This study was aimed to observe effect of pomegranate leaf tannin and ellagic acid on primary rat adipocyte differentiation and lipid metabolism-related factor expression. Primary rat preadipocyte was in vitro cultured to observe the effect of pomegranate leaf tannin and ellagic acid on lipid fat cells as well as mRNA expression of its related factor. The results showed that pomegranate leaf tannin and ellagic acid had obvious inhibition effect on fat formation in fat cells. It had certain inhibition effect on activities of lipoprotein lipase (LPL) and glucose-3-phosphate dehydrogenase (GPDH). It promoted fat decomposition and reduced intracellular lipid content. It upregulated PPARγ and fatty acid-binding protein (aP2). It downregulated obese (ob) gene level. It was concluded that pomegranate leaf tannin can inhibit fat generation of fat cells and promote fat metabolism. Ellagic acid was its main active ingredient, which had the same effect as pomegranate leaf tannin.

Objective To evaluate the therapeutic effect of low-dose herringtonine in lupus nephritis.Methods Harringtonine1mg,which was dissolved in500ml normal saline,was infused intravenously everyday for5to7days as a single course.The total therapeutic period was consisted of3to6courses.Each course was given every2to3weeks.12patients with lupus nephritis were treated in this study.Results Seven patients(58%)achieved complete clinical remission while5(42%)had partial clinical remission.The overall response rate was100%.Conclusion This study suggests that low-dose harringtonine is an effective therapeutic regimen for lupus nephritis with relatively low toxicity and low price.It has the potential to be used widely in treating lupus nephri-tis.

Objective To observe DNA damage and morphological changes of lung cells in rats with acute carbonyl nickel poisoning. Methods SD rats were exposed to 20, 135 and 250 mg/m3 carbonyl nickel for 30 min by inhalation. On the third day and 7th day after exposure, DNA damage of lung cells were examined by single cell gel electrophoresis (SCGE) and the pathological changes in lung tissues and the changes of microstructure in lung cells were observed. Results The DNA damage of the rat lung cells were obviously found in all exposure groups at different exposure times and there was the most obvious DNA damage at 72 hour after exposure to nickel carbonyl but the damage appeared later than chlorine exposure group. The inflammatory infiltration and hyperplasia in lung tissue, bronchiolar damage and the bronchial mucosa defulvium appeared in the rats exposed to carbonyl nickel. The results of microstructure examination indicated that the organelles of type I pneumocytes was swellen, the body of type Ⅱ pneumocytes was emptied. The cytoplasm empty bubbles increased, the mitochondria was swellen, and alveolar mediastinum inside the collagen fiber in alveolar mediastinum increased. Conclusion The acute carbonyl nickel exposure could induce the injury of lung tissues and cells with dose-effect relationship and time-effect relationship.

Objective To observe DNA damage and morphological changes of lung cells in rats with acute carbonyl nickel poisoning. Methods SD rats were exposed to 20, 135 and 250 mg/m3 carbonyl nickel for 30 min by inhalation. On the third day and 7th day after exposure, DNA damage of lung cells were examined by single cell gel electrophoresis (SCGE) and the pathological changes in lung tissues and the changes of microstructure in lung cells were observed. Results The DNA damage of the rat lung cells were obviously found in all exposure groups at different exposure times and there was the most obvious DNA damage at 72 hour after exposure to nickel carbonyl but the damage appeared later than chlorine exposure group. The inflammatory infiltration and hyperplasia in lung tissue, bronchiolar damage and the bronchial mucosa defulvium appeared in the rats exposed to carbonyl nickel. The results of microstructure examination indicated that the organelles of type I pneumocytes was swellen, the body of type Ⅱ pneumocytes was emptied. The cytoplasm empty bubbles increased, the mitochondria was swellen, and alveolar mediastinum inside the collagen fiber in alveolar mediastinum increased. Conclusion The acute carbonyl nickel exposure could induce the injury of lung tissues and cells with dose-effect relationship and time-effect relationship.

