The endophytic fungus Nigrospora sphaerica S5 derived from the semi-mangrove plant Myoporum bontioides was fermented. Its metabolites were purified by column chromatography. Nine compounds were obtained and identified as terezine P(1), 3-(1-hydroxyethyl)-4-methyl dihydrofuran-2(3H)-one(2), methylhydroheptelidate(3), hydroheptelidic acid(4), 5, 7-dimethoxy-4, 6-dimethylphthalide(5),(3R,4S)-(-)-4-hydroxymellein(6), pestalopyrone(7), indole-3-formaldehyde(8) and p-hydroxybenzaldehyde(9) by spectroscopic techniques. Terezine P(1) was a new alkaloid belonging to the terezine class with a pyrazine ring. Compounds 2-7 were lactones, of which 3 and 4 belonged to sesquiterpenes. Compounds 8 and 9 were indole alkaloids and phenols, respectively. Compounds 3-6 were purified from Nigrospora sp. for the first time. These compounds showed different degrees of antibacterial activity against Staphylococcus aureus, Escherichia coli of O6 serotype and E. coli of O78 serotype.
Alkaloids ; Anti-Bacterial Agents/pharmacology* ; Ascomycota/chemistry* ; Escherichia coli ; Formaldehyde ; Indoles/pharmacology* ; Lactones ; Molecular Structure ; Myoporum/microbiology* ; Phenols ; Pyrazines ; Sesquiterpenes

OBJECTIVE: To unravel the underlying mechanism of minocycline in formalin-induced inflammatory pain, and to investigate the effects of minocycline on synaptic transmission in substantia gela-tinosa (SG) neurons of rat spinal dorsal horn. METHODS: Behavioral and immunohistochemistry experiments: 30 male Sprague-Dawley (SD) rats (3-5 weeks old) were randomly assigned to control (n=8 rats), model (n=8 rats), saline treatment model (n=6 rats) and minocycline treatment model (n=8 rats) groups. The control group was subcutaneously injected with normal saline on the right hindpaws. Acute inflammatory pain model was established by injecting 5% (volume fraction) formalin into the right hindpaws. The rats in the latter two groups received intraperitoneal injection of saline and minocycline 1 h before the formalin injection, respectively. The time of licking and lifting was recorded every 5 min within 1 h after the subcutaneous injection of normal saline or formalin for all the groups, which was continuously recorded for 1 h. One hour after the pain behavioral recording, the spinal cord tissue was removed following transcardial perfusion of 4% paraformaldehyde. The expression of c-Fos protein in spinal dorsal horn was observed by immunohistochemistry. Electrophysiological experiment: In vitro whole-cell patch-clamp recordings were performed in spinal cord parasagittal slices obtained from 26 male SD rats (3-5 weeks old). Two to five neurons were randomly selected from each rat for patch-clamp recording. the effects of minocycline, fluorocitrate and doxycycline on spontaneous excitatory postsynaptic currents (sEPSCs) or spontaneous inhibitory postsynaptic currents (sIPSCs) of SG neurons were investigated. RESULTS: Compared with the control group, both the licking and lifting time and the expression of c-Fos protein in ipsilateral spinal dorsal horn of the model group were significantly increased. Intraperitoneal injection of minocycline largely attenuated the second phase of formalin-induced pain responses (t=2.957, P<0.05). Moreover, c-Fos protein expression was also dramatically reduced in both the superficial lamina (I-II) and deep lamina (III-IV) of spinal dorsal horn (t_I-II=3.912, t_III-IV=2.630, P<0.05). On the other side, bath application of minocycline significantly increased the sIPSCs frequency to 220%±10% (P<0.05) of the control but did not affect the frequency (100%±1%, t=0.112, P=0.951) and amplitude (98%±1%, t=0.273, P=0.167) of sEPSCs and the amplitude (105%±3%, t=0.568, P=0.058) of sIPSCs. However, fluorocitrate and doxycycline had no effect on the frequency [(99%±1%, t=0.366, P=0.099); (102%±1%, t=0.184, P=0.146), respectively] and amplitude [(98%±1%, t=0.208, P=0.253); (99%±1%, t=0.129, P=0.552), respectively] of sIPSCs. CONCLUSION Minocycline can inhibit formalin-induced inflammatory pain and the expression of c-Fos protein in spinal dorsal horn. These effects are probably due to its enhancement in inhibitory synaptic transmission of SG neurons but not its effect on microglial activation or antibiotic action.
Animals ; Anti-Bacterial Agents/pharmacology* ; Formaldehyde ; Inflammation/complications* ; Inhibitory Postsynaptic Potentials ; Male ; Minocycline/pharmacology* ; Pain/prevention & control* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord

OBJECTIVETo investigate the neuroprotective effects of icariin on formaldehyde (FA)-treated human neuroblastoma SH-SY5Y cells and the possible mechanisms involved.

METHODSSH-SY5Y cells were divided into FA treatment group, FA treatment group with icariin, and the control group. Cell viability, apoptosis, and morphological changes were determined by cell counting kit-8 (CCK 8), flow cytometry, and confocal microscopy, respectively. The phosphorylation of Tau protein was examined by western blotting.

RESULTSFA showed a half lethal dose (LD50) of 0.3 mmol/L in SH-SY5Y cells under the experimental conditions. Icariin (1-10 µmol/L) prevented FA-induced cell death in SH-SY5Y cells in a dose-dependent manner, with the optimal effect observed at 5 µmol/L. After FA treatment, the absorbance in FA group was 1.31±0.05, while in the group of icariin (5 µmol/L) was 1.63±0.05. Examination of cell morphology by confocal microscopy demonstrated that 5 µmol/L icariin significantly attenuated FA-induced cell injury (P <0.05). Additionally, Icariin inhibited FA-induced cell apoptosis in SH-SY5Y cells. Results from western blotting showed that icariin suppressed FA-induced phosphorylation at Thr 181 and Ser 396 of Tau protein, while having no effect on the expression of the total Tau protein level. Furthermore, FA activated Tau kinase glycogen synthase kinase 3 beta (GSK-3β) by enhancement of Y216 phosphorylation, but icariin reduced Y216 phosphorylation and increased Ser 9 phosphorylation.

CONCLUSIONIcariin protects SH-SY5Y cells from FA-induced injury poßsibly through the inhibition of GSK-3β-mediated Tau phosphorylation.

Blotting, Western ; Cell Death ; drug effects ; Cell Line, Tumor ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; DNA Fragmentation ; drug effects ; Flavonoids ; pharmacology ; Formaldehyde ; Glycogen Synthase Kinase 3 beta ; antagonists & inhibitors ; metabolism ; Humans ; Neuroprotective Agents ; pharmacology ; Phosphorylation ; drug effects ; tau Proteins ; metabolism

The aim of this study was to explore the mechanism of the nervous system lesions induced by formaldehyde (FA). Male Balb/c mice were exposed to gaseous formaldehyde for 7 days (8 h/d) with three different concentrations (0, 0.5 and 3.0 mg/m(3)). A group of animals injected with the nitric oxide synthase inhibitor L-NMMA (0.01 mL/g) was also set and exposed to 3.0 mg/m(3) FA. The concentrations of cAMP, cGMP, NO and the activity of NOS in cerebral cortex, hippocampus and brain stem were determined by corresponding assay kits. The results showed that, compared with the control (0 mg/m(3) FA) group, the cAMP contents in cerebral cortex and brain stem were significantly increased in 0.5 mg/m(3) FA group (P < 0.05), but decreased in 3.0 mg/m(3) FA group (P < 0.05); The concentration of cAMP in hippocampus was significantly decreased in 3.0 mg/m(3) FA group (P < 0.05). In comparison with the control group, L-NMMA group showed unchanged cAMP contents and NOS activities in different brain regions, but showed increased cGMP contents in hippocampus and NO contents in cerebral cortex (P < 0.05). In addition, compared with 3.0 mg/m(3) FA group, L-NMMA group showed increased contents of cAMP and reduced NOS activities in different brain regions, as well as significantly decreased cGMP contents in cerebral cortex and brain stem and NO content in brain stem. These results suggest that the toxicity of FA on mouse nervous system is related to NO/cGMP and cAMP signaling pathways.
Animals ; Brain Stem ; chemistry ; drug effects ; Cerebral Cortex ; chemistry ; drug effects ; Cyclic AMP ; chemistry ; Cyclic GMP ; chemistry ; Formaldehyde ; toxicity ; Hippocampus ; chemistry ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Nitric Oxide ; chemistry ; Nitric Oxide Synthase ; antagonists & inhibitors ; omega-N-Methylarginine ; pharmacology

Formaldehyde (FA) is a ubiquitous toxic organic compound, and it has been regarded as a leukemogen. However, the mechanisms by which FA induces bone marrow toxicity remain unclear. The present study was aimed to examine the bone marrow toxicity caused by FA and the mechanism involving the expression changes of peroxiredoxin3 (Prx3) in this process. The mice were divided into four groups with 6 mice per group. Animals in the control group were exposed to ambient air and those in the FA groups to different concentrations of FA (20, 40, 80 mg/m(3)) for 15 days in the separate inhalation chambers, 2 h a day. At the end of the 15-day experimental period, all mice were killed. Bone marrow cells were obtained. The level of hydrogen peroxide (H2O2), the apoptosis rate, and the activities and protein expression levels of caspase-3 and caspase-9 were determined by biochemical assay, flow cytometry and immunohistochemistry, respectively; DNA damage and Prx3 expression levels were measured by single cell gel eletrophoresis immunohistochemistry and Western blotting, respectively. The results showed that the H2O2 level and cell apoptosis rate were significantly increased in FA groups relative to the control group. Caspase-3 and caspase-9 activities and their protein expression levels were markedly increased as well. Additionally, FA also increased the rate of DNA damage and the expression level of Prx3 compared with control group. Our study suggested that a certain concentration of FA causes the bone marrow toxicity by regulating the expression of Prx3.
Animals ; Blotting, Western ; Bone Marrow ; drug effects ; metabolism ; Formaldehyde ; pharmacology ; Homeodomain Proteins ; metabolism ; Male ; Mice

PURPOSE: The aim of this study was to investigate the effect of epidural dexamethasone on analgesia and cytosolic phospholipase A2 (cPLA2) expression in the spinal cord in a rat formalin test. MATERIALS AND METHODS: Epidural dexamethasone injection was performed to Sprague-Dawley rats with a 25 gauge needle under fluoroscopy. Following the epidural injection, a formalin induced pain behavior test was performed. Next, the spinal cords corresponding to L4 dorsal root ganglion was extracted to observe the cPLA2 expression. RESULTS: There were no differences in pain response during phase I among the groups. The phase II pain response in 300 microg of epidural dexamethasone group decreased as compared to control, 30 microg of epidural dexamethasone, 100 microg of epidural dexamethasone, and 300 microg of systemic dexamethasone groups. The expression of cPLA2 decreased in Rexed laminae I-II in 300 microg of the epidural dexamethasone group compared with the ones in the control group. CONCLUSION: Taken together, these results suggest that 300 microg of epidural dexamethasone has an attenuating effect on the peripheral inflammatory tissue injury induced hyperalgesia and this effect is mediated through the inhibition of intraspinal cPLA2 expression and the primary site of action is the laminae I-II of the spinal cord.
Animals ; Anti-Inflammatory Agents/*pharmacology ; Dexamethasone/*pharmacology ; Formaldehyde/*adverse effects ; Group IV Phospholipases A2/*metabolism ; Hyperalgesia/*drug therapy ; Injections, Epidural ; Male ; Pain/chemically induced/*metabolism ; Pain Measurement ; Rats ; Rats, Sprague-Dawley ; Spinal Cord/*metabolism

