Store-operated Ca²⁺ channels (SOCs) are plasma membrane Ca²⁺ permeable channels activated by depletion of intracellular Ca²⁺ store. Ca²⁺ entry through SOCs is known as store-operated Ca²⁺ entry (SOCE), which plays an important role in the functional regulation of airway smooth muscle cells (ASMCs). Protein kinase C (PKC) has been shown to have an activating or inhibiting effect on SOCE, depending on cell types and PKC isoforms that are involved. In ASMCs, the effect of PKC on SOCE has not been elucidated so far. In this study, the role of PKC in the activation of SOCE in rat ASMCs was examined by using Ca²⁺ fluorescence imaging technique. The results showed that acute application of PKC activators PMA and PDBu did not affect SOCE induced by the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor thapsigargin. The non-selective PKC inhibitor chelerythrine significantly inhibited thapsigargin- and bradykinin-induced SOCE. RT-PCR assay identified PKCα, δ and ɛ isoforms in rat ASMCs. PKCα-selective inhibitor Gö6976 and PKCɛ-inhibiting peptide Epsilon-V1-2 had no effect on SOCE; by contrast, PKCδ-selective inhibitor rottlerin attenuated SOCE dramatically, suggesting that PKCδ was the major PKC isoform involved in the activation of SOCE in ASMCs. Moreover, PKC down-regulation by extended exposure to high doses of PMA or PDBu also reduced SOCE, confirming the essential role of PKC in the activation of SOCE in ASMCs. In addition, PKC down-regulation did not influence the expression of stromal interaction molecule 1 (STIM1) and Orai1, two elementary molecules in the regulation and activation of SOCs. These results identified PKCδ as an essential PKC isoform involved in the activation of SOCE, and confirmed that PKC regulates the function of ASMCs in a SOCE-dependent manner.

This study examined the anti-hepatitis B virus (HBV) effect of wild-type (WT) vacuolar protein sorting 4B (VPS4B) and its dominant negative (DN) mutant VPS4B-K180Q in vivo in order to further explore the relationship between HBV and the host cellular factor VPS4. VPS4B gene was amplified from Huh7 cells by RT-PCR and cloned into the eukaryotic expression vector pXF3H. Then, the VPS4B plasmid and the VPS4B-K180Q mutation plasmid were constructed by using the overlap extension PCR site-directed mutagenesis technique. VPS4B and HBV vectors were co-delivered into mice by the hydrodynamic tail-vein injection to establish HBV vector-based models. Quantities of HBsAg and HBeAg in the mouse sera were determined by ElectroChemiLuminescence (ECL). HBV DNA in sera was measured by real-time quantitative PCR. Southern blot analysis was used to assay the intracellular HBV nuclear capsid-related DNA, real-time quantitative PCR to detect the HBV-related mRNA and immunohistochemical staining to observe the HBcAg expression in the mouse liver tissues. Our results showed that VPS4B and its mutant VPS4B-K180Q could decrease the levels of serum HBsAg, HBeAg and HBV-DNA. In addition, the HBV DNA replication and the mRNA level of HBV in the liver tissues of treated mice could be suppressed by VPS4B and VPS4B-K180Q. It was also found that VPS4B and VPS4B-K180Q had an ability to inhibit core antigen expression in the infected mouse liver. Furthermore, the anti-HBV effect of mutant VPS4B-K180Q was more potent than that of wild-type VPS4B. Taken together, it was concluded that VPS4B and its DN mutant VPS4B-K180Q have anti-HBV effect in vivo, which helps develop molecular therapeutic strategies for HBV infection.

Staphylococcus aureus (S. aureus) is an important human pathogen which can cause a chronic condition with a high relapse rate despite the aggressive antimicrobial treatment. Recent studies showed that intracellular pattern recognition receptors (including NOD) in response to bacteria or bacterial products play a proinflammatory role by activating nuclear transcription factor-κB (NF-κB). But how NOD2 mediates the proinflammatory response to S. aureus in mast cells (MCs) is unclear. So, in this study, we attempted to examine the role of NOD2 in inflammatory responses of MCs to S. aureus. P815 cells (a mouse mast cell line) were cultured. Real-time PCR was used to detect the NOD2 mRNA expression in P815 cells during S. aureus infection. The siRNA against NOD2 gene was synthesized and transfected into S. aureus-infected P815 cells. By using the methods of ELISA and flow cytometry, the effects of NOD2 gene silencing on cell phagocytosis, cytokine secretion, NF-κB activation and cell apoptosis of the S. aureus-infected P815 cells were examined. It was found that S. aureus infection could increase the expression of NOD2 mRNA in P815 cells. NOD2 gene interference in P815 cells reduced the number of S. aureus engulfed by P815 cells, the level of cytokines and the activation of NF-κB. In addition, S. aureus could induce the apoptosis of P815 cells, but NOD2 gene silencing did not affect the cell apoptosis rate. Our data suggested that NOD2 plays a key role in pathogen recognition, signal transduction, and NF-κB activation in the inflammatory responses of MCs infected by S. aureus.

