Objective To establish a closed-chest pulmonary embolism-reperfusion animal model by Swan-Ganz catheter and to explore the mechanisms of pulmonary embolism(PE)-reperfusion injury(RI). Methods Experiments were made on 14 mongrel dogs,ranging in weight from 15 to 18 kg,anesthetized with 3% pentobarbital sodium.The dogs were intubated with I.D.7 endotracheal tubes.Under sterile conditions,a 7 F Swan-Ganz catheter via the external jugular vein was positioned in the unilateral pulmonary diaphragmatic lobe(DL)artery.Occlusion/reperfusion of the DL artery was controlled with 1.2 ml diluted contrast agent filled into/drawn from the balloon.After the 24 h PE,the balloon was deflated to result in 4 h reperfusion of the DL.Measurements of blood gases and tumor necrosis factor-?(TNF-?)were made at normal condition,at 24 h PE and at 4 h reperfusion.Thin-section CT scans were performed at normal condition,24 h PE,30 min,1,2,3 and 4 h reperfusion,respectively.At the end of each experiment, tissue specimens of bilateral diaphragmatic lobes were obtained for both wet/dry(W/D)weight ratio and for pathological study.Results Reperfusion pulmonary edema(RPE)was an acute,mixed,noncardiogenic edema that was observed in all 14 dogs who had been successfully established as PE/RI animal models.RPE demonstrated heterogeneous ground-glass opacifications that predominated in the areas distal to the recanalized vessels.It manifested pathologically as an edematous lung infiltrated by inflammatory cells.The mean ofPaO_2 and TNF-? of 4 h reperfusion was(81?4)mm Hg(l mm Hg=0.133 kPa)and(16.0? 2.5)pg/ml,which were significantly different(P

Objective To study the effects of nuclear factor-?B(NF-?B)decoy oligodeoxynucleotide(ODN)on the preeclamptic umbilical serum induced expression of precollagen Ⅰ,Ⅲ mRNA and tumor necrosis factor-?(TNF-?)in cultured human umbilical artery smooth muscle cells (HUASMC).Methods Primary cultured HUASMC of normal pregnancy were divided into four groups: group A(HUASMC were incubated with umbilical serum of normal pregnancy);group B(HUASMC were incubated with umbilical serum of preeclampsia);group C(HUASMC were transfected with NF-?B cis decoy ODN 48 h before incubation with umbilical serum of preeclampsia);group D(HUASMC were transfected with NF-?B scramble ODN 24 h before incubation with umbilical serum of preeclampsia).NF-?B cis decoy ODN and NF-?B scramble ODN were transfected with cationic lipofectamine to the latter two groups,respectively.The proliferation of human umbilical artery smooth muscle cells was evaluated by methyl thiazolyl tetrazolium and the apoptosis was analyzed by flow cytometry.The expression levels of precollagen Ⅰ,Ⅲ mRNA were detected by RT-PCR,the expression levels of TNF-? were detected by western blot.Results(1)The proliferation of group B(0.19?0.02)and group D(0.18?0.03)was significantly increased as compared with those of group A(0.11?0.02)and group C(0.14?20.02)(P0.05).(5)The expression of TNF-? of group B(0.74?0.11),group C(0.36?0.09)and group D(0.79?0.12)were significantly higher than that of group A(0.15?0.03)(P0.05).Conclusions NF-?B cis decoy ODN could down-regulate the proliferation,as well as the expression levels of precollagen and TNF-? of HUASMC induced by umbilical serum of preeclampsia.NF-?B may play an important role in the pathogenesis of placental artery abnormalities in preeclampsia.

