Aim To study the therapeutic effect of Huangqi Jianzhong decoction ( HQJZ) on duodenal ul-cer(DU) in rats and its effect on intestinal mucosal immune barrier function mediated by TLR-2. Methods SD rats were divided into normal group, model group, positive drug group and HQJZ group. DU model was established by methods of multiple factors. The body weight, food intake, and rectal temperature were measured. The ulcer index (UI) was calculated. The pathological changes of duodenal mucosa were observed by HE staining. The contents of cytokines were detec-ted by EL1SA. The expression of mRNx^ and protein were detected by qRT-PCR and immunohistochemistry, respectively. Results Compared with normal group, the body weight, food intake, and rectal temperature of model group decreased significantly. The UI significantly increased. The contents of serum IL-4 and IL-10 markedly decreased, and TNF-a content evidently in creased. The expressions of TLR-2 and MyD88 mRNA and protein significantly increased. Compared with model group, after treatment with HQJZ, the body weight, food intake, and rectal temperature were significantly up-regulated, while the UI was significantly down-regulated. The morphology of duodenal mucosa returned to normal status, and the villus height and crypt depth increased significantly. The levels of serum IL-4 and IL-10 were significantly elevated, while TNF- A significantly declined. The expressions of TLR-2 and MyD88 mRNA and protein were apparently down-regu-lated. Conclusions HQJZ can promote the healing of DU, and the mechanism may be related to the intervention of TLR-2 mediated intestinal mucosal immune barrier function.

Objective: To investigate the molecular mechanism of Salvia miltiorrhiza (SM) in the treatment of retinitis pigmentosa (RP) by interfering with the expression of characteristic genes and key protein in Müller cells (MC) based on the methods of network pharmacology and bioinformatics. Methods: Retrieval and screening of active ingredients and therapeutic targets of SM in blood was performed by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Differentially expressed genes of MC in normal and RP mice were obtained by searching GEO database. RP-related gene targets were retrieved through disease database. Cytoscape was used to construct protein-protein interaction networks of differentially expressed MC genes, disease targets and component targets and the intersection was extracted. Gene Ontology and KEGG signaling pathway analysis of characteristic genes were carried out by DAVID. CytoHubba was used to analyze and screen the key protein targets. Results: A total of 202 chemical constituents related to SM were retrieved, 65 active ingredients were screened according to ADME parameters, of which 13 were active ingredients in blood. A total of 117 possible targets were obtained by further searching and matching. A total of 242 differentially expressed genes in MC of normal and RP mice were obtained from chip data. A total of 206 targets closely related to RP were obtained from disease databases. A total of 85 characteristic genes of SM affecting MC in RP pathological process were extracted and intersected. These genes were mainly involved in transcriptional regulation, apoptotic signaling pathway regulation, DNA nuclear replication regulation and other biological processes. Molecular functions mainly include transcriptional coactivator activity, protein kinase activity, core promoter binding, etc. They were enriched in nuclear, nucleoplasm, transcription factor complex, Rb-E2F complex and other regions. The signaling pathways involved include splicer signaling pathway, actin cytoskeleton signaling pathway, cell cycle signaling pathway and so on. A total of eight key protein targets of SM on MC in RP pathological process were analyzed and screened. Conclusion: The substance basis of the pharmacodynamics of SM is 13 chemical constituents, such as cryptotanshinone, luteolin, tanshinone IIA, etc. The MC characteristic genes involved in the pathological process of RP intervened by SM are related to spliceosome signaling pathway, actin cytoskeleton signaling pathway, cell cycle regulation pathway, etc. The key targets include eight protein such as RB1, E2F1, TFDP1, etc.

Objective To establish a method for the determination of protocatechuic acid, salidroside, and chlorogenic acid in Sargentodoxa cuneata. Methods The separation was performed on a Waters XSELECT CSH C18 (150 mm × 4.6 mm, 5 μm) with methanol-acetonitrile-0.2 % phosphoric acid as the mobile phase in a gradient elution at a flow rate of 0.8 ml/min. The detection wavelength was 260 nm and the column temperature was 35 ℃. Results The linear ranges of protocatechuic acid, salidroside, and chlorogenic acid were 0.0020-0.0120, 0.0600-0.3602, 0.0750-4.5006 mg/ml, respectively. The average recoveries were 98.01% (RSD=0.07%), 98.53 % (RSD=0.12%), and 101.10 % (RSD=1.92%), respectively. Conclusions The method is simple, accurate, and highly reproducible, which could provide the scientific evidence for the quality control of Sargentodoxa cuneata.

