AIM: To investigate the effects of pioglitazone , a peroxisome proliferator-activated receptor γ( PPARγ) agonist, on the cognitive impairments and inflammatory cytokine production induced by isoflurane in aged mice . METHODS:Male C57BL/6J mice ( 11-month-old, n =136 ) were assigned randomly into 5 groups: control group ( Con) , isoflurane group ( Iso) , 10 mg/kg pioglitazone +isoflurane group ( Pi10+Iso) , 20 mg/kg pioglitazone +isoflu-rane group (Pi20+Iso) and 20 mg/kg pioglitazone alone group (Pi20).The mice in all isoflurane-treated groups were ex-posed to oxygen mixed with 1.4%isoflurane for 2 h.The mice in Con group and in Pi20 group were exposed to oxygen only for 2 h.Pioglitazone was suspended in 1% carboxymethyl cellulose (CMC).Pioglitazone (10 mg/kg or 20 mg/kg) was gavaged 2 h prior to the exposure of isoflurane or oxygen alone .The same volume of 1% CMC was gavaged in Con group and in Iso group.Fear conditioning tests were performed to determine the learning and memory abilities 48 h after isoflurane exposure.Fresh cerebral cortice and hippocampi were dissected to measure the protein expression of PPAR γby Western blotting, and the contents of IL-1βand TNF-αwere analyzed by ELISA 6 h after isoflurane exposure .RESULTS: Com-pared with Con group, the response of freezing behavior decreased (P<0.05) and IL-1βcontent in the hippocampus in-creased ( P<0.05) in Iso group.Compared with Iso group , the response of freezing behavior and PPARγprotein expres-sion level had no significant change ( P>0.05) in Pi10+Iso group, but the response of freezing behavior and PPARγpro-tein expression level increased significantly (P<0.05) and IL-1βcontent in the hippocampus decreased (P<0.05) in Pi20+Iso group.IL-1βcontent in the cerebral cortex and TNF-αlevels both in the cerebral cortex and the hippocampus showed no significant difference among all groups (P>0.05).CONCLUSION:Pioglitazone attenuates cognitive impair-ments and the elevates the level of IL-1βin the hippocampus induced by isoflurane in aged mice .

Objective To investigate if ketamine protects cultured rat cerebellar granule neurons (CGNs) from apoptosis induced by glutamate and its possible mechanism of signal transduction. Methods Primarily cultured CGNs prepared by enzymatical digestion of cerebellum isolated bom 7-8 day old SD rats were randomly divided into 8 groups: (1) control group (C); (2) glutamate group (G) in which CGNs were incubated with 300 ?mol?L-1 glutamate; (3) (4) (5) ketamine groups (K1 , K2, K3) in which CGNs were incubated with ketamine 10 (K1 ) 100 (K2) or 1 000 (K3) ?mol ? L-1 for 1h before glutamate was added; (6) Ly 294002 - K3 group (LK3) in which CGNs were incubated with 20 ?mol?L-1 Ly 294002 (a specific phosphatidyl-inositol 3-kinase inhibitor) for 30 min before ketamine 1 000 ? mol ? L-1 and glutamate were added as in group K3; (7) Ly 294002 group (L) in which CGNs were incubated with Ly 294002 20?mol ? L-1 and (8) Ly 294002-glutamate group (LG) in which CGNs were incubated with Ly 294002 20 ?mol ? L-1 for 30 min before glutamate was added. After 20 h incubation the apoptosis in CGNs was detected by Hoechst 33258 nucleus staining, phase-contrast microscopy and DNA fragmentation agarose gel electrophoresis. The neuronal survival was determined by fluorescein diacetate (FDA) staining.Results Glutamate 300 ?mol ? L-1 induced apoptosis in CGNs as characterized by cytoplasmic blebbing, heterochromatic clumping, condensation of nuclear chromatin and a typical apoptotic DNA ladder revealed by agarose gel electrophoresis. Ketamine improved the survival of CGNs incubated with glutamate (300 ?mol ? L-1) and blocked the glutamate-induced apoptosis in a concentration-dependent manner. Ly 294002 (20 ?mol ? L-1) antagonized the anti-apoptotic effects of ketamine. Conclusion Ketamine can protect CGNs from apoptosis induced by glutamate. Phosphatidyl-inositol 3-kinase/Akt pathway may be involved in the antiapoptotic action of ketamine.