Day-old chick is unique animal model in brain development and behavior study. The intermediate medial mesopallium (IMM), a region of the chick forebrain, is intimately involved in the early learning processes, which offers the ideal opportunity to study the neural changes that underlie behavioral plasticity. In this paper, the intracellular recordings were conducted from IMM neurons in chick forebrain slices, in which electrophysiological properties, synaptic responses and long-term potentiation (LTP) were observed. Coronal sections of left forebrains (500 mum thick), containing IMM, were prepared from domestic chicks, aged 2-10 days. In 69 IMM neurons, the resting membrane potential was measured to be (-59.4+/-5.3) mV, slope membrane resistance (70.8+/-27.2) MΩ, and time constant (10.2+/-4.3) ms. The amplitude, threshold, overshoot, half-width, max rise slope and max decay slope of action potential evoked by intracellular current injection were (85.2+/-9.4) mV, (-38.7+/-7.6) mV, (25.6+/-8.9) mV, (2.1+/-0.5) ms, (150.5+/-41.2) mV/ms and (-64.3+/-14.0) mV/ms, respectively. Spike-firing frequency was increased with depolarizing current intensity in 32 of 69 tested cells [linear regression slope was (21.5+/-10.9) Hz/nA, P<0.05 in all cells]. The depolarizing synaptic responses (i.e. EPSPs), with stimulus intensity- and membrane potential-dependent properties, were elicited by dorsal (n=25) or ventral (n=62) focal electrical stimuli at 0.1 Hz in all tested IMM neurons and could be nullified reversibly by perfusion with 100 mumol/L AP5 (NMDA receptor antagonist) and 3 mumol/L DNQX (non-NMDA receptor antagonist), but enlarged by 6 mumol/L bicuculline (GABA(A) receptor antagonist). The EPSPs evoked by ventral stimulation were persistently increased after tetanic stimulation (5 Hz, 300 pulses/train, 2 trains, train interval 10 min) in 6 of 12 tested IMM neurons. The amplitude of EPSPs was potentiated to more than 120% of control level (when analyzed at 45 min of enhancement, P<0.05, n=5), which lasted at least 30 min and then could be referred to as LTP. Moreover, area under curve, duration and max rise slope of EPSPs were also enhanced (P<0.05), while no significant changes were observed in the electrophysiological parameters of IMM neurons following induction of LTP (P>0.05). These results suggest that the intracellular recording techniques in the chick brain slices can be used to perform multi-parameter analysis of synaptic responses and their LTP.
Animals ; Brain ; physiology ; Chickens ; In Vitro Techniques ; Long-Term Potentiation ; Membrane Potentials ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors

OBJECTIVETo observe the effect of Shenluotong Decoction (SD) on serum levels of aldosterone, monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle protein (α-SMA), and nuclear factor-KB (NF-κB) in obstructive nephropathy rats, and to explore the initial mechanism of SD for inhibiting renal interstitial fibrosis.

METHODSTotally 48 healthy Wistar rats were randomly divided into the sham-operation group (n =12) and the model group (n =36). Renal interstitial fibrosis rat model was established by unilateral ureteral obstruction (UUO). After successful modeling, 36 rats were randomly divided into the model group, the Chinese medicine group, and the Western medicine group, 12 in each group. Eplerenone was added in the forage at the daily dose of 100 mg/kg for rats in the Western medicine group. Chinese medicine was added in the forage at the daily dose of 26 g/kg for rats in the Chinese medicine group. Equal volume of normal saline was administered to rats in the sham-operation group and the model group. All medication was performed once daily. The obstructive kidneys were extracted ten days after medication. The pathomorphological changes were observed. The contents of serum aldosterone and MCP-1, and the protein or mRNA expression of MCP-1, α-SMA, and NF-KB were detected.

RESULTSCompared with the sham-operation group, infiltration of a large amount of inflammatory cells and collagen deposition significantly increased, serum contents of aldosterone and MCP-1 obviously increased (P < 0.01), the expression of MCP-1 mRNA and protein were significantly up-regulated (P <0.01), the protein expression of α-SMA and NF-KB were significantly enhanced in the model group (P <0.01). Com- pared with the model group, infiltration of inflammatory cells and renal collagen deposition were attenua- ted in the Chinese medicine group and the Western medicine group, the serum MCP-1 level were reduced, and the mRNA and protein expression of MCP-1 were significantly down-regulated (P <0.01), the protein expression of α-SMA and NF-KB were obviously inhibited (P <0. 01). At the same time, serum aldosterone level was reduced in the Chinese medicine group (P <0.01).

CONCLUSIONSinflammatory lesions of the renal tissue could promote the progress of interstitial fibrosis in rats with obstructive nephropathy. SD could attenuate interstitial fibrosis through reducing serum contents of aldosterone and MCP-1, down-regulating MCP-1/ NF-KB, and inhibiting the expression of α-SMA.

Animals ; Chemokine CCL2 ; drug effects ; metabolism ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Kidney ; drug effects ; Kidney Diseases ; drug therapy ; genetics ; NF-kappa B ; drug effects ; metabolism ; RNA, Messenger ; biosynthesis ; Rats, Sprague-Dawley ; Ureteral Obstruction ; drug therapy ; genetics

Background::The use of microRNAs in the therapy of kidney disease is hampered by the difficulties in their effective delivery. Microvesicles (MVs) are known as natural carriers of small RNAs. Our prior research has demonstrated that MVs isolated from mesenchymal stem cells (MSCs) are capable of attenuating kidney injuries induced by unilateral ureteral obstruction and 5/6 sub-total nephrectomy in mice. The present study aimed to evaluate the effects of miR-34a-5p (miR-34a)-modified MSC-MVs on transforming growth factor (TGF)-β1-induced fibrosis and apoptosis in vitro. Methods::Bone marrow MSCs were modified by lentiviruses over-expressing miR-34a, from which MVs were collected for the treatment of human Kidney-2 (HK-2) renal tubular cells exposed to TGF-β1 (6 ng/mL). The survival of HK-2 cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Annexin V-Light 650/propidium iodide (PI) assays. The expression levels of epithelial markers (tight junction protein 1 [TJP1] and E-cadherin) and mesenchymal markers (smooth muscle actin alpha (α-SMA) and fibronectin) in HK-2 cells were measured using Western blot analysis and an immunofluorescence assay. In addition, changes in Notch-1/Jagged-1 signaling were analyzed using Western blotting. Data were analyzed using a Student’s t test or one-way analysis of variance. Results::MiR-34a expression increased three-fold in MVs generated by miR-34a-modified MSCs compared with that expressed in control MVs ( P < 0.01, t= 16.55). In HK-2 cells, TJP1 and E-cadherin levels decreased to 31% and 37% after treatment with TGF-β1, respectively, and were restored to 62% and 70% by miR-34a-enriched MSC-MVs, respectively. The expression of α-SMA and fibronectin increased by 3.9- and 5.0-fold following TGF-β1 treatment, and decreased to 2.0- and 1.7-fold after treatment of HK-2 cells with miR-34a-enriched MSC-MVs. The effects of miR-34a-enriched MSC-MVs on epithelial-mesenchymal transition (EMT) markers were stronger than control MSC-MVs. The effects of miR-34a-enriched MSC-MVs on these EMT markers were stronger than control MSC-MVs. Notch-1 receptor and Jagged-1 ligand, two major molecules of Notch signaling pathway, are predicted targets of miR-34a. It was further observed that elevation of Notch-1 and Jagged-1 induced by TGF-β1 was inhibited by miR-34a-enriched MSC-MVs. In addition, TGF-β1 exposure also induced apoptosis in HK-2 cells. Although miR-34a-mofidied MSC-MVs were able to inhibit TGF-β1-triggered apoptosis in HK-2 cells, the effects were less significant than control MSC-MVs (control:TGF-β1 :miR-nc-MV:miR-34a-MV = 1.3:0.6:1.1:0.9 for MTT assay, 1.8%:23.3%:9.4%:17.4% for apoptosis assay). This phenomenon may be the result of the pro-apoptotic effects of miR-34a. Conclusions::The present study demonstrated that miR-34a-over-expressing MSC-MVs inhibit EMT induced by pro-fibrotic TGF-β1 in renal tubular epithelial cells, possibly through inhibition of the Jagged-1/Notch-1 pathway. Genetic modification of MSC-MVs with an anti-fibrotic molecule may represent a novel strategy for the treatment of renal injuries.

OBJECTIVETo characterize molecular and cytogenetic abnormalities in six 46, XX males, and to investigate the clinical manifestations and underlying mechanisms in such patients.

METHODSClinical data of six XX male patients were collected. Karyotyping, multiple polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) were utilized to detect and locate the sex determining region (SRY) gene.

RESULTSPCR and FISH showed that all patients were SRY-positive XX males. All patients have their SRY gene located at the tip of derivative X chromosomes, which have resulted from translocation between short arms of X and Y chromosomes. High resolution karyotyping at 550-750 band level has revealed that the translocation breakpoints were at Xp22.33 and Yp11.2 in three patients. In the remaining patients, the breakpoints were either at Xp22.32 and Yp11.31 or Xp22.31 and Yp11.2. The breakpoints at Xp22.32, Xp22.31 and Yp11.31 were rarely reported. Genotype-phenotype correlation analysis indicated that the clinical manifestations were age-specific. Four adult patients have come to clinical attention due to infertility, with typical features including azoospermia and testis dysgenesis, whereas poorly developed secondary sexual characteristics and short stature were main complaints of adolescence patients, and short stature was the sole symptom in a child patient.

CONCLUSIONCombined karyotyping, PCR and FISH are important for the analysis of XX males. Particularly, high resolution karyotyping is valuable for the refinement of chromosome breakpoints and detailed analysis of genotype-phenotype correlation.

46, XX Disorders of Sex Development ; genetics ; Adolescent ; Adult ; Child, Preschool ; Chromosomes, Human, X ; Chromosomes, Human, Y ; Genetic Association Studies ; methods ; Humans ; Karyotyping ; methods ; Male ; Sex Chromosome Aberrations ; Translocation, Genetic ; Young Adult

Bovine adrenal medulla 22 (BAM22), an endogenous opioid peptide, is one of the cleavage products of proenkephalin A. It potently activates opioid receptors and sensory neuron-specific receptor (SNSR). The present study was aimed at investigating the effect of BAM22 on morphine tolerance. Intrathecal (i.t.) administration of morphine for 7 d produced morphine tolerance in rats. Then the rats were divided into three groups in which morphine, saline or BAM22 were administered i.t., respectively, on day 8, and morphine was given to all of the animals on day 9. It was found that morphine administered on day 9 resumed antinociceptive effects in BAM22 group, but not in saline or morphine group. The potency of morphine in BAM22 group was 48.5% of the maximal possible effect (MPE) detected by paw withdrawal test and the antinociception persisted for approximately 1 h. Following the similar treatment, morphine administered on day 9 reduced nocifensive behaviors by 3.2 min and 24 min in BAM22 group in the first and second phases, in the formalin test, respectively. The decreases were 45% and 82% of the corresponding values observed in saline group. Furthermore, following the treatment with BAM22 (10 nmol) on day 8 in morphine-tolerance rats, morphine administered on day 9 decreased the expressions of the heat-evoked c-Fos-like immunoreactivity (FLI) protein by approximately 80% in laminae I-II, III-IV and V-VI in the spinal cord at L4-L5 compared with that in saline or morphine group. The present study provided evidence at behavioral and cellular levels showing that BAM22 resumed antinociception of morphine. The results that the reversal effect of BAM22 on morphine tolerance was more efficient in persistent pain model than in acute pain may indicate that BAM22 differentially modulates morphine tolerance. The present study suggests that SNSR is involved in the modulation of morphine tolerance.
Animals ; Drug Tolerance ; Enkephalins ; pharmacology ; Morphine ; pharmacology ; Pain ; drug therapy ; Peptide Fragments ; pharmacology ; Rats ; Receptors, G-Protein-Coupled ; metabolism

OBJECTIVESTo investigate the protective effects of Sapindus saponins in spontaneously hypertensive rats, and the possible cellular and molecular mechanisms.

METHODSThirty-two 16-week-old spontaneously hypertensive rats were randomly divided into four groups (8 in each group): model group (placebo), positive control group (27 mg/kg of Captopril Tablets), Sapindus saponins groups (27 mg/kg and 108 mg/kg, respectively). Another 8 healthy Wistar-Kyoto strain (WKY) rats were used as the normal group. The animals were treated for 8 weeks. Blood pressure of rats was determined by non-invasive blood pressure meter (BP-6). Furthermore, the contents of angiotensin II (Ang II) in plasma and myocardial tissue were determined by enzyme-linked immunosorbent assay (ELISA), the gene expression of receptor angiotensin type 1 (AT1R) in aorta was determined by quantitative realtime polymerase chain reaction (qRT-PCR). The protein expression of transforming growth factor-β1 (TGF-β1) and AT1R in heart was determined by immunohistochemical staining. The protein expression of p-phosphorylation of p38 mitogen-activated protein kinase (p-p38MAPK) was determined by Western blotting. The contents of interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) in serum were determined by radioimmunoassay. And the histopathological and morphological changes of aorta and heart tissue samples were assessed semi-quantitatively by hematoxylin-eosin (HE) or Masson staining.

