BACKGROUND:Intervertebral disc degeneration is one of the most common underlying factors causing low back pain.Recent studies have shown that melatonin has a positive effect on alleviating intervertebral disc degeneration.However,the underlying mechanism of melatonin remains to be elucidated. OBJECTIVE:To explore the biological effect and potential mechanism of melatonin in inhibiting hydrogen peroxide(H2O2)-induced injury of human nucleus pulposus cells. METHODS:Human nucleus pulposus cells insolated from degenerative intervertebral disc were cultured in vitro.Cell proliferation and the optimal intervention concentration of melatonin and H2O2 were detected by cell counting kit-8.The Human nucleus pulposus cells treated with H2O2 were used as a model group;the cells treated with H2O2 and intervened with melatonin were used as a melatonin group;the cells cultured in simple medium were used as a control group.The reactive oxygen species levels were detected by 2',7'-dichlorofluorescin diacetate(DCFH-DA),the expression levels of BAX and Caspase3 were detected by immunofluorescence,and the mRNA expression levels of BAX,BCL-2,Casepase3,PI3K and AKT were detected using the real-time fluorescent quantitative reverse transcription PCR. RESULTS AND CONCLUSION:The results of cell counting kit-8 experiment showed that the optimal intervention concentration of H2O2 was 400 μmol/L and the optimal intervention concentration of melatonin was 5 μmol/L.The reactive oxygen species level in the melatonin group was significantly lower than that in the model group.The average fluorescence intensity of BAX and Caspase3 in the melatonin group was significantly lower than that in the model group.The mRNA expressions of BAX and Caspase3 in the melatonin group were lower than those in the model group,while the mRNA expression of Bcl-2 was increased.In addition,the mRNA expressions of PI3K and AKT were also higher in the melatonin group compared with the model group.To conclude,melatonin may protect human nucleus pulposus cells from H2O2-induced oxidative damage through the PI3K/AKT signaling pathway.

Objective:To explore the effect and underlying mechanism of casein kinase 2 interacting protein-1 (CKIP-1) on hepatocyte apoptosis in nonalcoholic fatty liver disease (NAFLD).Methods:Experimental study. An NAFLD cell model was established by inducing human hepatoma cell line, HepG 2 cells, with oleic acid (OA). Flag-CKIP-1 expression vector and shRNA-CKIP-1 were transfected into HepG 2 cells. Flow cytometry was used to detect the effect of CKIP-1 on the activity and apoptosis of NAFLD hepatocytes. The levels of apoptosis-related proteins were detected by Western blot. CKIP-1 knockout mice in C57BL/6 back-ground were fed with either standard or high-fat diet for 8 weeks. Apoptosis-related signal proteins in NAFLD hepatocytes were detected by immunohistochemistry. Results:After CKIP-1 was transfected into HepG 2 cells, the degree of OA induced cell liposis was significantly reduced ( P<0.05). Annexin V-FITC/PI flow cytometry showed that CKIP-1 reduced the apoptosis of steatotic hepatocytes. Overexpression of CKIP-1 could significantly inhibit the expression of caspase-3 and caspase-9 and increase the expression of Bcl-2/Bax ( P<0.05). Knockdown of CKIP-1 could increase the expression of caspase-3 and caspase-9 ( P<0.05). CKIP-1 knockout could further increase the expression of caspase-3 and caspase-9 in NAFLD mice ( P<0.01, P<0.05), and further decrease the expression of Bcl-2/Bax ( P<0.05). Conclusion:CKIP-1 inhibited the apoptosis of steatotic hepatocytes by up-regulating the expression of apoptosis inhibitor gene, Bcl-2/Bax, and affecting the proteases, caspase-3 and caspase-9.

Objective To investigate the effect of exogenous vascular endothelial growth factor (VEGF) on bone activity of rabbit heterotopic allograft decalcified bone.Methods 140 adult healthy China white rabbits were selected,no limitation with sex,20 rabbits as the donor preparation of allogenic decalcified bone,according to the random number table,the rest was divided into the experimental group (allograft decalcified bone ± VEGF) and the control group (Allograft decalcified bone),each group contained 60 rabbits.For the experimental group,the prepared 1.5 cm long homologous decalcified tibia was placed in rabbit right thigh of rectus femoris and vastus medialis muscle gap near by saphenous artery,and fixed on the femur with two 0.8 mm Kirschner wire.In the vicinity of the skin,implanted an osmotic pump which contain the VEGF solution 200 μl with concentration was 0.5 μg/ml.In the control group,implanted the isometric allograft decalcified bone in rabbit right thigh corresponding parts with the same method.Each group respectively at 0,2,4,6,8,10 weeks to death 10 white rabbits,By specimen observation,HE dyeing observation and detection of type Ⅰ glue protein fluorescence intensity,Analysis the bone activation degree of two groups of bone allograft decalcified.Results Experimental allograft decalcified bone gradually wrapped by connective tissue membrane,its surface appear different size of the pits and gradually increased and become deep,while the control group pits relatively little and shallow.In the experimental group and control group,the fluorescence intensity of type Ⅰ collagen reached its peak respectively at 8 weeks (47.57 ±3.50) and 10 weeks (45.07±6.02),with no statistically significant (P ＞ 0.05).Conclusion Rabbit allograft decalcified bone implanted in the muscle clearance with abundant blood supply can be transformed into activated bone after 10 weeks,and after applying exogenous VEGF,allograft decalcified bone can be transformed into activated bone after 8 weeks,the bone activation process obviously speed up.The reaults confirmed the exogenous VEGF can obviously promote the ectopic rabbit bone allograft decalcified bone activation process.