Herein we reported a case of follicular lymphoma with 50.26% clonal malignant lymphocytes and 50% tumor cells positive for the immunoglobulin heavy chain gene and B-cell lymphoma 2 gene (IGH-BCL2). To determine whether endothelial cells (ECs) within the tumor share the feature of advanced malignancy, we isolated and purified the ECs from the tumor by using the immunomagnetic beads conjugated with a monoclonal antibody against CD34, a surface marker of ECs. Thereafter, we identified ECs according to their morphology and found that ECs presented consistently flat and elongated appearance with a lot of Weibel-Palade bodies in the cytoplasm. Results of flow cytometry confirmed that ECs isolated from the follicular lymphoma expressed high level of both vWF and CD34 and the purity of the ECs fraction was more than 90%. Additionally, we used FISH to check chromosomal aberration in the purified ECs and found that some of the ECs had only one fusion signal for the green IGH probe and the red BCL2 probe in contrast to typical t(14;18)(q32;q21) translocation with two fusion signals. This phenomenon was also observed in the tumor cells. It might be a different breakpoint of IGH in this case, which induced the loss of the fusion signal, indicating t(14;18)(q32;q21) translocation. The positive cells accounted for 18% of the isolated ECs from the tumor, indicating that a proportion of ECs from follicular lymphoma had the same chromosome aberration as the neoplastic cells.

Leukemia seems to depend on a small population of “leukemia stem cells (LSCs)” for its growth and metastasis. However, the precise surviving mechanisms of LSCs remain obscure. Cellular senescence is an important obstacle for production and surviving of tumor cells. In this study we investigated the activated state of a pathway, in which reactive oxygen species (ROS) induces cellular senescence through DNA damage and phophorylation of p38 MAPK (p38), in myeloid leukemic CD34⁺CD38⁻ cells. Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34⁺CD38⁻ cells were isolated from mononuclear cells from these bone marrow samples, and K562 and KG1a cells (two kinds of myeloid leukemia cell lines) by mini-magnetic activated cell sorting. Hematopoietic stem cells (HSCs) from human cord blood served as controls. Intracellular ROS level was detected by flow cytometry. DNA damage defined as the γH2AX level was measured by immunofluorescence staining. Real-time RT-PCR was used to detect the expression of p21, a senescence-associated gene. Western blotting and immunofluorescence staining were employed to determine the p38 expression and activation. The proliferation and apoptosis of CD34⁺CD38⁻ cells were detected by MTT assay and flow cytometry. Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34⁺CD38⁻ cells as compared with HSCs and H₂O₂-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34⁺CD38⁻ cells from K562 and KG1a cell lines. These findings suggested that, although excessive accumulation of oxidative DNA damage was present in LSCs, the relatively decreased phosphorylation of p38 might help leukemic cells escape senescence and apoptosis.

The purpose of the present study was to examine the effects of oxidative stress on ventricular arrhythmias in rabbits with adriamycin-induced cardiomyopathy and the relationship between oxidative stress and ventricular arrhythmia. Forty Japanese white rabbits were randomly divided into four groups (n=10 in each): control group, metoprolol (a selective β1 receptor blocker) group, carvedilol (a nonselective β blocker/α-1 blocker) group and adriamycin group. Models of adriamycin-induced cardiomyopathy were established by intravenously injecting adriamycin hydrochloride (1 mg/kg) to rabbits via the auri-edge vein twice a week for 8 weeks in the adriamycin, metoprolol and carvedilol groups. Rabbits in the control group were given equal volume of saline through the auri-edge vein. Rabbits in the metoprolol and carvedilol groups were then intragastrically administrated metoprolol (5 mg/kg/d) and carvedilol (5 mg/kg/d) respectively for 2 months, while those in the adriamycin and control groups were treated with equal volume of saline in the same manner as in the metroprolol and carvedilol groups. Left ventricular end diastolic diameter (LVEDd) and left ventricular ejection fraction (LVEF) were measured by echocardiography. Plasma levels of N-terminal pro B-type natriuretic peptide (NT-proBNP), malondialdehyde (MAD) and superoxide dismutase (SOD) were detected. The left ventricular wedge preparations were perfused with Tyrode’s solution. The transmural electrocardiogram, transmural action potentials from epicardium (Epi) and endocardium (Endo), transmural repolarization dispersion (TDR) were recorded, and the incidences of triggered activity and ventricular arrhythmias were obtained at rapid cycle lengths. The results showed that TDR and the serum MDA and NT-proBNP levels were increased, and LVEF and the serum SOD level decreased in the adriamycin group compared with the control group. The incidences of triggered activity and ventricular arrhythmia were significantly higher in the adriamycin group than those in the control group (P<0.05). In the carvedilol group as compared with the adriamycin group, the serum SOD level and the LVEF were substantially increased; the TDR, and the serum MDA and NT-proBNP levels were significantly decreased; the incidences of triggered activity and ventricular arrhythmia were obviously reduced (P<0.05). There were no significant differences in the levels of MDA and SOD, LVEF, TDR and the incidences of triggered activity and ventricular arrhythmia between the adriamycin group and the metoprolol group. It was concluded that carvedilol may inhibit triggered activity and ventricular arrhythmias in rabbit with adriamycin-induced cardiomyopathy, which is related to the decrease in oxygen free radials.