RESULTSThirty minutes after single or continuous treatment, systolic blood pressure (SBP) was reduced significantly in Sapindus saponins groups. And the contents of AngII, IL-1, IL-6 and TNF-α in serum, the expression of AT1R mRNA, p-p38MAPK and TGF-β1 were significantly suppressed dose-dependently (P<0.05 or P<0.01). With the Sapindus saponins treatment, compared with those of the model group, the cardiac and aortic pathological changes were ameliorated significantly.

CONCLUSIONSOur findings suggest that Sapindus saponins might have protective effects in spontaneously hypertensive rats, the cellular and molecular mechanisms of which might be relevant to the regulation of inflammatory responses mediated by p-p38MAPK signal pathway based on activated Ang II and AT1R.

Angiotensin II ; metabolism ; Animals ; Aorta ; drug effects ; pathology ; physiopathology ; Blood Pressure ; drug effects ; Collagen ; metabolism ; Female ; Hypertension ; blood ; drug therapy ; enzymology ; physiopathology ; Interleukin-1 ; blood ; Interleukin-6 ; blood ; Male ; Phosphorylation ; drug effects ; Protective Agents ; pharmacology ; therapeutic use ; Rats, Inbred SHR ; Receptor, Angiotensin, Type 1 ; metabolism ; Renin-Angiotensin System ; drug effects ; Sapindus ; chemistry ; Saponins ; pharmacology ; therapeutic use ; Transforming Growth Factor beta1 ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo explore the possible mechanism of cyclovirobuxine D (CVB-D) in countering and inducing arrhythmia, by way of studying its electro-physiological effect on ventricular papillary muscles of rats in vitro.

METHODSThe transmembrane potential of rat's isolated right ventricular papillary muscles were recorded using conventional glass micro-electrode technique.

RESULTS(1) CVB-D in concentration of 13.3-63.3 micromol/L, showed prolonging effect on the action potential repolarization time, mainly the action potential duration 50 (APD50), APD70 and APD90, in dose-dependent manner, in concentration of 33.3-63.3 micromol/L, it could inhibit the resting potential, action potential amplitude (APA) and maximum depolarization velocity (Vmax) in dose-dependent manner. (2) CVB-D also showed time-dependent effect, the effect initiated 10 min after 20 micromol/L was perfused in ventricular muscle, the APD50, APD70 and APD90 were potentiated gradually along with prolongation of action time and reached the peak at 30-40 min, without any potentiation thereafter. (3) CVB-D could markedly prolong the effective refractory period (ERP) of action potential, increase the ratio of ERP/APD. (4) CVB-D in concentration of 33.3 micromol/L could induce frequent, multifocal spontaneous arrhythmia in some cells when the action time was longer than 45 min.

CONCLUSIONCVB-D has the action of anti-ventricular arrhythmia, the mechanism is correlated with the prolongation of APD and ERP of ventricular muscle as well as the increase of ERP/APD ratio, while it also has the effect of inducing arrhythmia, the mechanism might be concerned with excessive prolongation of APD and the inhibition on RP, APA and Vmax.

Action Potentials ; drug effects ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; Arrhythmias, Cardiac ; chemically induced ; physiopathology ; Drugs, Chinese Herbal ; pharmacology ; Electrophysiologic Techniques, Cardiac ; Heart Ventricles ; drug effects ; In Vitro Techniques ; Male ; Myocytes, Cardiac ; cytology ; Papillary Muscles ; drug effects ; Rats ; Rats, Sprague-Dawley ; Refractory Period, Electrophysiological ; drug effects ; Ventricular Function

Adult ; Aged ; Aged, 80 and over ; Female ; Hepatic Encephalopathy ; diagnosis ; Humans ; Male ; Middle Aged ; Neuropsychological Tests ; Psychometrics ; methods ; Young Adult