BackgroundWith the number of diabetics increases,people pay more attention to the diabetic keratopathy.The major mechanism leading to diabetic keratopathy is diabetic corneal neuropathy.So it is significant to observe pathologic mechanism of diabetic corneal neuropathy. Objective To investigate the protective effects of edaravone( a free radical scavenger) on corneal nerve of rats with experimental diabetic corneal neuropathy,then explain the effects of oxidative stress in the pathologic mechanism of diabetic corneal neuropathy. Methods Seventy Sprague-Daxley male rats were taken as experimental subjects and 20 of them were used as normal control group.The remaining 50 were induced to be diabetic mellitus by a single intraperitoneal injection of streptozotocin and divided into 2 groups randomly:edaravone treated group and diabetic control group.In the edaravone treated group,edaravone(0.2 g/L) eye drops were used 3 times a day until the animal was killed.Five rats in each group were sacrificed at 6,8,10 and 12 weeks respectively.Then the corneal sensation,number of corneal nerve fibers,morphology,content of malondialdehyde(MDA) and activity of superoxide dismutase(SOD) in the corneal tissue were detected.ResultsIn the diabetic control group,the corneal sensation and the number of corneal nerve fibers were decreased,the density of neural network for cluster was sparse,the nerve activity was decreased,the content of MDA in the corneal tissue was significantly increased,the activity of SOD in the corneal tissue was significantly decreased (P＜0.01 ).Accompany with the course of disease,the above change was obvious day after day.Compared with the diabetic control group,the corneal sensation and the morphological abnormalities in corneal nerve of edaravone group were improved significantly which had the partial branches to the 12th week,the content of MDA in the corneal tissue was significantly decreased,the activity of SOD in the corneal tissue was significantly increased (P＜0.01).Conclusions Edaravone can lower diabetic corneal nerve of rats with experimental diabetic corneal neuropathyinjury,Oxidative stress may be a critical pathologic mechanism of diabetic corneal neuropathy.

PurposeTo study the patterns of the expression of MMP-2 and MMP-9 in human salivary adenoid cystic carcinoma and evaluate the relationship between the expression and nerve invasion and lymph node metastasis. MethodsThe immunohistochemical location of MMP-2 and MMP-9 was evaluated by using Envision immunoperoxidase technique. Studies were performed in 53 surgical excised specimens. The immunohistochemical staining results were densitometrically semiquantitatived. ResultsThe expressions of MMP-2 and MMP-9 in salivary adenoid cystic carcinoma were 67.92 % and 79.25 % respectively. The MMP-2 and MMP-9 levels in salivary adenoid cystic carcinoma with nerve invasion were much higher than those without invasion(P＜ 0.05, P＜ 0.05 respectively), and the percentage of lymph node metastasis rose with the change of MMP-2 and MMP-9 (P ＜ 0.05, P ＜ 0.05 respectively). ConclusionsThe results indicate that the rise of MMP-2 and MMP-9 correlate with the nerve invasion and lymph node metastasis.

OBJECTIVE: To evaluate the effect of Ac-hE-18A-NH2 on TNF-α secretion and mRNA expression in ox-LDL-stimulated RAW264.7 macrophages and to elucidate the possible mechanisms. METHODS: Macrophages were incubated in the medium containing various concentrations of Ac-hE18A-NH2 (1-50 μg/mL) with ox-LDL (50 μg/mL) stimulated. The TNF-α level and intracellular cholesterol content were measured by commercially available quantitation kits following the manufacturer's instructions. TNF-α and ATP-binding cassette transporter A1 (ABCA1) mRNA expression were detected by real-time PCR. ABCA1 and IκB protein -expression in the macrophages were determined by Western blot. NF-κB activity was evaluated by electrophoretic mobility shift assay (EMSA). RESULTS: Ox-LDL stimulation induced a significant increase in TNF-α secretion, mRNA expression, cholesterol accumulation and nuclear factor-κB (NF-κB) activity in RAW264.7 macrophages. Ac-hE-18A-NH2 reduced TNF-α secretion and mRNA expression, up-regulated the ABCA1 mRNA and protein expression, reduced the intracellular cholesterol content, and inhibited NF- κB activation in a dose-dependent manner. Under the same condition and the same concentration, Ac-hE-18A-NH2 was more efficient than D-4F (apoA-I mimetic peptide) in inhibiting the inflammatory response induced by ox-LDL in the macrophages. CONCLUSION Ac-hE-18A-NH2 may suppress TNF-α secretion and mRNA expression in ox-LDL stimulated RAW264.7 macrophages via IκB-NF-κB signaling pathway. The anti-inflammatory effect of Ac-hE-18A-NH2 is better than that of apoA-I mimic peptide D-4F.
ATP Binding Cassette Transporter 1 ; metabolism ; Animals ; Cell Line ; Cholesterol ; metabolism ; Gene Expression Regulation ; I-kappa B Proteins ; metabolism ; Inflammation ; metabolism ; Lipoproteins ; pharmacology ; Lipoproteins, LDL ; Macrophages ; metabolism ; Mice ; NF-kappa B ; metabolism ; Peptide Fragments ; pharmacology ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo observe improved effects of Jingjin acupuncture on fine activity of hemiplegic hand in recovery period of stroke.