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.

Maspin belongs to the serine protease inhibitor (serpin) family and has been proven to be a suppressor of tumor growth and metastasis in many types of tumors. The purpose of this study was to investigate the expression of maspin in non-small cell lung cancer (NSCLC) and its relationship to vasculogenic mimicry (VM). A total of 160 specimens of NSCLC were involved in this study and 20 specimens of normal lung tissue served as controls. VM, microvessel density (MVD) and the expression of maspin were detected by using immunohistochemical staining. The results showed that the positive rates of maspin and VM in the NSCLC group were 48.1% (77/160) and 36.9% (59/160), respectively, which were significantly different from those in the control group with the positive rates of maspin and VM being 100% and 0% respectively (P<0.05). VM, MVD and the expression level of maspin were significantly related to tumor differentiation, lymph node metastasis, clinical stages and postoperative survival time (all P<0.05). The maspin expression in patients with squamous cell carcinoma was significantly higher than that in those with adenocarcinoma (P<0.05). The maspin expression was negatively correlated with VM and MVD, and there was a positive correlation between VM and MVD. Maspin-negative expression, VM and high MVD score were negatively related to the 5-year-survival rate. PTNM stages, VM, MVD and maspin expression were independent prognostic factors for NSCLC (P<0.05). It was suggested that the loss of expression of maspin may participate in the invasion and metastasis of NSCLC and it has a positive relationship to VM in NSCLC. Combined detection of maspin, VM and MVD may help predict the progression and prognosis of NSCLC.

Metastasis contributes to the poor prognosis of hepatocellular carcinoma (HCC). However, the mechanism through which a primary HCC cell develops into a metastatic phenotype is not well understood. The purpose of this study was to examine the correlation between metadherin (MTDH)/astrocyte elevated gene-1 (AEG-1) expression in HCC cell lines of different metastatic potentials and such metastatic phenotypes as orientation chemotaxis and adhesion. MTDH/AEG-1 expression was detected by RT-PCR and western blotting in HCC cell lines (HepG2, Huh7, Sk-HEP-1, MHCC-97H). Distribution of MTDH/AEG-1 was observed by immunofluorescence staining and confocal laser scanning microscopy. The abilities of orientation chemotaxis and adhesion and the index of interaction between HCC cell lines and microvascular endothelial cell lines (MVECs, including HUVECs and HPMECs) were measured by chemotaxis assay and adhesion assay, respectively. The results showed that MTDH/AEG-1 protein expression was significantly higher in high metastatic potential cancer cell lines (Sk-HEP-1, MHCC-97H) than in low metastatic potential cell lines (HepG2, Huh7) (P<0.05). The MTDH/AEG-1 protein was localized in the perinuclear region of HCC cells. Furthermore, the abilities of orientation chemotaxis and adhesion of HCC cells to HPMECs were increased as compared with those of HCC cells to HUVECs (P<0.05). The abilities of orientation chemotaxis and adhesion were much stronger in Sk-HEP-1 and MHCC-97H cells with MTDH/AEG-1 highly expressed than in HepG2 and Huh7 cells with MTDH/AEG-1 lowly expressed (P<0.05). These results suggested that the expression of MTDH/AEG-1 gene in HCC cell lines of different metastatic potentials was closely positively related to the abilities of orientation chemotaxis and adhesion of HCC cells. It was deduced that MTDH/AEG-1 might play a pivotal role in the lung-specific metastasis of HCC, which may be mediated through orientation chemotaxis and adhesion abilities of HCC cells. MTDH/AEG-1 may serve as a potential therapeutic target for HCC.

