ObjectiveTo find out if the immune system derived thyroid stimulating hormone(TSH) β splice variant(TSHβ-Ⅴ) would be regulated by circulating thyroid hormone levels to get a further understanding of the function and mechanism of this TSHβ-Ⅴ in thyroid homeostasis.MethodsA total of 20 weaning Balb/c mice (half male and half female) were selected and randomly divided into two groups according to their body mass and gender(n =10).Mice of control group were fed with common diet and deionized water.Mice of the low-iodine(LI) group were fed with low-iodine diet(containing iodine 20 - 40 μg/kg,iodine-intake about 0.25 μg/d) and deionized water.The experimental period was 3 months.At the end of the experiment,mice were executed and the blood was collected to observe the levels of TSH and thyroid hormone by chemiluminescence immunoassay (CIA) ; bone marrow (BM),peripheral blood(PBL),thyroid gland and pituitary were collected to assay the TSHβ-Ⅴ mRNA expression by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR).ResultsThe serum free thyroxine(FT4) and total thyroxine(TT4) levels in LI group of mice[(0.47 ± 0.70)nmol/L,(2.41 ± 0.28)pmol/L] were significantly lower than that of the control group of mice [(55.2 ± 3.68) nmol/L, (32.72 ± 1.02) pmol/L,t =43.81,86.04 、all P ＜ 0.01 ] and the serum total triiodothyronine(TT3) and free triiodothyronine(FT3) reduction in LI group of mice[ (0.76 ± 0.08)nmol/L,(4.01 ± 0.40)pmol/L] were significantly lower than that of the control group of mice [ (1.10 ± 0.06)nmol/L,(5.40 ± 0.38)pmol/L,t =9.81,7.5 1,P ＜ 0.01 ].Iodine insufficiency strongly elevated the serum TSH in LI group of mice[ (35.67 ± 17.39)mU/L] than that in control group of mice[ (0.24 ± 0.10)mU/L,t =- 6.11,P ＜ 0.01 ].The mRNA levels of TSH β-Ⅴ in BM (9.62 ± 0.60) and in PBL( 9.25 ± 0.83 ) of LI group of mice were lower than those in control group of mice (7.69 ± 0.36,7.11 ± 0.41,t =6.77,5.64,P ＜ 0.01),while the mRNA level of TSH β-Ⅴ in pituitary of LI group of mice (1.99 ± 0.61) was increased compared with that in control group of mice (5.75 ± 0.98,t =- 8.02,P＜ 0.01).Compared with control group of mice(9.12 ± 0.62),the level of thyroid TSH β-Ⅴ mRNA in LI group of mice (9.32 ± 0.91 ) was not significantly changed (t =0.45,P ＞ 0.05).There was no detectable native TSHβ in BM,PBL and thyroid.The mRNA level of native TSHβ in pituitary in LI group of mice( - 7.17 ± 1.78) was dramatically elevated compared to that in control group of mice( - 1.43 ± 0.51,t =- 7.60,P ＜ 0.01 ).ConclusionsThe mRNA levels of TSHβ-Ⅴ are suppressed in BM and PBL in low iodinediet induced hypothyroidism mice,which suggest that immune system derived TSHβ-Ⅴ may be more important thannative TSHβ in immune-thyroid regulation.

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.