AIM: To investigate the contribution of angiotensin-converting enzyme inhibitor (ACEI) to the regulation of calpain system in infarcted myocardium. METHODS: Rat myocardial infarction (MI) model was established by permanent ligation of the left coronary artery. The treatment with the ACEI inhibitor rampril (1 mg?kg~-1 ?d~-1 ) was started 7 days prior to surgery. On day 1, 3, 7 and 14 after MI, protein levels of calpainⅠ, Ⅱ and calpastatin were determined in left ventricular free wall (LVFW), interventricular septum (IS) and right ventricule. RESULTS: CalpainⅠprotein level was increased in IS 14 d post MI, whereas the protein level of calpainⅡ was maximally increased in LVFW 3 d post MI. Rampril decreased protein up-regulation of calpainⅠ and Ⅱ, and reduced infarct size and interstitial fibrosis. Calpastatin protein expression was not affected by ACEI. CONCLUSIONS: CalpainⅠ is involved in cardiac remodelling in the late and calpainⅡ contributes to cardiac tissue damage in the early phase of MI. The heart protective effect of ACEI may be related to the inhibition of calpain system in the pathogenesis of myocardial infarction.

AIM: To confirm the differential expression genes in the rat ischemic brain. METHODS: The middle cerebral artery occlusion ischemic model was set up in rats. Fluorescence differential display reverse transcriptase-polymerase chain reaction (FDD RT-PCR) and reverse Northern blotting were used to fast confirm the differential expression genes. RESULTS: Nine differential expression sequence tags, including 6 known sequences and 3 unknown sequences, were confirmed. Among the known sequences, mus musculus ab1-interactor1,homo sapiens CGI-99 protein, tissue inhibitor of metalloproteinase 3 and homosapiens nuclear receptor co-repressor were up-regulated while homo-sapiens nuclear matrix protein p84 and coatomer protein complex, subunit gamma 2 were down-regulated. CONCLUSIONS: ① Combination of fluorescence differential display reverse transcriptase-polymerase chain reaction (FDD RT-PCR) and reverse Northern blotting is a method to fast-confirm the differential expression genes; ② There are differential expression genes in ischemic brain regions compared to non-ischemic parts. [

AIM To investigate the protection of plicamycin on apoptosis in cerebellar granule neurons (CGN) of rat. METHODS TUNEL, Hoechst 33258 staining, agarose gel electrophoresis and fluorescein diacetate staining were used to detect morphological and biochemical characteristics of apoptosis in primary rat CGN. RESULTS Being pre-incubated with plicamycin for 1 h and lasting for 24 h, rat CGN apoptosis induced by low potassium basal modified Eagle′s medium for 24 h was inhibited in a plicamycin concentration-dependent manner. This effective concentrations of plicamycin were from 50 to 200 nmol·L-1, and the maximum inhibitory rate of plicamycin on CGN apoptosis was near 80% at 200 nmol·L-1. CONCLUSIONPlicamycin inhibits rat CGN apoptosis induced by low potassium.

AIM To construct a non-normalized cDNA library from Agkistrodon acutus venom gland as an imtial step to develop new and more effective venom by genetic engineering technique for screening and expressing target genes. METHODS The total RNA was extracted from fresh venom gland using Trizol. mRNA was reversely transcripted to cDNA using superscriptⅡ reverse transcriptase. Second-strand synthesis was performed using DNA polymeraseⅠ. After adding EcoRⅠ adaptor, phosphorylating the end and digesting with XhoⅠ, the cDNA was collected in five fractions (<0.25 kb, 0.25-0.5 kb, 0.5-1 kb, 1-2 kb and >2 kb) using the QIAquick Gel Extraction kit and ligated to pBluescriptⅡ vectors. The five libraries obtained were plated by infecting E.coli DH10B, constructing a cDNA library of Agkistrodon acutus venom gland. Sequencing clones at random, 8696 high quality 5′ end expressed sequenced tags (ESTs) were obtained and analyzed. The initial sequences were assembled into 2855 clusters. Among which, one of the clusters (Agkihagin) consisting of 74 ESTs was identified as a novel metalloprtoteinase based on RT-PCR and sequence analysis. RESULTSThe titers of library were 2.048×106. The novel metalloproteinase belonged to PⅢ type metalloproteinase. Its open reading frame was composed of 1827 nucleotides and coded a pre-zymogen of 608 amino acid with zinc-binding domain for metalloproteinase and Asp-Glu-Cys-Asp(DECD) domain for disintegrin. CONCLUSION The capacity of cDNA library of venom gland is above the general level of cDNA library. It would be a helpful platform to construct a catalog for transcripts in the venom gland of the Agkistrodon acutus. The sequence analysis indicates that the deduced amino acid sequence of the identified gene for metalloproteinase share the highest 87% identity with the metalloproteinase genes of other snakes in the GenBank. It lays a good foundation for the study of structure-function relationships of snake venom metalloproteinases.

【Objective】To study the effect of the specific p38 mitogen-activated protein kinase(p38 MAPK) inhibitor SB203580 on apoptosis of cerebellar granule neurons induced by low potassium.【Methods】Apoptosis was induced by switching the cultured cerebellar granule neurons from a culture medium containing K+ 25 mmol*L-1 to a medium containing K+ 5 mmol*L-1 (cLK).Fragmentation of DNA was analyzed using agarose gel eletrophoresis.SAPK/JNK activity was measured by SAPK/JNK assay kit.【Results】Low potassium resulted in apoptosis as characterized by morphological and biochemical features,but the specific p38 MAPK inhibitor SB203580 improved the survival of cerebellar granule neurons cultured in cLK medium by blocking apoptosis in a concentration-dependent manner.The expression and phosphorylation of c-Jun increased and the activity of c-Jun N-terminal protein kinase (JNK) elevated when cerebellar granule neurons were cultured in cLK medium.But when the cerebellar granule neurons cultured in cLK medium were exposed to 25 μmol*L-1 SB203580,the expression and phosphorylation of c-Jun and the activity of JNK were both decreased evidently.【Conclusions】These results indicate that SB203580 inhibits the activation of JNK and phosphorylation of c-Jun,and therefore protects granule neurons from apoptosis induced by low potassium.

【Objective】To investigate the molecular mechanism of dopamine (DA)-induced apoptosis in cultured cerebellar granule neurons (CGNs) and the effect of muscarinic cholinergic receptor (mAChR) agonist carbachol on it.【Methods】The apoptosis of neurons was measured by phase-contrast microscopy,Hoechst 33258 nucleus staining and DNA fragmentation agarose gel electrophoresis.The neuronal viability was measured by fluorescein diacetate (FDA) staining.The activation of extracellular signal-regulated protein kinase (ERK) was determined by Western blot.【Results】Dopamine increases the phosphorylation of ERK and induces apoptosis in CGNs,which is blocked by both carbachol and PD 98059.The protective effect and the inhibiting ERK phosphorylation of carbachol were blocked by atropine.【Conclusion】DA-induced apoptosis in CGNs may be mediated by activation of ERK.Carbachol protects CGNs from DA-induced apoptosis by activating mAChR and subsequent inhibition of activation of ERK.

Aim To investigate the contribution of cardiac L-and L/T-type Ca~2+ channels in the calpain mediated myocardial damage following myocardial infarction(MI).Methods Rat MI model was established by permanent ligation of the left coronary artery, infarcted rats were orally treated with placebo, amlodipine(L-channel blockade, 4 mg?kg~-1 ?d~-1 ) or mibefradil(L/T-Channel blockade, 10 mg?kg~-1 ?d~-1 ) beginning 7 d before induction of myocardial infarction. Protein levels of u-calpain and m-calpain were measured 1,3,7 and 14 d post coronary occlusion in the noninfarcted and infarcted myocardium.Infarcted size,left ventricular dilation were determined in picrosirius red stained hearts.Results Myocardial infarction induced an up regulation of u-calpain protein and activity in the noninfarcted myocardium(maximum day 14 days post infarction), whereas protein and activity of m-calpain were increased in the infarcted myocardium 3 d post infarction. Amlodipine inhibited protein up-regulation of u-calpain and decreased left ventricular dilation and interventricular septal thickness. Mibefradil attenuated protein up regulation of m-calpain 14 days post infarction, reduced infarct size more obviously.Conclusions Infarction-induced cardiac hypertrophy was accompanied by an up-regulation of u-calpain, whereas m-calpain was up-regulated in the infarcted myocardium in the processing of cardiac infarcted pathogensisi. Cardiac L and L/T-type Ca~2+ channel blockade differentially reduced post infarction remodeling associated with selective inhibition of cardiac u-calpain and m-calpain, respectively.

AIM: To analyze the subcellular localization of Arnt2 in rat cerebellar granule neurons (CGNs). METHODS: Based on the amino acids sequence of Arnt2 (LOCUS:NP_036913), the subcellular localization of Arnt2 in eukaryotic cells and the nuclear export signals (NES) of Arnt2 were predicted in CBS bioinformatics database. The subcellular localization of Arnt2 in rat cerebellar granule neurons was detected by the method of laser scanning confocal microscopy (LSM) analysis. RESULTS: It was predicted that Arnt2 located in nuclei of eukaryotic cells with the most probability, while located in cytoplasmic mitochondria with a slight possibility. A nuclear export signal was found in Arnt2 amino acids sequence, it was identified to be the leucine of No.143 that located in N-terminal of Arnt2 amino acids sequence. Finally, the result of LSM analysis shows nuclear localization of Arnt2 in rat CGNs. CONCLUSION: Arnt2 is located in nuclei of normal rat CGNs, it suggests that Arnt2 has the tendency to translocate into mitochondria after induced by some of inducible factors, for both the possibility of mitochondria localization and NES exist in Arnt2 amino acids sequence.