PURPOSE: Nociceptin/orphanin FQ (N/OFQ) as an endogeneous hexadecapeptide is known to exert antinociceptive effects spinally. The aims of this study were to demonstrate the antinociceptive effects of i.t. N/OFQ and to investigate the possible interaction between N/OFQ and endogenous opioid systems using selective opioid receptor antagonists in rat formalin tests. MATERIALS AND METHODS: I.t. N/OFQ was injected in different doses (1-10 nmol) via a lumbar catheter prior to a 50 microL injection of 5% formalin into the right hindpaw of rats. Flinching responses were measured from 0-10 min (phase I, an initial acute state) and 11-60 min (phase II, a prolonged tonic state). To observe which opioid receptors are involved in the anti-nociceptive effect of i.t. N/OFQ in the rat-formalin tests, naltrindole (5-20 nmol), beta-funaltrexamine (1-10 nmol), and norbinaltorphimine (10 nmol), selective delta-, micro- and kappa-opioid receptor antagonists, respectively, were administered intrathecally 5 min after i.t. N/OFQ. RESULTS: I.t. N/OFQ attenuated the formalin-induced flinching responses in a dose-dependent manner in both phases I and II. I.t. administration of naltrindole and beta-funaltrexamine dose-dependently reversed the N/OFQ-induced attenuation of flinching responses in both phases; however, norbinaltorphimine did not. CONCLUSION: I.t. N/OFQ exerted an antinociceptive effect in both phases of the rat-formalin test through the nociceptin opioid peptide receptor. In addition, the results suggested that delta- and micro-opioid receptors, but not kappa-opioid receptors, are involved in the antinociceptive effects of N/OFQ in the spinal cord of rats.
Analgesics/administration & dosage/*pharmacology ; Animals ; Formaldehyde/toxicity ; Injections, Spinal ; Male ; Naltrexone/administration & dosage/analogs & derivatives/pharmacology ; Narcotic Antagonists/administration & dosage/*pharmacology ; Opioid Peptides/administration & dosage/*pharmacology ; Pain Measurement ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid/*agonists/drug effects

OBJECTIVETo evaluate the potential role of p38 mitogen-activated protein kinase (MAPK) in the orofacial inflammatory pain.

METHODSSD rats received subcutaneous injection of 2.5% formalin 50 µl in the left vibrissa pad to establish the inflammatory pain model. The rats were grouped into the control group, the formalin group (FOR group), the formalin + saline group (FOR + NS group) and the formalin + SB203580 group (FOR + SB group). SB203580 or saline was inserted into the rat's cisterna magna 20 minutes prior to the formalin injection, then the behavioral changes were tested. The immunofluorescence staining and Western blotting analysis were performed to examine c-fos, p38MAPK and phosphorylated p38 (p-p38) activity in Vc at 20, 60, 120, 180 minutes after formalin injection.

RESULTSp38MAPK was constitutively expressed in Vc (P > 0.05) and p38MAPK was activated following formalin injection.Compared with the control group at 20 min (0.12 ± 0.01), the level of p-p38 in FOR group (0.66 ± 0.04) and FOR + NS group (0.64 ± 0.04) increased significantly (P < 0.001). The expression of p-p38 peaked at 20 minutes, and then declined in each group. Intracisterna magna pretreatment of p38MAPK inhibitor SB203580 resulted in potent attenuation of phase II of pain behavior (P < 0.05), while the expression of c-fos was also inhibited, especially at the point of 120 min (P < 0.01).

CONCLUSIONSActivation of p38 mitogen-activated protein kinase played a major role in the development of orofacial inflammatory pain and it was verified by the experimental result that p38MAPK inhibitor SB203580 inhibited the formalin-induced orofacial pain.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Behavior, Animal ; Enzyme Inhibitors ; pharmacology ; Facial Pain ; chemically induced ; metabolism ; Formaldehyde ; Imidazoles ; pharmacology ; Male ; Phosphorylation ; Proto-Oncogene Proteins c-fos ; metabolism ; Pyridines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Trigeminal Caudal Nucleus ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo observe whether formalin inflammatory pain can induce neuron apoptosis in rats spinal cord or not and the effects of nitric oxide on the spontaneous pain reaction and neuron apoptosis in the spinal cord of rats with formalin inflammatory pain.