Objective: To study the effect of Astragalus membranaceus polysaccharides (AMP) on migration, intracellular polyamines content, cytosolic free Ca2+ concentration ([Ca2+]cyto), and RhoA protein expression of intestinal epithelial (IEC-6) cells, so as to explore the repairing mechanism of Astragalus membranaceus (AM) on gastrointestinal injury. Methods: AM was extracted by water and precipitated by ethanol, AM crude polysaccharides were obtained after removing protein. AMP I, II, III, and IV were obtained by DEAE cellulose column chromatography. AMP V was further obtained by Sephadex LH-20 gel column chromatography from AMP I. Cell migration model was established by Tips scratch method; High performance liquid chromatography was used to determine the polyamines content; Flow cytometry was used to detect the [Ca2+]cyto; Western blotting analysis was used to detect RhoA protein expression. The improving effect of AMP on migration, [Ca2+]cyto, and RhoA protein expression of normal and polyamine-depleted IEC-6 cells was observed. Results: AM crude polysaccharides, AMP I, and AMP V (100 mg /L or 200 mg /L) promoted cell migration and reversed the inhibition of cell migration induced by DFMO (P < 0.01); AMP V increased the intracellular polyamines content in normal and polyamine-deficient IEC-6 cells; AMP V also enhanced [Ca2+]cyto in the process of IEC-6 cell migration and reversed the reduction of [Ca2+]cyto induced by DFMO (P < 0.01); Further study suggested that AMP V increased RhoA protein expression in normal and polyamine-deficient IEC-6 cells (P < 0.01). Conclusion: These results indicate that the repairing effect of AM on the gastrointestinal mucosa damage may be related to its role of increasing polyamines content, then improving [Ca2+]cyto and RhoA protein expression and thereby promoting cell migration.

OBJECTIVETo investigate the relationship of Fas mRNA and protein expression and apoptosis in human oral squamous cell carcinoma.

METHODSNorthern blot and flow cytometry (TUNEL method) were used to detect the expression of Fas mRNA and Fas protein, cell cycle and apoptotic level in oral squamous cell carcinoma. The relationship between Fas gene expression and OSCC apoptosis was analyzed statistically.

RESULTSFas mRNA and protein could be detected in all five normal oral mucosa specimens. There was positive correlation between expression of Fas mRNA/protein and cell differentiation as well as apoptosis in OSCC (P < 0.005).

CONCLUSIONThe expression of Fas gene was highly correlated with the differentiation and apoptosis in OSCC.

Apoptosis ; Carcinoma, Squamous Cell ; metabolism ; Humans ; Mouth Neoplasms ; metabolism ; fas Receptor ; metabolism

OBJECTIVETo investigate the effect of fas gene transfection and monoclonal anti-fas antibody on tumorigenicity and proliferation of transplanted tumor of Tca8113 cell.

METHODSPlasmid including fas gene was transfected into Tca8113 cell by lipofectamine kit. Some transfected cells were treated by monoclonal anti-fas antibody after 48 hours since transfection. Untransfected cell (control), fas-tansfected cell and fas-transfected cell treated with antibody were transplanted to nude mice subcutaneously. Growth of transplanted tumor was observed and recorded regularly. Animals were sacrificed and tumor samples were harvested at the end of experiment. Fas expression in each neoplasm was assessed by RT-PCR. Apoptosis, proliferation and expression of fas protein in tumor tissue were measured by flow cytometry (FCM).

RESULTSTumor occurred much later in fas-transfected group and fas-transfected plus antibody treated group. Growth arrest was found in them. RT-PCR and FCM suggested that fas-transfection up-regulated the expression of fas mRNA and protein, increased apoptosis index (AI). But no effect on proliferation index (PI) was observed. Monoclonal anti-fas antibody did not effect the expression of fas mRNA and protein, but increased AI and decreased PI.

CONCLUSIONFas-transfection suppressing tumorigenesis of Tca8113 cell transplanted in nude mice might be caused by up-regulation of expression of fas gene and enhancement of apoptosis. However, anti-fas antibody suppressing tumorigenesis might be associated with activation of apoptosis and repression of proliferation.

Animals ; Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Fas Ligand Protein ; biosynthesis ; genetics ; pharmacology ; Humans ; Mice ; Mice, Inbred BALB C ; Mouth Neoplasms ; metabolism ; pathology ; RNA, Messenger ; genetics ; Transfection

OBJECTIVEThe purpose of this study was to establish transfergeneic Tca8113 cell and evaluate the expression of human endostatin (hES) gene in the cell colone in vitro.

METHODSTo transfect hES gene into Tca8113 cells, lipofectamin was complexed with plasmid encoding hES gene, and blasticidin S antibiotic was adopted to select Tca8113--hES cell clone. Immunohistochemistry S-P method was adopted to detect the expression of hES in the transfergenic Tca8113 cell in vitro.

RESULTSTransfected by hES, the transfergenic Tca8113 cells could grow and proliferate in RPMI--1640 culture medium containing blasticidin S antibiotic. The expression rate of hES reached 100%.

CONCLUSIONhES gene can express in hES-transfected Tca8113 cell in vitro.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Division ; Cloning, Molecular ; Endostatins ; biosynthesis ; genetics ; Humans ; Lipids ; pharmacology ; Tongue Neoplasms ; genetics ; metabolism ; pathology ; Transfection ; Tumor Cells, Cultured

Objective: To establish the method for determination of titanium dioxide in the air of workplace by inductivehy coupled plasma optical emission spectrometry (ICP-OES) . Methods: The titanium dioxide was collected by filter membrane and then digested by microwave digestion apparatus in the mixed solvents (HNO₃∶HF∶H₂O=4∶1∶1) , dilutedto 25 ml and detected by ICP-OES. Results: The sampling efficiency was higher than 95%; the linearity of ICP-OES was good at the range of 10-500 μg/ml, the minimum quantitation concentration was 0.72 mg/m³ (as collecting 150 L air sample) , the maximum quantitation concentration was 21.7 mg/m³ (as collecting 960 L air sample) , the recovery was ranged from 99.0%-102.0%, the RSD of intra- and inter-batch precision were 0.5%-3.2% and 1.7%-3.5%, respectively. Conclusion The sampling method and determination method meet the requirements of guide for establishing occupational health standards-part 4: determinatin methods of air chemicals in workplace (GBZ/T 210.4-2008) , and areapplys to the collection and determination of TiO₂ in the air of workplace.

OBJECTIVETo investigate the expression of transfected human endostatin (hES) gene in tongue squamous cell carcinoma (TSCC) and its inhibitory effects on the growth of tumor cells in vivo.

METHODSLipofectamine-mediated hES gene was transferred into Tca8113 cells, selected with Blasticidin S; The stable transfected cells were inoculated in BALB/c mice, and then the growth of xenografts was observed. The hES and vascular endothelial growth factor (VEGF) protein expression of xenografts was detected by S-P immuno-histochemical assay. We also detected the microvessel density (MVD) of xenografts with Weidern's method and apoptotic index of the tumor cells by flow cytometry (FCM).

RESULTSThe hES protein expression of xenografts in experimental group was significantly higher than that in control group (P < 0.01), while the expression of VEGF protein was on the other way round (P < 0.01). MVD counting of xenografts in experimental group was lower than that in control group (P < 0.01). The mean apoptotic level of the tumor cells in control group was also lower than in experimental group (P < 0.01). In addition, the inhibitory rate to growth of xenografts induced by hES transfection was 78.9%.

CONCLUSIONShES gene can be transferred into TSCC cells and then induce corresponding protein expression efficiently in xenograft model, resulting in significantly inhibitory effects on the xenografts in vivo.

Animals ; Carcinoma, Squamous Cell ; blood supply ; genetics ; prevention & control ; Endostatins ; biosynthesis ; genetics ; Female ; Genetic Therapy ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neovascularization, Pathologic ; prevention & control ; Tongue Neoplasms ; blood supply ; genetics ; prevention & control ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein.

METHODSTIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs.

RESULTSThe prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein.

CONCLUSIONThe TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.

Animals ; Antibodies, Monoclonal ; immunology ; Cloning, Molecular ; Humans ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; immunology