Objective To observe the different effects of iodine excess on inducing two strain mice thyroiditis. Methods NOD and Balb/c mice, each having 14 mice, were divided into NaI and control group. The mice were given 0.05% NaI water for 8 weeks in NaI group. RIA and ELISA were used respectively to detect TT4, TgAb, TPOAb and TSH level in serum. Morphology changes of thyroid and apoptosis of thyrocytes stained by immunohistochemistry were observed under light microscope. Lymphocytic proliferation of cervical lymph node and spleen to responding to Tg were detected by MTr method. Results After intake of iodine water for 8 weeks, NOD and Balb/c mice showed relative quality of thyroid in Nal group[(104.83±14.52), (155.79±20.77)mg/kg]obviously increased compared with control group[(71.80±20.42), (105.15±21.98)mg/kg, t values:-3.293,-4.429, all P< 0.01)], enlarged follicular lumen with colloid accumulation were observed in thyroid. Serum level of TT4 in Nal group [(29.52±4.42), (19.53± 2.35)nmol/L]to control group[33.40±5.38), (23.47±6.22)nmol/L]of NOD and Balb/c mice showed a decreasing tendency(t values: 1.374,1.567, all P > 0.05). TSH of Nal group showed an increasing tendency in Balb/c mice[(4.14±1.71)μg/L, compared with control [(3.55±1.41)μg/L, t values:-0.705, P > 0.05]and obviously increased in NOD mice [(6.98±0.66)μg/L, compared with control[(555±056)μg/L, t values:-3.562, P< 0.01], but no change of TgAb and TPOAb level in Nal group(1281,1364 cpm, 2.50×103, 0.14×103U/L were observed, compared with control(1297,1220 cpm, 3.17×103,0.03×103 U/L; Zvalues:-0.081,-0.703, -0.244,-1.293, all P > 0.05). In NOD mice NaI group, apoptosis of thyrocytes was more intense than Balb/c mice, obvious infiltration of lymphoeytes, disorganization and focus fibrosis was seen in thyroid. The cell amount of NaI group increased in NOD mice lymph node and spleen cells[(1.100±0.014), (1.076±0.033)]were more than that in the control group [(0.993±0.011), (1.005±0.003), t value:-11.672,-4.314, P < 0.01). Conclusions Iodine leads to enlargement of thyroid and malfunction of thyroid in Balb/c mice. Besides, NOD mice have generate inflammatory reaction in thyroid and produced sensitized lymphocytes to Tg. Iodine excess can induce NOD mice to occur autoimmune thyroiditis.

This study aimed to explore the impact of depression caused by chronic unpredictable mild stress (CUMS) on in vivo activity of six kinds of CYP450 isoforms in rats. According to 'Katz' method, the model of CUMS was established. Tolbutamide, chlorzoxazone, theophylline, midazolam, omeprazole and dextromethorphan were chosen as probe substrates of CYP2C6, CYP2E1, CYP1A2, CYP3A2, CYP2D1 and CYP2D2 of rats. Plasma concentration of six kinds of CYP450 in control group and model group were determined by LC-MS/MS and computed pharmacokinetic parameters. Consequently, metabolism of theophylline and chlorzoxazone accelerated significantly (P < 0.01), but tolbutamide, dextromethorphan, omeprazole and midazolam had no significant difference. The present study proved that depression caused by CUMS had strong induction to CYP1A2 and medium induction to CYP2E1.
Animals ; Chlorzoxazone ; metabolism ; Chromatography, Liquid ; Cytochrome P-450 Enzyme System ; metabolism ; Depression ; Dextromethorphan ; metabolism ; Liver ; enzymology ; Midazolam ; metabolism ; Omeprazole ; metabolism ; Rats ; Stress, Physiological ; Tandem Mass Spectrometry ; Theophylline ; metabolism ; Tolbutamide ; metabolism

This study is to establish the characteristic HPLC chromatogram of phenols in Ephedrae Herba, from which to pick out the marker peaks, followed by the analysis of the regularity of their distribution and content in the herbaceous stems of Ephedra sinica, E. intermedia and E. equisetina. The HPLC-DAD method for the characteristic chromatogram as well as quantitative analysis was established. The separation was carried out on a YMC-Pack ODS-A column (4.6 mm x 250 mm, 5 µm), eluted with the mobile phases as 0.01% formic acid aqueous solution (A) and acetonitrile (B) in a linear gradient (0-10 min, 17% B; 10-25 min, 17%-19% B; 25- 33 min, 19%-48% B; 33-35 min, 48%-51% B; 35-44 min, 51% B). The flow rate was kept at 1.0 mL · min⁻¹. The column tem- perature was 40 °C, and the detection wavelength was set at 350 nm (0-16 min) and 330 nm (16-44 min). Forty-six batches of collected samples from three official origins of Ephedrae Herba were detected, whose liquid chromatograms proven to be helpful to the differentiation of different origins. With principal component analysis and the analysis of distribution of peak area, twelve key peaks from the chromatogram were discussed in details on their contributions to the characteristics and differences of three official origins of the herb: peak area of peak 10, 11, 12 were found out to be significantly higher in E. equisetina than in other two origins, whose sum (higher than 146 mAU in E. equisetina) was useful for the discrimination between E. equisetina and the other two origins; peak area of 1 and 4 were respectively higher in E. sinica and E. intermedia than in other official origins, indicating their important effect on the differen- tiation of corresponding origins; peak 8 and 9 were picked out as two characteristic common peaks in three official origins of the herb, whose peak area showed little difference among different origins; further, peak area of other key peaks in the chromatogram also showed some difference among three origins, which make contributions to the differentiation of origins as well. Then, four phenols as 2"-O-α- L-rhamnosyl-isovitexin (1), vitexin (2), pollenitin B (5) and herbacetin-7-O-β-D-glucoside (6) were quantitative analyzed with the above-mentioned method, with good linear relationship and accuracy (recoveries in a range of 97.8%-102.5%). The content of the four phenols were firstly reported in Ephedrae Herba from official origins, which were respectively trace-1.55 (1), trace-0.160 (2), trace-0.284 (5) and trace-0.620 (6) mg · g⁻¹ in all of the tested samples. In addition, the content of these phenols showed differences in three official origins, especially 1, whose content in E. sinica [(0.670 ± 0.88) mg ± g⁻¹] were significantly higher than in other two origins (lower than 0.16 mg ± g⁻¹ besides sample Ei-060630-2-2), and 6, whose average content in E. equisetina [(0.260 ± 0.039 2) mg · g⁻¹] were twice as high as in E. sinica [(0.120 ± 0.270) mg · g⁻¹] and E. intermedia [(0.136 ± 0.485) mg g⁻¹], indicating the important effects of the two constituents on the differentiation among three official origins of the herb. The method established for the characteristic HPLC chromatogram and quantitative analysis of phenols was simple and accurate, and the marker constituents selected may provide new guides for the discrimination of official origins as well as the improvement of quality criteria of EphedraeHerba.
Chromatography, High Pressure Liquid ; methods ; Ephedra ; chemistry ; Phenols ; analysis

OBJECTIVETo construct two recombinant plasmids of mdr1 and mcl1 shRNA, and to investigate their reversal effect on drug resistance in K562 adriamycin resistant cell lines (K562/A02).

METHODSTwo oligonucleotides of mdr1 and mcl1 gene were designed referring to that of GenBank, double-stranded DNA was derived through annealing, and cloned into pRNAT vector digested by two restricted endoenzymes. K562/A02 cells were transfected with the recombinant plasmids. The mdr1 mRNA expression and its protein product P-glycoprotein (P-gp) were detected by RT-PCR and flow cytometry. The expression of mcl1 gene was detected by RT-PCR. 50% inhibition concentration (IC50) of adriamycin (ADM) on K562/A02 cells was determined by MTT method. Cells apoptosis was analyzed by flow cytometry.

RESULTSComparing with K562/A02 cells, the shRNA of mdrl or mcl1 gene in vitro can remarkably increase the sensitivity of K562/A02 to adriamycin, down-regulate mdr1 or mcl1 gene expression, increase the K562/A02 cells apoptosis rates induced by adriamycin. Cotransfection of mdrl and mcl1 genes shRNA can also down-regulate the expression of their gene, more remarkably increase the sensitivity and apoptosis of K562/ A02 to adriamycin.

CONCLUSIONTransfection of mdrl or mcl1 gene shRNA can promote the sensitivity of K562/A02 to adriamycin and cotransfection of the two shRNA can more remarkably do so. The mel1 gene might be involved in adriamycin resistant in K562/A02 cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Apoptosis ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Flow Cytometry ; Gene Expression ; Humans ; K562 Cells ; Myeloid Cell Leukemia Sequence 1 Protein ; Neoplasm Proteins ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

The aim of the present study was to investigate the alterations in thoracic aortic vasomotor function in rats with chronic heart failure (CHF) post myocardial infarction (MI), and then explored the possible mechanism of pathological changes. Male Sprague-Dawley rats were divided into sham and CHF groups randomly. The CHF model group of rats was generated by ligating the left anterior descending artery. In sham-operated rats the ligation was placed but not tightened. A total of 20 rats underwent either sham-operated (n=8) or surgery for MI (n=12). All sham-operated rats survived the surgical procedure and the post-surgical period, whereas total mortality among MI-rats was 25% (3 out of 12). Only MI-rats with infarct-size >30% of the left ventricle (LV) were included for analysis (8 out of 9). Ten weeks after surgery, rats were anaesthetized for hemodynamic measurements, which contains systolic pressure, diastolic pressure, left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LV+dp/dt(max) and LV-dp/dt(max). After that hearts were rapidly excised and weighed. Myocardial infarct size was determined by triphenyltetrazolium chloride (TTC) staining method. Isolated thoracic artery ring preparations were studied in a wire-myograph. The arterial constrictive responses to KCl, CaCl2, phenylephrine (PE), and caffeine and the arterial diastolic responses to acetylcholine (ACh) were recorded by the Multi Myograph System. To explore the possible mechanism, nitric oxide synthase (NOS) inhibitor N-nitrl-L-arginine methylester (L-NAME) and non-selective cyclooxygenase (COX) inhibitor indomethacin (Indo) were used. The results obtained were as follows: (1) CHF group showed an increased contraction response to KCl (5-100 mmol/L) and PE (1x10(-8)-3x10(-4) mol/L), and a reduced endothelium-dependent relaxation response to ACh (1x10(-12)-1x10(-4) mol/L) compared with those observed in sham group (P<0.01, P<0.05); (2) In the presence of L-NAME (1 mmol/L), the endothelium-dependent cumulative contractions to ACh (1x10(-7)-1x 10(-4) mol/L) was significantly enhanced in CHF group (P<0.05), and this effect was reversed by pretreatment with Indo (10 mumol/L); (3) In CHF group, the vessels incubated with Indo (10 mumol/L) showed an increased vasodilation induced by ACh (1x10(-12)-1x10(-4) mol/L) (P<0.05); (4) In the Ca(2+)-free K-H solution, calcium-dependent contraction curves induced by CaCl2 (1x10(-4)-3x10(-2) mol/L) in CHF group significantly shifted to the left compared with sham group (P<0.05); while the vascular contraction induced by caffeine (30 mmol/L) had no significant changes. These findings suggest that thoracic arteries of rats with CHF have endothelial dysfunction, and the contribution of endothelial dilation and contraction was significantly altered in CHF rats. The mechanism could be partly associated with the increased endothelium-dependent contracting factors by COX pathway, or the increased extracellular Ca(2+) influx through voltage-operated channels, thus leading to elevated vasoconstriction.
Animals ; Aorta, Thoracic ; physiopathology ; Chronic Disease ; Endothelins ; metabolism ; Endothelium, Vascular ; physiopathology ; Heart Failure ; etiology ; physiopathology ; Male ; Myocardial Infarction ; complications ; Prostaglandin-Endoperoxide Synthases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vasomotor System ; physiopathology

Objective To observe the effect of iodine excess(HI),polyinosinic-polycytidylic acid[Poly(I:C),Poly]and thyroglobulin(TG)on the thyroid of mice by the expression of Toll-like receptor 3(TLR3)to reveal the functional role of TLR3 in autoimmune thyroiditis.Methods Forty-two non-obese diabetic mice,body weight (20±3)g,were divided into six groups:control group,HI group,Poly group,TG group,HI+TG group,HI+Poly group. Fed with deionized water and injected intraperitoneally with physiological saline 0.1 ml each day for a week, the mice in control group were injected with physiological saline every other day at the same dose for 1 week before they were sacrificed; HI group drank 0.05% NaI water and were injected intraperitoneally with physiological saline same as control group; Poly group drank deionized water and were injected intraperitoneally with poly 0.1 ml (1 g/L)each day of the week, then the mice were injected with Poly every other day at the same dose for 1 week before they were sacrificed; TG group drank deionized water and were injected intraperitoneally with physiological saline same as control group, immunized with 0.1 mg TG by subcutaneously injecting and the immunization was enhanced after they were fed half dose for 4 and 8 weeks separately. In HI + Poly group, the treatment was the same as HI group and Poly group; HI + TG group: the treatment was the same as HI group and TG group. Eight weeks later, mice were sacrificed and thyroids were taken to make frozen sections, Hematoxylin-Eosin (HE) staining was employed to observe the morphological change of the thyroids. The expression of TLR3 of thyroids was observed under fluorescence microscope after Immumofluorescence using TLR3 antibody and TR3-positive cells were analyzed in the thyroid density. Results HE staining showed thyroids of Poly group had no inflammation under microscope.There were different degrees of inflammatory cell infiltration in HI group and TG group. The inflammatory cell infiltration and the damage of follicular thyroid of HI + TG group and HI + Poly group were serious, and the degrees of inflammation were higher over "++". Thyroid follicular epithelial cell with TLR3 expression could be seen in Poly group and HI group, meanwhile, there were TLR3 strong positive inflammatory cells in HI group under fluorescent microscope. Using stereological analysis of TLR3-positive cell density in the thyroid, the difference between groups was statistically significant(F=7.870, P<0.01 ). TLR3-positive cell density in the thyroid of HI + Poly group was higher[ (9.287 ± 0.522)mm2] than control group[ (0.062 ± 0.025)mm2, P < 0.01] significantly, meanwhile, the density in HI + Poly group was higher than HI group [ (2.574 ± 0.257 )mm2] and Poly group[ (1.361 ± 0.148 )mm2, all P < 0.01]. The density in HI + TG group[ (4.843±0.405)mm2] was higher than HI group and TG group[(1.601 ±0.268)mm2, all P < 0.01 )]. Conclusions Excessive iodine and thyroglobulin can induce thyroiditis, and stimulate the expression of TLR3 in the thyroid follicular epithelial, Poly aggravated thyroiditis induced by iodine excess in NOD mice; TLR3 positive inflammatory cells also appeared in inflammatory region, suggesting that TLR3 is involved in the pathogenesis of autoimmune thyroiditis

Objective To investigate the influence of iodine excess on expression of TRAIl/TRAIL-sR1 in NOD and Balb/c mice and to study the effect of TRAIl/TRAIL-sR1 on the pathogenesis of experimental autoimmune thyroiditis(EAT). Methods Both Balb/c and NOD mice were divided randomly into control and iodine excess group by feeding with water containing no NaI or 0.05% Nal. The mice were sacrificed after 8 weeks. TRAIL and TRAIL-sR1 mRNA levels were detected by RT-PCR. The function, morphology and apoptosis of thyroids were also observed by ELISA and Tunnel stain. Results Treated by HI, enlarged follicles and flattened epithelium by accumulation of colloid were found in thyroids of both NOD and Balb/c mice. But significant lymphoid cell infiltration and local fibrosis were only found in thyroids of NOD HI group. The relative weight of thyroids of NOD mice in HI group[(104.8±14.5)mg/kg]was heavier than that of control group [(71.8±20.4)mg/kg]. The level of TT4 declined in HI group[(30.77±3.59)mmol/L]compared with control group[(36.43±2.66)mmol/L], meanwhile, the level of TSH was higher in HI group[(6.98±0.66)μg/L]than that in control group [(5.55±0.56)μg/L]. The difference being statistically significant(t=7.773,-9.526,-4.458, all P < 0.05). The relative weight of thyroids of Balb/c mice of HI group[(155.8±20.8)mg/kg]also heavier than that of control group [(105.1±22.0) mg/kg]. The level of TT4 droped in HI group [(19.75±3.32) mmoL/L]was higher than that in control group[(23.46±6.21)mmoL/L], the level of TSH in HI group[(4.14±1.71)μg/L]was higher than that in control group[(3.55±1.41)μg/L], the difference being statistically significant(t=7.554,-7.239,3.140, all P< 0.05). A great deal of apoptotie ceils observed in NOD (3.97±0.91) and Balb/c mice (1.05±0.45) by Tunnel stain were greater than control groups (0.21±0.15, 0.10±0.03), the difference being statistically significant in beth of the two species(t=-7.167,-17.772, both P < 0.05). The apoptosis index of thyroid follicular epithelium in NOD was obviously higher than Balb/c(t=-7.625, P<0.05). The level of TRAIL mRNA did not remarkably change in Balb/c between control group(0.000 59±0.000 39) and HI group(0.001 24±0.000 46, t=-1.940, P>0.05), but it increased apparently in NOD mice HI group(0.018 88±0.005 77) than that of control group(0.009 61± 0.00591, t=-2.71, P<0.05). The level of the expression of TRAIL-sR1 mRNA increased in HI groups of NOD (0.000 53±0.000 15) and Balb/c mice(0.000 42±0.000 09) than that in control groups of NOD(0.000 28± 0.000 05) and Balb/c mice (0.000 17±0.000 06) and the differences were statistically significant between the two species(t=3.050,3.990, all P<0.05). The differences of the expression of TRAIL and TRAIL-sR1 mRNA between the two species were significant(t=-3.37,-4.76, all P<0.05). Conclusions Iodine excess induces colloid goiter in beth species of mice and thyroiditis in NOD mice. The increase of TRAIL and TRAIL-sR1 influenced by iodine excess is one of the molecular bases of follicular epithelium apoptosis and inflammation in thyroids. Genetic factor is a key factor in the pathogenesis of thyroiditis.

OBJECTIVETo evaluate the efficacy and safety of hepatitis B immunoglobulin (HBIG) by different medicating ways in patients with liver transplantation and to explore the methods for calculating the intravenous loading dosage of HBIG.

METHODSThe patients enrolled were randomized into three groups (i.v group, i.m group and domino group). Under the combined utilization with Lamivudine, HBIG was given in different ways during anhepatic phase and the postoperative six days. The physical examination was done, the serum conversion rate of HBsAg was studied, the serum level of HBsAb titer, WBC, PLT, AST, GGT, TBIL, DBIL, CR, PT and PTA were tested daily within the postoperative seven days. The preoperative body weight, serum HBsAg and HBeAg titer were analyzed with the intravenous loading dosage of HBIG by multiple-factor linear regression (Stepwise).

RESULTSBoth the average negative-conversion rate of serum HBsAg and the average increasing rate of serum HBsAb titer are significantly faster in i.v group and domino group than that in i.m group within the postoperative four days (P < 0.05). The regression equation to calculate the i.v loading dosage of HBIG (IU) by preoperative criteria was drawn as 1123 + 3.4 x serum HBsAg titer (IU/L) +73 x body weight (kg). There was no linear correlation found between the level of HBeAg and the loading dosage of HBIG. There were no significant difference in body temperature, pulse rate, respiratory rate, blood pressure, WBC, PLT, AST, GGT, TBIL, DBIL, CR, PT and PTA among the three groups within the postoperative seven days (P < 0.05). The rate of the second elevation of serum ALT was 10.3% (3/29), 3.4% (1/29) and 6.7% (2/30) in i.v group, i.m group and domino group, respectively (P < 0.05), and the rate of the local complications (sclerosis, edema, pain) at the injection site was 0, 89.6% (26/29) and 0, respectively (P < 0.05).

CONCLUSIONSBased on the combined utilization of lamivudine and HBIG, the qualified intervention efficacy, less complications could be obtained by medicating HBIG in a domino way (i.v first, followed by i.m), which is worthy to be promoted.

Alanine Transaminase ; blood ; Antiviral Agents ; administration & dosage ; therapeutic use ; Combined Modality Therapy ; Drug Therapy, Combination ; Hepatitis B ; blood ; prevention & control ; therapy ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Humans ; Immunization, Passive ; methods ; Immunoglobulins ; administration & dosage ; therapeutic use ; Lamivudine ; therapeutic use ; Linear Models ; Liver Transplantation ; Secondary Prevention ; Treatment Outcome

This study was aimed to clone mouse adam10 gene promoter and construct its dual luciferase report vector, and to investigate its transcriptional activity. Total DNA was extracted from mouse brain and used for amplifying the fragment containing adam10 gene promoter by PCR. The amplified product was inserted into pGL-4.10 vector to construct pGL4.10-adam10. The pGL4.10-adam10 and control plasmid pGL4.74 were co-transfected into HEK293 FT cells by lipofectamine 2000. The activity of adam10 gene promoter was assayed by luciferase system. The results showed that the recombinant plasmid pGL4.10-adam10 containing promoter of mouse adam10 was correctly constructed. The method was optimized by changing ratio of two plasmids. Moreover, the transcriptional activity of pGL4.10-adam10 stimulated by ionomycin increased. It is concluded that the dual luciferase reporter system is successfully established, which is useful in bioluminescence imaging technology in vitro. The effect of ionomycin can enhance the transcriptional activity of adam10 gene promoter.
ADAM Proteins ; genetics ; ADAM10 Protein ; Amyloid Precursor Protein Secretases ; genetics ; Animals ; Cell Line ; Cloning, Organism ; Genes, Reporter ; Genetic Vectors ; Luciferases ; genetics ; Membrane Proteins ; genetics ; Mice ; Mice, Inbred BALB C ; Plasmids ; Promoter Regions, Genetic

OBJECTIVETo investigate the general pattern of cholinergic nerve distribution and M(2) receptors in adult rat heart.

METHODSKarnovsky-Roots histochemical staining combining point counting method and immunochemical SABC method with image analysis were used to identify the cholinergic nerves and M(2) receptors, respectively, in adult rat heart.

RESULTSPositive staining of cholinergic nerves and M(2) receptors was found in all regions of the rat heart, and the point count of cholinergic nerves in the atria was 4.6 times as much as that in ventricles, and the area of immunoreactive substance for M(2) receptors two-fold higher in the atria than in the ventricles. The point counts of the cholinergic nerves in the medial-layer myocardium were fewer than that in subepicardial and endocardial tissues of the left ventricular free wall. However, M(2) receptors were comparable among the 3 layers of the left free ventricular wall.

CONCLUSIONCholinergic nerves and M(2) receptors are located in both rat atria and ventricles, but their density is much higher in the atria than in the ventricles. Transmural heterogeneity characterizes cholinergic nerve innervation in the left ventricular free wall without significant differences in M(2) receptor density.

Animals ; Cholinergic Fibers ; metabolism ; Female ; Heart ; innervation ; Heart Atria ; innervation ; metabolism ; Heart Ventricles ; innervation ; metabolism ; Immunohistochemistry ; Male ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Muscarinic M2 ; analysis