The aim of the present study was to examine the effects of suppression of EphB4 and/or mTOR on the biological behaviors of ovarian cancer cells, and the potential regulatory pathways. Antisense EphB4 vectors and shRNA vectors targeting mammalian target of rapamycin (mTOR) were constructed and transfected into A2780 and SKOV3 cells (two ovarian cancer cell lines). The effects of the antisense EphB4 vectors and the shRNA vectors on the proliferation, apoptosis and invasion of ovarian cancer cells were measured, and the expression of EphB4, mTOR and Akt detected. The results showed that transfection with mTOR shRNA could inhibit growth, induce apoptosis, and reduce invasive ability of ovarian cancer cells, which was accompanied by downregulation of EphB4, mTOR and Akt. The inhibitory effects on cell growth caused by mTOR shRNA alone were weaker than those by antisense pEGFP-C1-EphB4. In the antisense pEGFP-C1-EphB4-transfected cells, it was found that EphB4 knockdown could decrease the mTOR expression and slightly reduce the Akt phosphorylation. Significant suppressive effects on cell growth were observed in cells co-transfected with antisense pEGFP-C1-EphB4 and mTOR shRNA. In co-transfection group, the expression levels of EphB4, mTOR and Akt were distinctly lower than those in other groups. It was concluded that suppression of EphB4 may inhibit the growth of ovarian cancer cells by downregulation of the PI3K/Akt/mTOR pathway, and reverse Akt phosphorylation induced by mTOR shRNA. Inhibition of EphB4 and mTOR combined may cooperatively suppress the biological behaviors of ovarian cancer cells.

The formation of osteolytic bone lesions is a key process for osteolytic cancer to metastasize to the bone and is under the control of a set of transcription factors. Recently, the inhibitor of differentiation 1 (Id1) has been linked with angiogenesis, tumorigenesis, metastasis and bone formation. However, the function of Id1 during the process of bone destruction caused by cancer in vivo has not yet been elucidated. We, therefore, examined whether and how Id1 affects the ability of cancer to form osteolytic lesion in vivo. The study used a lentiviral vector overexpressing short hairpin RNA (shRNA) targeting Id1 gene. PC3 cells, a prostate cancer cell line, were transduced with Id1 shRNA or negative control (NC) shRNA before implantation in BALB/c mice. Cells were implanted in a tibial injection model. Tumor formation in bone was monitored by X-ray. The relationship between parathyroid hormone-related protein (PTHrP), an osteolytic factor, and Id1 was analyzed by using immunohistochemistry in tissue sections from osteolytic lesion of the BALB/c mice. Our results showed that Id1 shRNA delivery to PC3 cells by lentivirus caused efficient and stable Id1 gene silencing. In the intratibial model, PC3 cells produced primarily osteolytic lesions in the bone. Eleven of 14 mice in Id1 shRNA group but only 4 of 14 mice in the NC shRNA group developed osteolytic lesions with cortical destruction at 4th week. Mice treated with Id1 shRNA had larger tumor volume in the bone and larger cortical destruction. The expression of PTHrP protein in PC3 cells was not affected by Id1 knockdown in vivo. These results indicate that Id1 may down-regulate the ability of PC3 cells to form osteolytic lesions in vivo and the signal pathway needs to be further investigated.

The effects of microRNA-34a (miR-34a)-regulated Notch1 gene on the proliferation and apoptosis of the human glioma cell line U87 were investigated in this study. The U87 cells were divided into miR-34a mimics, negative control, mock transfection and blank control groups in terms of different treatments. In miR-34a mimics group, human U87 glioma cells were transfected with miR-34a mimics by using lipofectamine 2000. The cells transfected with nonsense microRNA were set up as negative control group. Those treated with lipofectamine 2000 only were designated to the mock tranfection group. In the blank control group, the cells were cultured routinely and no treatment was given. The expression of miR-34a and Notch1 was detected by using real-time RT-PCR. Western blotting was employed to monitor the change in Notch1 protein. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry. The results showed that the proliferative ability of U87 cells was significantly reduced and the apoptotic cells increased in miR-34a mimics group relative to control groups. The expression of miR-34a was significantly up-regulated in mimics group as compared with control groups (P<0.05). Furthermore, Notch1 protein levels were significantly decreased in miR-34a mimics group when compared with control groups (P<0.05), but the mRNA expression of Notch1 showed no significant difference among these groups. It was concluded that miR-34a may suppress the proliferation and induce apoptosis of U87 cells by decreasing the expression of target gene Notch1, suggesting that miR-34a may become a promising gene therapeutic target for brain glioma.

