Background::Heterotaxy (HTX) is a thoracoabdominal organ anomaly syndrome and commonly accompanied by congenital heart disease (CHD). The aim of this study was to analyze rare copy number variations (CNVs) in a HTX/CHD cohort and to examine the potential mechanisms contributing to HTX/CHD.Methods::Chromosome microarray analysis was used to identify rare CNVs in a cohort of 120 unrelated HTX/CHD patients, and available samples from parents were used to confirm the inheritance pattern. Potential candidate genes in CNVs region were prioritized via the DECIPHER database, and PNPLA4 was identified as the leading candidate gene. To validate, we generated PNPLA4-overexpressing human induced pluripotent stem cell lines as well as pnpla4-overexpressing zebrafish model, followed by a series of transcriptomic, biochemical and cellular analyses. Results::Seventeen rare CNVs were identified in 15 of the 120 HTX/CHD patients (12.5%). Xp22.31 duplication was one of the inherited CNVs identified in this HTX/CHD cohort, and PNPLA4 in the Xp22.31 was a candidate gene associated with HTX/CHD. PNPLA4 is expressed in the lateral plate mesoderm, which is known to be critical for left/right embryonic patterning as well as cardiomyocyte differentiation, and in the neural crest cell lineage. Through a series of in vivo and in vitro analyses at the molecular and cellular levels, we revealed that the biological function of PNPLA4 is importantly involved in the primary cilia formation and function via its regulation of energy metabolism and mitochondria-mediated ATP production. Conclusions::Our findings demonstrated a significant association between CNVs and HTX/CHD. Our data strongly suggested that an increased genetic dose of PNPLA4 due to Xp22.31 duplication is a disease-causing risk factor for HTX/CHD.

Objective To explore dendritic cells (DCs)-mediated antigen presentation for radiation-injured cells by using the in vitro cell co-culture technology to simulate the in vivo microenvironment of the lung tissue. Methods ⁶⁰Co γ-irradiated mouse lung epithelial cells (MLE-12) were cultured with bone marrow-derived DCs and/or splenic T lymphocytes for 48 hours. Flow cytometry was used to measure the expression levels of costimulatory molecules (CD80/86) and antigenic peptide recognition complexes (the major histocompatibility complex [MHC] class Ⅰ/Ⅱ) on DCs and T cell activation markers (CD69/28/152) as well as the numbers of CD4⁺ and CD8⁺ T cells. Results ⁶⁰Co γ irradiation significantly increased the apoptosis rate of MLE-12 cells in a dose-dependent manner, and significantly stimulated the expression of CD80/86 and MHC Ⅱ on DCs, without direct activation of T cells. After γ (6 Gy)-irradiated MLE-12 cells were co-cultured with DCs and T lymphocytes for 48 h, there were significant increases in the expression of CD69 and CD28 on T cells, the numbers of CD4⁺ and CD8⁺ T cells, and the expression of CD86 and MHC I on DCs, as compared with the control groups. Conclusion Radiation-injured cells can stimulate antigen presentation by DCs and activate T cells.

Objective To investigate the role of complement in radiation-induced lung injury in mice after chest irradiation with ⁶⁰Co γ-rays at a single dose of 20 Gy. Methods C57BL/6 mice underwent chest irradiation with ⁶⁰Co γ-rays at a single dose of 20 Gy, followed by observation for the inflammatory reaction of the lung tissue in the early stage (within 15 d) and pulmonary fibrosis in the later stage (30 and 180 d). Enzyme-linked immunosorbent assay was used to measure the levels of C2, C3a, C4, and C5b-9 in the lung tissues at 1, 3, 7, 15, 30, and 180 d after irradiation. The expression of complement mRNA in BEAS-2B cells after irradiation was determined using RT-PCR. Results Radiation-induced lung injury in micepresented as inflammatory response in the early stage and fibrosis in the late stage. Complement C2, C4, and C5b-9 complexes were increased in the early period (3 or 7 d) after irradiation (P < 0.05), which might be associated with the inflammatory response induced by irradiation. During 3 to 180 d, complement C3a was significantly higher in the irradiated mice than in the control mice, suggesting a close relationship between C3a and radiation-induced lung injury. The irradiated cells showed increased mRNA expression of C2 and C3, with no changes in the mRNA levels of C4 and C5. Conclusion Different complement proteins have varying responses to radiation-induced lung injury, among which C3a is closely related to radiation-induced lung injury, suggesting that regulating C3a and its receptors may be a new way to prevent and treat radiation-induced lung injury.