METHODSFifty cases were randomly divided into an observation group and a control group, 25 cases in each one. Regular western medicine treatment, rehabilitation training and regular acupuncture (in which Shuigou (GV 26), Baihui (GV 20), Neiguan (PC 6), etc. were selected) were applied in both groups. Additionally, muscles in palm side of affected hand, dorsal metacarpophalangeal joints and proximal interphalangeal joints were treated with acupuncture in the observation group, once every other day and electroacupuncture was applied when arrival of qi was acquired. Baxie (EX-UE 9) in the affected hand were needled in the control group, and electroacupuncture was added when arrival of qi was acquired. Ten days of treatment was considered a treatment course, and after two courses Lindmark score, Brunnstrom movement function grade, joint range of hand and Barthel index (BI) were observed in two groups.

RESULTSCompared before the treatment, the Lindmark score in two groups were both improved after the treatment (both P < 0.01). Compared with the control group, the motor coordination ability, sensory function and total score of Lindmark in observation group were obviously improved (differences before and after treatment: 8.24 +/- 3.07 vs 6.84 +/- 2.43, 3.52 +/- 2.33 vs 2.16 +/- 2.12, 11.76 +/- 3.55 vs 9.00 +/- 3.62, all P < 0.05). The Brunnstrom movement function grade was significantly improved in both groups after treatment (both P < 0.01), which was more obvious in the observation group (P < 0.05). The joint range of hemiplegic hand was improved in both groups after treatment (both P < 0.01), which was more obvious in the observation group [differences before and after treatment: (25.35 +/- 10.91) degrees vs (18.65 +/- 7.86) degrees, p < 0.05]. The score of BI was also significantly improved after treatment in two groups (both P < 0.01).

CONCLUSIONThe Jingjin acupuncture could effectively improve fine activity of hemiplegic hand in recovery period of stroke prove daily life ability.

Acupuncture Therapy ; Aged ; Female ; Hand ; physiopathology ; Hemiplegia ; etiology ; physiopathology ; therapy ; Humans ; Male ; Middle Aged ; Movement ; Recovery of Function ; Stroke ; complications ; physiopathology

OBJECTIVETo investigate the effects of marine collagen peptide on adenine-induced chronic renal function impairment in rats.

METHODSAdenine suspension (100 mg/kg ) was given to Sprague-Dawley rats to made the model of renal function impairment. Marine collagen peptide 1.125 g x kg(-1) x d(-1) and 2.25 g x kg(-1) x d(-1) were administered intragastricly in two intervention groups. In addition, adenine suspension (100 mg/kg ) was given. Experiment was kept 12 weeks. Time-dependent levels of serum creatinine (Cr), urea nitrogen (BUN) and creatinine clearance rate were monitored. Kidney ultramicrostructure was checked through transmission electron microscope.