This study was purposed to investigate the mutational status of DNA methyltransferase (DNMT3a) gene and the clinical features of AML patients with DNMT3a mutations. Using PCR combined with directly sequencing, the somatic mutations of DNMT3a involving residue of amino acid 882 were detected in 77 AML patients. Furthermore, the clinical features of these patients were also studied. The results showed that the DNMT3a mutation were detected in 7 out of 59 patients with de novo AML (11.9%), which included 4 patients with DNMT3a R882C, 2 patients with DNMT3a R882H and 1 patient with DNMT3a Y874C. Morphology examination indicated that 2 patients were M(2), 1 patient was M(4) and 4 patients were M(5). Cytogenetic analysis revealed that karyotype in 5 out of 7 patients with DNMT3a mutation were normal. In total of 27 patients with normal karyotype 5 patients (22.7%) were found harboring DNMT3a mutation, while no DNMT3a mutation was found in 21 patients with abnormal karyotype. The mutation rate in patients with positive CEBPA was obviously higher than that in patients with negative CEBPA (p = 0.002). Immunophenotype analysis showed that 4 patients (4/7, 57.1%) with DNMT3a mutation expressed lymphoid antigens including CD4 or/and CD7. There were no statistical significance in age, gender, blast cells of bone marrow, white blood cell and platelet counts, hemoglobin level, ratio of CR, mutations of FLT3-ITD, NPM1 and c-kit between patients with DNMT3a mutation and patients with wild DNMT3a (p > 0.05). It is concluded that the DNMT3a mutations are more prevalent in AML patients with normal karyotype accompanying with positive NPM1 and/or CEBPA mutation, the role of DNMT3a mutation in AML prognosis needs to be further studied.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; CCAAT-Enhancer-Binding Proteins ; genetics ; Child ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Young Adult

OBJECTIVETo express and purify the fusion protein of extracellular domain of human Ig domain-containing, neurite outgrowth inhibitor (Nogo) receptor-interacting protein-1 (LINGO-1(aa76-319)) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody (pAb).

METHODSThe 732 bp DNA sequence of hLINGO-1(aa76-319) was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319), which was subsequently transformed into E.coli. The target fusion protein was expressed with IPTG induction and purified by Ni(2+)-NTA affinity chromatography column. The antiserum against hLINGO-1(aa76-319) was obtained from the rabbits immunized with hLINGO-1(aa76-319), and the titer of the pAb was determined using enzyme linked immunosorbent assay (ELISA) and its specificity identified using Western blotting.

RESULTSThe prokaryotic expression plasmid pET30a(+)-hLINGO-1(aa76-319) was constructed successfully. Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 degrees celsius; for 2.5 h. The hLINGO-1(aa76-319) fusion protein was effectively expressed in E.coli as inclusion bodies, and the soluble protein was obtained through denaturation and refolding procedures, and the purified fusion protein showed a purity above 90%. The titer of the anti-hLINGO-1(aa76-319) pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6x10(6), and Western blotting confirmed its good specificity.

CONCLUSIONThe fusion protein hLINGO-1(aa76-319) with high purity has been obtained and the anti-hLINGO-1(aa76-319) pAb obtained shows a high titer and good specificity, which provide important experimental basis for further functional investigation of LINGO-1.

Animals ; Antibodies ; immunology ; isolation & purification ; Antibody Specificity ; Escherichia coli ; genetics ; metabolism ; Humans ; Immune Sera ; immunology ; Membrane Proteins ; biosynthesis ; genetics ; immunology ; Nerve Tissue Proteins ; biosynthesis ; genetics ; immunology ; Plasmids ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

BACKGROUNDLocal hypothermia induced by intravascular infusion of cold saline solution effectively reduces brain damage in stroke. We further determined the optimal temperature of local hypothermia in our study.