MicroRNAs (miRNAs or miRs) are a class of short, non-coding RNAs that participate in various oncological processes. This study aims to explore the roles of microRNA-34a (miR-34a) in invasive urothelial bladder carcinoma. miR-34a was transfected into bladder cancer cell lines 253J and J82. The miR-34a expression levels in tissues and cells were detected by using qRT-PCR. The Notch1 expression was detected by qRT-PCR and Western blotting. Cell migratory and invasive abilities were measured by Transwell chamber assay. Bioinformatics and luciferase assay were performed to predict and analyze the binding sites between miRNA-34a and Notch1. It was found that there was aberrant expression of miR-34a in bladder cancer tissues. Moreover, we revealed that ectopic expression of miR-34a suppressed cell migration and invasion, while forced expression of Notch1 increased cell migratory and invasive abilities. Finally, we observed that miR-34a transfection significantly down-regulated luciferase activity and reduced the mRNA and protein levels of Notch1. Our study concluded that microRNA-34a antagonizes Notch1 and inhibits cell migration and invasion of bladder cancer cells, which indicates the tumor-suppressive function of microRNA-34a in bladder cancer.

Over-expression of CD151 was found to be associated with metastasis and poor prognosis of prostatic carcinoma. This study was designed to examine the mechanism by which CD151 promotes the proliferation and migration of prostatic cancer cells. The pAAV-CD151, pAAV-GFP and pAAV-CD151-AAA mutant plasmids were constructed and used to transiently transfect PC3 cells (a prostatic carcinoma 3 cell line) by the mediation of Fugene HD. Then, the cells were assigned to control group, pAAV-GFP group, pAAV-CD151 group, and pAAV-CD151-AAA group respectively. Cell proliferation was evaluated by using the 3-[4,5-dimet-hylthiazol-2-yl]-2,5, diphenyltetrazolium bromide (MTT) method. Cell migration assay was performed by using Boyden chambers. The formation of CD151-integrin α3/α6 complex was determined by the method of co-immunoprecipitation. The protein expression levels of CD151 and extracellular signal-regulated kinase (ERK) were measured by Western blotting. The results showed that transfection of pAAV-CD151 or pAAV-CD151-AAA mutant increased the expression of CD151 protein in PC3 cells. Co-immunoprecipitation showed that more CD151-integrin α3/α6 complex was formed in the pAAV-CD151 group than in the control group, the pAAV-GFP group and the pAAV-CD151-AAA mutant group. Furthermore, the proliferative and migrating capacity of PC3 cells was substantially increased in the pAAV-CD151 group but inhibited in the pAAV-CD151-AAA mutant group. CD151 transfection increased the expression of phospho-ERK. Taken together, it was concluded that CD151 promotes the proliferation and migration of PC3 cells through the formation of CD151-integrin complex and the activation of phosphorylated ERK.

In the present study, we investigated the roles of TGF-β signaling pathway in a rat benign prostatic hyperplasia (BPH) model treated with cetrorelix. TGF-β1 and c-Myc expression were measured by qRT-PCR and Western blotting in the proximal and distal region of ventral prostatic lobes, respectively. We observed that treatment with cetrorelix led to a significant reduction of ventral prostate weight in a dose-dependent manner. In the proximal region, after cetrorelix treatment, the expression of TGF-β1 was dramatically increased (P<0.05), while the expression of c-Myc was significantly decreased (P<0.05). In comparison with the control group, the cetrorelix groups had more TUNEL-positive cells. Our findings strongly suggest that the TGF-β signaling pathway may be one of the major causes responsible for prostate volume reduction in BPH rats after cetrorelix treatment.

This study was aimed to investigate the influence of coital frequency and masturbation on erectile dysfunction (ED) in Chinese patients. A total of 332 male outpatients with or without ED and volunteers were recruited from Zhongnan Hospital, Wuhan University, China. ED was assessed by using the five-item International Index of Erectile Function scale and the frequency of intercourse by patients’ self-report. After adjusting for lifestyle factors and diseases-related factors, the analyses showed that coital and masturbation in a certain frequency tended to decrease the risk of ED. Men reporting intercourse once a week had lower risk of ED than those did less than once a week, with age, hyperlipidemia, hypertension, diabetes, body mass index, smoking, and drinking as covariates (P=0.67, adjusted odds ratio [OR] = 0.84; 95% confidence interval [CI]: 0.37–1.88). For those reporting coital frequency two times per week and three or more times per week, there were 63% (adjusted OR = 0.37; 95% CI: 0.18–0.77) and 85% (adjusted OR = 0.15; 95% CI: 0.07–0.35) lower risk of ED than those reporting intercourse frequency less than once per week, respectively (P<0.05). Results indicated that maintaining a regular frequency of intercourse can reduce the risk of ED for males aged among 30 to 75 years.