RESULTSIn model group, level of serum Cr, BUN and Ccr of 5, 8, 12th week respectively were: (182.2 +/- 119.52, 308.17 +/- 88.37, 347.57 +/- 68.24; 29.20 +/- 16.48, 63.03 +/- 18.68, 95.53 +/- 24.88; 0.53 +/- 0.23, 0.17 +/- 0.13, 0.14 +/- 0.08). Serum Cr, BUN levels in marine collagen peptide 2.25 g x kg(-1) x d(-1) treated rats were lower and Ccr was higher significantly than that of model group. Level of serum Cr, BUN and Ccr of 5, 8, 12th week in marine collagen peptide treatment group respectively were: (105.60 +/- 11.84, 175.40 +/- 73.93, 240.14 +/- 71.53; 23.62 +/- 3.89, 41.90 +/- 23.78, 72.93 +/- 26.12; 0.99 +/- 0.35, 0.45 +/- 0.28, 0.26 +/- 0.06). Besides, kidney ultramicrostructure damage was ameliorated. Favorable effect of marine collagen peptide 1.125 g x kg(-1) x d(-1) was also observed on renal function impairment, but the difference compared to model group was not significant.

CONCLUSIONMarine collagen peptide at a dose of 2.25 g x kg(-1) x d(-1) might slow down the progression of chronic renal function impairment induced by adenine in rats.

Adenine ; adverse effects ; Animals ; Collagen ; administration & dosage ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Male ; Marine Biology ; Peptides ; administration & dosage ; Rats ; Rats, Sprague-Dawley ; Renal Insufficiency, Chronic ; chemically induced ; prevention & control

OBJECTIVETo explore the therapeutic efficacy of patients with ulcerative colitis (UC) treated by retention enema and per-colonoscopic spraying of Zhikang Compound Liquid (ZKCL).

METHODSEighty-six patients with UC were divided into two groups. The 52 patients in the treated group were treated for 4 courses of retention enema, the drug for enema used in the 1st course was ZKCL-A (consisted of normal saline, Zhikang capsule, gentamycin and dexamethasone) and smecta, in the 2nd course ZKCL-A alone, in the 3rd and 4th course, ZKCL-B (with the same contents of ZKCL-A but without dexamethasone), the enema was carried out once a day in the evening, 15 days as one course. Besides, local spraying of ZKCL-A and smecta were given once by colonoscopy before the 1st and 3rd course. The 34 patients in the control group were treated by salicylazosulfapyridine orally.

RESULTSIn the treated group, 32 patients got complete remitted, 15 were treated effectively, 5 ineffectively, the total effective rate being 90.38% while the corresponding number in the control group were 8, 14, 12, and 64.71%, respectively. Significant difference was seen when compared with the therapeutic effects of the two groups. CONCLUSION Good efficacy was got in treating patients with UC by retention enema and per-colonoscopic spraying with ZKCL.

Administration, Rectal ; Adult ; Aged ; Aged, 80 and over ; Colitis, Ulcerative ; drug therapy ; pathology ; Colonoscopy ; Dexamethasone ; administration & dosage ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Gentamicins ; administration & dosage ; Humans ; Male ; Middle Aged ; Phytotherapy

Objective To study the exact function of human gene 5 transactivated by pre-S1 protein of hepatitis B virus(PS1TP5)by investigating the gene expression of PS1TP5 in yeast cells. Methods Reverse transcription-polymerase chain reaction(RT-PCR)was performed to amplify the gene of PS1TP5 using the mRNA of HepG2 cells as template and the gene was cloned into pGEM-T vector.The gene of PS1TP5 was cut from pGEM-T-PS1TP5 vector and cloned into yeast expressive plasmid pGBKT7,then pGBKT7-PS1TP5 was transformed into yeast cell AH109.The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western hybridization.Results PS1TP5 gene was successfully amplified and identified by DNA sequencing.The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109.The results of SDS-PAGE and Western assay showed that the relative molecular weight of the expressed product was about 36 950,and PS1TP5 protein existed in yeast cells.Conclusion The findings suggest that PS1TP5 can be successfully expressed in yeast cell.