METHODSSeventy-eight adult male Sprague Dawley rats (260 - 300 g) were randomly divided into 3 groups: group A, ischemia/reperfusion without cold saline infusion (n = 26) (control group); group B, infusion with 20 degrees C saline before reperfusion (n = 26); group C: infusion with 10 degrees C saline before reperfusion (n = 26). In each group, we chose 15 rats for monitoring physical indexes and the temperature of the brain (cortex and striatum) and body (anus), measurement of brain infarction volume, assessment of neurological deficits and the survival rate of reperfusion at 48 hours. Another 8 rats from each group was chosen for examining brain edema, another 3 from each group for histological observation by electron microscopy (EM) and light microscopy (LM) at 48 hours after reperfusion.

RESULTSThere was no significant difference among the 3 groups for physical indexes during the examination (F((2, 45)) = 0.577, P = 0.568; F((2, 45)) = 0.42, P = 0.78 for blood pressure and blood gas analysis, respectively). The brain temperature was significantly reduced in the group C compared to the other groups (F((2, 45)) = 37.074, P = 0.000; F((2, 45)) = 32.983, P = 0.000, for cortex and striatum temperature respectively), while the difference in rectal temperature between group A and B or C after reperfusion was not significant (F((2, 45)) = 0.17115, P = 0.637). And the brain infarct volume was significantly reduced in group C (from 40% +/- 10% in group A, 26% +/- 8% in group B, to 12% +/- 6% in group C, F((2, 45)) = 43.465, P = 0.000) with the neurological deficits improving in group C (chi(2) = 27.626, P = 0.000). The survival rate at 48 hours after 10 degrees C and 20 degrees C saline reperfusion was increased by 132.5% and 150%, respectively, as compared to the control group (chi(2) = 10.489, P = 0.005). The extent of the brain edema showed no significant difference (F((2, 21)) = 0.547, P = 0.587) after cold saline infusion compared to the control group. No obvious vascular injury was found by electron or light microscopy in either infusion group.

CONCLUSIONSRegional hypothermia with 10 degrees C cold saline infusion can significantly decrease the infarction volume, improve the neurological deficits, and 10 degrees C seems to be the optimal temperature in inducing a cerebral protection effect during stroke. This procedure could be adopted as a further treatment for acute stroke patients.

Animals ; Body Temperature ; Brain ; pathology ; Cerebral Infarction ; pathology ; Hypothermia, Induced ; Male ; Rats ; Rats, Sprague-Dawley ; Stroke ; mortality ; pathology ; physiopathology ; therapy ; Survival Rate ; Temperature

OBJECTIVETo compare the effects of instant electroacupuncture (EA) at the different acupoints on IP3 in the uterus tissue of dysmenorrhea model rats so as to investigate the specificity of acupoints.

METHODSFifty female SD rats were randomly divided into a saline group, a model group, a Sanyinjiao (SF 6) group, a Xuehai (SP 10) group and a Hegu (LI 4) group, 10 rats in each group. The rats were given subcutaneous injection of Estradiol Beozoate injection for 10 consecutive days except those in the saline group, and intraperitoneal injection of 2U Oxytocin at 1 h after the last administration to create the dysmenorrhea rats model, and the saline group was given the same dose of saline every day. On the 10th day the rats in each EA group were given EA 20 min, and the rats in the saline group and model group were bound 20 min, and the writhing response was observed at the same time. The uterine IP3 contents were detected with enzyme-linked immunosorbent assay method.

RESULTS(1) Compared with (0.311+/- 0.253) in the saline group, the writhing scores per minute of (5.867 +/- 3.442) in the model group and (2.311 +/- 0.957) in the Xuehai (SP 10) group were both increased significantly (P < 0.01, P < 0.05), and (1.833 +/- 1.355) in the Sanyinjiao (SP 6) group and (0.743 +/- 0.306) in the Hegu (LI 4) group showed no significant differences (P > 0.05). Compared with that in the model group, the writhing scores per minute decreased significantly (all P < 0.01) in all the EA groups, with no significant differences among all the EA groups (all P > 0.05). (2) Compared with (2.698 +/- 1.491) ng/mg in the saline group, IP3 contents of the uterus of (0.813 +/- 0.899) ng/mg in the model group, (1.740 +/- 0.375) ng/mg in the Sanyinjiao (SP 6) group and (0.692 +/- 0.212) ng/mg in the Hegu (LI 4) group were all lower significantly (P < 0.05, P < 0.01), and (0.743+/- 0.306) ng/mg in the Xuehai (SP 10) group showed no significant differences (P > 0.05). Compared with that in the model group, IP3 content of the uterus in the Hegu (LI 4) group showed no significant difference (P > 0.05), and those in the Sanyinjiao (SP 6) group and in the Xuehai (SP 10) group increased significantly (both P < 0 05), which were significantly higher than that in the Hegu (II 4) group (P < 0.05, P < 0.01).

