Effects of coenzyme Q10 supplementation on inflammatory markers: A systematic review and meta-analysis of randomized controlled trials.

The aims of this meta-analysis were to evaluate the effects of coenzyme Q10 (CoQ10) supplementation on inflammatory mediators including C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) by analyzing published randomized controlled trials (RCTs). A systematic search in PubMed, Cochrane Library and Clinicaltrials.gov was performed to identify eligible RCTs. Data synthesis was performed using a random- or a fixed-effects model depending on the results of heterogeneity tests, and pooled data were displayed as weighed mean difference (WMD) and 95% confidence interval (CI). Seventeen RCTs were selected for the meta-analysis. CoQ10 supplementation significantly reduced the levels of circulating CRP (WMD: -0.35mg/L, 95% CI: -0.64 to -0.05, P=0.022), IL-6 (WMD: -1.61pg/mL, 95% CI: -2.64 to -0.58, P=0.002) and TNF-α (WMD: -0.49pg/mL, 95% CI: -0.93 to -0.06, P=0.027). The results of meta-regression showed that the changes of CRP were independent of baseline CRP, treatment duration, dosage, and patients characteristics. In the meta-regression analyses, a higher baseline IL-6 level was significantly associated with greater effects of CoQ10 on IL-6 levels (P for interaction=0.006). In conclusion, this meta-analysis of RCTs suggests significant lowering effects of CoQ10 on CRP, IL-6 and TNF-α. However, results should be interpreted with caution because of the evidence of heterogeneity and limited number of studies.

[Construction of a Mouse Model for Myeloproliferative Neoplasms and an Evaluation System].

OBJECTIVE: To construct a myeloproliferative neoplasms (MPN) transplanted mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation, and establish a systematic evaluation system to verify the success of model construction. METHODS: The bone marrow c-kit+ cells of the mice were obtained by the following steps: The mice were killed by cervical dislocation, the femur, tibia and ilium were separated, and the bone marrow cells were collected. The c-kit+ cells were sorted after incubation with CD117 magnetic beads. The method of constructing mouse primary mutant cells is as follows: A gene mutation vector with a GFP tag was constructed by the retroviral system, and the retroviral vector was packaged into the Platinum-E cells to obtain the virus supernatant, and then used it to infect the c-kit+ cells of mice. The MPN mouse model was constructed as follows: the mouse primary c-kit+ cells containing the mutant genes were collected after infection, and then transplanted them via the tail vein into the female recipient mice of the same species which were irradiated with a lethal dose of gamma rays (8.0 Gy). The MPN mouse model was evaluated as follows: After transplantation, the peripheral blood of the mice was regularly collected from the tail vein to perform the complete blood count test, and the size of spleen and the degree of bone marrow fibrosis were estimated. RESULTS: The mouse c-kit+ cells with the mutant genes were successfully obtained from the bone marrow. MPN mouse model was successfully constructed: The peripheral blood cells of the MPN-transplanted mice carried exogenous implanted GFP-positive cells, and the white blood cells (WBC), platelet (PLT) and hematocrit (HCT) were all increased; the body weight loss, and the water and food intake were reduced in the transplanted mice; further pathological analysis showed that the transplanted mice displayed splenomegaly and bone marrow fibrosis. These results suggested that the MPN mouse model was successfully constructed. According to the common and different characteristics of the three MPN mouse model, a preliminary evaluation system for judging the success of MPN mouse model construction was summarized, which mainly included the following indicators, for example, the proportion of GFP-positive cells in the peripheral blood of mice; WBC, PLT and HCT; the degree of spleen enlargement and the bone marrow fibrosis. CONCLUSION: The MPN mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation is successfully established by retroviral system, which can provide an important experimental animal model for the research of MPN pathogenesis and drug-targeted therapy.

[Detection of Cryptosporidium parvum in human stool using TaqMan real-time polymerase chain reaction].

