Objective To study the effect and mechanism of estradiol (E

A bioassay method for inhibiting platelet aggregation in vitro was established to quantify the pharmacological effects of Compound Danshen Tablets and support its quality control. The inhibition of platelet aggregation in rabbit plasma in vitro by Compound Danshen Tablets was used as the experimental system. The titer was calculated by using the method of dose-response parallel lines. As a result, a bioassay for the inhibition of platelet aggregation in vitro by Compound Danshen Tablets was established. Linearity was good in the concentration range of 0.128 g·mL^-1 to 0.205 g·mL^-1, and the titer of standard Compound Danshen tablets was 7 659 U·g^-1 according to the titer definition. This in vitro assay was simple, reliable, reproducible and convenient. The activity of Compound Danshen Tablets in inhibiting platelet aggregation was quantified by potency assay and the quality of different batches was evaluated. The method can be applied for the quality control of Compound Danshen Tablets.

To investigate whether the protection of rutaecarpine against bleomycin-induced pulmonary fibrosis is mediated by inhibiting Notch1/eukaryotic initiation factor 3a (eIF3a) signaling pathway, and whether these effects are related to the synthesis and release of calcitonin gene-related peptide (CGRP) and inhibition of epithelial-mesenchymal transition (EMT) of alveolar epithelial cells, male Sprague-Dawley rats were randomly divided into five groups (=12), respectively, Control group, bleomycin group, rutaecarpine (100, 300 mg·kg⁻¹) group and capsaicin plus rutaecarpine (300 mg·kg⁻¹) group. Bleomycin (5 mg·kg⁻¹) was used to induce pulmonary fibrosis rat model. Rats were given capsaicin (50 mg·kg⁻¹) by subcutaneous injections 1 days before and 7, 14, 21 days after induce pulmonary fibrosis rat model to deplete endogenous CGRP. At the end of experiments, blood was collected from carotid artery to determinate the plasma levels of CGRP by ELISA. Pulmonary tissue change was observed by HE staining. Masson's trichrome stain was used to demonstration collagen deposition. The collagen I expression in pulmonary tissue was measured by immunohistochemisty. The expression of CGRP, Notch1, eIF3a, collagen I, vimentin, alpha-smooth muscle actin (α-SMA), E-cadherin and zonula occludens-1 (ZO-1) was detected by qPCR or Western blot. Compared with the control group, the pulmonary tissue of the bleomycin group showed significant fibrosis, including significant disturbed alveolar structure, marked thickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells and fibroblasts, and concomitantly with the decrease in plasma CGRP and expression of CGRP. Importantly the expression of E-cadherin and ZO-1 was decreased and expression of Notch1, eIF3a, collagen I, vimentin and α-SMA was increased in bleomycin group (<0.05 or <0.01). Compared with the bleomycin group, rutaecarpine (100, 300 mg·kg⁻¹) group significantly reduced bleomycin-induced pulmonary injury concomitantly with the increase in plasma CGRP and expression of CGRP. Importantly the expression of E-cadherin and ZO-1 was increased and expression of Notch1, eIF3a, collagen I, vimentin and α-SMA was decreased by rutaecarpine treatment (<0.05 or <0.01). All these effects of rutaecarpine were abolished by capsaicin.These results suggest that rutaecarpine protects against bleomycin-induced pulmonary fibrosis by inhibiting Notch1/eIF3a signaling pathway, alleviating EMT process, which is related to the increased synthesis and release of CGRP.

OBJECTIVE To explore the inhibitory effects of epalrestat (EPS) on platelet-derived growth factor (PDGF)-induced rat pulmonary artery smooth muscle cells proliferation by inhibiting aldose reductase (AR) expression.METHODS Primary rat pulmonary arterial smooth muscle cells (PASMCs) were prepared from the pulmonary artery of male 10-week-old Sprague-Dawley rats using explant method.PDGF 30 mg·L-1was given to induce cell proliferation.After PASMCs grew to 70%-80% conflu?ence, AR small-interferring RNA(ARsiRNA) was transfected with Lipofectamine 3000 into PASMCs. After 24 h,the expression and activity of AR were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR),Western blotting and spectrophotometric method,respectively to investigate EPS on PASMCs proliferation and proliferating cell nuclear antigen (PCNA) and collagenⅠexpression induced by PDGF from in vitro. PASMCs (normal control, PDGF 30 mg·L-1, PDGF+EPS 1, 10 and 100 μmol·L-1,EPS 100 μmol·L-1)were treated according to groups.Cell proliferation was measured by BrdU marking and flow cytometry. The expressions of AR, PCNA and collagenⅠwere analyzed with RT-qPCR and Western blotting.RESULTS In cultured PASMCs,compared with normal control group, the application of exogenous PDGF-induced cell proliferation concomitantly up-regulated AR expres?sion and activity (P<0.01), and such effect was abolished by ARsiRNA. Compared with PDGF group, EPS attenuated PDGF-induced proliferation of PASMCs,expression of PCNA,and collagenⅠ(P<0.05, P<0.01),and the inhibitory effect of EPS was accompanied by inhibition of AR expression(P<0.05,P<0.01).CONCLUSION EPS inhibits PDGF-induced proliferation of PASMCs via inhibiting AR expression.

BACKGROUNDSepsis is a major cause of mortality in Intensive Care Units. Anesthetic dose isoflurane and 100% oxygen were proved to be beneficial in sepsis; however, their application in septic patients is limited because long-term hyperoxia may induce oxygen toxicity and anesthetic dose isoflurane has potential adverse consequences. This study was scheduled to find the optimal combination of isoflurane and oxygen in protecting experimental sepsis and its mechanisms.

METHODSThe effects of combined therapy with isoflurane and oxygen on lung injury and sepsis were determined in animal models of sepsis induced by cecal ligation and puncture (CLP) or intraperitoneal injection of lipopolysaccharide (LPS) or zymosan. Mouse RAW264.7 cells or human peripheral blood mononuclear cells (PBMCs) were treated by LPS to probe mechanisms. The nuclear factor kappa B (NF-κB) signaling molecules were examined by Western blot and cellular immunohistochemistry.

RESULTSThe 0.5 minimum alveolar concentration (MAC) isoflurane in 60% oxygen was the best combination of oxygen and isoflurane for reducing mortality in experimental sepsis induced by CLP, intraperitoneal injection of LPS, or zymosan. The 0.5 MAC isoflurane in 60% oxygen inhibited proinflammatory cytokines in peritoneal lavage fluids (tumor necrosis factor-alpha [TNF-β]: 149.3 vs. 229.7 pg/ml, interleukin [IL]-1β: 12.5 vs. 20.6 pg/ml, IL-6: 86.1 vs. 116.1 pg/ml, and high-mobility group protein 1 [HMGB1]: 323.7 vs. 449.3 ng/ml; all P< 0.05) and serum (TNF-β: 302.7 vs. 450.7 pg/ml, IL-1β: 51.7 vs. 96.7 pg/ml, IL-6: 390.4 vs. 722.5 pg/ml, and HMGB1: 592.2 vs. 985.4 ng/ml; all P< 0.05) in septic animals. In vitro experiments showed that the 0.5 MAC isoflurane in 60% oxygen reduced inflammatory responses in mouse RAW264.7 cells, after LPS stimulation (all P< 0.05). Suppressed activation of NF-κB pathway was also observed in mouse RAW264.7 macrophages and human PBMCs after LPS stimulation or plasma from septic patients. The 0.5 MAC isoflurane in 60% oxygen also prevented the increases of phospho-IKKβ/β, phospho-IκBβ, and phospho-p65 expressions in RAW264.7 macrophages after LPS stimulation (all P< 0.05).

CONCLUSIONCombined administration of a sedative dose of isoflurane with 60% oxygen improves survival of septic animals through reducing inflammatory responses.

Adult ; Anesthesia ; methods ; Animals ; Blotting, Western ; Bronchoalveolar Lavage Fluid ; Disease Models, Animal ; Female ; Humans ; Inflammation ; drug therapy ; Isoflurane ; therapeutic use ; Leukocytes, Mononuclear ; metabolism ; Lipopolysaccharide Receptors ; metabolism ; Lipopolysaccharides ; pharmacology ; Lung Injury ; drug therapy ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NF-kappa B ; metabolism ; Oxygen ; therapeutic use ; Peroxidase ; metabolism ; RAW 264.7 Cells ; Rats, Sprague-Dawley ; Sepsis ; drug therapy ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo identify a novel HLA allele DRB1 * 16:36 from a Uygur woman.

METHODSPCR-SBT technology was applied to the extracted DNA for genotyping, and a possible new gene was sequenced by using sequence specific primers and single stranded SBT. This novel allele was compared with known most homologous gene sequences and their difference was analyzed.

RESULTSThis novel allele was different from HLA alle DRB1 * 16:23, and had highest similarity in 2 nucleotides at position 227 A→T and 236 T→C in exon 2, resulting in 3 amino acid changes from Tyr to Phe at codon 47 and Val to Ala at codon 50. The sequence of this novel allele had been submitted to GenBank.

CONCLUSIONThis HLA allele DRB1 * 16:23 has been confirmed to be a novel allele, and has been officially named DRB1 * 16:36 by the World Health Organization (WHO) Nomenclature Committee in May 2015.

Alleles ; Base Sequence ; DNA Primers ; Exons ; Female ; Genotype ; HLA-DRB1 Chains ; genetics ; Humans ; Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo establish the in vitro model of PGE2 released by hypothalamic neurocytes under rrIL-1beta in vitro interference, and investigate the correlation of the PGE2 content and the effect of the drug effect concentration in the model under the effect of Bupleurum injection.

METHODHypothalamic neurocytes were cultured in vitro, and added with rrIL-1beta (40 microg x L(-1)) stimulation. Cell sap was collected at different time points. ELISA was adopted to determine the content of PGE2 in cell sap collected at different time points. Hypothalamic neurocytes were cultured in vitro, added with rrIL-1beta (40 microg x L(-1)) stimulation and then different concentrations of Bupleurum injection. The changes in the content of PGE2 in cell supernatant were detected by ELISA. An analysis was made on the linear relationship between the sample concentration and the inhibition rate of PGE2.

RESULTThe rrIL-1 cells could stimulate in vitro cultured hypothalamic neurocytes to release PGE2 and reach the peak at 10 h. Bupleurum injection could significantly interfere the release of PGE2 in the in vitro model (P < 0.01, P < 0.05), with a certain linear relationship between the interference effect and the effect concentration of Bupleurum injection (r = 0.911, P < 0.01).

CONCLUSIONThe rrIL-1 cells could stimulate in vitro cultured hypothalamic neurocytes to release PGE2, with a good correlation between the inhibition and generation effects of PGE2 and the drug concentration.

Animals ; Biological Assay ; Bupleurum ; chemistry ; Cells, Cultured ; Dinoprostone ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hypothalamus ; cytology ; drug effects ; metabolism ; Neurons ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the prevalence of viral infections and putative association of viral infection with illness severity in young children with severe lower respiratory tract infection (LRTI) in Chongqing.

METHODRespiratory secretion specimens were collected from 119 hospitalized patients with severe pneumonia from December 2006 to March 2008.After being processed, the samples were detected for respiratory viruses including respiratory syncytial virus (RSV), adenovirus (ADV), human metapneumovirus (hMPV), human bocavirus (HBoV), parainfluenza virus 1, 2, 3 (PIV 1, 2, 3), influenza virus A and B (IVA and IVB) either by PCR or RT-PCR. Clinical data were analyzed along with virological data by using appropriate statistical methods.

RESULTViral pathogens were identified in specimens of 86 (72.3%) cases, among which RSV was detected in 49 (41.2%) patients. More than one virus was detected in 23 individual (26.7%) samples, of which 19 were dual positive for RSV and another virus. Bacterial cultures were performed for 69 patients. Both bacterial and viral pathogens were identified in 53 (76.8%) patients. Bacterial and viral coinfection was demonstrated in samples from 41 (59.4%) cases.

CONCLUSIONViral pathogens are the main etiology of severe pneumonia in young children in Chongqing area during the study period. RSV was the most frequent viral pathogens, followed by ADV and hMPV. Coinfection with respiratory common viruses was relatively common, though co-infection with viruses did not appear to aggravate the patients' condition.

Adenoviridae ; isolation & purification ; Child, Preschool ; China ; epidemiology ; Human bocavirus ; isolation & purification ; Humans ; Infant ; Infant, Newborn ; Influenza A virus ; isolation & purification ; Metapneumovirus ; isolation & purification ; Pneumonia, Bacterial ; microbiology ; virology ; Pneumonia, Viral ; microbiology ; virology ; Respiratory Syncytial Viruses ; isolation & purification ; Virus Diseases ; virology

OBJECTIVEThe SH2 domain containing inositol 5'-phosphatase (SHIP) is predominately expressed in hematopoietic cells, and is a crucial negative regulator in the development of hematopoietic cells. This paper is to evaluate the role of the SHIP gene in human leukemogenesis.

METHODSExpression of SHIP gene in bone marrow and/or peripheral blood from 32 patients with acute myeloid leukemia (AML), 9 with acute lymphoblastic leukemia (ALL), as well as human hematopoietic cell lines was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and DNA sequencing.

RESULTSRT-PCR showed that all samples expressed SHIP gene. Mutations of SHIP gene were detected in 7 (22%) of 32 AML patients and one (12%) of 9 ALL patients. Interestingly, two missense mutations that had been observed in a AML patient at diagnosis disappeared after complete remission (CR). In addition, in vitro Akt phosphorylation was prolonged and increased following IL-3 stimulation of this patient's cells.

CONCLUSIONOur data demonstrate for the first time the mutation of SHIP gene in acute leukemia and suggest a possible role of the mutation of this gene in the development of acute leukemia. SHIP may serve as a tumor suppressor by negatively regulating the PI3K/Akt signaling pathway in hematopoietic cells.

Blotting, Western ; Cell Line, Tumor ; DNA Mutational Analysis ; HL-60 Cells ; Humans ; Inositol Polyphosphate 5-Phosphatases ; Interleukin-3 ; pharmacology ; K562 Cells ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Mutation ; Oncogene Protein v-akt ; metabolism ; Phosphoric Monoester Hydrolases ; genetics ; metabolism ; Phosphorylation ; drug effects ; Polymorphism, Single-Stranded Conformational ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; U937 Cells

Recently, a mitochondrial ceramidase has been identified and cloned, whose mitochondrial localization strongly suggests the existence of an unexpected mitochondrial pathway of ceramide metabolism that may play a key role in mitochondrial functions, especially in the regulation of apoptosis. To explore the biological effect of mitochondrial ceramidase on cells, pcDNA 3.1/His-CDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transducted into K562 cells mediated by liposome, and G418 was used to screen for positive colonies. A stable transfected K562 cell line was established and named as 'K562TC'. The difference between K562 and K562TC cells in chemotheraputic cytotoxicity response and serum-withdrawal resistance and Bcl-2 protein expression were evaluated by MTT assay, annexin V/PI test, flow cytometry or Western blotting, respectively. The results showed that although survival was comparable between K562 and K562TC cells after exposed to adriamycin, etoposide or arsenious acid, K562TC cells with elevated Bcl-2 protein expression level as identified by FCM or Western blotting revealed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N-dimethylsphingosine, a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate production, also abrogated Bcl-2 protein expression in K562TC cells, while Bcl-2 protein level in K562 cells was up-regulated by exogenous sphingosine-1-phosphate. It is concluded that mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form. This is the first evidence that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.
Amidohydrolases ; physiology ; Apoptosis ; Arsenites ; pharmacology ; Ceramidases ; Doxorubicin ; pharmacology ; Etoposide ; pharmacology ; Humans ; K562 Cells ; Lysophospholipids ; physiology ; Mitochondria ; enzymology ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Sphingosine ; analogs & derivatives ; physiology ; Up-Regulation