CONCLUSIONThere are no significant differences among the instant EA groups in improving the dysmenorrhea symptoms, but there is obvious specificity of acupoint effects in the regulation of IP3. Electroacupuncture at "Sanyinjiao (SP 6) " Xuehai (SP 10)" has more marked effect in dysmenorrhea model rats.

Acupuncture Points ; Animals ; Disease Models, Animal ; Dysmenorrhea ; metabolism ; therapy ; Electroacupuncture ; Female ; Humans ; Inositol 1,4,5-Trisphosphate ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Uterus ; metabolism

Objective To analyze the feasibility of local LINGO-1 polyclonal antibody administration for treatment of spinal cord injury in adult rats. Methods Twenty-four adult female SD rats were randomized into sham-operated group, rabbit IgG group and LINGO-1 antibody group. In the latter two groups, partial transaction of the T9 segment of the spinal cord was performed to completely sever the dorsal eorticospinal tract, followed immediately by administration of rabbit IgG and anti-LINGO polyclonal antibody via a mini-osmotic pump, respectively. At 3 and 28 days after the operation, the T8～10 segments of the spinal cord were harvested to prepare cryosections, and immunofluorescence staining was used to analyze the penetration of LINGO-1 polyclonal antibody into the spinal cord tissue and its specific binding to LINGO-1 molecules. Results In LINGO-1 antibody group, the presence of rabbit antibodies was detected at the injured sites of the spinal cord at 3 and 28 days after the operation. The mean immunofluorescence density was significantly lower in L1NGO-1 antibody group than in rabbit IgG group at 3 days after the operation (P<0.05). In rabbit IgG group, the mean immunofluorescence density for LINGO-1 in the crysections pre-treated with LINGO-1 polyclonal antibody was significantly lower than that in sections pre-treated with rabbit IgG(P<0.05). Conclusion Locally administered LINGO-1 polyclonal antibody can penetrate into the injured sites in the spinal cord in a wide time window and recognizes LINGO-1 molecule specifically, suggesting the feasibility of passive immunotherapy for spinal cord injury.

Objective To detect the immunogenicity of the recombinant DNA vaccine that encoded for neurite growth inhibitors: Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), tenascin-R (TN-R) and myelin-associated glycoprotein (MAG) after the nerve injury under the help of pAdEasy, a kind of adenovirus plasmid being the vector of the DNA. Methods Sixteen 5-w-old Lewis rats were randomized into DNA vaccination group (vaccine group) and pAdEasy group. Rats in the vaccine group were immunized once weekly for a consecutive 8 w by bilateral injection of the recombinant plasmid into the musculus tibialis. The immunized animals in the 2 groups were exsanguinated each time before the vaccination for sera collection, and the qualitation and quantitation of the antibodies in the serum were detected by Dot-blot analysis and ELISA. Results The vaccine group could produce fusion-protein antibodies against Nogo-A, MAG, OMgp and TN-R at the 6th w of vaccine injection, while pAdEasy group could not. The valency of antiserum was shown by ELISA as 1:1 000 000 at the 6th w of vaccine injection and kept this level stably. Conclusion The DNA vaccine exclusively induces the generation of the fusion-protein antibodies against Nogo-A, MAG, OMgp and TN-R in vivo, which controls the favorable immunogenicity.