord, and EVs were isolated from the medium. The expression levels of miRNAs in MSCs and EVs were determined by microarray analysis, and gene ontology (GO) was used to analyze the functions of their target genes. RESULTS: MSCs and EVs had similar miRNA expression profiles, with the exception of a small number of selectively enriched miRNAs. GO analysis indicated that, unlike MSCs, the target genes of EV-enriched miRNAs were associated with calcium channel regulation and cell junction activities, which may indicate that MSC and EVs have different regulatory properties. Angiogenesis, oxidative stress, and inflammatory signaling pathways related to the repair of renal injury were also analyzed, and EV-enriched miRNAs targeted genes associated with oxidative stress, T cell activation, and Toll-like receptor signaling. The miRNAs enriched in both MSCs and EVs targeted different genes in signaling pathways regulating angiogenesis and chemokine release. CONCLUSION: MSCs and their EVs shared similar miRNA component, and some selectively enriched miRNAs observed in MSCs and EVs may affect different target genes through some specific signaling pathways.

Microvesicles derived from human umbilical cord mesenchymal stem cells facilitate tubular epithelial cell dedifferentiation and growth via hepatocyte growth factor induction.

During acute kidney injury (AKI), tubular cell dedifferentiation initiates cell regeneration; hepatocyte growth factor (HGF) is involved in modulating cell dedifferentiation. Mesenchymal stem cell (MSC)-derived microvesicles (MVs) deliver RNA into injured tubular cells and alter their gene expression, thus regenerating these cells. We boldly speculated that MVs might induce HGF synthesis via RNA transfer, thereby facilitating tubular cell dedifferentiation and regeneration. In a rat model of unilateral AKI, the administration of MVs promoted kidney recovery. One of the mechanisms of action is the acceleration of tubular cell dedifferentiation and growth. Both in vivo and in vitro, rat HGF expression in damaged rat tubular cells was greatly enhanced by MV treatment. In addition, human HGF mRNA present in MVs was delivered into rat tubular cells and translated into the HGF protein as another mechanism of HGF induction. RNase treatment abrogated all MV effects. In the in vitro experimental setting, the conditioned medium of MV-treated injured tubular cells, which contains a higher concentration of HGF, strongly stimulated cell dedifferentiation and growth, as well as Erk1/2 signaling activation. Intriguingly, these effects were completely abrogated by either c-Met inhibitor or MEK inhibitor, suggesting that HGF induction is a crucial contributor to the acceleration of cell dedifferentiation and growth. All these findings indicate that MV-induced HGF synthesis in damaged tubular cells via RNA transfer facilitates cell dedifferentiation and growth, which are important regenerative mechanisms.

Determination of polychlorinated biphenyls and organochlorine pesticides in human serum by gas chromatography with micro-electron capture detector.

A method for determination of concentrations of polychlorinated biphenyl congeners (PCB-28, 52, 101, 118, 138, 153, 156, and 187) and organochlorine pesticides (hexachlorobenzene, alpha-hexachlorocyclohexane, beta-hexachlorocyclohexane, gamma-hexachlorocyclohexane, delta-hexachlorocyclohexane, p,p'-dichlorodiphenyl dichloroethylene, o,p'-dichlorodiphenyl trichloroethane, p,p'-dichlorodiphenyl dichloroethane, p,p'-dichlorodiphenyl trichloroethane, alpha-chlordane, gamma-chlordane, heptachlor, heptachlor epoxide, and aldrin) in human serum is developed. Recovery is assessed with artificial serum, in which PCBs and OCPs could not be detected. The method is then confirmed with pooled human serum. Experiments are performed by adding two concentrations of analytes (0.5 µg/L and 1.0 µg/L) to both matrices. The sample pretreatment process involves denaturing with a mixture of water-1-propanol (v:v, 85:15), extraction with a C-18 cartridge, and cleanup with an Alumina B cartridge. This process required about 2 mL of serum. The limit of detection ranged from 0.05-0.35 µg/L for all the analytes. Recovery of analytes at low and high spiking concentrations varied from 63-122% and 61-124% for artificial serum and pooled human serum, respectively. Relative standard deviation was lower than 16% and 18% for artificial serum and pooled human serum, respectively. Stability of the method, expressed as relative standard deviation, was lower than 14%. The method has been applied in epidemiological research.

The role of metabotropic glutamate receptor 5 in developmental lead neurotoxicity.