The special DnaJ-like protein gene of Cryptosporidium parvum was amplified through designing special primers and TaqMan probes within the conserved and specific regions for this gene. method of real-time PCR assay for the detection of C. parvum was established. The specificity and sensitivity of PCR were also analyzed. By adding standard culture fluid in blank fecal sample, the sensitivity of the method was evaluated. The results showed that the detection limit of pure culture with real-time PCR assay was 26 oocysts/ml. The detection limit for C. parvum in artificially contaminated fecal sample was 2 600 oocysts/ml. The specificity of the method was verified with no amplification on DNA from other enteric parasites and bacteria. These results indicated that the real-time PCR method for C. parvum detection in fecal sample is simple, rapid, with high specificity and sensitivity.

A novel single-chain variable fragment antibody against FGF-1 inhibits the growth of breast carcinoma cells by blocking the intracrine pathway of FGF-1.

The fibroblast growth factors (FGFs) are important for embryo development, wound healing, hematopoiesis, and angiogenesis. FGF-1, a member of FGF family, is involved in both receptor-dependent pathways and an intracrine pathway. Studies have recently shown that FGF-1 is overexpressed in the early stages of several kinds of cancer. Thus, FGF-1 is a candidate for cancer immunotargeting. To study the potential use of therapeutic antibodies against FGF-1, a monoclonal hybridoma 1C9 secreting monoclonal antibody specific for FGF-1 was developed. Then, a single-chain variable fragment (scFv) antibody was genetically engineered from hybridama 1C9. The binding of the scFv1C9 to the antigen FGF-1 was demonstrated by ELISA and immunoprecipitation assays. Functional analysis showed that the overexpressed scFv1C9 in MCF-7 cells targeted endogenous FGF-1 and prevented the translocation of FGF-1 into the nucleus, resulting in the blockade of the intracrine pathway of FGF-1, which caused the G1 arrest by p21 up-regulation. These results suggest that the generated scFv1C9 is an effective inhibitor of the intracrine pathway of FGF-1 and has a potential application as anti-tumoral agent in breast cancer.

Screening of differential expressive genes in murine cells following radon exposure.

This study was designed to construct and identify the subtracted cDNA library in peripheral blood cells of BALB/c mice and tracheal-bronchial epithelial cells of Wistar rats following exposure to radon inhalation. Two groups of the animals were exposed in a radon chamber at an accumulative dose of 100 WLM, while control animals were housed in a room at a background dose of 1 WLM. To construct a subtracted cDNA library enriched with differentially expressed genes, the SMART technique and suppression subtractive hybridization (SSH) assay were performed. The obtained forward and reverse cDNA fragments were directly inserted into a pMD-18 vector and transformed into Escherichia coli JM109. In total, 593 white bacterial clones were selected from both forward- and reverse-subtracted libraries. Among them, 81 clones were chosen for their differential expressions based on reverse Northern blot. Portions of these cDNA clones were also verified by a quantitative real-time polymerase chain reaction (PCR). The screening resulted in 14 upregulative and 8 downregulative known function/annotation genes, which were revealed to be functionally related to cell proliferation, cell oxidative and DNA damage, apoptosis, and tumor promotion. Access numbers were obtained from the GenBank for 11 unknown expressed sequence tags (EST). Analysis of biological roles of these cDNA fragments may provide further insight into mechanisms underlying adverse molecular events induced by high-dose radon exposure.

PTEN deletion leads to deregulation of antioxidants and increased oxidative damage in mouse embryonic fibroblasts.

Despite the significance of oxidative damage in carcinogenesis, the molecular mechanisms that lead to increased susceptibility to oxidative stress are not well understood. We now report a link between loss of protection against oxidative damage and loss of function of PTEN, a highly mutated tumor suppressor gene in a variety of human tumors. Using two-dimensional gel electrophoresis, combined with Western and Northern blot analyses, we found that PTEN deficiency in mouse embryonic fibroblasts (MEFs) displays deregulated expression of several antioxidant enzymes, including peroxiredoxins 1, 2, 5, and 6 and Cu, Zn superoxide dismutase. In these Pten-deleted MEFs, the basal levels of reactive oxygen species (ROS) were increased, and both the basal level and the ROS-induced oxidative damage of DNA were increased, as evidenced by increased levels of hydrogen peroxide (H2O2), superoxide anion, 8-hydroxy-2'-deoxyguanosine, and DNA double-strand breaks. We further show that Pten deletion is correlated with resistance to H2O2-induced expression of several antioxidants. These findings suggest an essential role for PTEN in maintaining the normal redox state of mouse embryonic fibroblasts against oxidative damage. They also provide a molecular link between PTEN, whose inactivation is known to be involved in a variety of human tumors, and antioxidants, whose perturbation leads to oxidative damage of cells.

Screening of differential expression genes in bone marrow cells of radon-exposed mice.

This study was designed to screen for differential expression genes in bone marrow cells of mice exposed to radon inhalation. Based upon established pathological findings in mouse, differential screening of gene expressions was conducted by using the SSH method. Among 285 cDNA clones selected from both forward- and reverse subtracted libraries, 45 were chosen for their differential expressions based on reverse Northern blot and quantitative real-time PCR analysis. Of these, up-regulation of the mRNA levels of E-cadherin and down-regulation of the replication protein A1 (RPA1) and casein kinase 1 delta (CKI delta) were also verified by a quantitative real-time PCR. Biological roles of these obtained cDNAs are described and the results of the screening may provide important clues for further investigations of the adverse molecular events induced by radon exposure.

Lipolysis and thermogenesis in adipose tissues as new potential mechanisms for metabolic benefits of dietary fiber.

OBJECTIVE: Dietary fiber consumption is associated with reduced risk for the development of noncommunicable diseases. The aim of the present study was to evaluate the effects of cereal dietary fiber on the levels of proteins involved in lipolysis and thermogenesis in white adipose tissue (WAT) and brown adipose tissue (BAT) of C57 BL/6 J mice fed a high-fat diet (HFD). METHODS: Male C57BL/6 J mice were fed normal chow diet (Chow), HFD, HFD plus oat fiber (H-oat), or HFD plus wheat bran fiber (H-wheat) for 24 wk. Body weight and food intake were recorded weekly. Serum adiponectin was assayed by an enzyme-linked immunosorbent assay kit. Western blotting was used to assess the protein expressions of adipose triacylglycerol lipase (ATGL), cAMP protein kinase catalytic subunit (cAMP), protein kinase A (PKA), perilipin A, hormone-sensitive lipase (HSL), uncoupling protein 1 (UCP1), fibroblast growth factor 21 (FGF-21), ß3-adrenergic receptor (ß3AR), and proliferator-activated receptor gamma coactivator-1 α (PGC-1 α) in the WAT and BAT. RESULTS: At the end of the feeding period, body and adipose tissues weight in both H-oat and H-wheat groups were lower than in the HFD group. Mice in the H-oat and H-wheat groups showed an increasing trend in serum adiponectin level. Compared with the HFD group, cereal dietary fiber increased protein expressions involved in the lipolysis and browning process. Compared with the H-wheat group, H-oat was more effective in protein expressions of PKA, PGC-1 α, and UCP1 of the WAT samples. Compared with the H-oat group, H-wheat was more effective in protein expressions of PKA, ATGL, UCP1, ß3AR, and FGF-21 of the BAT samples. CONCLUSIONS: Taken together, our results suggested that cereal dietary fiber enhanced adipocyte lipolysis by the cAMP-PKA-HSL pathway and promoted WAT browning by activation of UCP1, and consequently reduced visceral fat mass in response to HFD feeding.

Circadian rhythms and different photoresponses of Clock gene transcription in the rat suprachiasmatic nucleus and pineal gland.

The aim of this study was to observe and compare the endogenous circadian rhythm and photoresponse of Clock gene transcription in the suprachiasmatic nucleus (SCN) and pineal gland (PG) of rats. With free access to food and water in special darkrooms, Sprague-Dawley rats were housed under the light regime of constant darkness (DD) for 8 weeks (n=36) or 12 hour-light: 12 hour-dark cycle (LD) for 4 weeks (n=36), respectively. Then, their SCN and PG were dissected out every 4 h in a circadian day, 6 rats at each time (n=6). All animal treatments and sampling during the dark phases were conducted under red dim light (<0.1 lux). The total RNA was extracted from each sample and the semi-quantitative RT-PCR was used to determine the temporal mRNA changes of Clock gene in the SCN and PG at different circadian times (CT) or zeitgeber times (ZT). The grayness ratio of Clock/H3.3 bands was served as the relative estimation of Clock gene expression. The experimental data were analyzed by the Cosine method and the Clock Lab software to fit original results measured at 6 time points and to simulate a circadian rhythmic curve which was then examined for statistical difference by the amplitude F test. The main results are as follows: (1) The mRNA levels of Clock gene in the SCN under DD regime displayed the circadian oscillation (P<0.05). The endogenous rhythmic profiles of Clock gene transcription in the PG were similar to those in the SCN (P>0.05) throughout the day with the peak at the subjective night (CT15 in the SCN or CT18 in the PG) and the trough during the subjective day (CT3 in the SCN or CT6 in the PG). (2) Clock gene transcription in the SCN under LD cycle also showed the circadian oscillation (P<0.05), and the rhythmic profile was anti-phasic to that under DD condition (P<0.05). The amplitude and the mRNA level at the peak of Clock gene transcription in the SCN under LD were significantly increased compared with that under DD (P<0.05), while the value of corresponding rhythmic parameters in the PG under LD were remarkably decreased (P<0.05). (3) Under LD cycle, the circadian profiles of Clock gene transcription induced by light in the PG were quite different from those in the SCN (P<0.05). Their Clock transcription rhythms were anti-phasic, i.e., showing peaks at the light phase ZT10 in the SCN or at the dark time ZT17 in the PG and troughs during the dark time ZT22 in the SCN or during the light phase ZT5 in the PG. The findings of the present study indicate a synchronous endogenous nature of the Clock gene circadian transcriptions in the SCN and PG, and different roles of light regime in modulating the circadian transcriptions of Clock gene in these two central nuclei.

[Establishment of Human Chronic Lymphocytic Leukemia Model in the BALB/c Nude Mice by Using MEC-1 and HG3 Cell Lines].

OBJECTIVE: To explore the the optimal condition for establishing immune deficiency mouse(BALB/c) model with CLL via subcutaneous inoculation of human chronic lymphocytic leukemia (CLL) cells at different inoculative locations and different cell concentrations. METHODS: Firstly, Two CLL cell lines (MEC-1-GFP and HG3-GFP)with the green fluorescent protein (GFP) were established by lentivirus system respectively, and then the MEC-1-GFP cells (5×107/ml) were inoculated into forelimb, hindlimb and abdomen to observe the tumorigenesis. Secondly, the MEC-1-GFP and HG3-GFP cells with same density (5×107/ml) were inoculated into forelimb to compare the time and rate of tumor formation. Thirdly, the MEC-1-GFP cells (1×107/ml) and HG3-GFP cells (5×107/ml) were inoculated into forelimb to compare the time and rate of tumor formation at different inoculative density. After observation for 5 weeks, the peripheral blood was collected and treated with EDTA and erythrocytolysin, then the of GFP positive cells were detected by flow cytomety. Meanwhile, the tumor-bearing mice were killed, and the tumors were isolated and cut into slices for histopathological examination. RESULTS: MEC-1-GFP and HG3-GFP cell lines were successfully established, and after inocutation of MEC-1-GFP cells with 5×107/ml the xenograt tumors were formated in forelimb, hindlimb and abdomen of mice, especially in the forelimb with a higher tumorigenic rate. In addition, the inoculation of same density of MEC-1-GFP and HG3-GFP cells (5×107/ml) also resulted in xenograft in forelimb, and the tumorigenic rate reached to 80% after 5 weeks. Moreover, the inocutation of MEC-1-GFP and HG3-GFP cells with 1×107 and 5×107/ml respectively also effectively resulted to xenograft tumor in forelimb. The flow cytometry showed that there was no MEC-1-GFP and HG3-GFP cells in peripheral blood, while histopathological examination demonstrated CLL cell metastasis towards peritoneal cavity. CONCLUSION: The BALB/c nude mouse model is successfully established by subcutaneous injection of MEC-1-GFP and HG3-GFP cells. This model is a useful tool to explore the pathogenic mechanism.

Antithymocyte globulin combined with cyclosporine A down-regulates T helper 1 cells by modulating T cell immune response cDNA 7 in aplastic anemia.

Antithymocyte globulin (ATG) combined with cyclosporine A (CsA) has been widely used as a standard regimen in the treatment of aplastic anemia (AA), especially in severe aplastic anemia (SAA). Abnormally activated T cells might be the immune pathogenesis of AA. T cell immune response cDNA 7 (TIRC7) has been demonstrated its essential role in T cell activation; however, little is known about the role of TIRC7 in AA. In this study, we documented that TIRC7 levels in CsA group were higher than that in ATG + CsA (AC) group only in the follow-up phase (P < 0.05; P < 0.05); nevertheless, TIRC7 levels in SAA group were elevated than non severe aplastic anemia group not only in the treatment phase (P < 0.05; P < 0.05) but also in the follow-up phase (P < 0.05; P < 0.01). The trend of changes of T helper (Th) 1, Th17 and Th22 levels before and after treatment was similar to the changes of TIRC7 levels in either AC group or CsA group. Thus, TIRC7 might be involved in the pathogenesis of AA and AC might down-regulate Th1 cells by modulating the expression of TIRC7 in AA.

[Roles of the histaminergic receptors in the locus ceruleus in stress-induced carotid baroreflex resetting in rats].

AIM: To explore the roles of H1 and H2 receptors in the locus ceruleus (LC) in the carotid baroreflex (CBR) resetting resulted from foot-shock stress. METHODS: Male SD rats were divided into two groups (n=18) at random: unstressed and stressed group. The latter were subjected to unavoidable electric foot-shock twice daily for a week and each session of foot-shock lasted 2 hours. The left and right carotid sinus regions were isolated from the systemic circulation in all animals anesthetized with pentobarbital sodium. The intracarotid sinus pressure (ISP) was altered in a stepwise manner in vivo. ISP-mean arterial pressure (MAP), ISP-Gain relationship curves and reflex characteristic parameters were constructed by fitting to the logistic function with five parameters. The changes in CBR performance induced by stress and the effects of microinjection with histaminergic receptors antagonists into the LC on the responses of CBR to stress were examined. RESULTS: Stress significantly shifted the ISP-MAP relationship curve upwards (P < 0.05) and obviously moved the middle part of ISP-Gain relationship curve downwards (P < 0.05), and decreased the value of the MAP range and maximum gain (P < 0.05), but increased the threshold pressure, saturation pressure, set point and ISP at maximum gain (P < 0.05). Microinjection of selective H1 or H2 receptor antagonist, chlorpheniramine (CHL, 0.5 microg/microl) or cimetidine (CIM, 1.5 microg/microl) into the LC, significantly attenuated the above-mentioned changes in CBR performance induced by stress and the alleviate effect of CIM was less remarkable than that of CHL (P < 0.05). The responses of CBR under stress to H1 or H2 receptor antagonist generally occurred 20 min after the administration and lasted approximately for 16 min. Microinjection with the same dose of CHL or CIM into the LC in the unstressed group did not change CBR performance significantly (P > 0.05). However, microinjection of CHL or CIM into the LC could not completely abolish the stress-induced changes in CBR. CONCLUSION: The stress results in a resetting of CBR and a decrease in reflex sensitivity. The stress-induced changes in CBR may be mediated, at least in part, by activating the brain histaminergic system. The H1 and H2 receptors in the LC, especially, Hi receptors may play an important role in the resetting of CBR under stress. The descending histaminergic pathway from the hypothalamus to LC may be involved in these effects. Moreover, the effects of stress on CBR also have other mechanisms.