Using whole-cell patch clamp technique this study investigated the effects of adenosine (Ado) on action potential, L-type calcium current (I(Ca.L)), delayed afterdepolarizations (DADs), and transient inward current (I(ti)) induced by isoproterenol (Iso) in guinea pig isolated single ventricular myocytes. The results showed: (1) Ado alone had no significant direct effects on action potential and I(Ca.L) in guinea pig ventricular myocytes at 20-100 micromol/L. However, Ado significantly attenuated the prolongation of action potential duration (APD) and the increase of the peak amplitude of I(Ca.L) induced by Iso. Iso (10 nmol/L) markedly increased APD(50) and APD(90) from 340+/-21 ms to 486+/-28 ms and from 361+/-17 ms to 501+/-29 ms, respectively (P<0.01), and increased the amplitude of I(Ca.L) from 6.53+/-1.4 pA/pF to 18.28+/-2.4 pA/pF (P<0.01). The peak potential of current-potential relationship shifted to the left. Ado (50 micromol/L) abbreviated APD(50), APD(90) to 403+/-19 ms and 419+/-26 ms (P<0.01), and decreased the peak amplitude of I(Ca.L) to 10.2+/-1.5 pA/pF (P<0.01 vs Iso), but did not change resting membrane potential (RMP), action potential amplitude (APA), and overshoot (OS). (2) Iso (30 nmol/L) reproducibly elicited DADs with 100% incidence of DADs under this condition. Ado (50 micromol/L) completely inhibited Iso from inducing DADs. Iso (30 nmol/L) elicited I(ti) with 2-second depolarizing voltage-clamp pulses rising to +20 mV from a holding potential of -40 mV, the incidence of I(ti) being 100%, and the I(ti) was suppressed in the presence of Ado (50 micromol/L) with the incidence of I(ti) decreased to 14.3% (P<0.05). These data indicate that Ado antagonizes the stimulatory effect of Iso, and that the antiarrhythmic mechanism of Ado preventing Iso-induced DADs is due to the inhibition of intracellular Ca(2+) overload through attenuating the prolongation of APD, the enhance of I(Ca.L) and I(ti).
Action Potentials ; drug effects ; Adenosine ; pharmacology ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; Arrhythmias, Cardiac ; physiopathology ; Calcium Channels, L-Type ; drug effects ; Female ; Guinea Pigs ; Heart Ventricles ; cytology ; Isoproterenol ; antagonists & inhibitors ; Male ; Myocytes, Cardiac ; physiology ; Patch-Clamp Techniques

The purpose of this study was to investigate the different effects of ACh on the action potential and force contraction in guinea pig atrial and ventricular myocardium by using standard microelectrodes and force transducer. The results showed that the duration of the action potential (APD) of atrial myocardium was shortened from 208.57+/-36.05 to 101.78+/-14.41 ms (n=6, P<0.01), and the APD of the ventricular myocardium was shortened from 286.73+/-36.11 to 265.16+/-30.06 ms (n=6, P<0.01).The amplitude of the action potential (APA) of the atrial myocardium was decreased from 88.00+/-9.35 to 62.62+/-20.50 mV (n=6, P<0.01), while the APA of the ventricular myocardium did not change significantly.The force contraction of atrial myocardium was inhibited completely (n=6, P<0.01), while the force contraction of ventricular myocardium was inhibited by 37.57+/-2.58% (n=6, P<0.01). The ACh effects correlated with its concentration. The K(D) of the APD shortening effects in the atrial and ventricular myocardium were 0.275 and 0.575 micromol/L. The K(D) of the negative inotropic in the atrial and ventricular myocardium were 0.135 and 0.676 micromol/L, respectively. The corresponding data points were compared using t test between the atrial and ventricular myocardium, and the differences were significant when the ACh concentration was above 10 nmol/L. Furthermore, atropine (10 micromol/L) and CsCl (20 mmol/L) blocked the effects of 10 micromol/L ACh on the APD of ventricular myocardium, while CdCl2 (0.1 mmol/L) had no influence on these effects. In conclusion, ACh could shorten the action potential duration and inhibit the force contraction of atrial and ventricular myocardium in a concentration-dependent manner. There are differences in the effects of ACh on the atrial and ventricular myocardium. The atrial myocardium is more sensitive to ACh than the ventricular myocardium. It is probable that the muscarinic receptor and the potassium channel, but not the calcium channel, are involved in the ACh-induced shortening of the ventricular APD.
Acetylcholine ; pharmacology ; Action Potentials ; drug effects ; Animals ; Calcium Channels ; metabolism ; Guinea Pigs ; Heart Atria ; drug effects ; Heart Ventricles ; drug effects ; Microelectrodes ; Potassium Channels ; metabolism ; Receptors, Muscarinic ; metabolism

AIMTo observe the regulatory volume decrease (RVD) process of human intestine cells and investigate its ion channel mechanism.

