Objective To explore the effects of hypericin on inhibition of hepatitis 15 virus (HBV) replication.Methods The concentration gradient hypericin was added to HepG2.2.15 cell culture system and lamivudine was used as control.Enzyme-linked immunosorbent assay (ELISA) and Southern blot were used to examine HBsAg,HBeAg and HBV DNA level in the culture supernatant,respectively.Half inhibitory concentration (IC50) and half effective concentration (EC50) of hypericin were calculated.The effects of hypericin on HBV DNA polymerase were detected by 32p marked deoxy-ribonucleoside triphosphate as the substrate. Independent sample t-test and single factor analysis of variance were used to compare the data between two groups and among multiple groups.Results The inhibition rates of hypericin on HBsAg, HBeAg were enhanced with hypericin concentration increasing and those were higher than lamivudine control group when the concentration was higher than 0.5 μmol/L (t=-0.127,P＜0.05).Southern blot confirmed that hypericin was stronger in inhibition of HBV DNA (EC50=0.2 μmol/L) than lamivudine (t=-0.058,P＜0.05).Hypericin was non-toxic on HepG2.2.15 cells in the range of test with EC50 of 0.2 μmol/L and IC50 of 200 μmol/L.Hypericin did not act on HBV DNA polymerase which was quite different from lamivudine.Conclusions Hypericin can effectively inhibit HBV replication as well as antigen synthesis and is non-toxic on HepG2.2.15 cell.The anti-HBV target of hypericin is different from nucleos(t)ide analogues.

AIM:To compare the effects of alcohol and aqueous extracts from Huiyang shengji gao(HYSJG),a Chinese medicine,on proliferation of human fibroblasts derived from normal and ulcer margin skin.METHODS:Using explant culture techniques,we obtained populations of normal human fibroblast(nHFB)and ulcer marginal fibroblast(uHFB).Alarmar blue assay was used for observing cell proliferation,and flow cytometry for cell cycle analysis.RESULTS:After cultured with aqueous extracts and alcohol extracts of HYSJG for 24 hours,the proliferation rates of the two cell lines were both elevated.For alcohol extract,obvious proliferation-promoting concentrations on nHFB and uHFB were 0.027-0.425 mg/L and 0.027-0.213 mg/L,respectively(P

0.05).Conclusion There is no significant correlation between ACE gene polymorphism and type 2 diabetic nephropathy.

Sailuotong (SLT) is a compound preparation composed of ginseng, ginkgo and saffron for the treatment of vascular dementia In order to identify its material foundation and provide evidence for therapeutic regimen, the pharmacokinetics and brain distribution of ginsenosides were investigated after intragastric administration of SLT. An LC-MS/MS method was developed for the determination of 7 ginsenosides in rat plasma simultaneously. Statistical analysis of obtained data demonstrated that the method has achieved the desired linearity, precision, accuracy and sensitivity. After administration of SLT at 60 mg x kg(-1) dose, 7 ginsengosides were all absorbed into systematic circulation. The quantitative and statistical analysis of gensenosides in plasma showed that protopanaxdiol saponins exhibited higher concentration and longer half life than protopanaxatriol saponins. The mean value of half life of ginsenosides Rg1, Re, Rb1, Rb2/b3, Rc and Rd were 15.26, 2.46, 18.41, 27.70, 21.86 and 61.58 h respectively. The peak concentration of them were 7.15, 2.83, 55.32, 30.22, 21.42, 8.81 microg x L(-1) respectively. The determination of brain distribution at different time after dosing revealed ginsenosides entered into brain promptly but the concentration declined along with time rapidly. The ginsenosides with higher concentration in brain were Rg1, Re, Rb1 and Rc. These findings demonstrated ginsenosides could be absorbed in blood and penetrated into brain rapidly. Some ginsenosides, especially Rg1 and Re, might be the main components directly effecting neurocyte in brain taking advantage of their better brain distribution. While ginsenosides of mostly protopanaxdiol saponins might protect brain mainly depending on peripheral efficacy in virtue of their long residence in blood, by which higher concentration could be reached after multiple dosing.
Animals ; Brain ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Ginsenosides ; pharmacokinetics ; Male ; Rats ; Rats, Wistar ; Time Factors

The aerolysin genes (aerA) of BZ and NK isolates were cloned and sequenced. The sequence analysis showed that the partial aerA of BZ and NK isolates consisted of 1393 bp, encoding a protein of 464 amino acids. The similarity of nucleotide and amino acid sequences of aerA between BZ and NK isolates was 97.6% and 98.3% respectively. The nucleotide sequence of aerA of BZ strain exhibited 71.6% to 97.5% homology with other Aeromonas isolates, and the amino acid sequence exhibited 68.0% to 98.9% homology. The phylogenetic tree based on aerA nucleotide sequences from Aeromonas isolates was constructed with neighbor-joining method. It showed that there were three branches of aerolysin genes, and a close relation- ship among Aeromonas hydrophila isolates which were clustered into the same branch.

