Objective To investigate the effect of extracts from rabbit skins inflamed by Viccinia virus vaccine (analgecine) on the proliferation of human cancer cells and on the cytokine secretion in mouse spleen lymphocytes in vitro. Methods Five human tumor cell lines, HepG2, LM3, H460, A549 and HeLa were used and the effect of analgecine (1.63, 0.815, 0.326, 0.163 and0.0815 U/ml) on the cell proliferation was evaluated by the CCK-8 assay. The mouse spleen was isolated aseptically, and the spleen lymphocyte suspension was prepared and cultured with PRMI-1640 medium containing 10％ fetal bovine serum (FBS). For detection of the cytokine IL-2, IFN-γ, IL-4 and IL-12 level, the stimulant concanavalin A (ConA) or lipoplysaccharide (LPS) was added into the lymphocyte suspension, and the lymphocytes were cultured under the presence of analgecine at the final concentration of 0.815, 0.163 and 0.0815 U/ml for 24 hours. Then, the level of the cytokines in the supernatant was detected by the ELISA kit. On the other hand, the effect of supernatant of the spleen lymphocyte cultures under the presence of analgecine at 0.815 U/ml on the proliferation HepG2 cells was also evaluated by the CCK-8 assay. The CCK-8 assay was performed after cultivation of the HepG2 cells in the whole supernatant or in its dilution with fresh medium for 24 hours. Results Analgecine showed a dose-dependent inhibitory effect on the five tested cancer cell lines, with the inhibition rate of 58.95％, 55.08％, 57.28％, 45.80％ and 48.18％ at the 1.63 U/ml on the HepG2, LM3, H460, A549 and HeLa cells, respectively. Compared with the control group, the secretion of IL-2, IFN-γ and IL-4 was significantly increased in the 0.163 and 0.815 U/ml analgecine groups (P＜0.01), while the secretion level of IL-12 was increased in the 0.0815, 0.163 and 0.815 U/ml analgecine groups (P＜0.01). The supernatant of the mouse spleen lymphocyte cultures under the presence of0.163 U/ml analgecine could inhibit the HepG2 cell proliferation in a dose-dependent manner, and the inhibitory effect of the whole supernatant was significantly stronger than the effect of the same concentration analgecine 0.163 U/ml (P＜0.01). Conclusion Analgecine could inhibit the cell proliferation of the tested five human cancer cell lines, increased the secretion of IL-2, IFN-γ, IL-4 and IL-12 cytokines in mouse spleen lymphocytes, all in vitro, and its effect on the cytokine secretion may be related to the inhibitory effect on the human cancer cell proliferation.

To study the effect of Tibetan medicine Zuotai on the activity, protein and mRNA expression of CYP1A2 and NAT2, three different doses (1.2, 3.8 and 12 mg x kg(-1)) of Zuotai were administrated orally to rats once a day or once daily for twelve days, separately. Rats were administrated orally caffeine (CF) on the second day after Zuotai administration, and the urine concentration of CF metabolite 5-acetylamino-6-formylamino-3-methyl-uracil (AFMU), 1-methyluric acid (1U), 1-methylxanthine (1X), 1, 7-dimethylxanthine (17U) at 5 h after study drug administration was determined by RP-HPLC. The activity of CYP1A2 and NAT2 was evaluated by the ratio of metabolites (AFMU+1X+1U)/17U and the ratio of AFMU/(AFMU+1X+1U), respectively. The protein and mRNA expression of CYP1A2 and NAT2 were determined by ELISA and RT-PCR method, respectively. After single administration of Zuotai 3.8 mg x kg(-1) and repeated administration of Zuotai 3.8 and 12 mg x kg(-1), the activity of CYP1A2 and NAT2 decreased significantly compared with control group and there was no significant difference between other dose group and control group. The protein expression of CYP1A2 was significant lower than that in control group after repeated administration of Zuotai 12 mg x kg(-1), and the mRNA expression of CYP1A2 decreased significantly compared with that of control group after single administration of Zuotai 3.8 mg x kg(-1) and repeated admistration of Zuotai 12 mg x kg(-1), separately. The protein expression of NAT2 decreased significantly compared with that of control group after single and repeated administration of Zuotai 3.8 mg x kg(-1), respectively, and the mRNA expression of CYP1A2 decreased significantly compared with control group after single administration of Zuotai 3.8 mg x kg(-1). This study found that Tibetan medicine Zuotai had significant effect on the activity, protein and mRNA expression of CYP1A2 and NAT2.
Administration, Oral ; Animals ; Arylamine N-Acetyltransferase ; genetics ; metabolism ; Caffeine ; metabolism ; urine ; Cytochrome P-450 CYP1A2 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Male ; Medicine, Tibetan Traditional ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Theophylline ; urine ; Uracil ; analogs & derivatives ; urine ; Uric Acid ; analogs & derivatives ; urine ; Xanthines ; urine

A(p.G888S)were detected in POLG1 gene.Sequence analysis of parental blood DNA revealed that her father carried L83P and her mother carried G888S.Conclusions The characteristics of clinical manifestation,electrophysiology,pathology and POLG1 gene mutation of the patient were highly consistent with Alpers syndrome.The prominent white matter change and increased immunological factors in CSF were first reported in Alpers syndrome.Alpers syndrome should be considered for those patients whose liver function were severely impaired after exposure to valproic acid.