One of the most common causes of male infertility is asthenospermia, whose pathogenesis, however, is not yet clear. Recent researches have found that some genes (such as tektin-2, DNAI1, DNAH5, DNAH11, AKAP4, SEPT4 and Smcp) and proteins (such as sperm proteins ACTB, ANXA5, PRM1, PRM2 and SABP and seminal proteins Tf, PSA, PAP and Fractalkine) are associated with asthenospermia. The finding of these molecular markers has provided a base for the explanation of the molecular mechanism of asthenospermia, and these markers may become the diagnostic and therapeutic targets of the disease.
A Kinase Anchor Proteins ; genetics ; Animals ; Asthenozoospermia ; genetics ; metabolism ; Cytoskeletal Proteins ; genetics ; DNA Methylation ; genetics ; GTP Phosphohydrolases ; genetics ; Humans ; Male ; Mutation ; Septins

OBJECTIVETo screen proteins in leukocytes interacting with PS1TP2 by yeast-two hybrid and to view their subcellular localization in HepG2 cells.

METHODSThe function and structure of PS1TP2 were studied by bioinformatic analysis. PS1TP2 gene was amplified and cloned into plasmid pET32a (+) and pGBKT7 to construct recombinant expression vectors pET32a (+)-PS1TP2 and pGBKT7-PS1TP2. They were transduced into E. coli Rosetta strain and yeast AH109. The transformed yeast mated with yeast Y187 containing leukocyte cDNA library plasmid in a 2xYPDA medium. Diploid yeast cells were plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) for selecting twice and then screening. Then a green fluorescent protein (GFP) expression vector pEGFP-C1-PS1TP2 was established, transduced into HepG2, and its subcellular localization was studied by fluorescence microscopy and confocal microscopy.

RESULTSBioinformatic analysis showed that the PS1TP2 gene was located at 6q24.1, the protein was unstable and the aliphatic index was very high. After transformation of the E. coli and yeast AH109, the expression protein showed: (1) the molecular weight of the expressed product was about 41000 Da, and (2) PS1TP2 existed within the cells. Diploid yeast cells were plated on the synthetic dropout nutrient medium containing X-a-gal for selecting twice and then screening. Twenty-six colonies from blue colonies were sequenced, pEGFP-C1-PS1TP2 was successfully expressed in the HepG2 cells, and PS1TP2 was located in the cell plasma.

CONCLUSIONA prokaryotic expression vector pET32a(+)-PS1TP2 was constructed successfully and the PS1TP2 was successfully expressed in the yeast system. Genes of PS1TP2 interact with leukocyte proteins. These results bring some new clues for studying the biological functions of HBV.

Base Sequence ; Cloning, Molecular ; Dipeptides ; Gene Expression ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Humans ; Protein Precursors ; metabolism ; Proteins ; genetics ; metabolism ; Two-Hybrid System Techniques

OBJECTIVETo appraise the effect of single- and two-layer Percoll density gradient centrifugation in sperm separation.

METHODSTwenty semen specimens underwent single-(50%) and two-layer (90% and 45%) density gradient centrifugation, respectively. The sperm class analyzer (SCA) was used to analyze sperm density, motility and dynamic parameters and round cell density before and after the treatment.

RESULTSAfter separation, the sperm recovery rate of the single-layer method was (65.5 +/- 12.8)%, significantly higher than that of the two-layer method (P < 0.01). The percentages of grade a sperm of the single- and two-layer method were significantly higher than pre-treatment (P < 0.05, P < 0.01), that of the single-layer was significantly lower than that of the two-layer method (P < 0.05), but the percentage of grade c sperm of the former was significantly higher than that of the latter (P < 0.05). Compared with pre-treatment, the percentage of grade a + b sperm of the two-layer method was significantly higher (P < 0.05), while that of the single-layer method showed no significant difference (P > 0.05), and the round cell density of both the methods was significantly lower (P < 0.05, P < 0.01), with no significant differences between the two methods (P > 0.05).

CONCLUSIONThe single-layer method yields a higher rate of sperm recovery and causes little change in the sperm motility, while the two-layer method effects a lower rate and significantly improves sperm motility. Both the methods can efficiently separate sperm from round cells, and each has its own advantages and its application value in in vitro treatment of sperm.

Cell Separation ; methods ; Centrifugation, Density Gradient ; methods ; Humans ; Male ; Povidone ; Silicon Dioxide ; Sperm Count ; methods ; Spermatozoa ; cytology