A complete explanation of the mechanisms of lead-induced developmental neurotoxicity remains unknown. The glutamate receptor is one of the most important targets of lead. More recently, metabotropic glutamate receptor 5 (mGluR5) has been shown to have a functional relationship with learning and memory. We investigated the impact of developmental lead exposure on hippocampal mGluR5 expression and its potential role in lead neurotoxicity. Both in vitro model of lead exposure with Pb(2+) concentrations of 0, 10 nM, 1 microM, and 100 microM in cultured rat embryonic hippocampal neurons, and the in vivo model of rat maternal lead exposure involving both gestational and lactational exposure with 0, 0.05%, 0.2%, and 0.5% lead acetate were utilized. Immunoperoxidase and immunofluorescent analyses, quantitative PCR and western blotting were used. In vitro studies revealed that expression of mGluR5 mRNA and protein was decreased dose-dependently after lead exposure, which was further confirmed by the results of in vivo studies. These data suggest that mGluR5 might be involved in lead-induced neurotoxicity by disturbing mGluR5-induced long-term depression and decreasing N-methyl-D-aspartic acid receptor (NMDAR)-dependent or protein synthesis-dependent long-term potentiation. These results might improve the understanding of the mechanism and potential treatments for moderate to severe lead poisoning in children.

[Effects of chelation therapy with succimer in young rabbits of moderate lead poisoning].

OBJECTIVE: To explore the effect of chelation therapy with succimer (DMSA) in male rabbits of moderate lead poisoning during juvenile stage. METHODS: Twenty-four 45-day-old male New Zealand rabbits were randomly divided into three groups (therapy group, TG; positive control group, PG and negative control group, NG, n=8). The TG and PG were orally exposed to lead acetate (5 mg x kg(-1) x d(-1)) for 6 weeks. Rabbits in TG were orally supplied DMSA 1050 mg/m2 in the first week and 700 mg/m2 in the next two weeks, while the other two groups wren't blood and urinary samples of all rabbits were collected per week. The tissues and organs of all rabbits were collected after 12 weeks. The blood lead levels (BLLs) were determined by atomic absorption spectrometer. The urine lead levels and the lead contents of tissue and organ were determined by inductively coupled plasma-mass spectrometry. Histopathology of tissue and organ was observed by light microscope. RESULTS: Compared with PG, the lead level in the morning urine of TG with DMSA chelating was increased significantly. The level was peaked at (1246.96 +/- 157.91) microg/L on the first day after chelating. While the base line was (40.97 +/- 1.77) microg/L before chelating. Meanwhile, the BLLs were sharply declined from (429.63 +/- 10.82) microg/L to (238.50 +/- 11.82) microg/L. The urine lead levels of TG decreased through the 3-week chelating and 3-week discontinuation. The urine lead levels of these two groups were significantly different (F=2934.35, P<0.01). Compared to each two groups in these three groups, there were significant difference (P<0.01). The authors found the reversion of BLLs in first week after stop chelating. The BLLs of PG presented the slow course of declining in the same time, were (135.50 +/- 7.09) microg/L, very close to the level of TG for (149.88 +/- 11.39) microg/L. Compared with treatment discontinuation for 3 weeks, the urine lead levels and the body weight gain of the therapy group increased more than that of PG, and the BLLs and the lead concentrations in tissues and organs decreased more than that of PG, and histopathology in the liver tissues and testicle tissues were improved. CONCLUSION: DMSA chelating for the rodent models of moderate lead poisoning might reduce the BLLs and soft tissue lead contents quickly and effectively, decrease toxic effects of lead in a short period of time, thus alleviate the impairment of lead poisoning on tissues and organs by decreasing lead burden, and bring out improvement on the growth retardation caused by lead poisoning.

[Effects of moderate lead poisoning on the hippocampus tissue of rabbits in juvenile stage].

OBJECTIVE: To understand the effects of moderate lead poisoning on the hippocampus tissue of rabbits in juvenile stage. METHODS: Sixteen 45-day-old male New Zealand rabbits were randomly divided into blank group and lead-exposed group,8 for each group. Rabbits in the lead-exposed group were treated with 5 mg x kg(-1) x d(-1) lead acetate in their forage for 6 weeks to establish a moderate lead poisoning animal model. The blood lead levels and the lead contents in the hippocampus were determined by atomic absorption spectrometer and inductively coupled plasma-mass spectrometry respectively. Histopathology and ultra-microstructure in the hippocampus tissue were observed by light microscope and electron microscope. The NR1, NR2A and NR2B protein expressions in the CA1 hippocampal region were analyzed through immunohistochemical method. RESULTS: Compared with those of blank group, the blood lead levels of lead-exposed group were significant increased, (428.63 +/- 9.46) vs (66.38+/-3.93) microg/L (t = 100.08, P<0.01); and lead contents of hippocampus was significantly increased, (44.57+/-2.03) vs (21.20+/-1.53) ng/g, (t = 26.05, P<0.01); the hippocampus wet weight were significant decreased, (0.735 +/-0.012) vs (0.808+/-0.010), (t =12.97, P<0.01); the coefficient of hippocampus wet weight, was (0.458 +/-0.004) vs (0.476+/-0.005), (t =7.87, P<0.01). The significant declines in both the positive rate of NR1 and NR2A in the CA1 hippocampal region for NR1: (37.44 +/- 2.05)% vs (41.81+/-2.50)% (t = 3.82, P<0.01) and for NR2A: 21.97+/-1.08 vs 25.48+/-1.30 (t =5.89, P<0.01) were also observed. With light microscope and electron microscope, the histopathology and ultra-microstructure of neuron and glial cell in the hippocampus tissue were changed. CONCLUSION: The impairment of hippocampus of rabbits in juvenile stage with chronic moderate lead poisoning were observed, and the histopathology and N-methyl-D-aspartate receptor protein expressions in the hippocampus tissue were changed.

[Effects of prenatal exposure to stress and lead on spatial learning and memory development in rats].

OBJECTIVE: To explore the effects of prenatal exposure to stress and lead on spatial learning and memory development in rats. METHODS: All 32 Sprague-Dawley (SD) pregnant rats were divided randomly into 4 groups, 8 per group in line with the Random Number Table. The four groups were: no maternal stress, no Pb exposure (NS/C); non-maternal stress, Pb exposure (NS/L), maternal stress, no Pb exposure (S/C), and maternal stress plus Pb exposure (S/L). The spatial learning and memory ability, the serum corticosterone level both pre and post-testing of 30-day old offsprings, and lead concentration in hippocampus were tested by means of Morris Water Maze, radioimmunoassay and Inductively Coupled Plasma Mass Spectrometry (ICP-MS). RESULTS: The residence time of male and female in NS/L was (16.08+/-3.41) s, (15.72+/-3.33) s, which were significantly shorter than NS/L (25.42+/-4.76) s, (24.55+/-4.43) s and S/C (20.96+/-3.45) s, (20.65+/-2.98) s, and significant difference was observed in the joint exposure effect (F=5.478, P<0.05). The effect of the joint exposure was significant on post-testing serum corticosterone. The hippocampus lead concentrations of NS/L and S/L were (0.4378+/-0.1041) microg/g and (0.4679+/-0.1243) microg/g without significant differences (F=0.298, P>0.05). CONCLUSION: Prenatal joint exposure to restraint stress and lead might increase the effects of single exposure on the spatial learning and memory ability and serum corticosterone level of offsprings, and the joint influence on corticosterone level might be one of the reasons of further impairment of learning and memory.

[Study on maternal-fetal status of Pb, As, Cd, Mn and Zn elements and the influence factors].

OBJECTIVE: Trace and toxic elements have great influences on the fetus growth during the pregnancy. The status of Pb, As, Cd, Mn and Zn in maternal and umbilical cord blood and influence factors were analyzed. METHODS: From September 2006 to April 2007, 130 pairs of maternal blood and cord blood in total were collected at the time of spontaneous delivery or cesarean section. At the same time, the development of newborn was measured immediately. The concentrations of elements were determined by inductively coupled plasma mass spectrometry, the relationship of these elements between maternal and cord blood were also analyzed. RESULTS: The median (microg/L) concentration of blood Pb, As, Cd, Mn and Zn in maternal blood were 64.32, 3.81, 0.84, 54.26 and 6312.50. And the median (microg/L) of those elements in cord blood were 35.72, 2.84, 0.32, 78.99 and 2250. The levels of Cd (r=0.341, P=0.000) and As (r=0.552, P=0.000) in maternal blood were positively correlated with the elements in the cord blood. From the questionnaire we conclude that the occupational hazardous factors and room decorated were the risk factors for the blood As and Zn levels. After multilinear regression analysis we also found mother weight, occupational hazardous factors and mother systolic pressure might affect the levels of blood Mn, Zn, As and Cd. CONCLUSIONS: The levels of these elements were affected by environmental and maternal factors. In this study, although the levels of all heavy metals in pregnant women were below those considered hazardous, however, they were still higher than those in the developed countries. The effects of heavy metals of maternal exposure on developing fetuses should deserve attention further.