Subject(s)
Blood Platelets , Fibrin/therapeutic use , Mouth Mucosa/drug effects , Humans , Mouth Mucosa/pathologyABSTRACT
The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.
Subject(s)
Animals , Rats , Anti-Inflammatory Agents/therapeutic use , Glutamic Acid/toxicity , Glycyrrhizic Acid/therapeutic use , Neuroprotective Agents/therapeutic use , /drug effects , Signal Transduction/drug effects , Apoptosis/drug effects , /isolation & purification , Cell Differentiation/drug effects , Cell Survival/drug effects , Chromones/pharmacology , Cytochromes c/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , MAP Kinase Signaling System/drug effects , Mitochondria/drug effects , Morpholines/pharmacology , /classification , /cytology , Proto-Oncogene Proteins c-akt/drug effects , /isolation & purification , /isolation & purificationABSTRACT
The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Glutamic Acid/toxicity , Glycyrrhizic Acid/therapeutic use , Neuroprotective Agents/therapeutic use , PC12 Cells/drug effects , Signal Transduction/drug effects , Animals , Apoptosis/drug effects , Caspase 3/isolation & purification , Cell Differentiation/drug effects , Cell Survival/drug effects , Chromones/pharmacology , Cytochromes c/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , MAP Kinase Signaling System/drug effects , Mitochondria/drug effects , Morpholines/pharmacology , PC12 Cells/classification , PC12 Cells/cytology , Proto-Oncogene Proteins c-akt/drug effects , Proto-Oncogene Proteins c-bcl-2/isolation & purification , Rats , bcl-2-Associated X Protein/isolation & purificationABSTRACT
To evaluate the possibility of establishing an in vivo baculovirus expression system in a silk gland specific secretory way, the recombinant Autographa californica nucleopolyhedrovirus (AcserpegfpDeltaEGT) carrying the reporter gene egfp downstream of silkworm ser1 promoter and signal peptide coding sequence was generated. The purified recombinant baculovirus AcserpegfpDeltaEGT was injected into the haemocoel of newly ecdysed 5thHendekl) instar silkworm larvae at the amount of 10(6) pfu per larva. At 5 days post injection, green fluorescence derived from EGFP could be observed with fluorescent microscope in only the silk gland but not other tissues after dissection of the silkworm. By making an opening on the silk gland wall, green fluorescence could be observed in the outflow of silk gland indicating the secretion of EGFP and the effectiveness of ser1 signal peptide. Western blotting assay confirmed that EGFP exists in the water-soluble part of cocoon silk too. We also established a simple protocol to purify EGFP from the secreted silk proteins.
Subject(s)
Bombyx/metabolism , Exocrine Glands/metabolism , Green Fluorescent Proteins/metabolism , Animals , Bombyx/genetics , Bombyx/growth & development , Bombyx/virology , Gene Expression Regulation , Genes, Reporter , Genetic Vectors , Green Fluorescent Proteins/genetics , Larva/genetics , Larva/growth & development , Larva/metabolism , Larva/virology , Nucleopolyhedroviruses/genetics , Nucleopolyhedroviruses/metabolism , Promoter Regions, Genetic , Protein Sorting Signals , Recombinant Fusion Proteins/metabolism , Sericins/genetics , Sericins/metabolism , SilkABSTRACT
Several strains of silkworm Bombyx mori were tested for the gene delivery feasibility of Autographa californica nucleopolyhedrovirus (AcNPV) in vivo. In contrast to the general belief that silkworms were non-permissive to AcNPV, we found that 3 of 7 tested strains were AcNPV permissive. To dispel the physiological influence of the ecdysteroid UDP-glucosyltransferase (EGT) on the silkworm, we modified the AcNPV bacmid by disruption of that gene. Expression pattern of EGFP in tissues of silkworm larvae after injection of EGT-null AcNPV vector carrying EGFP cassette was revealed by green fluorescence and Western blot analysis. Viral DNA was detected and semi-quantified in various kinds of tissues by dot blot assay. Active recombinant virus from larval hemolymph was detectable by TCID(50). Our results indicate that some strains of silkworm were permissive to AcNPV, which could serve as a novel gene deliver tool to silkworm in vivo.