Neuro-osteological interactions have an important role in the regulation of bone metabolism and regeneration. Neuropeptides combined with porous biphasic calcium phosphates (BCP) using protein adsorption may contribute to the acceleration of bone formation. In the present study, we investigated the effect of BCP combined with nerve growth factor (NGF) on the growth of osteoblasts in vitro and the combinational therapeutic effect on the repair of calvarial defects in vivo. NGF was separated and purified from Chinese cobra venom using a simplified three-step chromatography method. BCP combined with NGF exerted a potent effect on osteoblast differentiation, as evidenced by enhanced cell proliferation, increased ALP activity and the up-regulated expression of osteogenesis-related genes and proteins. Further, combinational therapy with BCP and NGF improved calvarial regeneration, which was superior to treatment with therapy alone, as observed using imageological and morphological examination and histological and immunohistochemical staining. The results confirmed the effect of neuro-osteological interactions through combinatorial treatment with NGF and BCP to promote osteogenesis and bone formation, which may provide an effective and economical strategy for clinical application.
Acceleration ; Adsorption ; Asian Continental Ancestry Group ; Calcium Phosphates ; Cell Proliferation ; Ceramics* ; Chromatography ; Cobra Venoms ; Elapidae* ; Humans ; In Vitro Techniques ; Metabolism ; Methods ; Nerve Growth Factor* ; Neuropeptides ; Osteoblasts ; Osteogenesis ; Regeneration ; Therapeutic Uses*

BACKGROUNDNajanalgesin, a toxin isolated from the venom of Naja naja atra, has been shown to exert significant analgesic effects in a neuropathic pain model in rats. However, the molecular mechanism underlying this protective effect of najanalgesin is poorly understood. The present study sought to evaluate the intracellular signaling pathways that are involved in the antinociceptive effect of najanalgesin on neuropathic pain.

METHODSThe antinociceptive properties of najanalgesin were tested in hind paw withdrawal thresholds in response to mechanical stimulation. We analyzed the participation of the mitogen-activated protein kinase p38, extracellular-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) by western blot analysis. This inhibition of JNK was confirmed by immunohistochemistry.

RESULTSThe phosphorylation levels of JNK (as well as its downstream molecule c-Jun), p38, and ERK were significantly increased after injury. Najanalgesin only inhibited JNK and c-Jun phosphorylation but had no effect on either ERK or p38. This inhibition of JNK was confirmed by immunohistochemistry, which suggested that the antinociceptive effect of najanalgesin on spinal nerve ligation-induced neuropathic pain in rats is associated with JNK activation in the spinal cord.

CONCLUSIONThe antinociceptive effect of najanalgesin functions by inhibiting the JNK in a neuropathic pain model.

Animals ; Elapid Venoms ; therapeutic use ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Immunohistochemistry ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Male ; Neuralgia ; drug therapy ; enzymology ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

PURPOSE: Breast cancer is a significant health problem worldwide, accounting for a quarter of all cancer diagnoses in women. Current strategies for breast cancer treatment are not fully effective, and there is substantial interest in the identification of novel anticancer agents especially from natural products including toxins. Cytotoxins are polypeptides found in the venom of cobras and have various physiological effects. In the present study, the anticancer potential of cytotoxin-II against the human breast adenocarcinoma cell line (MCF-7) was investigated. METHODS: The cytotoxic effects of cytotoxin-II were determined by morphological analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The mode and mechanism of cell death were investigated via acridine orange/ethidium bromide (AO/EtBr) double staining, flow cytometric analysis of cell death, detection of mitochondrial membrane potential, measurement of intracellular reactive oxygen species (ROS), annexin V/propidium iodide staining, and caspase-9 activity assays. RESULTS: The half maximal inhibitory concentration (IC50) of cytotoxin-II in MCF-7 cells was 4.18+/-1.23 microg/mL, while the value for cisplatin was approximately 28.02+/-1.87 microg/mL. Morphological analysis and AO/EtBr double staining showed typical manifestations of apoptotic cell death (in doses lower than 8 microg/mL). Dose- and time-dependent ROS generation, loss of mitochondrial membrane potential, caspase-9 activation, and cell cycle arrest were observed in their respective tests. CONCLUSION: In conclusion, cytotoxin-II has potent anticancer effects in the MCF-7 cell line, which are induced via the intrinsic pathways of apoptosis. Based on these findings, cytotoxin-II is a suitable choice for breast cancer treatment.
Adenocarcinoma* ; Antineoplastic Agents ; Apoptosis* ; Biological Products ; Breast Neoplasms ; Breast* ; Caspase 9 ; Cell Cycle Checkpoints ; Cell Death ; Cell Line* ; Cisplatin ; Cobra Venoms* ; Cytotoxins ; Diagnosis ; Elapidae ; Female ; Humans ; MCF-7 Cells ; Membrane Potential, Mitochondrial ; Peptides ; Reactive Oxygen Species ; Snakes ; Venoms

OBJECTIVETo study the protective effects of naja naja atra venom (NNAV) in a rat model of diabetic nephropathy (DN).