This study examined the effect of intensive insulin therapy on immune function and inflammatory factors at the early phase after severe trauma. At day 1, 3, 5, 7 after admission, subsets of CD4⁺ helper T lymphocytes (Th1/Th2) and human leukocyte antigen (HLA)-DR expression on CD14⁺ monocytes were flow cytometrically measured. Levels of cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10) and other immunity markers, such as IgA, IgG, IgM, C3, C4 and C reaction protein (CRP) were examined in two groups. The results showed that TNF-α, IL-6 and CRP levels in the intensive insulin therapy group were significantly lower than those in the conventional therapy group, whereas IL-10 levels were substantially increased after intensive insulin therapy. C3 level at day 3, 5, 7 and C4 levels at day 5, 7 were lower in the intensive therapy group than in the conventional therapy group. Th1/Th2 ratios decreased gradually over time in both groups, and were much lower at day 3, 5, 7 in intensive therapy group. There were significant differences among day 3 to day 7 after admission in HLA-DR expression in CD14⁺ monocytes. It was concluded that the intensive insulin therapy could decrease pro-inflammatory cytokines and increase anti-inflammatory cytokines in the elderly suffering from severe trauma, at the same time, with complement recovery being delayed. Moreover, intensive insulin therapy promoted immune suppression and, therefore, measures need be taken to address the issue.

Embryos with a poor morphological score at cleavage stage are usually discarded because they are considered unsuitable for transfer and cryopreservation. This study examined the in vitro blastocyst development after extended culture of these embryos and the clinical outcomes after transfer of these blastocysts in warming cycles. A total of 597 blastocysts (24.7%) were obtained from 2421 embryos with low morphological scores after extended culture. One hundred and sixty blastocysts (6.6%) with optimal morphology were vitrified. Embryo utilization rate was increased from 30.8% to 32.6%. After warming, 61 out of 92 blastocysts (66.3%) survived and were transferred in 44 cycles. The clinical pregnancy rate and the implantation rate were 40.9% (18/44) and 32.8% (20/61) respectively. Thirteen healthy babies were born, and 5 pregnancies aborted spontaneously. Our study suggested that some blastocysts derived from embryos with a poor morphological score can be successfully vitrified and give rise to live births. Selection and vitrification of viable embryos after extended culture of embryos with a poor morphological score may constitute a proposal to avoid embryo wastage.

Although empirically well understood in their clinical administration, volatile anesthetics are not yet well comprehended in their mechanism studies. A major conundrum emerging from these studies is that there is no validated model to assess the presumed candidate sites of the anesthetics. We undertook this study to test the hypothesis that the single-celled Paramecium could be anesthetized and served as a model organism in the study of anesthetics. We assessed the motion of Paramecium cells with Expert Vision system and the chemoresponse of Paramecium cells with T-maze assays in the presence of four different volatile anesthetics, including isoflurane, sevoflurane, enflurane and ether. Each of those volatiles was dissolved in buffers to give drug concentrations equal to 0.8, 1.0, and 1.2 EC50, respectively, in clinical practice. We could see that after application of volatile anesthetics, the swimming of the Paramecium cells was accelerated and then suppressed, or even stopped eventually, and the index of the chemoresponse of the Paramecium cells (denoted as I_che) was decreased. All of the above impacts were found in a concentration-dependent fashion. The biphasic effects of the clinical concentrations of volatile anesthetics on Paramecium simulated the situation of high species in anesthesia, and the inhibition of the chemoresponse also indicated anesthetized. In conclusion, the findings in our studies suggested that the single-celled Paramecium could be anesthetized with clinical concentrations of volatile anesthetics and therefore be utilized as a model organism to study the mechanisms of volatile anesthetics.

This study examined the neuroprotective effect of cluster of differentiation molecule 200 (CD200) against methamphetamine (METH)-induced neurotoxicity. In the in vitro experiment, neuron-microglia cultures were treated with METH (20 μmol/L), METH (20 μmol/L)+CD200-Fc (10 μg/mL) or CD200-Fc (10 μg/mL). Those untreated served as control. Microglia activation expressed as the ratio of MHC-II/CD11b was assessed by flow cytometry. The cytokines (IL-1β, TNF-α) secreted by activated microglia were detected by enzyme-linked immunosorbent assay (ELISA). In the in vivo experiment, 40 SD rats were divided into control, METH, METH+CD200-Fc and CD200-Fc groups at random. Rats were intraperitoneally injected with METH (15 mg/kg 8 times at 12 h interval) in METH group, with METH (administered as the same dose and time as the METH group) and CD200-Fc (1 mg/kg at day 0, 2, 4 after METH injection) in METH+CD200-Fc group, with CD200-Fc (1 mg/kg injected as the same time as the METH+CD200-Fc group) or with physiological saline solution in the control group. The level of striatal dopamine (DA) in rats was measured by high-performance liquid chromatography (HPLC). The microglial cells were immunohistochemically detected for the expression of Iba-1, a marker for microglial activation. The results showed that METH could increase the microglia activation in the neuron-microglia cultures and elevate the secretion of IL-1β and TNF-α, which could be attenuated by CD200-Fc. Moreover, CD200-Fc could partially reverse the striatal DA depletion induced by METH and reduce the number of activated microglia, i.e. Iba-1-positive cells. It was concluded that CD200 may have neuroprotective effects against METH-induced neurotoxicity by inhibiting microglial activation and reversing DA depletion in striatum.

This study examined the effects of combined administration of tyrosine, lecithin, L-glutamine and L-5-hydroxytryptophan (5-HTP) on heroin withdrawal syndromes and mental symptoms in detoxified heroin addicts. In the cluster-randomized placebo-controlled trial, 83 detoxified heroin addicts were recruited from a detoxification treatment center in Wuhan, China. Patients in the intervention group (n=41) were given the combined treatment with tyrosine, lecithin, L-glutamine and 5-HTP and those in the control group (n=42) were administered the placebo. The sleep status and the withdrawal symptoms were observed daily throughout the study, and the mood states were monitored pre- and post-intervention. The results showed that the insomnia and withdrawal scores were significantly improved over time in participants in the intervention group as compared with those in the control group. A greater reduction in tension-anxiety, depression-dejection, anger-hostility, fatigue-inertia and total mood disturbance, and a greater increase in their vigor-activity symptoms were found at day 6 in the intervention group than in the control group (all P<0.05). It was concluded that the neurotransmitter-precursor-supplement intervention is effective in alleviating the withdrawal and mood symptoms and it may become a supplementary method for patients’ recovery from heroin addiction.

This study examined the change of reported incidence rate for viral hepatitis in Hubei province, China, between 2004 to 2010 to provide scientific evidence for viral hepatitis control. Reported viral hepatitis infection cases were queried from Centre for Disease Control of Hubei Province, China. The incidence of viral hepatitis A decreased steadily across the study period. Viral hepatitis B composed 85% of the viral hepatitis cases. When reported incidence rates for chronic hepatitis B increased, the rates of acute and unclassified cases dropped from 2005 to 2010. The reported viral hepatitis B incidence rate for males was around 1.5–2 times higher than for females. The average annual percentage change of reported viral hepatitis B incidence rates was 4%. The same index for viral hepatitis C was 28%. The reported viral hepatitis B incidence rate of people under 20 years old declined over the period. This decrease was mainly attributed to the recent implementation of vaccination plan. Reported incidence rate of viral hepatitis E also rose in those years. Having a better understanding on reported incidence rates of the present surveillance system is important for developing strategies for further prevention of viral hepatitis. In addition, the data showed that a surveillance system that differentiates new and former infected cases will be more effective in providing evidence for disease control.

Respirator breathing resistance impacts performance of wearers during constant work load. However, it is less clear as to how breathing resistance affects the tolerant capacity of users during graded work load. The present study investigated the tolerant capacity of 8 individuals during incremental work load. The 8 subjects were required to wear two matched respirators (respirators I and II which were designed to have different breathing resistances and the same dead space) respectively on separate days and then work to end points. Minute ventilation (V_E), breathing frequency (BF), oxygen consumption (VO₂) and heart rate (HR) were recorded during exercise, while tolerant time, response time and breathing discomfort were measured at the end of each test trial. The test variables were compared between the two respirators by using matched-pairs t-test. The results showed that the tolerant time was significantly reduced for the respirator I with higher level of breathing resistance when compared with its counterpart with lower breathing resistance (respirator II) (P<0.05). The same changes occurred for response time. Results also showed a significant increase in V_E and BF for respirator I wearers when the work load was above 125 W. The O₂ consumption was similar under the two breathing resistance conditions. These findings suggested that the respiratory resistance caused by self-contained breathing apparatus (SCBA) has an impact on the tolerant capacity of users.

Over one half the patients with rheumatoid arthritis (RA) are being treated with methotrexate (MTX). Although well proven, the efficacy of MTX varies in individual patients. This study examined the metabolic biomarkers that can be used to predict the therapeutic effect of MTX by using metabolomic analysis. Rats were immunized with collagen to rapidly cause collagen-induced arthritis (CIA) and then treated with 0.1 mg/kg MTX for 4 weeks. The clinical signs and the histopathological features of CIA were observed to evaluate the therapeutic effects. Urine samples of CIA rats were collected, and analyzed by using 600 M ¹H-nuclear magnetic resonance (¹H-NMR) for spectral binning after the therapy. The urine spectra were divided into spectral bins, and 20 endogenous metabolites were assigned by Chenomx Suite. Multivariate analyses were performed to identify the spectral pattern of endogenous metabolites related to MTX therapy. The results showed that the clustering of the spectra of the urine samples from the responsive rats (n=20) was different from that from the non-responsive rats (n=11). Multivariate analysis showed difference in metabolic profiles between the responsive and non-responsive rats by using partial least squares-discrimination analysis (PLS-DA) (R²=0.812, Q²=0.604). In targeted profiling, 13 endogenous metabolites (uric acid, taurine, histidine, methionine, glycine, etc.) were selected as putative biomarkers for predicting therapeutic response to MTX. It was suggested that ¹H-NMR-based metabolomic analysis can be used to predict the therapeutic effect of MTX, and several metabolites were found to be related to the therapeutic effects of MTX.

This study compared a new type of polysaccharide-coated magnetic nanoparticles (in which the polysaccharide is derived from Angelica sinensis) with the dextran magnetic nanoparticles in terms of preparation, biocompatibility and tissue distribution in vivo and in vitro in order to examine the potential application of Angelica polysaccharide as a novel carrier in magnetic drug targeting (MDT). Magnetic nanoparticles were prepared by chemical co-precipitation. Their physical and chemical properties were determined by using the transmission electron microscope (TEM), laser particle size analyzer (DLS) and vibrating sample magnetometer (VSM), and their purity and structure by using X-ray diffractometer (XRD) and Fourier transform infrared spectroscopy (FTIR). The atomic absorption spectrometric method was performed for quantification of the iron content in different tissues. Histological sections were stained by Prussian blue staining to observe the disposition of magnetic nanoparticles in the liver and kidney. The results showed that both kinds of magnetic nanoparticles possessed small particle size, good dispersion and good magnetic properties. XRD showed the main component of the two magnetic nanoparticles was Fe₃O₄ crystals, and FTIR proved Fe₃O₄ was successfully coated by each polysaccharide, respectively. In vivo, Fe₃O₄-dextran accumulated in the liver, spleen and lung and Fe₃O₄-Angelica polysaccharide only in the spleen and lung. It was concluded that Angelica polysaccharide may be applied as a novel carrier in the preparation of magnetic nanoparticles.

Oral lichen planus (OLP) is considered a T cell-mediated autoimmune disease with unknown aetiology. T helper cells appear to play an important role in the pathogenesis of OLP. We investigated the possible role of T helper cells, Th1 and Th17, in the lesions and circulation of patients with OLP. Forty patients with OLP and 15 healthy volunteers were recruited. Double immunofluorescence staining was used to detect Th1 and Th17 cells in the OLP lesions, and intracellular cytokine staining and flow cytometry to evaluate the proportion of Th1 and Th17 cells in peripheral blood. The levels of serum interferon (IFN)-γ and interleukin (IL)-17 were assessed by using an enzyme-linked immunosorbent assay. It was found that Th17 cells, as well as Th1 cells, were present in OLP lesions. The proportion of peripheral Th1 and Th17 cells was significantly increased in patients with OLP. The proportion of Th17 cells in atrophic-erosive OLP was elevated as compared with that in reticular OLP. Serum IL-17 levels in OLP patients were significantly higher than in controls, and those in the atrophic-erosive OLP group were increased as compared with the reticular OLP group. However, the levels of serum IFN-γ were slightly decreased in OLP patients. Our data suggested that Th1 and Th17 cells in the local lesions and peripheral blood may be associated with the pathogenesis of OLP, and that IL-17 may be an important proinflammatory cytokine in OLP. These findings enhance our understanding of OLP pathogenesis.