AIM:To study the anti-tumor effect and mechanism of fusion vaccine on DCs and C6 glioma cells.METHODS: PEG was used to fuse DCs with C6 glioma cells.Immunofluorescence with GFAP-FITC was used to identify the DC/C6 fusion cells.Rat brain glioma models were made by stereotactic technique.After 5 days of inoculation of C6,107 fusion cells were injected through tail vein in group A.The same number of DCs and the same volume of PBS were used in group B and group C.The survival time of rats in these three groups was analyzed by Log-rank survival analysis.Tumor samples were checked by HE staining and immunohistochemical staining with CD8Mcab.RESULTS: Positive result of GFAP-FITC immunofluorescence was observed in DC/C6 fusion cells.The Log-rank survival analysis showed that statistically significant difference in group A was observed compared to that in group B and group C(P

ObjectiveTo rapidly detect the mutation frequency of DBC2 gene 7776C ＞ T in breast cancer and breast fibroadenoma by applying the mutation-sensitive molecular switch ( comprised of high-fidelity DNA polymerase and phosphorothioate-modified allele-specific primers) and agarose gel electrophoresis.Methods Allelic specific primers targeting mutation type and wild type were designed with the primers'3'terminal phosphorothioate modification.When the primers matched with the tissue DNA,the primers could be extended with highfidelity polymerase; when they mismatched with the tissue DNA,the primers could not be extended.DNA samples from 85 cases of breast cancer and 10 cases of breast fibroadenoma tissues were chosen and analyzed by PCR amplifications mediated by high-fidelity DNA polymerase.Gel imaging system was employed to make analysis of PCT products.ResultsThe mutation-sensitive molecular switch system showed that the mutation rate of 7776C ＞ T was 2.4％ ( 2/85 ) in the 85 cases of breast cancer,and no mutation was found in the 10 cases of breast fibroadenoma.ConclusionsThe mutation-sensitive molecular switch combined with agarose gel electrophoresis can rapidly detect the mutation of breast cancer DBC2 gene 7776C ＞ T.It is applicable in single nucleotide polymorphisms assay and has enormous application value in detecting gene mutation.

OBJECTIVETo observe the effect of formula of removing both phlegm and blood stasis (TYTZ) in inhibiting the inflammatory reaction in Chinese mini-swine with coronary atherosclerosis.

METHODTotally 36 Chinese mini-swine were randomly divided to six groups: the normal control group, the model group, the Shujiangzhi group and TYTZ groups with does of 2.0, 1.0 and 0.5 g x kg(-1), and six each in every group. Except for the normal control group, all of other groups were fed with high-fat diet for 2 weeks. Interventional balloons are adopted to injure their left anterior descending artery endothelium. After the operation, they were fed with high-fat diet for 8 weeks to prepare the coronary atherosclerosis model. In the 8th week after the operation and administration, the intravascular ultrasound was adopted to observe the coronary artery plaque burden of each group and the pathological morphology of coronary artery. Such inflammatory factors as high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were detected by ELISA. The expression of NF-kappaB p65 nuclear translocation was observed by the immunohistochemical method.

RESULTCompared with the normal control group, the model group showed significant increase in the coronary artery plaque burden at the end of the experiment (P < 0.01), notably abnormal structural changes in atherosclerotic vascular tissues, luminal stenosis, a large number of foam cells and inflammatory cell infiltration, remarkable growth of hs-CRP, TNF-alpha and IL-6 levels (P < 0.01). The immunohistochemical staining also showed the significant increase in the NF-kappaB p65 nuclear translocation of coronary artery of Chinese mini-swine in the model group. Compared with the model group, TYTZ could significantly attenuate atherosclerotic plaque burden (P < 0.01), inhibit the coronary luminal stenosis, reduce inflammatory cell infiltration, decrease such inflammatory cell factors as hs-CRP, TNF-alpha and IL-6 in serum, and inhibit the NF-kappaB p65 nuclear translocation of coronary artery (P < 0.05 or P < 0.01).

CONCLUSIONTYTZ can reduce the downstream inflammatory reaction by controlling NF-kappaB p65 nuclear translocation, so as to inhibit the occurrence and development of coronary atherosclerotic plaque in Chinese mini-swine.