With the economic growth and better standards of living,the prevalence of non-alcoholic fatty liver disease (NAFLD) is expected to increase dramatically worldwide.NAFLD is a common chronic inflammation disease.NF-κB is a transcription factor that plays crucial roles in inflammation,immunity,cell proliferation and apoptosis.It can facilitate the occurrence and development of NAFLD,and the underlying mechanisms are related to insulin resistance,oxidative stress,alteration of intestinal flora,activation ofrenin angiotensin system,etc.

Objective To investigate the effect of erythropoietin(EPO)on the expression of aquaporin - 2 (AQP2)in the kidney of young SD rats after release of bilateral ureter obstruction(BUO - R). Methods Thirty - two young SD rats were equally divided into 4 groups randomly(BUO group,BUO - R group,BUO - R ﹢ EPO group and Sham group,8 rats in each group). The BUO model was built through bilateral ureteral ligation. EPO(500 U/ kg)was given to BUO - R ﹢ EPO rats at 2 h after release of BUO,and then repeated 6 h,12 h,24 h and 36 h thereafter and the same volume of 9 g/ L saline was simultaneously given to BUO - R rats. The Sham group was prepared in parallel by laparotomy and free dissection of bilateral ureters but not ligated. Both side kidneys were harvested 48 h(72 h for Sham group)after release of BUO to examine the effect of EPO on the expression of AQP2 in inner medulla by immunohisto-chemistry,Real - time PCR and Western blot. The urine samples were collected by using metabolic cage before death. Results The osmotic pressure of BUO - R ﹢ EPO group was higher than that of BUO - R group,but lower than that of Sham group(P ﹤ 0. 05). Immunohistochemistry showed that the collecting duct wall thinned and lumen enlarged. After the pictures were analysized by using Image - Pro Plus software,it showed that the expression of AQP2 in collecting duct in BUO group was significantly down - regulated compared with that in Sham group,whereas,it was slightly weaker in BUO - R group and BUO - R ﹢ EPO group than Sham group(P ﹤ 0. 05). These results were further confirmed by a-dopting Western blot,and the relative quantity of AQP2 in BUO group was also the lowest of the four groups(P ﹤0. 05). Real - time PCR showed that the level of AQP2 mRNA in Sham group was(24. 30 ± 1. 03)folds of BUO group,(10. 60 ± 1. 05)folds of BUO - R group and(5. 70 ± 1. 01)folds of BUO - R ﹢ EPO group,respectively. Conclusion EPO could promote not only the recovery of AQP2 mRNA and protein expression but also the recovery of AQP2 function in young BUO - R rats.

OBJECTIVE: To screen differentially expressed genes and gene pathways in L02 cell line stably expressing hepatitis C virus (HCV) Ib type nonstructural protein 4B (NS4B) mediated by lentiviral system, and to provide a basis for further research of molecular biological mechanism of NS4B gene in chronic hepatitis C and hepatocarcinogenesis. METHODS: NS4B stably overexpressed L02 cell line and negative control stable L02 cell line, designated as L02-NS4B and L02-mkate2 respectively, were resurrected and amplified in vitro. The differentially expressed genes between L02-NS4B and L02-mkate2 were determined by gene expression microarray from Human Gene 1.0ST. The significant pathways of the differential genes were selected by the Fisher's exact test and χ2 test according to kyoto encyclopedia of genes and genomes (KEGG) database. The differential expression levels of 5 selected genes including protein kinase C delta binding protein (PRKCDBP), tumor protein p53 (TP53), v-akt murine thymoma viral oncogene homolog 1 (AKT1), baculoviral IAP repeat containing 3 (BIRC3) and B-cell lymphoma 2-like1 (BCL2L1) from cDNA microarray data were further verified by real-time quantitative polymerase chain reaction (real-time QPCR). RESULTS: Between L02-NS4B and L02-mkate2, the genes with flurescence intensity ratio >1.2 or <0.8 were considered as differentially expressed genes. A total of 2 682 differentially expressed genes in the known 28 869 human genes were detected in L02-NS4B, 1 446 genes were upregulated and 1 236 genes were downregulated. A total of 41 involved pathways of up-regulated differential genes were identified by KEGG database, mainly including apoptosis, extracellular matrix receptor interaction, cell cycle, pathways in cancer and Toll-like receptor signaling pathway; and 20 involved pathways of down-regulated differential genes were identified, mainly including pathways in cancer, Wnt signaling pathway and cell cycle pathway. Of the 5 upregulated genes selected from cDNA microarray data, 3 genes showed the same differential expression pattern by real-time QPCR as that shown in cDNA microarray data, namely AKT1, BIRC3 and BCL2L1. The confirmation rate of real-time QPCR was 60%. CONCLUSION HCVNS4B can up-regulate or down-regulate the expression of many genes in L02 cells, thus affecting multiple signaling pathways relevant to cell apoptosis, cell cycle and carcinogenesis.
Cell Cycle ; Cell Line ; Gene Expression Profiling ; Hepacivirus ; genetics ; Humans ; Oligonucleotide Array Sequence Analysis ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transcriptional Activation ; Viral Nonstructural Proteins ; genetics

Objective To investigate the effect of hepatitis C virus (HCV) core protein and nonstructural protein 4B(NS4B) on the proliferation of HepG2 cells and its possible mechanism.Methods The two recombinant plasmid pcDNA3.1 (-)Core and pcDNA3.1 (-) NS4B were transiently transfected respectively and co-transfected into HepG2 cells by lipofectamine, simultaneously HepG2 cells transfected with pcDNA3.1 (-) and untransfected HepG2 cells were used as control.The expression of mRNA and protein of HCV Core,NS4B,Wnt1,β-catenin,c-myc and CyclinDl in the cells of each group were detected by RT-PCR and Western blot respectively.Cell proliferation changes were measured by MTT assay and plate colony formation assay.The cell cycle distribution was tested by flow cytometry.Results ①HCV Core or/and NS4B mRNA and protein were expressed successfully in the HepG2 cells transfected with pcDNA3.1 (-)Core,pcDNA3.1 (-)NS4B alone or in combination.② The relative expression levels of mRNA and protein of Wnt1,β-catenin,c-myc and CyclinD1 were higher in the cells transfected with pcDNA3.1 (-) Core,pcDNA3.1 (-) NS4B alone or in combination than those in the cells transfected with pcDNA3.1 (-) and untransfected HepG2 cells(P ＜0.01).③Compared with the HepG2 cells transfected with pcDNA3.1 (-)and untransfected,cell viability,cloning efficiency and the percentage of cells at S and G2/M phases were significantly increased in the cells transfected with pcDNA3.1 (-) Core,pcDNA3.1 (-) NS4B alone or in combination (P ＜ 0.01).Conclusion HCV core protein and NS4B can accelerate cell cycle progression and promote cell proliferation of HepG2 cells probably through enhancement of Wntl,β-catenin,c-myc and CyclinD1 expression.

Objective To investigate clinical features and antifungal therapeutic effect of chronic severe hepatitis (CSH) patients with invasive fungal infection (IFI), and to improve the diagnosis and treatment.Methods Clinical manifestation, blood routine, imageology and mycetology characteristic, antifungal treatment perscription and therapeutic effect of 79 CSH patients with IFI were retrospectively analyzed. Antifungal therapeutic effect was compared between fluconazole and voriconazole. Results Thirteen (16.5%) patients received glucocorticoid or other immunodepressants for a relatively long time, 40 (50.6%) patients had invasive operation, and 61 (77.2 %) patients were administered 1-6 kinds of broad-spectrum antibiotics. Seventy-three patients had fever. Leucocytes and neutrophilic granulocyte increased in 96.2% of the patients. Lung (31.6%), intestinal tract (26.2%) and oral cavity (14%) infections were common. Fungus was found in 70.9% of the patients. Candida albicans (40.9%) and aspergillus (21.1%) were often seen. Halo signs and crescent signs on lung CT were relatively specific in 40% of the patients with fungal pneumonia. Voriconazole was more effective than fluconazole(71.4% vs. 39.0%, P<0.05). Twelve patients with lung aspergillus infection were administered voriconazole, 8 (66.7%) patients of whom was effective, and the other 4 (33.3%) patients died. Conclusion There are high risk factors in major CSH patients with IFI. The most common clinical manifestations of CSH patients with IFI are fever, leukocytosis, lung and intestinal tract infection. Candida albicans and aspergillus infection are common. Voriconazole is more effective than fluconazole, and can increase the survival rate of CSH patients with IFI.

arkably increases with the development of cirrhosis,which may play an important role in the development of PHG.AG may remarkably ameliorate the degree of PHG，mainly by inhibiting the expression of iNOS in gastric musosa.

Objective To evaluate the in vitro inhibiting activities of interferon-alpha (INF-?) against hepatitis B virus (HBV). Methods HepG2 2 2 15 cells were treated with INF-?. At 3rd, 6th and 9th days after treatment, the supernatant was collected for HBsAg quantitative assay, and total RNA of the cells was isolated for RT-PCR and realtime-PCR assay of HBV mRNA. Results There were no significant differences in HBsAg titer and virus mRNA level at 3rd and 6th days between the experimental and control groups. At 9th day, HBsAg titer and virus mRNA level in INF-? group were significantly lower than those in control group. And 500IU/ml INF-? could get maximal inhibiting effect against HBV. Conclusion INF-? can inhibit transcription and expression of HBV gene in HepG2 2 2 15 cells, which indicated that INF-? can inhibit HBV replication directly.