METHODSThe rat diabetes model was induced by intraperitoneal injection of streptozotocin (STZ). Thirty-two model rats were randomly divided into one DN group (n=8) and three treatment groups (n=8 each) that received NNAV at doses of 30, 90, or 270 μg/(kg·day) via oral gavage, another eight rats as normal controls. After 12 weeks, all rats were sacrificed and the changes in serum and urine biological index levels were determined by colorimetric assay. Microalbumin (mALB), N-acetyl-β- glucosaminidase (NAG) and cystatin C (CysC) concentrations were measured by ELISA. Renal tissues were sliced for pathological and immunohistochemical observations.

RESULTSComparied with the DN group, serum glucose was decreased by 31.04%, total cholesterol 21.96%, triglyceride 23.78%, serum creatinine 19.83%, blood urea nitrogen 31.28%, urinary protein excretion 45.42%, mALB 10.42%, NAG 20.65%, CysC 19.57%, whereas albumin increased by 5.55%, high-density lipoprotein-cholesterol 59.09%, creatinine clearance 19.05% in the treatment group by NNAV administration at dose of 90 μg/(kg·day). NNAV also reduced the levels of malondialdehyde in serum (22.56%) and kidney tissue (9.79%), and increased superoxide dismutase concentration in serum (15%) and decreased it in renal tissue (8.85%). In addition, under light microscopy kidney structure was improved and glomerular hypertrophy decreased by 8.29%. As shown by immunohistochemistry, NNAV inhibited transforming growth factor-β1 by 6.70% and nuclear actor-κB by 5.15%.

CONCLUSIONNNAV improves biological indexes in DN, and it may exert renoprotective effects in rats with STZ-induced diabetes.

Animals ; Body Weight ; Diabetes Mellitus, Experimental ; complications ; Diabetic Nephropathies ; drug therapy ; pathology ; Dose-Response Relationship, Drug ; Elapid Venoms ; administration & dosage ; pharmacology ; Elapidae ; physiology ; Kidney ; drug effects ; pathology ; Male ; Malondialdehyde ; Organ Size ; Rats ; Rats, Wistar ; Superoxide Dismutase

Dendroaspis natriuretic peptide (DNP), a new member of the natriuretic peptide family, is structurally similar to atrial, brain, and C-type natriuretic peptides. However, the effects of DNP on the cardiac function are poorly defined. In the present study, we examined the effect of DNP on the cardiac L-type Ca2+ channels in rabbit ventricular myocytes. DNP inhibited the L-type Ca2+ current (ICa,L) in a concentration dependent manner with a IC50 of 25.5 nM, which was blocked by an inhibitor of protein kinase G (PKG), KT5823 (1 microM). DNP did not affect the voltage dependence of activation and inactivation of ICa,L. The alpha1c subunit of cardiac L-type Ca2+ channel proteins was phosphorylated by the treatment of DNP (1 microM), which was completely blocked by KT5823 (1 microM). Finally, DNP also caused the shortening of action potential duration in rabbit ventricular tissue by 22.3 +/- 4.2% of the control (n = 6), which was completely blocked by KT5823 (1 microM). These results clearly indicate that DNP inhibits the L-type Ca2+ channel activity by phosphorylating the Ca2+ channel protein via PKG activation.
Action Potentials/drug effects ; Animals ; Biological Transport/drug effects ; Calcium/metabolism ; Calcium Channels, L-Type/*metabolism ; Carbazoles/pharmacology ; Cells, Cultured ; Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors ; Elapid Venoms/*metabolism/pharmacology ; Enzyme Activation ; Heart ; Heart Ventricles/drug effects ; Myocytes, Cardiac/drug effects ; Patch-Clamp Techniques ; Peptides/*metabolism/pharmacology ; Phosphorylation/drug effects ; Rabbits

OBJECTIVETo investigate the influence of najanalgesin on glutamate-glial transporter 1(GLT-1) in spinal cord of rats after L5 spinal nerve ligation and transection (SNL), and explore the spinal analgesic mechanism of najanalgesin.

METHODOne hundred male SD rats were randomly divided into 6 groups: sham(A), SNL(B), SNL + najanalgesin(C), SNL + saline (D), SNL + najanalgesin + liposome (E), SNL + najanalgesin + liposome + GLT-1 As-ODNs(F) and treated with intrathecal injections of 10 p.L saline (A and D), 40 ng X kg(-1) najanalgesin (C, E and F), qd, respectively. Besides intrathecal administration of najanalgesin the rats were intrathecally injected with 10 microL of GLT-1 antisense oligodeoxynucleotides (As-ODNs) (F) and 10 micdroL of liposome(E) once daily on day 3. The L4-L6 segments of the spinal cord were isolated in 1, 4 and 7 d(A,B,C and D), 7 d(E and F) after surgery. The mRNA and protein of GLT-1 were determined.

RESULTThe SNL model has successfully been set up. Compared to sham group, the expression of GLT-1 mRNA and protein level in group B and D both increased firstly and decreased later, the expression of GLT-1 in group C was significantly increased and kept stable, which were also higher when compared to group D in day 7th. Compared to SNL + najanalgesin group, after intrathecal injection of GLT-1 As-ODNs the GLT-1, expression of GLT-1 in F group significantly decreased. While intrathecal administration of liposome had no significant effect on the spinal GLT-1 expression.

CONCLUSIONNajanalgesin could increase the mRNA and protein expression of GLT-1 in spinal cord, which may be one of its spinal mechanisms of analgesia.

Animals ; Elapid Venoms ; pharmacology ; Elapidae ; Excitatory Amino Acid Transporter 2 ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; Male ; Neuralgia ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; drug effects ; metabolism

Voltage-gated K+ (Kv) channels have been considered to be a regulator of membrane potential and neuronal excitability. Recently, accumulated evidence has indicated that several Kv channel subtypes contribute to the control of cell proliferation in various types of cells and are worth noting as potential emerging molecular targets of cancer therapy. In the present study, we investigated the effects of the Kv1.1-specific blocker, dendrotoxin-kappa (DTX-kappa), on tumor formation induced by the human lung adenocarcinoma cell line A549 in a xenograft model. Kv1.1 mRNA and protein was expressed in A549 cells and the blockade of Kv1.1 by DTX-kappa, reduced tumor formation in nude mice. Furthermore, treatment with DTX-kappa significantly increased protein expression of p21Waf1/Cip1, p27Kip1, and p15INK4B and significantly decreased protein expression of cyclin D3 in tumor tissues compared to the control. These results suggest that DTX-kappa has anti-tumor effects in A549 cells through the pathway governing G1-S transition.
Adenocarcinoma/drug therapy/genetics/pathology ; Animals ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Disease Models, Animal ; Elapid Venoms/*pharmacology ; Elapidae ; Humans ; Kv1.1 Potassium Channel/*antagonists & inhibitors/deficiency/genetics/metabolism ; Lung Neoplasms/*drug therapy/genetics/pathology ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Potassium Channel Blockers/*pharmacology ; RNA, Messenger/genetics ; Transplantation, Heterologous

BACKGROUND AND OBJECTIVES: Hyperacute rejection (HAR) is a major obstacle to successful xenotransplantation of vascularized organs. This study was conducted to observe the effect of hemolysis of perfused human whole blood on pig heart function, and determine the major risk factors for preservation of xenoperfused cardiac function using ex-vivo pig to human xenogeneic cardiac perfusion model. MATERIALS AND METHODS: Harvested pig hearts were perfused with normal human whole blood (group 1), two different types of pre-treated human whole blood (group 2: immunoglobulins were depleted by plasmapheresis, group 3: pre-treated with plasmapheresis, GAS914, cobra venom factor (CVF) and steroid), and normal porcine whole blood as control (group 4) for 3 hours. RESULTS: Duration of heart beat was significantly prolonged in group 2 and group 3. Histological examination showed widespread HAR features but was gradually delayed in groups 2 and 3 compared to group 1. The absolute levels of serum creatine kinase-MB and Troponin I increased gradually, and was lower in group 3. Serum hemoglobin levels were rapidly increased in groups 3 and 4, compared to group 1. Extracellular potassium level increased sharply from the beginning of blood perfusion in groups 1, 2 and 3, compared to group 4. CONCLUSION: Pretreatment of human whole blood, including immunoglobulin depletion, CVF and steroid reduced and delayed the destruction of pig myocardium by HAR. However, the increased extracellular potassium levels in groups 1, 2 and 3 reflected that these treatments could not prohibit myocardial injury by HAR.
Cobra Venoms ; Creatine ; Diphtheria Toxoid ; Extracorporeal Circulation ; Haemophilus Vaccines ; Heart ; Hemoglobins ; Hemolysis ; Humans ; Hyperkalemia ; Immunoglobulins ; Myocardium ; Perfusion ; Plasmapheresis ; Potassium ; Rejection (Psychology) ; Risk Factors ; Transplantation, Heterologous ; Trisaccharides ; Troponin I

Since Willis described 'fatigable weakness' in 1672, most physicians consider it as a kind of hysteria due to the inconsistent fluctuation of symptoms. Erb presented three cases of 'bulbal palsy' in the 1870s, and Oppenheim and Hopper considered myasthenia gravis as a disease similar to curare poisoning and as a disease induced by attack of the motor centers by intrinsic toxins, respectively. In 1903, Elliot suggested that a 'chemical substance' mediates the nerve impulses at synapse. However, it was not until 1921 that this was demonstrated by Loewi, who provided evidence from the famous two-frog-hearts experiment. Dale later revealed the substance to be acetylcholine, and he also suggested that myasthenia gravis is due to a problem with the motor end plate. In 1934, Walker was prompted by the resemblance between myasthenia gravis and curare poisoning to apply physostigmine, a curare-poisoning antidote, to a patient, which produced a dramatic result. Since then the use of anticholinesterase inhibitors has been adopted for standard therapeutic modality. Some prominent surgeons have also applied thymectomy as a surgical modality. The most recent focus of myasthenia gravis has been immunological. In 1960, Simpson proposed the autoimmune hypothesis, and Chang et al. showed that snake venom contained a selective antagonist of the nicotinic acetylcholine receptor, alpha-bungarotoxin. The immunization of rabbits with acetylcholine receptor purified from the electrical organs of electric eels by Patrick et al. induced myasthenic symptoms and signs, and these were reversed by acetylcholinesterase inhibitors. The role of the autoimmune system has led to the introduction of an immunosuppressive modality and plasma exchange to the field of clinical neurology.
Acetylcholine ; Action Potentials ; Bungarotoxins ; Cholinesterase Inhibitors ; Curare ; Electrophorus ; History of Medicine ; Humans ; Hysteria ; Immunization ; Motor Endplate ; Myasthenia Gravis ; Physostigmine ; Plasma Exchange ; Rabbits ; Receptors, Nicotinic ; Snake Venoms ; Synapses ; Thymectomy

The purpose of this paper is to encapsulate neurotoxin-I (NT-I), a kind of analgesic peptide, into polylactic acid (PLA) nanoparticles (NPs) and to evaluate their transport into the brain after intranasal administration (in) by use of microdialysis sampling technique developed in our laboratory recently. NT-I-NPs (NT-Iradiolabeled with sodium 125I-Iodide) were prepared by a double emulsification solvent evaporation method, and were characterized in terms of surface morphology, particle size distribution, zeta potential and entrapment efficiency. Then, NT-I-NPs were administered intranasally or intravenously to rats and the radioactivities in periaqueductal gray (PAG) were monitored up to 240 min utilizing the microdialysis sampling technique. Nanoparticles prepared were spherical with homogenous size distribution. Their mean particle size and zeta potential measured were (65.3 +/- 10.8) nm and (-28.6 +/- 2.3) mV, respectively. The entrapment efficiency of NT-Iencapsulated into nanoparticles was (35.5 +/- 2.8)%. The brain transport results showed that the time to peak level (Tmax) of NT-I-NPs (in) was (65 +/- 10) min approximately, apparently shorter compared with NT-I-NPs [iv, (95 +/- 10) min] or NT-I [iv, (145 +/- 10) min]. The concentration to peak level (Cmax) and the area under the curves from zero to 4 h (AUC0-4h) of each group followed this order: NT-I-NPs (in) > NT-I-NPs (iv) > NT-I (iv). With nanoparticles as carriers and administered intranasally could be a potential way for centrally active peptides to improve their brain transport. Microdialysis is quite a good technique for the study of drug delivery to the brain.
Administration, Intranasal ; Animals ; Area Under Curve ; Cobra Neurotoxin Proteins ; administration & dosage ; pharmacokinetics ; Drug Delivery Systems ; Lactic Acid ; chemistry ; Male ; Microdialysis ; Nanoparticles ; Particle Size ; Periaqueductal Gray ; metabolism ; Polyesters ; Polymers ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley