Objective:To investigate the clinical features of primary biliary cholangitis (PBC) with thyroid disease (TD) and the association between TD and PBC.Methods:From 2005 to 2017, clinical data of PBC patients from the affiliated hospital of Qingdao university were retrospectively analyzed. All PBC patients were divided into 2 groups according to whether they have TD. The general conditionsand clinical manife-stations in the two groups were analyzed. T-test, nonparametric test, Chi-square test and Fisher's exact test-swere applied to compare datain subgroups. Results:A total of 148 PBC patients were involved in to our study, of which 45 cases (30.4%) had TD. PBC patients with TD showed a higher incidence of Sj?gren's synd- rome (SS) (33.3% vs 17.5%, χ2=4.545, P=0.033). Moreover, there was a higher positive rate of anti-SP100 and anti-SSB antibody in PBC patients with TD (20.0% vs 5.8%, χ2=5.440, P=0.020; 20.0% vs 2.9%, χ2=10.087, P=0.001) compared with patients without. PBC patients without TD presented a higher incidence of abdominal distension and jaundice (29.1% vs 11.1%, χ2=5.629, P=0.018; 23.3% vs 8.9%, χ2=4.241, P=0.039) compared to patients with TD. The ratio of patients with elevated total bilirubin (TBiL), direct bilirubin (DBiL), or increased alkaline phosphatase (ALP) was higher in PBC without TD group(40.8% vs 17.8%, χ2=7.405, P=0.007; 43.7% vs 17.8%, χ2=9.147, P=0.002; 69.9% vs 51.1%, χ2=4.811, P=0.028). Correspondingly, PBC patients without TD was associated with a higher probability of cirrhosis and portal hypertension (40.8% vs 22.2%, χ2=4.731, P=0.030; 25.2% vs 8.9%, χ2=5.183, P=0.023). Conclusion:TD has no effect on the natural history of PBC. PBC patients with TD are associated with a lower probability of liver fibrosis, portal hypertension and cholestasis symptoms but a higher incidence of SS when compared with PBC patients without TD. Multi-disciplinary approach should be implemented to the mag-nagement of PBC.

BACKGROUNDThe fat derived protein adiponectin plays an important role in the regulation of glucose metabolism. The aim of this study was to provide the experimental basis for further investigating on adiponectin (ADPN) function. Its eukaryotic recombinant was constructed and expressed in precursor cells of 3T3-L1 adipocytes. The effects of dexamethasone on peroxisome proliferator activated receptor-gamma (PPAR-gamma) mRNA expression in 3T3-L1 cells with human recombinant adiponectin were assessed.

METHODSThe recombinant plasmid pMD18-T-hADPN and eukaryotic expression vector pcDNA3.1(+) were digested by two restrictive endonucleases and adiponectin and linear pcDNA3.1(+) were obtained. Then, they were ligated and translated into JM109. The recombinant pcDNA3.1(+)-hADPN so obtained was identified by digestion by restrictive endonuclease and nucleotide sequencing. The 3T3-L1 precursor cells were transfected using SuperFect Transfection Reagent (Qiagen). Furthermore, 3T3-L1 cells with human recombinant adiponectin incubated with dexamethasone (0.5 mmol/L) for 24 hours, cells were collected and total RNA was extracted. The PPAR-gamma mRNA expression was quantified by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSAfter eukaryotic recombinant was digested by Hind III and EcoR I, fragments of 800 bp and 5.4 kb were identified by nucleotide sequence scanning and consistent with theoretical values. Electrophoretogram of RT-PCR in 3T3-L1 precursors showed only one band in front of 250 bp, which was consistent with theoretical value 234 bp. In the 3T3-L1 cells, 3T3-L1 cells with plasmid and 3T3-L1 cells human recombinant adiponectin, treatment with dexamethasone (0.5 mmol/L) decreased PPAR-gamma mRNA expression compared to untreated controls (P < 0.01). Effect of dexamethasone on PPAR-gamma mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.

CONCLUSIONThe 3T3-L1 cells stably transfected human recombinant adiponectin had increased PPAR-gamma mRNA expression. Dexamethasone suppressed PPAR-gamma mRNA expression in the 3T3-L1 cells. Effect of dexamethasone on PPAR-gamma mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.

3T3-L1 Cells ; Adiponectin ; physiology ; Animals ; Dexamethasone ; pharmacology ; Insulin Resistance ; Mice ; PPAR gamma ; genetics ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the effect of total flavonoids of Hedysarum polybotry on the proliferation, cell cycle, and expressions of p21(Ras) and proliferating cell nuclear antigen (PCNA) gene in erythroleukemia cell line K562.

METHODSThe effect of total flavonoids of Hedysarum polybotry on K562 cell line survival was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay. The time- and dose-dependent manner was also observed. The cell cycle and apoptosis were analyzed with flow cytometry (FCM). The immunocytochemistry method was applied to quantitatively analyze the effects of flavonoids of Hedysarum polybotry on changes p21(Ras) and PCNA gene expressions.

RESULTSFlavonoids of Hedysarum polybotry (20-100 μg/mL) significantly inhibited the proliferation of K562 cells in a time- and dose-dependent manner. After K562 cells were cultured for 48 h, total flavonoids of Hedysarum polybotry had no significant effect on the apoptosis of K562 cells but showed significantly inhibition (P<0.01), indicating that total flavonoids of Hedysarum polybotry could induce K562 cells arrested at G(0)/G(1) and G(2)/M phases. Compared with the control group, p21(Ras) and PCNA gene expressions were decreased significantly in K562 cells treated with total flavonoids of Hedysarum polybotry (40 and 80 μg/mL, respectively) for 48 h.

CONCLUSIONThe inhibitory effect on proliferation of K562 cells was observed in the groups treated with flavonoids of Hedysarum polybotry, which might be related to cells arresting.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Flavonoids ; pharmacology ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; K562 Cells ; Leukemia, Erythroblastic, Acute ; drug therapy ; genetics ; pathology ; Oncogene Protein p21(ras) ; genetics ; Proliferating Cell Nuclear Antigen ; genetics ; Ranunculaceae ; chemistry

OBJECTIVETo investigate the phosphorylation of KCNE2 protein in heart of old SHR rats.

METHODSThe membrane proteins from ventricular myocardium of old SHR were extracted, treated with or without alkaline phosphatase and tested binding with Ab2 (an anti-KCNE2 polyclonal antibody) by Western blot. A KCNE2 fusion protein with c-myc was obtained from in vitro translation system and treated with or without alkaline phosphatase. A series of mono- and double-point mutated fusion KCNE2 proteins with c-myc were obtained from an in vitro translation system, and Western blots with Ab2 or anti-myc antibody were performed.

RESULTSAfter alkaline phosphatase treatment, Ab2 significantly attenuated its binding with KCNE2. In vitro translation system confirmed that after alkaline phosphatase treatment, Ab2 weakened binding ability to KCNE2 while binding to c-myc was not changed. Point mutation experiments showed that serine residue in position 98 of KCNE2 proteins might be phosphorylated.

CONCLUSIONKCNE2 protein in heart of old SHR rats is phosphorylated and this phosphorylation takes place in serine residue of position 98.

Aging ; Animals ; Blotting, Western ; Hypertension ; genetics ; metabolism ; Myocardium ; metabolism ; Phosphorylation ; Point Mutation ; Potassium Channels, Voltage-Gated ; genetics ; metabolism ; Protein Binding ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo study the quality difference among Citrus grandis 'Tomentosas' of different cultivars, in order to provide scientific basis for seeking for fine breeds.

METHODThe HPLC fingerprints were established for C. grandis 'Tomentosa' of different cultivars in the GAP base of Huazhou Green Life Co., Ltd. The software of similarity evaluation system of traditional Chinese medicine HPLC fingerprints 2004A edition of Chinese Pharmacopoeia Commission was adopted for the similarity analysis and judgment of cultivars.

RESULTThe fingerprints showed similar general characteristics of samples of different cultivars. Specifically, the similarity of areas of the 18 common peaks ranged between 0.938-0.998. The success rate of judging cultivars using similarity software stood at 92%.

CONCLUSIONThis method can be applied to better identify quality and source of cultivars of C. grandis 'Tomentosa', and provide technical measures and scientific basis for seeking for fine breeds of Citri Grandis Exocarpium.

China ; Chromatography, High Pressure Liquid ; methods ; Citrus ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; standards ; Quality Control

OBJECTIVETo study the inhibitory effect of paeoniflorin (PAE) on TNF-α-induced TNF receptor type I (TNFR1)-mediated signaling pathway in mouse renal arterial endothelial cells (AECs) and to explore its underlying molecular mechanisms.

METHODSMouse AECs were cultured in vitro and then they were treated by different concentrations PAE or TNF-α for various time periods. Expression levels of intercellular cell adhesion molecule-1 (ICAM-1) were detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 6-h TNF-α 30 ng/mL), the low dose PAE group (cultured by 2-h PAE 0.8 μmo/L plus 6-h TNF-α 30 ng/mL), the middle dose PAE group (cultured by 2-h PAE 8 μmol/L plus 6-h TNF-α 30 ng/mL), the high dose PAE group (cultured by 2-h PAE 80 μmol/L plus 6-h TNF-α 30 ng/mL) with Western blot analysis. Nuclear translocation of transcription factor NF-κB (NE-κB) was detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 45-mm TNF-α 30 ng/mL), and the high dose PAE group (cultured by 2-h PAE 80 μmol/L plus 45-min TNF-α 30 ng/mL) by immunofluorescent staining. Expression levels of the phosphorylation of extracellular signal-regulated (protein) kinase (ph-ERK) and p38 (ph- p38) were detected in the normal group (cultured by serum-free culture media) and the high dose PAE group (2-h PAE 80 μmol/L culture) by Western blot. NF-κB inhibitor-α (IκBα) protein expressions were detected in the normal group (cultured by serum-free culture media), the TNF-α group (cultured by 2-h serum-free culture media plus 30-min TNF-α 30 ng/mL), the high dose PAE group (cultured by 2-h PAE 80 μmol/L plus 30-min TNF-α 30 ng/mL), the p38 inhibitor group (SB group, pretreatment with SB238025 25 μmol/L for 30 min, then treated by PAE 80 μmol/L for 2 h, and finally treated by TNF-α 30 ng/mL for 30 min), the ERK inhibitor group (PD group, treated by PD98059 50 μmol/L for 30 min, then treated by PAE 80 μmol/L for 2 h, and finally treated by TNF-α 30 ng/mL for 30 min) by Western blot.

RESULTSCompared with the normal group, ICAM-1 protein expression levels obviously increased (P < 0.01). Compared with the TNFα group, ICAM-1 protein expression levels were obviously inhibited in the high dose PAE group (P < 0.05). Protein expression levels of ph-p38 and ph-ERK were obviously higher in the hIgh dose PAE group (P < 0.05). Compared with the normal group, IκBα protein expression levels obviously decreased in the TNF-α group (P < 0.01). Compared with the TNFα group, TNF-α-induced IκBα degradation could be significantly inhibited in the high dose PAE group (P < 0.01); the inhibition of PAE on IκBα degradation could be significantly inhibited in the SB group (P < 0.05). NF-κB/p65 signal was mainly located in cytoplasm in the normal group. NF-κB/p65 was translocated from cytoplasm to nucleus after stimulated by 45 min TNF-α in the TNF-α group, while it could be significantly inhibited in the high dose PAE group.

CONCLUSIONSPAE inhibited TNF-α-induced expression of lCAM-1. Its action might be associated with inhibiting TNFR1/NF-κB signaling pathway. p38 participated and mediated these actions.

Animals ; Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; Glucosides ; pharmacology ; Intercellular Adhesion Molecule-1 ; metabolism ; Mice ; Monoterpenes ; pharmacology ; NF-kappa B ; metabolism ; Receptors, Tumor Necrosis Factor ; metabolism ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; pharmacology

Objective To observe the clinical efficacy and safety of paclitaxel injection combined with nedaplatin injection in the treatment of advanced recurrent metastatic cervical cancer.Methods Forty-five patients with advanced recurrent metastatic cervical cancer were given intravenous infusion of 150 mg · m-2 paclitaxel,day 1 + 80 mg · m-2 nedaplatin,day 1.The curative effect,adverse drug reactions and prognosis were observed.Results After treatment,complete remission was 3 cases (6.67％),partial response was 21 cases (46.67％),stable disease was 10 cases (22.22％),progress disease was 11 cases (24.44％),objective response rate was 53.33％ (24/45 cases).The clinical efficacy of time since the last chemotherapy (＞ 12 months or ≤ 12 months) were 20.00％ (3/15 cases) and 80.95％ (17/21 cases) with significant difference (P ＜ 0.05).Median overall survival time was 13.90 months with 42 patients died.Cox single regression showed that there were no significant differences between patients in terms of chemotherapy or not,and median overall survival rate since the last chemotherapy (P ＞0.05).The adverse drug reactions were based on bone marrow suppression,gastrointestinal reactions,fatigue and peripheral nerve sensory abnormalities.Conclusion Paclitaxel injection combined with nedaplatin injection has a definitive clinical efficacy and safety for the treatment of metastatic cervical cancer with good prognosis.

OBJECTIVETo evaluate the effects of rapamycin (RPM)-loaded poly (lactic-co- glycolic) acid (PLGA) nanoparticles (NPs) on the proliferation, distribution of cell cycle, and expression of p27 protein in human umbilical arterial vascular smooth muscle cell (HUASMC) in vitro.

METHODSThe primarily culture model of HUASMC was successfully established by explant-attached method in vitro. The cells were administrated with different doses of RPM, and RPM-PLGA NPs were observed as treat groups compared with PLGA NPs and M231-SMGs medium cultured group. The effect of RPM-PLGA NPs on proliferation of HUASMC was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetry method. The influences of RPM-PLGA NPs on the cell cycle and cellular growth kinetics of HUASMCs were tested by flow cytometry. The effect of RPM-PLGA NPs on the expression of p27 protein of HUASMCs was assessed through an immunohistochemical method.

RESULTSCompared with the control group, the proliferation of HUASMCs was inhibited by 50 microg/L and higher concentration of RPM-PLGA NPs in a dose-dependent manner (P < 0.05). The numbers of cells entering cell cycle of S/G2/M phases were significantly lower in RPM-PLGA NPs and RPM treated groups. Histologically, the expression of p27 were up-regulated in 500 microg/L RPM-PLGA NPs and 100 microg/L RPM treated group (all P < 0.01 ) when compared with the control group.

CONCLUSIONSRPM-PLGA NPs has a similar effects as RPM in inhibiting the growth of in vitro cultured HUASMC. It can remarkably suppress the expression of in vitro cultured HUASMC p27 protein, arrest its cell cycle at G1/S phase, and inhibit its proliferation.

Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Drug Carriers ; Humans ; Lactic Acid ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Nanoparticles ; Polyglycolic Acid ; Sirolimus ; administration & dosage ; pharmacology ; Umbilical Arteries ; cytology

BACKGROUND: Long-term complications of cesarean section include placenta praevia, placenta accreta, cesarean scar pregnancy and uterine rupture. These life-threatening complications in pregnant women maybe result from the defects of endometrium and uterine smooth muscle as well as poorly formed decidua in the scar of cesarean section. Mesenchymal stem cells have the function of repairing tissue injuries, and the amount of cells homing to the site of injury may affect the effect of tissue repair. OBJECTIVE: To explore the distribution and homing of bone marrow mesenchymal stem cells from male rats into rat models of cesarean section. METHODS: Bone marrow mesenchymal stem cells isolated from male rats in vitro were cultured and identified. Female Wistar rats were randomized into two groups: cesarean section group and control group. Rats in the cesarean section group were given intravenous administration of bone marrow mesenchymal stem cells from male rats via the tail vein on day 21 after cesarean section, and non-operative rats in the control groups were given the same amount of bone marrow mesenchymal stem cells from male rats after natural delivery. Rats in the two groups were sacrificed on days 7 and 28 after cell injection. The distribution of bone marrow mesenchymal stem cells from male rats in tissues (including heart, lungs, livers, kidneys, and uterine scar) was detected by measurement of the SRY mRNA level using SPY-PCR. RESULTS AND CONCLUSION: In the cesarean section group, the SRY gene was most abundant in the lung, followed by the liver and the kidney on day 7 after injection, although the distribution of SRY gene in the heart and uterine scar was low; on day 28 after injection, the levels of SRY gene in the lung, liver and kidney decreased (P < 0.05), but had no significant changes in the heart and uterine scar (P > 0.05). In the control group, the distribution of SRY gene was similar to that in the cesarean section group on both days 7 and 28 after injection, and the levels of SRY gene in the heart and uterus were low. These findings reveal that allogeneic bone marrow mesenchymal stem cells implanted mainly distribute in tissues with abundant blood flow, including lungs, livers and kidneys. And the cell number decreases gradually over time. Since the amount of implanted cells in the heart and uterus is very low, the change with time is not obvious. Cesarean section injury has no impact on the distribution of bone marrow mesenchymal stem cells in pregnant rats and there is of course no increase in the homing and colonization of bone marrow mesenchymal stem cells in a cesarean scar.

OBJECTIVETo identify differentially expressed genes related to asthma by using a rat model.

METHODSTotal RNA extracted from the asthmatic rats was taken as the tester and the total RNA from the control rats as the driver. Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pGEM-T Easy vector to establish the subtractive library. The library was amplified through E.coli transformation and positive clones of the transformants were screened. The white clones in selective medium from cDNA library were isolated and digested by EcoR I restriction endonuclease. Thirty-six positive clones were chosen randomly and sequenced. Nucleic acid similarity was subsequently analyzed by comparing with the data from GenBank (NCBI).

RESULTSThere were more than 300 white clones in the cDNA library. The clones were sequenced and similarity search (http://www.ncbi.hlm.nih.gov/BLAST) revealed 4 known genes, 2 ESTs without homologous genes and 3 potential new gene fragments.

CONCLUSIONThe forward-subtracted cDNA library for differentially expressed in the lung of asthmatic rats has been successfully constructed and the interesting candidate genes related to asthma have been identified.

Animals ; Asthma ; genetics ; DNA, Complementary ; genetics ; Female ; Gene Expression Profiling ; Gene Expression Regulation ; Gene Library ; Male ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; Rats ; Rats, Inbred Strains