Objective To observe the effect of PIF1 knockdown on cell growth and cell cycle arrest induced by ionizing radiation.Methods HeLa cell lines that consistently down-regulated PIF1 were prepared by the lentivirus granules interfering technology and confirmed by real-time PCR and Western blotting.The effect of down-regulation of PIF1 on cell growth and cell cycle arrest induced by ionizing radiation was evaluated by cell counting and flow cytometry.Results HeLa cell lines consistently down-regulating PIF1 were established.The growth of HeLa that down-regulated PIF1 was inhibited greatly after 4 Gy of γ-ray irradiation.There was little cell proliferation until the 5th day post 4 Gy γ-ray.Moreover, the S phase block and G2/M phase block of PIF1 knock-downed cell lines were significantly delayed after 8 Gy γ-ray irradiation.Conclusion Knockdown of PIF1 can significantly enhance the radiation sensitivity and delayes the S phase block and G 2 /M phase block induced by ionizing radiation.

Objective To construct the recombinant eukaryotic expression plasmids of human adenylyl cyclase-associated protein 1 (CAP1)and to explore its intracellular location and functions.Methods By using Hela cDNA as the template,the cDNAs encoding CAP1 was amplified by PCR and inserted into pCMV-Myc vector to construct the recombinant plasmid.The recombinant plasmid was transfected into 293 cells using lipofectamine 2000.The protein expression and the intracellular location of the inserted gene were confirmed by Western blotting and immunofluorescence,respectively.Scratch-repair experiment was used to detect the cancer cells’ migration ability.Results The recombinant eukaryotic expression plasmid of human CAP1 was successfully constructed and transfected into eukaryote cells.The recombinant plasmid was successfully expressed in eukaryote cells.CAP1 was located in the cytoplasm.The results of scratch-repair experiment showed that the overexpression of CAP1 could significantly inhibit the cells’ migration.Conclusion CAP1 recombinant plasmid was successfully expressed in eukaryotic cells.CAP1 protein was located in the cytoplasm.The overexpression of CAP1 inhibited cell migration. The present study provides important experimental evidence for further study on CAP1.

ObjectiveToexplorethedynamicchangesofoxidativestressandcytokinesinMongoliangerbilswith nonalcoholic fatty liver disease ( NAFLD) and their significance.Methods Forty-eight healthy male gerbils were randomly divided into normal group and model group , 24 in each group .Gerbils of the model group were fed with high fat diet while those of the normal group with normal diet .Eight gerbils in each group were killed at the end of 4 w, 8 w and 16 w, respectively .MDA content and SOD , GSH-PX and T-AOC activity in the liver tissue were detected by chemical method, and serum TNF-α, INF-γand IL-10 levels were determined using liquid suspension chip .Results With the development of NAFLD , MDA content in liver increased gradually , and the MDA contents were all significantly higher than those of the normal group ( P<0.01 ); T-AOC level slightly increased , and then decreased , the levels at 4 w and 16 w were markedly decreased compared with those of the normal group (P<0.05);SOD level was significantly increased and then markedly reduced, the level of the model group at 4 w was significantly increased (P<0.05), while that at 8 w and 16 w were significantly decreased (P<0.05, P<0.01).The level of GSH-PX was decreased gradually , the levels at 8 w and 16 w were significantly lower than those of the normal group (P<0.05).With the progression of NAFLD,serum TNF-αand IFN-γwere increased gradually , while the level of IL-10 decreased gradually , and the levels at 8 w and 16 w were significantly lower than those of the normal group ( P <0.05, P <0.01).Conclusions The oxidative stress-related indicators and inflammatory cytokines in the gerbil NAFLD models induced by high fat diet are significantly changed as simple fatty liver develops into steatohepatitis , liver fibrosis and cirrhosis , and participate in the development and progression of NAFLD .

Objective To explore the effect of high fat diet on serum biochemical parameters and histopathology of main organs in Mongolian gerbils.Methods Forty-eight healthy adult male Mongolian gerbils were randomly and equally divided into model and normal groups.The gerbils in the model group were fed with high fat diet while the normal group with standard diet.Eight gerbils in each group were killed at the end of 4th,8th and 16th week,respectively,and the body weight, serum levels of Glu, TG, CHOL, HDL-C, LDL-C, UA, CREA, BUN, TBil, TP, ALB, ALT, AST and AMS were determined.The histopathological changes of main organs were observed.Results Compared with the normal group,the blood lipid of the model gerbils was significantly increased, the liver function was impaired, the blood uric acid level was higher, and the blood glucose was decreased at the end of 16th week.The AMS was increased at the end of 16th week,but the renal function showed no significant changes.The liver tissue of the model group gradually showed steatosis, inflammation, fibrosis and cirrhosis, accompanied by splenomegalia. The lung tissue and myocardium showed fatty degeneration and obvious damages in the later period,the pancreatic islets were enlarged and the amount of endocrine cells was increased,and the small intestine and kidney didn’ t show any distinct changes.Conclusions A gerbil models of hyperlipidemia and nonalcoholic steatohepatitis and cirrhosis can be well established by high fat diet feeding,and may serve as good models for research of hyperlipidemia-related hyperuricemia, and lung and myocardial damages.

Objective To observe the expression of insulin-like growth factor 1 (IGF-1),insulin-like growth factor binding protein 3 ( IGFBP-3) in rats’ serum with non-alcoholic fatty liver disease ( NAFLD) and the impact of Polyene phosphatidyl choline .Methods NAFLD model was induced by feeding the SD rats with a high-fat diet, with Polyene phosphatidyl choline to intervene .Observe the pathological changes of the rats ’ liver tissue dynamically after HE staining . Detect the contents of IGF-1,IGFBP-3 in serum dynamically through the immuno radio metric assay ( IRMA).Results There were no obvious exception of the liver tissue pathology in the normal group at each time .With the extension of a high-fat diet,the liver tissue of the model group increased on fatty change ,the degree of inflammation ,the balloon sample change , the NAFLD activity score at 4,8,12weeks.IGF-1,IGFBP-3 in serum were decreased significantly ,and the model set was significantly lower than the normal group at the same phase .After the application of polyene phosphatidyl choline ,the degree of rat liver tissue inflammation and the NAFLD activity score were reduced significantly when compared with model group , while the level of IGF-1,IGFBP-3were significantly higher .Conclusion The levels of IGF-1,IGFBP-3 in the rats’ serum reduce in the development of non-alcoholic fatty liver disease .

OBJECTIVETo study the in vitro effect of herb components on scavenging harmful components of cigarette smoke such as radicals, polycyclic aromatic hydrocarbons, nitrosamines in vitro, and its reducing effect on cytotoxicity of cigarette smoke.

METHODspectrophotometry was used to examine the scavenging effect of herb components on DPPH free radicals, superoxide anion radical, and hydroxyl radical, and the results were compared with the anti-oxidation of ascorbic acid. Fluorescence spectroscopy was used to examine the scavenging effect of herb components on polycyclic aromatic hydrocarbons. UV spectrophotometry was used to examine the scavenging effect of herb components on volatile nitrosamines. MTT assay was used to examine cytotoxicity of cigarette smoke.

RESULTAll the herb components showed a certain scavenging effect on DPPH free radicals, superoxide anion radical, hydroxyl radical, polycyclic aromatic hydrocarbons and volatile nitrosamines, espacially the ginkgo biloba extract (GBE), flavonoids of glycyrrhiza, procyanidine, total saponins in ophiopogonin, total saponins of astragalus and epimediun flavonoids. After these six herb components were added to cigarette, the cytotoxicity of cigarette smoke on BEP2D cells was remarkably reduced, by increasing cell survival fraction (SF, %) and mean lethal dose (DML).

CONCLUSIONThe herb components can scavenge harmful components of cigarette smoke such as radicals, polycyclic aromatic hydrocarbons and nitrosamines, which reduce the damage of cigarette smoke on human being.

Cell Survival ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Free Radical Scavengers ; pharmacology ; Humans ; Smoke ; adverse effects ; Tobacco ; adverse effects

OBJECTIVETo study the expression of uncoupling protein 2 (UCP2) in liver of rats with nonalcoholic steatohepatitis (NASH) induced by fat-rich diet, and the effect of total flavones of hawthorn leafon (TFHL) on UCP2.

METHODThe NASH model of rat was induced by 12 weeks of fat-rich diet. Subsequently the rats were administrated with TFHL in accordance with 250, 125 mg x kg(-1) x d(-1) and the Essentiale N with 195.4 mg x kg(-1) x d(-1). The change of liver pathological. The levels of serum ALT and AST, the content of TG, CHOL, MDA and T-AOC activity of liver and were evaluated. The UCP2mRNA expression in liver was detected with RT-PCR, and the contents of UCP2 were examined with ELISA.

RESULTThere are severe steatosis, inflammatory cellular infiltration in the liver of the NASH models. The levels of serum ALT, AST and the contents of TG, CHOL, MDA and UCP2 in the model group were higher than those of in the normal groop. The expression of UCP2mRNA was obviously enhanced and the activity of T-AOC decreased. The expression of UCP2 mRNA of rats was positively correlation with the contents of MDA, TNF-alpha. The inflammation activity in rat liver, the contents of MDA and UCP2, the expression of UCP2 mRNA in the administrated groups were obviously lower than those in the model group, while the activity of T-AOC was higher than that of model.

CONCLUSIONTFHL may alleviate liver injury by means of the suppression of Oxidative stress/lipid peroxidation reaction and the overexpression of UCP2 in liver, which could prevent the further development of NASH.

Animals ; Crataegus ; chemistry ; Enzyme-Linked Immunosorbent Assay ; Fatty Liver ; drug therapy ; metabolism ; Flavones ; chemistry ; therapeutic use ; Gene Expression ; drug effects ; Ion Channels ; genetics ; Liver ; drug effects ; metabolism ; Male ; Mitochondrial Proteins ; genetics ; Plant Leaves ; chemistry ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley ; Uncoupling Protein 2