Abstract: Objective　To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results　A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions　The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.

Aim To evaluate the effects of salvianolic acid B ( Sal B ) on bone metabolism and its potential mechanism in high fat diet ( HFD) mice.Methods Thirty C57BL/6J male mice were divided into three groups with 10 mice each, namely normal , HFD and HFD+Sal B.HFD and HFD+Sal B mice were treated with HFD, and HFD+Sal B group mice were also with Sal B (125 mg· kg -1· d-1).After 12 weeks' treat-ment, femurs were harvested .The effects of Sal B on biomechanical strength were evaluated by biomechani-cal tests, and the effects of Sal B on bone microstruc-ture were evaluated by Safranin O/fast green staining and hematoxylin and eosin staining .The expression of nuclear factor-kappa B ( NF-κB)-p65 and NADPH ox-idase 4 ( Nox4 ) and cathepsin K in femurs was deter-mined by immunohistochemical staining . Results Maximum load and elastic load significantly decreased ,and the trabeculae became thinner and irregular in the femurs of HFD mice , while Sal B treatment could re-verse the descending biomechanical strength and the disorganized femurs bone micro-structures in HFD mice.In addition, the expressions of Nox4, NF-κB-p65 and cathepsin Kmarkedly increased in HFD mice , and Sal B possessed the ability to down-regulate the ex-pression of Nox4, NF-κB-p65, and cathepsin K in the femurs triggered by HFD .Conclusions Sal B treat-ment improves bone metabolism via regulating Nox 4/NF-κB/cathepsin K signaling pathway in HFD mice . The findings contribute to the understanding and exten-sion of the applications of Salvia miltiorrhiza and its constituents on osteoporosis .

OBJECTIVETo investigate the process of separating and purifying flavonoids from Smilax glabra.

METHODWith the yield of flavonoids as index, the optimum process of separating and purifying flavonoids from S. glabra Roxb was screened by static and dynamic adsorption tests.

RESULTThe static saturated adsorption capacity of D101 macroporous resin to flavonoids of S. glabra was 45.6 mg x g(-1) (dry resin). The optimum conditions of dynamic adsorption and elution were as that the pH, the concentration, the adsorption velocity of the extracting solution, and the adsorption capacity were 6.00 +/- 0.20, 4.2 mg x mL(-1), 2 mL x min(-1) and 15 mL, respectively. The adsorbed resin column was washed by 100 mL 60% ethanol with pH value of 8.00 +/- 0.20 at the eluting velocity of 3 mL x min(-1) after washed by 100 mL distilled water.

CONCLUSIONThe flavonoids of S. glabra was able to be easily separated and purified by D101 macroporous resin under the optimum conditions above, and the recovery rate was higher than 90%. The content of obtained flavonoids reached 62.6%, which was 2 times of the content before purification.

Adsorption ; Chromatography, Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Flavonoids ; chemistry ; isolation & purification ; Resins, Synthetic ; chemistry ; Smilax ; chemistry

OBJECTIVETo study the changes of serum leptin (LEP) and vascular endothelial growth factor (VEGF) in children with congenital heart disease(CHD) and their roles in CHD.

METHODSForty-eight children with acyanotic CHD (ACHD group), 20 age-matched children with cyanotic CHD (CCHD group) and 20 healthy children (control group) were enrolled. The ACHD group was subdivided into two groups with (n=20) or without concurrent heart failure (n=28). Serum LEP, VEGF, total protein and albumin levels and body mass index (BMI) were measured.

RESULTSSerum total protein and albumin levels were not apparently different in all CHD children from healthy controls, but there was a significant difference in the BMI between them (p<0.01). Serum LEP and VEGF levels and the ratio of LEP/BMI in all CHD children were significantly higher than those in healthy controls (p<0.01). Compared with the ACHD group without heart failure, the serum LEP and VEGF levels and the ratio of LEP/BMI in the CCHD and the ACHD with heart failure groups increased significantly (p<0.01). In the ACHD group, serum LEP level was positively correlated with BMI (p<0.01). In the CCHD group, there were positive correlations between serum LEP level and serum VEGF level (p<0.01) and between hemoglobin concentration and serum VEGF level (p<0.01). Arterial oxygen saturation was negatively correlated with serum VEGF (p<0.01) and LEP levels (p<0.01) in the CCHD group.

CONCLUSIONSBoth VEGF and LEP play roles in the pathophisiological process of CHD. VEGF and LEP are associated with the development of heart failure in children with ACHD.

Body Mass Index ; Child ; Child, Preschool ; Female ; Heart Defects, Congenital ; blood ; Hemoglobins ; analysis ; Humans ; Infant ; Leptin ; blood ; Male ; Oxygen ; blood ; Vascular Endothelial Growth Factor A ; blood

OBJECTIVEThe congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders with progressive muscle wasting and weakness that begin during neonatal or early infantile period. To study the clinical diagnosis, immunohistochemical feature and follow-up information of CMD, data of 8 cases with CMD were analyzed.

METHODSImmunohistochemical features of biopsied muscle specimens were summarized and analyzed by using anti-laminin alpha2 (merosin), anti alpha-dystroglycan (alpha-DG) and anti beta-dystroglycan (beta-DG) antibodies.

RESULTSThese patients mostly presented at birth or during the first six months of life with muscle weakness, hypotonia, contractures, and feeding difficulty or respiratory dysfunction. Hematoxylin-eosin staining of skeletal muscle specimens from these patients showed typical characteristics of CMD. Differences in fiber size, with predominantly small and round fibers, and dense connective tissue infiltration were seen. Four of the 8 patients were merosin-stain negative, which might be due to primary merosin deficiency. T2-weighted magnetic resonance imaging of the brain shows abnormalities of the white matter. Four cases were merosin-stain positive, and two of them also had hypoglycosylation of alpha-dystroglycan. Two patients had mental retardation. One of them had optic nerve atrophy and abnormal brain structure.

CONCLUSIONSTwo types of CMD were present in our group. Merosin-deficient congenital muscular dystrophy (congenital muscular dystrophy 1A, MDC1A) was more common, accompanied by abnormalities of the white matter. "Alpha-dystroglycanopathy" could be seen in merosin-positive cases.

Female ; Humans ; Infant ; Laminin ; deficiency ; Male ; Muscular Dystrophies ; congenital ; diagnosis ; metabolism

To define the molecular basis of ethanol dependence, changes in the phosphorylation of cAMP response element binding protein (CREB) in the nucleus accumbens of rats after acute and chronic ethanol administration were detected using immunohistochemistry. The results demonstrate that the expression of phospho-CREB (p-CREB) protein in the rat nucleus accumbens significantly increased after 15 min of acute ethanol exposure, reaching a peak at 30 min after ethanol administration. The increment remained after 1 or 6 h of ethanol exposure compared to the control rats. In contrast, chronic intake of ethanol solution obviously decreased the expression of p-CREB protein compared to the control rats. The decrement remained 24 h or 72 h after ethanol withdrawal, and returned to the control levels after 7 d of ethanol withdrawal. The results suggest that an acute ethanol administration led to an increase in the phosphorylation of CREB in the nucleus accumbens, but chronic ethanol administration produced a decrement, which is possibly one of the molecular mechanisms of alcohol dependence.
Alcoholism ; metabolism ; physiopathology ; Animals ; Cyclic AMP Response Element-Binding Protein ; chemistry ; metabolism ; Ethanol ; pharmacology ; Male ; Nucleus Accumbens ; metabolism ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; Substance Withdrawal Syndrome ; metabolism ; physiopathology ; Substance-Related Disorders ; metabolism ; physiopathology

OBJECTIVETo observe the effect of 2-methoxycinnamaldehyde (isolated from fraction A of Guizhi Tang) on activity of COX and PGE2 release in rat cerebral microvascular endothelial cells (rCMEC) stimulated by IL-1.

METHODrCMEC were cultured, and identified by immunohistochemistry for von Willebrand factor (VIII factor, a marker for all endothelial cells) in cytoplasm of the cells. Different concentrations of 2-methoxycinnamaldehyde were added respectively and incubated for 3 hours, then stimulated for another 12 hours by IL-1. Activities of COX-1 and COX-2 in rCMEC, and production of PGE2 in the conditioned media were measured by ELISA.

RESULTPositive immunostaining for VIII factor was present diffusely in the cytoplasm of > 90% rCMEC. After being exposed to 30 ng x mL(-1) IL, the activity of COX-2 in rCMEC and the production of PGE2 in conditioned media were higher than those of control group, while there was no difference on activity of COX-1 in the two groups. 2-methoxycinnamaldehyde could down-regulate them in concentration-dependently, and significant differences on the activity of COX-2 and amount of PGE2 were showed in 200 microg x mL(-1) concentration.

CONCLUSION2-methoxycinnamaldehyde can affect the PGE2 release in rCMEC induced by IL-1, which might be related with its inhibition on the activity of COX-2.

Acrolein ; administration & dosage ; analogs & derivatives ; isolation & purification ; pharmacology ; Animals ; Brain ; blood supply ; Cells, Cultured ; Cyclooxygenase 1 ; metabolism ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; metabolism ; Dose-Response Relationship, Drug ; Drug Combinations ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Endothelial Cells ; cytology ; metabolism ; Interleukin-1 ; antagonists & inhibitors ; Male ; Microcirculation ; cytology ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley