1.Involvement of MAPK pathways in NMDA-induced apoptosis of rat cortical neurons.
Xiao-Rong YANG ; Ping SUN ; Hua-Ping QIN ; Pei-Pei SI ; Xue-Fei SUN ; Ce ZHANG
Acta Physiologica Sinica 2012;64(6):609-616
NMDA-induced excitotoxicity cause severe neuronal damage including apoptosis and necrosis. The present study was aimed to evaluate the proportion of NMDA-induced apoptosis of rat cortical neurons and discover signal transduction mechanism. Caspase inhibitor and lactate dehydrogenase (LDH) assay were used to study the NMDA-induced apoptosis. To explore the involved signal pathways, the primary culture of rat cortical neurons were pretreated by the inhibitors of three MAPK pathways, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. With 2 h of NMDA treatment, cellular apoptosis was measured by caspase-3 activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and Annexin V staining. The results showed that: (1) Caspase-dependent apoptosis accounted for 22.49% in NMDA-induced neuronal death; (2) Pretreatment with p38 MAPK inhibitor SB203580 (10 μmol/L) significantly decreased NMDA-mediated caspase-3 activity by 30.43% (P < 0.05). However, ERK inhibitor PD98059 (20 μmol/L) or JNK inhibitor SP600125 (20 μmol/L) did not influence caspase-3 activity; (3) Pretreatment with SB203580 significantly reduced the number of NMDA-induced TUNEL-positive cells by 33.10% (P < 0.05). PD98059 (20 μmol/L) or SP600125 (20 μmol/L) did not show obvious effect; (4) Pretreatment with SB203580 (10 μmol/L) significantly reduced the number of NMDA-induced early apoptotic neurons by 55.56% (P < 0.05). Also, SP600125 (20 μmol/L) significantly decreased the amount of late apoptotic/dead cells by 67.59% (P < 0.05). There was no effect of PD98059 (20 μmol/L). These results indicate that: (1) NMDA induces neuronal apoptosis besides necrosis; (2) p38 MAPK, but not JNK and ERK, is involved in NMDA-induced neuronal apoptosis, and inhibition of the apoptotic signaling pathway contributes to neuroprotection; (3) JNK activation might contribute to NMDA-induced neuronal necrosis rather than apoptosis.
Animals
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Anthracenes
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pharmacology
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Apoptosis
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Caspase 3
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metabolism
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Cells, Cultured
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Extracellular Signal-Regulated MAP Kinases
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antagonists & inhibitors
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Imidazoles
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pharmacology
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JNK Mitogen-Activated Protein Kinases
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antagonists & inhibitors
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MAP Kinase Signaling System
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N-Methylaspartate
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pharmacology
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Neurons
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cytology
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Primary Cell Culture
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Pyridines
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pharmacology
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Rats
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p38 Mitogen-Activated Protein Kinases
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antagonists & inhibitors
2.Construction of recombinant gene adenovirus encoding enhanced green fluorecence protein-peroxisome proliferator-activated receptor gamma2 fusion protein and its expression in bone marrow mesenchymal stem cells.
Li-zi LIAO ; Jin-gang XIAO ; Miao-miao YANG ; Zi-ren KONG ; Qin-ce SUN ; Wei-dong TIAN
West China Journal of Stomatology 2010;28(4):430-434
OBJECTIVETo construct mouse enhanced green fluorecence protein (EGFP) -peroxisome proliferator-activated receptor (PPAR)gamma2, and to detect EGFP-PPARgamma2 expression in infected mouse bone marrow mesenchymal stem cells (BMSC).
METHODSCut the fragment of PPARgamma2 from the expression plasmid pcDNA flag PPARgamma2, then cloned the gene fragment into pEGFP-C1 and pEGFP-N1 vector. Subsequently, subclone the fragment EGFP-PPARgamma2 from pEGFP-C1-PPARgamma2 into the shuttle plasmid DC315. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid DC315-EGFP-PPARgamma2 and large adenovirus helper plasmid pBHGlox deltaE1, 3Cre in mediation of liposome. The obtained replication-defective recombinant adenovirus Ad-EGFP-PPARgamma2 was confirmed. Then it was propagated in HEK293 cells. After the BMSC were transfected for 72 h, adipogenic differentiation was demonstrated.
RESULTSHEK293 cells were transfected with the pEGFP-C1-PPARgamma2 or pEGFP-N1-PPARgamma2 in mediation of liposome. The former green fluorescence protein was better than the latter by fluorescence microscope. The recombinant plasmids were digested and identified. Western blot analysis showed the expression of EGFP-PPARgamma2 in vitro. EGFP-PPARgamma2 protein was detectable in the nucleus of BMSC.
CONCLUSIONThe recombinant adenovirus encoding EGFP-PPARgamma2 fusion protein was successfully constructed, which provided a basis for application of EGFP-PPARgamma2 gene to adenovirus-mediated gene therapy.
Adenoviridae ; Animals ; Bone Marrow Cells ; metabolism ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; HEK293 Cells ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Mice ; PPAR gamma ; metabolism ; Recombinant Proteins ; metabolism ; Transfection
3.Is oral microbiome of children able to maintain resistance and functional stability in response to short-term interference of ingesta?
Fangqiao WEI ; Xiangyu SUN ; Yufeng GAO ; Haoyu DOU ; Yang LIU ; Lili SU ; Haofei LUO ; Ce ZHU ; Qian ZHANG ; Peiyuan TONG ; Wen REN ; Zhe XUN ; Ruochun GUO ; Yuanlin GUAN ; Shenghui LI ; Yijun QI ; Junjie QIN ; Feng CHEN ; Shuguo ZHENG
Protein & Cell 2021;12(6):502-510