METHODSFormalin-induced paw licking time was used to reflect the degree of spontaneous pain of rats, and the flow cytometry was used to detecte neuron apoptosis rate of spinal cord.

RESULTSCompared with control group, the apoptosis ratio of spinal neuron was increased in the rats with formalin inflammatory pain, and peaked at 3d after formalin injection. Pre-intrathecal injection of NOS inhibitor L-NAME inhibited the nociceptive behavioural response in double phases induced by fonnrmalin injection and cut down the neuron apoptosis ratio of spinal cord of rats with formalin inflammatory pain. Nociceptive behavioural response and incraesed neuron apoptosis in the spinal cord were induced by intrathecal injection of L-Arg in normal rats.

CONCLUSIONThe results indicated that formalin inflammatory pain could induce the apoptosis of spinal neurons. The neurons apoptosis was the most significant on the third day after formalin injection. The increased pruduction of NO in spinal cord could promote the transmit of nociceptive information and participate the induction of neuronal apoptosis during the formalin inflammatory pain.

Animals ; Apoptosis ; drug effects ; Formaldehyde ; Male ; NG-Nitroarginine Methyl Ester ; pharmacology ; Neurons ; pathology ; Nitric Oxide ; metabolism ; physiology ; Nitric Oxide Synthase ; antagonists & inhibitors ; Nociceptors ; physiology ; Pain ; chemically induced ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; pathology ; physiopathology

OBJECTIVETo investigate the expression of protein kinase C (PKC) in the spinal dorsal horn of rats with formalin-induced pain and the effect of intrathecal ketamine on PKC expression.

METHODSThirty-two SD rats were randomly divided into 4 equal groups, namely the control group, intrathecal saline group (NS), 50 µg ketamine group (K1) and 100 µg ketamine group (K2). The rats were anesthetized with 10% chloral hydrate, and a microspinal catheter was inserted intrathecally into the lumbar region. Five days later, the rats in groups, K1 and K2 were subjected to intrathecal administration of 50 and 100 µg ketamine (10 µl), respectively, followed by 10 µl saline, and those in NS group received 20 µl saline only. Thirty minutes later, 5% formalin (50 µl) was subcutaneously injected into the left hindpaw. The pain intensity score (PIS) was utilized to assess antinociceptive behavior within 1 h after formalin injection. Twenty-four hours later, the left hindpaw thickness was measured and the expression of PKC in the spinal dorsal horn in the L5 segment was assayed using immunohistochemistry.

RESULTSCompared to group NS, groups K1 and K2 showed significantly decreased PIS (P<0.01) in the second phase of formalin-induced pain; 24 h later, the left hindpaw thickness of group NS increased obviously in comparison with that in the control group (P<0.01), whereas the thickness was significantly reduced in group K1 and K2 as compared to that in group NS (P<0.05). The number of immunoreactive cells and the immunohistochemical score of PKC in the spinal dorsal horn were significantly higher in group NS than in group C (P<0.01), but significantly lower in groups K1 and K2 than in group NS (P<0.05).

CONCLUSIONIntrathecal ketamine produces obvious antinociception against formalin-induced pain in rats and inhibits the enhanced PKC expression in the spinal dorsal horn in response to formalin-induced pain, suggesting the important role of PKC in nociceptive signal transmission and modulation in the spinal cord.

Animals ; Formaldehyde ; adverse effects ; Injections, Spinal ; Ketamine ; administration & dosage ; pharmacology ; Male ; Pain ; chemically induced ; metabolism ; Pain Measurement ; Posterior Horn Cells ; metabolism ; Protein Kinase C ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; drug effects ; metabolism