Objective To evaluate the effects of hydrogen-rich saline on the early diabetic neuropathic pain (DPN) in rats.Methods Healthy male Sprague-Dawley rats,aged 8 weeks,weighing 180-220 g,were used in the study.DPN model was made by intraperitoneal injection of 1％ streptozocin (STZ) 65 mg/kg.Twelve diabetic rats were randomly divided into 2 groups (n =6 each) using a random number table:DPN group and hydrogen-rich saline group (HRS group).Another 6 normal rats were randomly collected as control group (group C).At 14 days after STZ injection,hydrogen-rich saline 5 ml/kg was injected intraperitoneally once a day for 14 consecutive days in HRS group,while C and DNP groups received the equal volume of normal saline.Mechanical paw withdrawal threshold to yon Frey stimuli (MWT) and paw withdrawal latency to nociceptive thermal stimulation (TWL) were measured at 2 days before STZ injection (To) and 7,14,21 and 28 days after STZ injection (T1-4).The motor nerve conduction velocity (MNCV) of the right hindlimb and distal motor latency was measured after pain threshold was measured at T4.After measurement of neurological function was completed,tumor necrosis factor-α (TNF-α) and i nterleukin-6 (IL-6) contents (by ELISA) and nuclear factor kappa B (NF-κB) activity (by immuno-histochemistry) were detected.Results Compared with group C,MNCV was significantly decreased,the motor latency was prolonged,MWT was decreased at T1-T4,TWL was shortened at T2-T4,TNF-a and IL-6 contents were increased,and NF-κB activity was enhanced in DNP and HRS groups (P ＜ 0.05).Compared with group DNP,MNCV was significandy increased,the motor latency was shortened,MWT was increased at T3.4,TWL was prolonged at T4,TNF-a and IL-6 contents were decreased,and NF-κB activity was weakened in group HRS (P ＜ 0.05).Conclusion Hydrogen-rich saline can relieve the early DPN through inhibiting NF-κB signaling pathway in rats.

With the progess in studying gene structure and function of foot and mouth disease virus (FMDV), FMDV can express exogenous genes in different sites. Through transforming and modifying FMDV can achieve different application purposes such as improving virus titer, introducing tag, improving immune responses, and reducing pathogenicity. From the perspective of FMDV receiving inserted exogenous gene, this paper mainly describes the latest relevant developments of FMDV's expression to exogenous gene.
Foot-and-Mouth Disease Virus ; genetics ; Genetic Engineering ; Mutagenesis, Insertional

Objective To investigate the role of Rho kinase (ROCK) in the protective effects of hydrogen on intestinal epithelial barrier function in sepsis. Methods Caco-2 cells were cultured routinely, and divided into 6 groups randomly (n=3):control group (C group), hydrogen-rich medium group (H group), lipopolysaccharide (LPS)-treatment group (L group), hy?drogen+LPS-treatment group (HL group), Rho kinase inhibitor (Y-37632) treatment group (Y group) and Rho kinase inhibi?tor Y-27632+LPS-treatment group (YL group). H group was treated with 0.6 mmol/L hydrogen-rich media. The concentra?tion of LPS and Y-27632 were 50 mg/L and 25μmol/L separately. After the Caco-2 monolayer model was established, the transepithelial electrical resistance (TEER) values were measured regularly. When the TEER value reached 800Ω·cm2, the treatment was administered. Then TEER values were measured at 6 h, 12 h and 24 h, and FITC-dextran permeability was de?tected at 24 h. Cells were seeded on 6-well plates. After cell density reached 80%-90%, treatments were given randomly. The real time-polymerase chain reaction (RT-PCR) was conducted to assess mRNA levels of ZO-1 and ROCK mRNA. ZO-1 and ROCK protein expression levels were detected by Western blot assay. Results Compared with C group, TEER values were elevated in 12 h and 24 h in H group (P<0.05). There were no statistical significances in FITC-dextran permeability,
protein expression levels of ZO-1 and ROCK between C group and H group (P>0.05). TEER values were elevated at 6 h, 12 h and 24 h in Y group (P<0.05). There was no significant difference in FITC-dextran permeability between C group and Y group (P > 0.05). The mRNA expression of ZO-1 increased and mRNA expression of ROCK decreased in Y group (P <0.05). The TEER values reduced at 6 h, 12 h and 24 h in L group. The FITC-dextran permeability increased significantly, mRNA and protein expressions of ZO-1 significantly decreased, mRNA and protein expressions of ROCK significantly in?creased in L group (all P<0.05). Compared with L group, TEER values increased significantly at 6 h, 12 h and 24 h in YL group, FITC-dextran permeability decreased, mRNA expressions of ZO-1 increased, mRNA expressions of ROCK de?creased in YL group (P<0.05). Compared with L group, TEER values increased at 6 h, 12 h and 24 h in HL group, FITC-dextran permeability reduced markedly, protein expressions of ZO-1 increased at each time point, protein expressions of ROCK decreased at each time point in HL group (P<0.05). Conclusion Hydrogen can protect intestinal barrier function against sepsis, ameliorate the integrity and permeability of intestinal epithelium and increase the expressions of intercellular tight junction proteins. The suppression of Rho kinase over-expression induced by LPS may be involved in these protective effects of hydrogen.