Animals ; C-Reactive Protein ; metabolism ; Coronary Artery Disease ; blood ; complications ; drug therapy ; pathology ; Female ; Inflammation ; complications ; Interleukin-6 ; blood ; Male ; Medicine, Chinese Traditional ; methods ; Mucous Membrane ; drug effects ; secretion ; Swine ; Swine, Miniature ; Tumor Necrosis Factor-alpha ; blood

Objective To investigate the role of microRNA-129-5p (miR-129-5p) in the regulation of epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) isolated from peritoneal dialysate effluents and TGF-β1 induced HPMCs line.Methods The isolated cells were cultured from peritoneal dialysate effluents overnight of 10 patients just started PD and 12 patients with PD over 6 months.Taqman PCR assay was used to determine the expression of miR-129-5p in the HPMCs.Moreover,the expression of miR-129-5p in HPMCs induced by 5 μg/L TGF-β1 for 0-72 h was also detected by Taqman PCR.HPMCs were pre-transfected with miR-129-5p precursor (pre-mir-129-5p) to overexpress miR-129-5p,then incubated with TGF-β1 for 48 h,and the expression of EMT associated gene and protein was detected by real-time PCR,Western blotting and immunofluorescence,respectively.Furthermore,the effect of TGF-β1 on the expression of Smad interacting protein-1 (SIP1) and the regulation of pre-miR-129-5p on the SIP1 expression also were investigated.Results MiR-129-5p expression significantly down-regulated in the HPMCs isolated from PD patients over 6 months than from PD start patients(P ＜ 0.01).Similarly,TGF-β1 remarkably decreased miR-129-5p in HPMCs lines on time-dependent manner (P ＜ 0.01).Pre-mir-129-5p dramatically restored the expression of epithelial marker E-cadherin,while inhibited the expression of Vimentin,a mesenchymal marker,in HPMCs induced by TGF-β1 (all P ＜ 0.01).In addition,TGF-β1 increased SIP1 expression in HPMCs time dependently,while the high level of SIP1 protein was obviously repressed after transfected of pre-miR-129-5p (P ＜ 0.01),but there was no obvious change of its mRNA expression.Conclusion MiR-129-5p modulates EMT formation of HPMCs in PD process,possibly by posttranscriptional inhibition of SIP1.Targeting miR-129-5p/SIP1 may provide a new approach for the prevention and treatment of peritoneal fibrosis during PD.

Objective To construct a recombinant baculovirus dual expression vector containing NIS gene under the control of human telomerase reverse transcriptase (hTERT) promoter and plasminogen kringle 5 (K5) gene driven by early growth response 1 (Egr1) promoter,and to explore the feasibility of targeting both tumor and tumor vessel with combination of radioiodide and antiangiogenic therapy.Methods The hTERT-NIS gene and Egr1-K5 gene fragments were subcloned into baculovirus vector,then packaged and amplified in the sf9 cells to obtain recombinant baculovirus Bac-hTERT-NIS-Egr1-K5.Bac-CMV-NISEgr1-K5,Bac-hTERT-0-Egr1-K5 and Bac-hTERT-NIS-Egr1-0 were constructed as controls.The expression of NIS and K5 genes in human cervix cancers cells (HeLa) was examined by Western blot and quantitative real-time PCR.Functional NIS activity was confirmed by the uptake of 125I,the inhibition of NaClO4 and the cytotoxicity of 131I.The apoptotic effect of 131I-inducedK5 on human umbilical veins endothelial cells (HUVEC)was analyzed by an apoptosis assay using flow cytometry.Statistical analysis was performed using the analysis of variance.Results The recombinant baculovirus Bac-hTERT-NIS-Egr1-K5 was successfully constructed.The NIS gene under the control of hTERT promoter was specifically expressed in HeLa cells.The baculovirusinfected HeLa cells showed a significant increase of 125I uptake,which was significantly inhibited by NaClO4(F199.296,P＜0.05).Furthermore,a notable decreased cell survival rate (38.3％) was found after 131I treatment.The expression of K5 gene induced by 131I was elevated in a dose or time dependent manner and resulted in obvious inhibition with cell survival rate of 30.8％ in baculovirus-infected HUVEC cells,which was significantly higher than that in the control groups (11.2％ and 10.9％ respectively,F=19.926,45.409;both P＜0.05).Conclusions A recombinant baculovirus dual expression vector containing the NIS and K5 genes has been successfully constructed.This study suggests the feasibility of a synergistic strategy of NISbased raidoiodide therapy and K5-based antiangiogenic therapy in vitro,and make it possible to perform in vivo study in the near future.