METHODSCultured human intestine cells were exposed to hypotonic solution and the cell volume was measured using Coulter Counter System. RT-PCR was explored to detect the mRNA expression of Ca(2+) -activated K+ channel.

RESULTSHuman intestine cells showed a RVD process and this process could be blocked by Cl- channel blocker NPPB and K+ channel blocker TEA. Further results demonstrated the subtype of K+ channel involved in RVD was an intermediate-conductance, Ca(2+) -activated K+ channel (IK) because of its high sensitivity to clotrimazole. RT-PCR results also showed the expression of IK in this cell line.

CONCLUSIONThe RVD process of intestine cell was dependent on the parallel activation of Cl- channel and K+ channel. The subtype of K+ channel in volved in the RVD process was IK channel.

Cell Line ; Cell Size ; drug effects ; Chloride Channels ; antagonists & inhibitors ; metabolism ; Epithelial Cells ; cytology ; Humans ; Hypotonic Solutions ; Intestine, Small ; cytology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; metabolism ; Potassium Channels, Calcium-Activated ; metabolism

This study is to establish an HPLC-DAD-ELSD method for simultaneous determination of 5 flavones and saponins in Rhizoma Anemarrhenae including neo-mangiferin, mangiferin, timosaponin B II, timosaponin B III and timosaponin A III. Samples were analyzed on a Merck Purospher STAR column(4.6 mm x 250 mm, 5 μm). The mobile phase consisted of acetonitrile( A) and 0. 1% formic acid (B) with gradient elution at a flow rate of 1.0 mL · min(-1). The column temperature was set at 40 °C. The DAD detector wavelength was set at 254 nm. The ELSD conditions were as follows: the nebulizing gas flow rate was 2.0 L · min(-1) and temperature of drift tube was 105 °C. The volume was 10 μL. The five compounds were well separated with good linear correlations. The mean recoveries were between 102.0%-104.0%. This method was quick and reliable which provides a foundation for quality control of R. Anemarrhenae.
Anemarrhena ; chemistry ; Chromatography, High Pressure Liquid ; instrumentation ; methods ; Drugs, Chinese Herbal ; analysis ; Flavones ; analysis ; Rhizome ; chemistry ; Saponins ; analysis

Obecfive To study the clinical significance of detecting serum proeollagen type Ⅲ(PCⅢ) and hyaluronie acid(HA)in patients with autoimmune thyroid diseases(AITD).Methods According to the thyroid function,the 114 patients with AITD were divided into hyperthyroidism group(38),hypothyroidism group(35),and sub-hypothyroidism group(41).In addition,40 healthy persons were served as controls.The level of serum PCⅢ was determined with ELISA and that of serum HA with RIA.The level of FT3,FT4 and sTSH were detected by immumnofluorometric assay.Results Serum FT3(18.35±6.19)pmol/L]and FT4[(76.28±23.49)pmol/L]level of patients with hyperthyroidism were obviously higher than those of the controls[(4.75±0.31),(16.12±3.27) pmol/L],but serum sTSH[(0.15±0.07)mU/L]was obviously lower than that of the control[(3.78±0.15)mU/L],the differences were statically significant(P＜0.01).Serum FT3[(3.36±0.26)pmol/L]and FT4 [(6.37±2.19) pmol/L]level of patients with hypothyroidism were both lower than those of the controls(P＜0.05).but serum sTSH[(44.58±13.29)mU/L]was obviously higher than that of the control(P＜0.01).Serum FT3 [(4.86±0.45)pmol/L]and FT4[(15.26±2.78)pmol/L]level of patients with sub-hypothyroidism had no statistical difference compared with those of the controls(P＞0.05),but serum sTSH[(14.26±4.73)mU/L] was obviously higher than that of the controls(P＜0.01).The level of sernm PCⅢ[(4.63±1.22)μg/L]in pafients with hyperthyroidism was significantly higher than that of any other group(P＜0.05).There waB no statistical significant difference in PCⅢ among the patients with hypothyroidism,the patients with sub-hypothyroidism and controls [(3.64±1.12),(3.54±1.17)and(3.56±1.07)μg/L],respectively(P＞0.05).The level of serum HA [(31.13±10.28)μg/L]in patients with hypothyroidism was significantly higher than that of any other group(P＜0.05).There was no statistical significant difference in HA among the patients with hyperthyroidism,the patients with sub-hypothyroidism and controls[(22.24±7.22),(22.43±7.99)and(23.09±9.19)μg/L,respectively,P＞0.05].Conclusions It is very significant to understand myocardial fibrosis early through detecting sernm PCⅢ in patients with hyperthyroidism.Measurement of serum PCⅢ and HA will be useful to discovery hepatic fibrosisearly in patients with a long course of hyperthyroidism.

The fruit of Lycium ruthenicum is a common folk medicine in China. Now it is popular for its antioxidative effect and other medical functions. The adulterants of the herb confuse consumers. In order to identify a new adulterant of L. ruthenicum, a research was performed based on NCBI Nucleotide Database ITS Sequence, combined analysis of the origin and morphology of the adulterant to traceable varieties. Total genomic DNA was isolated from the materials, and nuclear DNA ITS sequences were amplified and sequenced; DNA fragments were collated and matched by using ContingExpress. Similarity identification of BLAST analysis was performed. Besides, the distribution of plant origin and morphology were considered to further identification and verification. Families and genera were identified by molecular identification method. The adulterant was identified as plant belonging to Berberis. Origin analysis narrowed the range of sample identification. Seven different kinds of plants in Berberis were potential sources of the sample. Adulterants variety was traced by morphological analysis. The united molecular identification-origin-morphology research proves to be a preceding way to medical herbs traceability with time-saving and economic advantages and the results showed the new adulterant of L. ruthenicum was B. kaschgarica. The main differences between B. kaschgarica and L. ruthenicum are as follows: in terms of the traits, the surface of B. kaschgarica is smooth and crispy, and that of L. ruthenicum is shrinkage, solid and hard. In microscopic characteristics, epicarp cells of B. aschgarica thickening like a string of beads, stone cells as the rectangle, and the stone cell walls of L. ruthenicum is wavy, obvious grain layer. In molecular sequences, the length of ITS sequence of B. kaschgarica is 606 bp, L. ruthenicum is 654 bp, the similarity of the two sequences is 53.32%.
Berberis ; classification ; cytology ; genetics ; China ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Drug Contamination ; Drugs, Chinese Herbal ; isolation & purification ; standards ; Lycium ; classification ; cytology ; genetics ; Medicine, Chinese Traditional ; Phylogeny ; Sequence Analysis, DNA ; Species Specificity

The gating mechanism of ClC-1 chloride channel was studied in this paper by heteroexpression of rat wild type ClC-1 gene in Xenopus oocytes and by two-electrode voltage clamping technique. The deactivation gating kinetic parameters were obtained by applying two exponential fitting of the deactivating currents at various extracellular chloride concentrations. It was found that decrease in extracellular chloride concentration increased the fractional amplitude of fast deactivating component, and depressed the fractional amplitude of slow deactivating component accompanied by a decrease in fast and slow deactivating time constants. These results demonstrate that the deactivation kinetic parameters of ClC-1 are largely dependent on the extracellular chloride concentration, which induces changes in channel gating.
Animals ; Chloride Channels ; drug effects ; physiology ; Chlorides ; pharmacology ; Electrophysiology ; Female ; In Vitro Techniques ; Ion Channel Gating ; drug effects ; physiology ; Oocytes ; physiology ; Rats ; Xenopus

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.