Objective To investigate the role of JNK in intestinal barrier dysfunction in severe septic mice treated by hydrogen. Methods Eighty male ICR mice were randomly divided into four groups (n=20 each):sham operation group, hydrogen control group, sepsis group and hydrogen treatment group. Severe sepsis rat model was reproduced by cecal ligation and puncture (CLP). Laparotomy without CLP was performed in sham operation group and hydrogen control group. The mice in hydrogen control group and hydrogen treatment group received 1-hour inhalation of 2%hydrogen at 1 hour and 6 hours after sham operation or CLP, respectively. Ten mice of each group were selected at 20 h after CLP operation and were gavaged with fluorescein-isothiocyanate-conjugated dextran (FITC-dextran). Blood samples were obtained by cardiac puncture to measure the serum concentration of FITC-dextran 4 h after treatment with FITC-dextran . Ten mice in each group were sacrificed at 24 h after CLP operation. The colony-forming unit (CFU) numbers in the peritoneal lavage fluid were counted. The middle intestinal tissues were obtained for the measurement of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1βand high mobility group box 1(HMGB1) by ELISA. The level of phosphorylated JNK (p-JNK) and the expression of tight junction protein ZO-1 and Occludin were detected by Western blot assay. The intestinal pathological changes and epithelial ultrastructure changes were observed by light microscope and transmission electron microscope (TEM). Results There was no statistical significance in clinical variables between sham operation group and hydrogen control group. Compared with sham operation group, the serum FITC-dextran concentration, the CFU numbers in the peritoneal lavage fluid, the levels of TNF-α, IL-1βand HMGB1 in intestine, and the expression of p-JNK were significantly increased, the expression of ZO-1 and Occludin were down-regulated in sepsis group(P < 0.05). There was a significant intestinal pathological injury along with epithelial ultrastrcture injury in sepsis group. Compared with sepsis group, the serum FITC-dextran concentration, the CFU numbers in the peritoneal lavage fluid, the levels of intestinal TNF-α, IL-1β and HMGB1, and the expression of p-JNK were significantly decreased, the expression of ZO-1 and Occludin were up-regulated in hydrogen treatment group(P < 0.05), and the pathological and ultrastructure damage was significantly reduced. Conclusion Hydrogen can decrease levels of proinflammatory factors and up-regulate the expression of tight junction to improve intestinal barrier dysfunction caused by severe sepsis, which is related with the inhibition of JNK signaling pathway.

Objective To investigate the effect of hydrogen on endotoxin-induced expression of zonula occludens-1 (ZO-1) in human colon epithelial cells (Caco-2 cells).Methods Caco-2 cells were cultured routinely,seeded in Transwell chambers or wells,and randomly divided into 4 groups (n =45 each) using a random number table:control group (group C);hydrogen-rich culture medium group (group H);endotoxin group (group E);hydrogen-rich culture medium + endotoxin group (group HE).The cells were cultured in high-glucose DMEM culture medium in group C.The cells were incubated in hydrogen-rich culture medium containing hydrogen 0.6 mmol/L in group H.The cells were incubated in highglucose DMEM culture medium containing 50 μg/ml lipopolysaccharide in group E.The cells were incubated in hydrogen-rich culture medium containing 50 μg/ml lipopolysaccharide and 0.6 mmol/L hydrogen in group HE.Transepithelial electrical resistance (TEER) was measured before incubation or culture,and at 6,12 and 24 h of incubation or culture.The viability of Caco-2 cells was measured by methyl thiazolyl tetrazolium assay at 24 h of incubation or culture.The expression of ZO-1 mRNA in Caco-2 cells was determined using real-time reverse transcriptase polymerase chain reaction at 6,12 and 24 h of incubation or culture.The distribution of ZO-1 in Caco-2 cells was observed by immunofluorescence at 24 of incubation or culture.Results Compared with group C,TEER was significantly decreased at 6,12 and 24 h of incubation or culture,and the expression of ZO-1 mRNA was significantly down-regulated in E and HE groups (P＜0.05),and no significant change was found in the parameters mentioned above in group H (P＞0.05).Compared with group E,TEER was significantly increased at 6,12 and 24 h of incubation or culture,and the expression of ZO-1 mRNA was significantly up-regulated in group HE (P＜0.05).The distribution of ZO-1 protein in cell membrane became discontinuous,and the distribution of ZO-1 protein in cytoplasm was significantly increased in group E.Compared with group E,the distribution of ZO-1 protein in cell membrane was significantly increased and gradually became continuous,and the distribution of ZO-1 protein in cytoplasm was significantly decreased in group HE.Conclusion The mechanism by which hydrogen reduces the damage to human colon epithelial cell barrier is related to up-regulation of ZO-1 expression and improvement in the redistribution of ZO-1 protein.

Objective To evaluate the effects of hydrogen on oxidative stress injury induced by high glucose in Schwann cells and its relationship with PARP-1-dependent cell death (parthanatos).Methods Primary rat Schwann cells were cultured in 96-well plate (1×104 cells/ml,200 μl/well) or in 6-well plate (1 × 106 cells/ml,2 ml/well) with RSM culture medium and were randomly divided into 5 groups (n=30 each):control group (group C),hydrogen group (group H2),high glucose group (group HG),high glucose plus hydrogen group (group HG+H2) and high osmotic control group (group M).The cells were cultured in the common culture medium in C,HG and M groups.The cells were cultured in hydrogen-rich culture medium in H2 and HG + H2 groups.In HG and HG + H2 groups,50 mmol/L of glucose was added to the culture medium.In C and H2 groups,the equal volume of normal saline was added to the culture medium.In M group,mannitol 44.4 mmol/L was added to the culture medium.The cells were then incubated for 48 h.After 48 h of incubation,the cell viability was measured using CCK-8 assay,intracellular reactive oxygen species (ROS) level was detected by flow cytometry,the concentration of 8-hydroxy-2-deoxy Guanosine (8-OHdG) was determined by ELISA,and the expression of poly (ADP-ribose)-polymerase-1 (PARP-1),cleaved-PARP-1,poly (ADP-ribose) (PAR),and apoptosis-inducing factor (AIF) in the total protein and nucleus was measured by Western blot.PARP-1 activity (cleaved-PARP-1/PARP-1) and AIF nuclear translocation were recorded.Results Compared with C and H2 groups,the cell viability was significantly decreased,and PARP-1 activity,expression of ROS,8-OhdG and PAR,and AIF nuclear translocation were increased in HG and HG + H2 groups.Compared with HG group,the cell viability was significantly increased,and PARP-1 activity,expression of ROS,8-OhdG and PAR,and AIF nuclear translocation were decreased in HG+H2 group.There was no significant difference in each parameter between M and C groups.Conclusion Hydrogen can reduce oxidative stress injury induced by high glucose in Schwann cells,and the mechanism is related to inhibition of parthanatos.

Objective To investigate the protective effects and underlying molecular mechanisms of hydrogen (H2) on high glucose-induced poly (ADP-ribose) polymerase-1 (PARP-1) dependent cell death (PARthanatos) in primary rat Schwann cells. Methods Cultured primary rat Schwann cells were randomly divided into five groups: blank control group (C group), H2 control group (H2 group), high osmotic control group (M group), high glucose treatment group (HG group), and H2 treatment group (HG+H2 group). The cells in H2 group and HG+H2 group were cultured with saturated hydrogen-rich medium containing 0.6 mmol/L of H2, and those in three control groups were cultured with low sugar DMEM medium containing 5.6 mmol/L of sugar, and the cells in HG and HG+H2 groups were given 44.4 mmol/L of glucose in addition (the medium containing 50 mmol/L of glucose), the cells in C group and H2 group were given the same volume of normal saline, and the cells in M group were given the same volume of mannitol. Cytotoxicity was evaluated using lactate dehydrogenase (LDH) release rate assays after treatment for 48 hours in each group. The contents of peroxynitrite (ONOO-) and 8-hydroxy-2-deoxyguanosine (8-OHdG) reflecting oxidative stress injury and DNA damage were detected by enzyme linked immunosorbent assay (ELISA). Poly (ADP-ribose) (PAR) protein expression was analyzed by Western Blot, and immunofluorescence staining was used to determine the nuclear translocation of the apoptosis-inducing factor (AIF). Results The cytotoxicity in HG and HG+H2 groups was significantly increased as compared with that of C group [LDH release rate: (61.40±2.89)%, (42.80±2.32)% vs. (9.92±0.38)%, both P < 0.01], the levels of ONOO- and 8-OHdG were markedly elevated [ONOO- (ng/L): 853.58±51.00, 553.11±38.66 vs. 113.56±14.22; 8-OHdG (ng/L): 1 177.37±60.97, 732.06±54.29 vs. 419.67±28.77, all P < 0.01], and the PAR protein expression was up-regulated (A value: 0.603±0.028, 0.441±0.010 vs. 0.324±0.021, both P < 0.01). The cytotoxicity, the levels of ONOO- and 8-OHdG, and PAR expression in HG+H2 group were significantly lower than those of the HG group (all P < 0.01). There were no significant differences in above parameters between H2 group as well as M group and C group. It was shown by immunofluorescence that AIF was expressed in the cytoplasm in C group, H2 group and M group, AIF was expressed in the whole cell in HG group, and the expression in the nucleus was particularly increased. A small amount of AIF expression was found in the nucleus of HG+H2 group, which indicated that high glucose could promote the AIF nuclear translocation, and that hydrogen-rich medium could prevent the process of translocation. Conclusions High glucose levels could enhance DNA damage that enhance PARthanatos in primary rat Schwann cells. However, H2 can not only reduce DNA damage of injured cells, but also inhibit the special death process, reduce the cell toxicity, all of which have protective effects.

Objective To investigate the effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction of human intestinal epithelial (Caco2) cells. Methods Caco2 cells (passages 28-35) were purchased from the Cell Bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences in Shanghai, China, and they were cultured in Dulbecco minimum essential medium (DMEM) containing 20% fetal bovine serum. These cells were randomly divided into four groups: control group (group A), hydrogen-rich medium group (group B), LPS group (group C) and LPS + hydrogen-rich medium group (group D). Cells were cultured with normal medium in group A and group C or with hydrogen-rich medium in group B and group D. Meanwhile, 1 g/L LPS was simultaneously added into group C and group D, while an equivalent volume of normal saline was added into group A and group B instead. In vitro intestinal epithelial models were reproduced with monolayer filter-grown Caco2 and intestinal epithelium. The trans-epithelial electrical resistance (TEER) in models of each group was measured at different incubation times (0, 3, 6, 12, 24 and 48 hours). Cell viability and cytotoxicity were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release assay, respectively, after incubation for 24 hours. The expression levels of claudin-1 and occludin were respectively determined at 6, 12 and 24 hours of incubation by Western Blot assay. The morphological structure of claudin-1 and occludin was respectively observed after incubation for 24 hours with immunofluorescence staining. Results There was no statistical significance in variables between group A and group B. Compared with group A, it was shown that TEER was time-dependently decreased in groups C and D after 6 hours. Compared with group C, TEER in group D was increased after 6 hours. Compared with group A, the cell viability was significantly reduced in group C [(67.2±7.9)% vs. (100.0±0.0)%, P < 0.05] and cell injury was obvious [LDH release rate: (38.5±2.1)% vs. (1.2±0.3)%, P < 0.05]; the expression levels of claudin-1 and occludin at 6, 12, 24 hours were significantly down-regulated [claudin-1 (gray value): 0.351±0.079, 0.272±0.075, 0.190±0.049 vs. 0.518±0.030; occludin (gray value): 0.416±0.044, 0.290±0.062, 0.226±0.019 vs. 0.602±0.038, all P < 0.05], and the structure of claudin-1 and occludin were profoundly disrupted. Compared with group C, it was shown that the cell viability was significantly increased in group D [(88.8±7.4)% vs. (67.2±7.9)%, P < 0.05] and cell injury was significantly abated [LDH release rate: (16.4±4.3)% vs. (38.5±2.1)%, P < 0.05]; the expression levels of claudin-1 and occludin were significantly up-regulated at 24 hours [claudin-1 (gray value): 0.428±0.046 vs. 0.190±0.049, occludin (gray value): 0.466±0.071 vs. 0.226±0.019, both P < 0.05]; the disrupted structures of claudin-1 and occludin were partially recovered. Conclusion Hydrogen-rich medium can effectively attenuate LPS-induced dysfunction of intestinal epithelial barrier in human Caco2 cells by ameliorating cell viability as well as regulating claudin-1 and occludin expression and structure.

Objective To retrospectively investigate the endoprosthetic survival and complications after custom-made tumor prosthesis replacement of knee joint.Methods From April 1996 to April 2007,85 patients with bone tumors around knee joints undergoing custom-prosthetic replacement were respectively analyzed,including 54 males and 31 females with an average of 31.33±15.3 years(range,11-72).The diagnoses were osteosarcoma(43 patients),giant cell tumor(31),chondrosarcoma(4),metastatic tumor(3),malignant fibrohistiocytoma(2),Ewing sarcoma(1),and fibrosarcoma(1).The distal femar was affected in 43 patients and the proximal tibia in 42.All patients in this group underwent wide resection and domestic custommade cemented endoprosthetic reconstruction.Results Mean following was 45.8 months.The overall complication rate was 27.1%(23/85),which was 25.6% in distal femar and 28.6% in proximal tibia.Thirteen complications occurred within 3 years after definite surgery,3 in 3-5 years,and 7 over 5 years.All of 6 operation-relation complications(wound dehiscence,periprosthetic infection,peroneal nerve injury,etc) occurred within 3 years.Seven prosthetic-related complications(prosthetic loosening or breaking,dislocation,periprosthetic fracture,etc)occurred within 3 years,3 occurred in 3-5 years,and 7 over 5 years after definite surgery.The overall endoprosthetic cumulative survival rate was 79.4%,67.7%and 62.3% after 3,5 and 7 years,respectively;which was 86.6%,79.2% and 73.5% in distal femur;66.1%,53.7% and 48.8% in proximal tibia.Conclusion All operation-related complications occurred within 3 years,prosthesis-related complications occurred mainly within 3 years and over 5 years postoperatively.Prosthesis-related complications could occur iu the early stage after using domestic custom-made prosthesis reconstruction.

To generate an epitope-mutated foot-and-mouth disease virus (FMDV) as a marker vaccine, the infectious clone pAsia 1-FMDV containing the complete genomic cDNA of Asia 1 type FMDV was used as backbone, the residues at positions 27 and 31 in the 3D gene were mutated (H27Y and N31R). The resulting plasmid pAsia 1-FMDV-3DM encoding a mutated epitope was transfected into BHK-21 cells and the recombinant virus rAsia 1-3DM was rescued. The recombinant virus showed similar biological characteristics comparable with the parental virus. In serological neutralization test the antisera against recombine virus have a good reactivity with parental virus. The antisera against the mutant virus were shown to be reactive with the mutated epitope but not the wild-type one. The results indicated that the two virus strains could be distinguished by western blotting using synthetic peptides. This epitope-mutated FMDV strain will be evaluated as a potential marker vaccine against FMDV infections.
Animals ; Cell Line ; DNA, Complementary ; Epitopes ; genetics ; Foot-and-Mouth Disease Virus ; genetics ; Immune Sera ; Neutralization Tests ; Transfection