Animals ; Antigens, Surface/genetics* ; Cell Communication/genetics* ; Copulation/physiology* ; Fallopian Tubes/metabolism* ; Female ; Fertilization/genetics* ; GPI-Linked Proteins/genetics* ; Gene Expression Regulation ; Genes, Reporter ; Green Fluorescent Proteins/metabolism* ; Litter Size ; Luminescent Proteins/metabolism* ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria/metabolism* ; Reproduction/genetics* ; Signal Transduction ; Sperm Count ; Sperm Motility/genetics* ; Spermatozoa/metabolism* ; Uterus/metabolism*

Voltage-gated sodium channels (VGSCs) in primary sensory neurons play a key role in transmitting pain signals to the central nervous system. BmK I, a site-3 sodium channel-specific toxin from scorpion Buthus martensi Karsch, induces pain behaviors in rats. However, the subtypes of VGSCs targeted by BmK I were not entirely clear. We therefore investigated the effects of BmK I on the current amplitude, gating and kinetic properties of Nav1.8, which is associated with neuronal hyperexcitability in DRG neurons. It was found that BmK I dose-dependently increased Nav1.8 current in small-sized (<25 μm) acutely dissociated DRG neurons, which correlated with its inhibition on both fast and slow inactivation. Moreover, voltage-dependent activation and steady-state inactivation curves of Nav1.8 were shifted in a hyperpolarized direction. Thus, BmK I reduced the threshold of neuronal excitability and increased action potential firing in DRG neurons. In conclusion, our data clearly demonstrated that BmK I modulated Nav1.8 remarkably, suggesting BmK I as a valuable probe for studying Nav1.8. And Nav1.8 is an important target related to BmK I-evoked pain.
Aniline Compounds ; pharmacology ; Animals ; Cell Size ; Cells, Cultured ; Electrophysiological Phenomena ; drug effects ; Furans ; pharmacology ; Ganglia, Spinal ; cytology ; Kinetics ; Male ; NAV1.8 Voltage-Gated Sodium Channel ; metabolism ; Rats ; Rats, Sprague-Dawley ; Scorpion Venoms ; antagonists & inhibitors ; pharmacology ; Scorpions ; Sensory Receptor Cells ; drug effects ; metabolism ; physiology ; Sodium Channel Blockers ; pharmacology ; Voltage-Gated Sodium Channel Agonists ; pharmacology

Pathological cardiac hypertrophy induced by angiotensin II (AngII) can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots of Nardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on AngII-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosinone (25, 50, 100, and 200 μmol/L) or AngII (1 μmol/L). Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by AngII. The mRNA expression of ANP, BNP and β-MHC was obviously elevated in AngII-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100 μmol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang II-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways.
Angiotensin II ; physiology ; Animals ; Cardiotonic Agents ; pharmacology ; Cell Line ; Cell Size ; drug effects ; Hypertrophy ; metabolism ; pathology ; MAP Kinase Signaling System ; Myoblasts, Cardiac ; cytology ; drug effects ; metabolism ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Sesquiterpenes ; pharmacology ; TOR Serine-Threonine Kinases ; genetics ; metabolism

This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type II collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at Gl phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G1 phase and failed to enter S phase. The expression of type II collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.
Animals ; Apoptosis ; physiology ; Cell Cycle ; physiology ; Cell Proliferation ; Cell Size ; Cell Survival ; physiology ; Cells, Cultured ; Intervertebral Disc ; cytology ; physiopathology ; Intervertebral Disc Degeneration ; pathology ; physiopathology ; Rabbits ; Reference Values

Higher levels of body fat are associated with an increased risk for development numerous adverse health conditions. FTY720 is an immune modulator and a synthetic analogue of sphingosine 1-phosphate (S1P), activated S1P receptors and is effective in experimental models of transplantation and autoimmunity. Whereas immune modulation by FTY720 has been extensively studied, other actions of FTY720 are not well understood. Here we describe a novel role of FTY720 in the prevention of obesity, involving the regulation of adipogenesis and lipolysis in vivo and in vitro. Male C57B/6J mice were fed a standard diet or a high fat diet (HFD) without or with FTY720 (0.04 mg/kg, twice a week) for 6 weeks. The HFD induced an accumulation of large adipocytes, down-regulation of phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha) and Akt (p-Akt); down-regulation of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and perilipin mRNA as well as up-regulation of phosphorylated HSL (p-HSL, Ser563) and glycogen synthase kinase 3 alpha/beta (p-GSK3alpha/beta). All these effects were blunted by FTY720 treatment, which inhibited adipogenesis and promoted lipolysis. Also, FTY720 significantly decreased lipid accumulation in maturing preadipocytes. FTY720 down-regulated the transcriptional levels of the PPARgamma, C/EBPalpha and adiponectin, which are markers of adipogenic differentiation. FTY720 significantly increased the release of glycerol and the expression of the HSL, ATGL and perilipin, which are regulators of lipolysis. These results show that FTY720 prevented obesity by modulating adipogenesis and lipolysis, and suggest that FTY720 is used for the treatment of obesity.
3T3-L1 Cells ; AMP-Activated Protein Kinases/metabolism ; Adipocytes/*drug effects/physiology ; Adipogenesis/drug effects ; Animals ; Anti-Obesity Agents/*pharmacology/therapeutic use ; Antigens, Differentiation/genetics/metabolism ; Carrier Proteins/genetics/metabolism ; Cell Size ; Diet, High-Fat/adverse effects ; Disease Models, Animal ; Enzyme Activation ; Gene Expression Regulation, Enzymologic/drug effects ; Glycogen Synthase Kinase 3/genetics/metabolism ; Lipase/genetics/metabolism ; Lipolysis/drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Obesity/etiology/metabolism/*prevention & control ; Phosphoproteins/genetics/metabolism ; Phosphorylation ; Propylene Glycols/*pharmacology/therapeutic use ; Protein Processing, Post-Translational ; Proto-Oncogene Proteins c-akt/metabolism ; Sphingosine/*analogs & derivatives/pharmacology/therapeutic use ; Sterol Esterase/metabolism

The control of organ size growth is one of the most fundamental aspects of life. In the past two decades, a highly conserved Hippo signaling pathway has been identified as a key molecular mechanism for governing organ size regulation. In the middle of this pathway is a kinase cascade that negatively regulates the downstream component Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ)/Yorkie through phosphorylation. Phosphorylation of YAP/TAZ/Yorkie promotes its cytoplasmic localization, leads to cell apoptosis and restricts organ size overgrowth. When the Hippo pathway is inactivated, YAP/TAZ/Yorkie translocates into the nucleus to bind to the transcription enhancer factor (TEAD/TEF) family of transcriptional factors to promote cell growth and proliferation. In this review, we will focus on the structural and functional studies on the downstream transcription factor TEAD and its coactivator YAP.
Animals ; Apoptosis ; Cell Proliferation ; Drosophila ; genetics ; metabolism ; Drosophila Proteins ; genetics ; metabolism ; Gene Expression Regulation, Developmental ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Mice ; Nuclear Proteins ; genetics ; metabolism ; Organ Size ; physiology ; PDZ Domains ; Phosphorylation ; Protein Binding ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Signal Transduction ; physiology ; Trans-Activators ; genetics ; metabolism

Fragments of testis tissue from immature animals grow and develop spermatogenesis when grafted onto subcutaneous areas of immunodeficient mice. The same results are obtained when dissociated cells from immature testes of rodents are injected into the subcutis of nude mice. Those cells reconstitute seminiferous tubules and facilitate spermatogenesis. We compared these two methods, tissue grafting and cell-injection methods, in terms of the efficiency of spermatogenesis in the backs of three strains of immunodeficient mice, using neonatal porcine testicular tissues and cells as donor material. Nude, severe combined immunodeficient (SCID) and NOD/Shi-SCID, IL-2Rgammacnull (NOG) mice were used as recipients. At 10 months after surgery, the transplants were examined histologically. Both grafting and cell-injection methods resulted in porcine spermatogenesis on the backs of recipient mice; the percentage of spermatids present in the transplants was 67% and 22%, respectively. Using the grafting method, all three strains of mice supported the same extent of spermatogenesis. As for the cell-injection method, although SCID mice were the best host for supporting reconstitution and spermatogenesis, any difference from the other strains was not significant. As NOG mice did not show any better results, the severity of immunodeficiency seemed to be irrelevant for supporting xeno-ectopic spermatogenesis. Our results confirmed that tubular reconstitution is applicable to porcine testicular cells. This method as well as the grafting method would be useful for studying spermatogenesis in different kinds of animals.
Animals ; Cell Transplantation ; methods ; Injections ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred NOD ; Mice, Nude ; Mice, SCID ; Organ Size ; Seminiferous Tubules ; cytology ; physiology ; transplantation ; Spermatids ; cytology ; transplantation ; Spermatogenesis ; physiology ; Subcutaneous Tissue ; surgery ; Swine ; Tissue Transplantation ; methods ; Transplantation, Heterologous ; methods

AIMTo detect the anti-follicle-stimulating hormone (FSH) antibody in idiopathic infertile patients and fertile subjects in order to determine the role of this antibody in patients with spermatogenic dysfunction.

METHODSThe anti-FSH antibody in serum was detected by an enzyme-linked immunosorbent assay (ELISA). The functional and structural integrity of the sperm membrane was evaluated with hypo-osmotic swelling (HOS) test and the ultrastructure of the spermatozoa was investigated by transmission electron microscopy (TEM).

RESULTSThe extent of positive FSH antibody in the patients with oligozoospermia and/or asthenozoospermia was significantly higher than that in the fertile subjects and infertile patients with normal sperm concentration and motility, but it was significantly lower than that in the patients with azoospermia. The extent of anti-FSH antibody in the patients with azoospermia was significantly greater than that in patients with oligospermia and/or asthenospermia, infertile people with normal sperm density and motility and fertile people. The hypo-osmotic swelling test showed that the percentage of HOS-positive spermatozoa (swollen) was 45.1 mu 3.5% in the FSH antibody-positive group and 59.1% micro 6.2% in the FSH antibody-negative control group. The percentage of functional membrane damage to spermatozoa was significantly higher in the anti-FSH antibody-positive group than in the control group. TEM showed that the outer acrosomal membrane was located far from the nucleus, and detachment of the acrosome was found in the FSH autoantibody-positive group.

