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

This study aims to observe the intervention effects of Chinese herbal medicine of supplementing Qi and activating blood circulation on chronic intermittent hypoxia(CIH) composite insulin resistance(IR) mediated atherosclerosis(AS) mice model,and to observe the mechanism of SREBP-1 c signaling molecule.IR Apo E-/-mice model was induced by high-fat diet combined with STZ injection.Then the mice were treated with hypoxic animal incubator for 8 h per day and 8 weeks to establish a CIH+IR-ApoE-/-mouse model.Model mice were randomly and averagely divided into normoxic control group(NC),model group(CIH) and SREBPs inhibitor group(betulin),atorvastatin group(WM),TCM low-dose group(TCM-L),TCM middle-dose group(TCM-M) and TCM high-dose group(TCM-H) group.Chinese herbal medicine of supplementing Qi and activating blood circulation including ginsenosides combined with ligustrazine(TMP) were used as intervention drugs.The study observed the effect of drugs on IR,serum lipid,inflammation,stress,AS and SREBP-1 c related molecules.The results showed that fasting blood glucose in TCM-H group decreased compared with other experimental groups(P<0.05).HDL-C level in betulin group,WM group,TCM-H group was higher than that in CIH group(P<0.05).LDL-C level in TCM-M group,TCM-H group is lower than that in CIH group(P<0.05).The level of CRP in CIH group was higher than that in other groups(P<0.05).The level of SOD in TCM-H group was higher than that in CIH group(P<0.05).NC group and CIH group showed obvious AS aortic plaque,while betulin group,WM group,TCM-H group showed reduction in AS plaque(P<0.05).For descending aorta,AS plaque in CIH group was multiple and large,while less and smaller in WM group and TCM-H(P<0.05).The expression of SREBP-1 c and FAS in aorta and skeletal muscle in TCM-H group was lower than that in CIH group(P<0.05).In aorta,the expression of TNF-α and CD106(VCAM-1) was lower in TCM-H group than that in CIH group(P<0.05).In aorta,skeletal muscle and liver,the level of p-IRS-1 in TCM-H group was significantly higher than that in CIH group(P<0.05).In aorta and liver,the expression of HIF-1α in TCM-H group was lower than that in CIH group(P<0.05).The study demonstrated that combination ginsenosides with TMP could improve IR and serum lipid level and inhibit inflammation and oxidative stress as well as ultimately alleviate AS to some extent.And the mechanism of its interventional effects might be related to the inhibition of CIH-induced upregulation of SREBP-1 c related molecules.
Animals ; Atherosclerosis ; drug therapy ; Blood Circulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Ginsenosides ; pharmacology ; Hypoxia ; pathology ; Insulin Resistance ; Mice ; Mice, Knockout, ApoE ; Pyrazines ; pharmacology ; Qi ; Random Allocation

OBJECTIVES: To investigate the role of tetramethylpyrazine(TMP) nitrone in proliferation and differentiation of neural stem cells (NSCs). METHODS: We separated and cultivated the original generation of NSCs from cerebral cortex of 14 days rat embryo, and the phenotype characteristics of the third-generation NSCs was tested by immunofluorescence. The experiment was divided into control group, β-mercaptoethanol positive control group, tetramethylpyrazine nitrone group and tetramethylpyrazine nitrone + ethylene glycol tetraacetic acid(EGTA) group (=4). The third-generation cultivation of NSCs was used in the experiment. The effect of tetramethylpyrazine nitrone on the number of NSCs proliferation was determined by BrdU and MTT, and the differentiation of NSCs was determined by Western blot. RESULTS: The primary NSCs was isolated successfully, neurospheres with typical NSCs morphology and expressing nestin was formed at 3-5 days. As BrdU and MTT assay results shown, compared with the control group andβ-mercaptoethanol positive control group, the NSCs proliferation numbers of tetramethylpyrazine nitrone group increased significantly(<0.05). The results of Western blot showed that the neuronal differentiation rate of NSCs was increased significantly in both the tetramethylpyrazine nitrone group and tetramethylpyrazine nitrone + EGTA group, and the differentiation rate of NSCs in tetramethylpyrazine nitrone + EGTA group increased more significantly(<0.05). CONCLUSIONS Tetramethylpyrazine nitrone can significantly enhance the proliferation and neuronal differentiation rate of NSCs. Decrease in extracellular Ca can promote the differentiation of NSCs into neurons induced by tetramethylpyrazine nitrone. Ca signaling plays an important role in the differentiation of NSCs into neurons.
Animals ; Calcium Signaling ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Neural Stem Cells ; cytology ; drug effects ; Nitrogen Oxides ; pharmacology ; Pyrazines ; pharmacology ; Rats

OBJECTIVETo investigate the effects of ligustrazine (LTZ) on airway inflammation in a mouse model of neutrophilic asthma (NA).

METHODSForty healthy C57BL/6 female mice were randomly divided into 4 groups using a random number table, including the normal control, NA, LTZ and dexamethasone (DXM) groups, with 10 rats in each group. The NA mice model was established by the method of ovalbumin combined with lipopolysaccharide sensitization. At 0.5 h before each challenge, LTZ and DXM groups were intraperitoneally injected with LTZ (80 mg/kg) or DXM (0.5 mg/kg) for 14 d, respectively, while the other two groups were given the equal volume of normal saline. After last challenge for 24 h, the aerosol inhalation of methacholine was performed and the airway reactivity was measured. The bronchoalveolar lavage fluid (BALF) was collected. The Wright-Giemsa staining was used for total white blood cells and differential counts. The levels of cytokines interleukin (IL)-17 and IL-10 were detected by enzyme-linked immunosorbent assay. The pathological change of lung tissue was observed by hematoxylin eosin staining.

RESULTSThe airway responsiveness of the NA group was signifificantly higher than the normal control group (P<0.05), while those in the LTZ and DXM groups were signifificantly lower than the NA group (P<0.05). The neutrophil and eosinophil counts in the LTZ and DXM groups were signifificantly lower than the NA group (P<0.05), and those in the LTZ group were signifificantly lower than the DXM group (P<0.05). There were a large number of peribronchiolar and perivascular inflammatory cells in fifiltration in the NA group. The airway inflflammation in the LTZ and DXM groups were signifificantly alleviated than the NA group. The infifiltration in the LTZ group was signifificantly reduced than the DXM group. Compared with the normal control group, the IL-17 level in BALF was signifificantly increased and the IL-10 level in BALF was signifificantly decreased in the NA group (P<0.05). LTZ and DXM treatment signifificantly decreased IL-17 levels and increased IL-10 levels compared with the NA group (P<0.05), and the changes in the above indices were more signifificant in the LTZ group (P<0.05).

CONCLUSIONLTZ could alleviate the airway inflflammation in the NA mice model through increasing the IL-10 level and decreasing the IL-17 level.

Animals ; Asthma ; blood ; complications ; drug therapy ; pathology ; Bronchoalveolar Lavage Fluid ; cytology ; Disease Models, Animal ; Female ; Interleukin-10 ; metabolism ; Interleukin-17 ; metabolism ; Leukocyte Count ; Lung ; drug effects ; pathology ; Mice, Inbred C57BL ; Neutrophils ; drug effects ; pathology ; Pneumonia ; blood ; complications ; drug therapy ; pathology ; Pyrazines ; pharmacology ; therapeutic use ; Respiratory Hypersensitivity ; blood ; complications ; drug therapy ; pathology

Trigeminal neuralgia (TN) is a kind of recurrent transient and severe pain that is limited to the trigeminal nerve in one or more branches. The clinical incidence of TN is high, which seriously affects the quality of life of the patients and is difficult to cure. The present study investigated the effects of tetramethylpyrazine (TMP) on TN induced by chronic constriction injury of the infraorbital nerve (ION-CCI) in rats. Adult male Sprague-Dawley rats were randomly assigned to four groups: sham, sham treated with TMP (Sham+TMP), TN model (TN), and TN treated with TMP (TN+TMP). The rat TN model was established by ION-CCI and TMP (50 mg/kg) was injected intraperitoneally once a day for 2 weeks after operation. The mechanical response threshold was tested by the electronic von Frey filaments. The expression of CGRP in the trigeminal ganglia (TGs) of rats on the operative side was detected by RT-PCR, immunohistochemical staining and Western blot. In 15 days after operation, TN group showed a robust decrease in mechanical response threshold as compared with sham group. From day 9 to day 15 after operation, TMP treatment significantly suppressed the TN-induced mechanical hyperalgesia (P < 0.05). On day 15 after operation, RT-PCR, immunohistochemical staining and Western blot analysis showed an obvious increase in expression level of CGRP in TGs of TN group compared with sham group, which was downregulated by TMP treatment (P < 0.05). These results suggested that TMP might have a therapeutic potential for the treatment of TN through regulating CGRP expression in the TGs.
Animals ; Constriction ; Hyperalgesia ; drug therapy ; Male ; Pyrazines ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Trigeminal Ganglion ; physiopathology ; Trigeminal Neuralgia ; drug therapy

OBJECTIVETo explore the effect of ligustrazine on the migration of bone marrow mesenchymal stem cells (BMSCs) and protein expressions of matrix metalloproteinase-2 and-9 (MMP-2 and MMP-9) in vitro.

METHODSBMSCs were in vitro isolated and cultured using whole bone marrow adherent method, and phenotypes [surface positive antigens (CD29 and CD90) and negative antigens (CD34 and CD45)] identified using flow cytometry. BMSCs were divided into the blank control group, 25, 50, 100 µmol/L ligustrazine group, and the GM6001 group (100 µmol/L ligustrazine +MMPs inhibitor GM6001 ). The migration of BMSCs was tested by Transwell chamber test and wound healing assay after treated with ligustrazine for 24 h. The protein expressions of MMP-2 and MMP-9 were detected by Western blot.

RESULTSThe third passage BMSCs grew well in uniform morphology. The expression rate of CD29, CD90, CD34, and CD45 was 96.9%, 97.3%, 0.2%, and 3.0%, respectively. Compared with the blank control group, the number of migrated cells and relative distance of cell invasion increased, and the protein expressions of MMP-2 and MMP-9 were elevated in each ligustrazine group (P < 0.05, P < 0.01). Compared with 100 µmol/L ligustrazine group, the number of migrated cells and relative distance of cell invasion decreased in 25 and 50 µmol/L ligustrazine groups and the GM6001 group (P < 0.01). Protein expression of MMP-2 decreased in 25 and 50 µmol/L ligustrazine groups (P < 0.01).

CONCLUSIONLigustrazine could promote the migration of BMSCs in vitro, and its mechanism might be related to up-regulating expression levels of MMP-2 and MMP-9 protein.

Cell Movement ; Cells, Cultured ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Pyrazines ; pharmacology ; Up-Regulation

OBJECTIVETo study the effect of ligustrazine nanoparticles nano spray (LNNS) on transforming growth factor β (TGF-β)/Smad signal protein of rat peritoneal mesothelial cells (RPMC) induced by tumor necrosis factor α (TNF-α), and the anti-adhesion mechanism of LNNS in the abdominal cavity.

METHODSThe primary culture and subculture of rat peritoneal mesothelial cells (RPMC) was processed by trypsin digestion method in vitro. The third generation was identifified for experiment and divided into 5 groups: a blank group: RPMC without treatment; a control group: RPMC stimulated with TNF-α; RPMC treated by a low-dosage LNNS group (2.5 mg/L); RPMC treated by a medium-dosage LNNS group (5 mg/L); and RPMC treated by a high-dosage LNNS group (10 mg/L). Reverse transcription-polymerase chain reaction was applied to test the expression of fifibronectin, collagen I (COL-I), TGF-β mRNA, and Western blot method to test the Smad protein 7 expression of RPMC.

RESULTSCompared with the blank group, a signifificant elevation in fifibronectin (FN), COL-I and TGF-β mRNA expression of RPMC were observed in the control group (P<0.05). Compared with the control group, LNNS suppressed the expressions of FN, COL-I and TGF-β mRNA in a concentrationdependent manner (P<0.05). The expression of Smad7 protein of RPMC was down-regulated by TNF-α stimulation, and up-regulated with the increase of LNNS dose (P<0.05).

CONCLUSIONSTNF-α may induce changes in RPMC's viability, leading to peritoneal injury. LNNS could reverse the induction of fifibrosis related cytokine FN, COL-I and TGF-β, up-regulating the expression of Smad7 by TNF-α in RPMC, thus attenuate peritoneal injury by repairing mesothelial cells.

Animals ; Collagen Type I ; genetics ; metabolism ; Epithelium ; drug effects ; metabolism ; Fibronectins ; metabolism ; Male ; Nanoparticles ; chemistry ; ultrastructure ; Particle Size ; Peritoneal Cavity ; cytology ; Pyrazines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

BACKGROUNDTwo recent whole-exome sequencing researches identifying somatic mutations in the ubiquitin-specific protease 8 (USP8) gene in pituitary corticotroph adenomas provide exciting advances in this field. These mutations drive increased epidermal growth factor receptor (EGFR) signaling and promote adrenocorticotropic hormone (ACTH) production. This study was to investigate whether the inhibition of USP8 activity could be a strategy for the treatment of Cushing's disease (CD).

METHODSThe anticancer effect of USP8 inhibitor was determined by testing cell viability, colony formation, apoptosis, and ACTH secretion. The immunoblotting and quantitative reverse transcription polymerase chain reaction were conducted to explore the signaling pathway by USP8 inhibition.

RESULTSInhibition of USP8-induced degradation of receptor tyrosine kinases including EGFR, EGFR-2 (ERBB2), and Met leading to a suppression of AtT20 cell growth and ACTH secretion. Moreover, treatment with USP8 inhibitor markedly induced AtT20 cells apoptosis.

CONCLUSIONSInhibition of USP8 activity could be an effective strategy for CD. It might provide a novel pharmacological approach for the treatment of CD.

Adrenocorticotropic Hormone ; metabolism ; Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; physiology ; Cell Survival ; drug effects ; physiology ; Endopeptidases ; metabolism ; Endosomal Sorting Complexes Required for Transport ; antagonists & inhibitors ; metabolism ; Enzyme Inhibitors ; pharmacology ; Humans ; Indenes ; pharmacology ; Mice ; Pyrazines ; pharmacology ; Receptor, Epidermal Growth Factor ; metabolism ; Ubiquitin Thiolesterase ; antagonists & inhibitors ; metabolism

OBJECTIVETo investigate the effects of perfluorocarbon and ligustrazine in protecting the lungs against ischemia-reperfusion injury in rats.

METHDSForty SD rats with ischemia-reperfusion lung injury were randomized equally into control, ligustrazine, perfluorocarbon, and perfluorocarbon plus ligustrazine groups and received the corresponding treatment via the tail vein 5 min before reperfusion. The lung tissues were harvested and the levels of malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD) and tumor necrosis factor-α (TNF-α) were detected 3 h after reperfusion. The pathological changes and pathological scores of the lung tissues were analyzed.

RESULTSMDA and MPO levels were significantly lower and SOD activities significantly higher in the lung tissues in the 3 treatment groups than in the control group (P<0.05). The rats in the combined treatment group showed a significantly lower MPO level and a significantly higher SOD activity than those treated with ligustrazine or perfluorocarbon alone (P<0.05). No significant difference was found in TNF-α levels in the lung tissues among the 4 groups (P>0.05). The lung tissues in the control group showed obvious edema and exudation, and the tissues in ligustrazine and perfluorocarbon groups showed no edema but with a few red blood cells and exudation; no edema was found in the combined treatment group with only a small amount of exudation. The pathological scores differed significantly among the 4 groups.

CONCLUSIONPerfluorocarbon and ligustrazine, especially in combined use, can promote endogenous oxygen free radical scavenging, decrease peripheral blood proinflammatory cytokines, and inhibit neutrophils filtration in the lungs of rats with ischemia/reperfusion lung injury.

Animals ; Cytokines ; Fluorocarbons ; pharmacology ; Lung Injury ; drug therapy ; Malondialdehyde ; metabolism ; Peroxidase ; metabolism ; Protective Agents ; pharmacology ; Pyrazines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; Superoxide Dismutase ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

To study the anti-inflammatory activity of glycyrrhizin acid, ligustrazine and puerarin. In the study, the liquichip-based high-throughput synchronous detection technique for 23 inflammatory factors, uniform design, comprehensive weight method were adopted to study the effect of different combined administration of glycyrrhizin acid, ligustrazine and puerarin in inhibiting the expression of lipopolysaccharide (LPS)-induced RAW264. 7 cells and multiple inflammatory cytokines. In the study, the uniform design table U₉ (9³) was adopted to design doses of glycyrrhizin acid, ligustrazine and puerarin. The liquichip technique was used to detect the effect of different combined administration of glycyrrhizin acid, ligustrazine and puerarin on the 23 cytokines expressed in LPS-induced mouse macrophage RAW264. 7 inflammation model. The traditional Chinese medicine component optimization software and the improved least angle regression algorithm were used to analyze the dose-effect relationship among the three components and the cytokine inhibition rate and produce the regression equation. The comprehensive weight method was applied to get the optimal dose ratio of glycyrrhizic acid, ligustrazine and puerarin with highest efficacy of 25:2:13 and verify the optimal dose ratio. The verification results were consistent with the prediction trend, indicating the accuracy of the mathematical model for predicting the experiment. The experimental results showed the multi-target and multi-level efficacies of glycyrrhizic acid, ligustrazine and puerarin and the high anti-inflammatory activity of their combined administration, which provides powerful basis for subsequent drug development.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Cytokines ; Glycyrrhizic Acid ; pharmacology ; Isoflavones ; pharmacology ; Lipopolysaccharides ; immunology ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Pyrazines ; pharmacology ; RAW 264.7 Cells