CONCLUSIONThese data suggest that the presence of anti-FSH antibody is strongly correlated with the sperm quantity and quality in idiopathic male infertility. Anti-FSH antibody may be an important factor causing spermatogenic dysfunction and infertility.

Adult ; Autoantibodies ; physiology ; Cell Membrane ; immunology ; ultrastructure ; Cell Size ; Enzyme-Linked Immunosorbent Assay ; Follicle Stimulating Hormone ; immunology ; Humans ; Infertility, Male ; immunology ; Luteinizing Hormone ; blood ; Male ; Microscopy, Electron, Transmission ; Osmotic Pressure ; Semen ; cytology ; Spermatogenesis ; immunology ; Spermatozoa ; immunology ; ultrastructure

BACKGROUNDBradykinin (BK) acts mainly on two receptor subtypes: B(1) and B(2), and activation of B(2) receptor mediates the most well-known cardioprotective effects of angiotensin converting enzyme inhibitors (ACEi), however, the role that B(1) receptor plays in ACEi has not been fully defined. We examined the role of B(1) receptor in the inhibitory effect of ACE inhibitor captopril on rat cardiomyocyte hypertrophy and cardiac fibroblast proliferation induced by angiotensin II (Ang II) and explored its possible mechanism.

METHODSNeonatal cardiomyocytes and cardiac fibroblasts (CFs) were randomly treated with Ang II, captopril, B(2) receptor antagonist (HOE-140) and B(1) receptor antagonist (des-Arg(10), Leu(9)-kallidin) alone or in combination. Flow cytometry was used to evaluate cell cycle, size and protein content. Nitric oxide (NO) and intracellular cyclic guanosine monophosphate (cGMP) level were measured by colorimetry and radioimmunoassay.

RESULTSAfter the CFs and cardiomyocytes were incubated with 0.1 micromol/L Ang II for 48 hours, the percentage of CFs in the S stage, cardiomyocytes size and protein content significantly increased (both P < 0.01 vs control), and these increases were inhibited by 10 micromol/L captopril. However, NO and cGMP levels were significantly higher than that with Ang II alone (both P < 0.01). 1 micromol/L HOE-140 or 0.1 micromol/L des-Arg(10), Leu(9)-kallidin attenuated the effects of captopril, which was blunted further by blockade of both B(1) and B(2) receptors.

CONCLUSIONSActing via B(2) receptor, BK contributes to the antihypertrophic and antiproliferative effects of captopril on cardiomyocytes and CFs. In the absence of B(2) receptor, B(1) receptor may act a compensatory mechanism for the B(2) receptor and contribute to the inhibition of cardiomyocyte hypertrophy and CFs proliferation by captopril. NO and cGMP play an important role in the effect of B(1) receptor.

Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Animals, Newborn ; Captopril ; pharmacology ; Cardiomegaly ; prevention & control ; Cell Proliferation ; drug effects ; Cell Size ; drug effects ; Cyclic GMP ; analysis ; DNA ; biosynthesis ; Fibroblasts ; drug effects ; physiology ; Myocytes, Cardiac ; drug effects ; pathology ; Nitric Oxide ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Bradykinin B1 ; physiology

This study was purposed to investigate the angiogenesis effect of platelet-derived membrane microparticles (PMPs) in chick chorioallantoic membranes (CAM). Thrombin were adopted to activate the platelets and then PMPs were obtained. PMPs were isolated by high speed centrifugation. Flow cytometry (FCM) was adopted to evaluate the efficiency of thrombin to produce PMPs and BCA method was adopted to evaluate the content of PMPs. PMPs were put into CAM and the effects of PMPs on angiogenesis in CAM were observed. The results indicated that after incubation for 72 hours at the concentration of 80 microg/ml PMPs, the vessel nets in a 'spoked-wheel' pattern were shown around mixed fibrous filter membranes, number of vessel ramification was 112.5 +/- 11.31 and ratio of vessel area/CAM area was 6.19 +/- 1.29%, but there were not localized allantoic vessels developing in the control group, the number of vessel ramification and ratio of vessel area/CAM area in control group were 82.6 +/- 8.05 and 1.78 +/- 0.33 respectively, so there was significant difference between PMP and control groups. In above mentioned conditions, the number of vessel ramification and ratio of vessel area/CAM area in VEGF group were 128.4 +/- 10.02 and 7.44 +/- 1.36 respectively, so there was no difference between PMP and VEGF groups. It is concluded that PMPs show promotive effect on the formation of capillaries in chick chorioallantoic membranes.
Animals ; Blood Platelets ; physiology ; Cell-Derived Microparticles ; physiology ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; Humans ; Neovascularization, Physiologic ; drug effects ; Particle Size