ABSTRACT
OBJECTIVE: To investigate the effect of trichostatin A (TSA),a specific inhibitor of histone acetyltransferase,on apoptosis of human hepotoma cell line HepG2,the expression of fragile histidine triad (FHIT) and apoptosis-related proteins in order to study apoptosis mechanisms.METHODS: Human hepatoma cell lines HepG2 were cultured and treated with different concentrations of TSA(125,250,500,1 000,2 000 nmol?L-1) for 24 and 48 hours.Human hepatoma cell lines HepG2 survival and apoptosis were determined by MTT assay with absorbance vale and inhibitory rate as index.Apoptotic percentage of HepG2 treated with 250 and 1 000 nmol?L-1 TSA were determined using TUNEL assay.The expressions of FHIT and caspase-3,bax,bcl-2 were analyzed by immunocytochemistry with solvent as control.RESULTS: As compared with control group,absorbance vale of human hepatoma cell lines HepG2 were decreased after treated with different concentration of TSA in dose-dependent and time-dependent manners.Positive cell rate in TUNEL was increased (P0.05).CONCLUSION: TSA may inhibit proliferation and promote apoptosis of hepatoma HepG2 cell by up-regulating the protein expression of FHIT,caspase-3 and bax.
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AIM: To observe the change of insulin receptor in rabbit kidney with acute ischemic-reperfusion injury. METHODS: 15 Japanese white rabbits were allocated randomly into control group, ischemic-reperfusion group(IR group). IR group received clamping for 1 h followed by 2 h or 48 h of reperfusion. At 2 h or 48 h after reperfusion, glucose and insulin in serum were determined. Insulin receptor in renal tissue was analyzed by radioligand binging assay(BAD). RESULTS: The level of serum glucose increased after 2 h reperfusion in 2 groups, but in IR group the value increased much more higher than those in control groups(P
ABSTRACT
AIM:To explore the intracellular signaling transduction pathways in mouse neurons stimulated by octapeptide cholecystokinin(CCK8)on the base of phosphoproteome analysis and to evaluate the types of receptor by the use of CCK8 receptor specific antagonists.METHODS:Cultured neurons were divided into CCK stimulation group,CCK receptor antagonist group and control group at random.All of them were labeled with [32P] orthophosphate(2.03?108 Bq/L)for 2 h in 5% CO2 at 37 ℃.Control and stimulation groups were stimulated with DMEM and CCK8(10-7 mol/L)respectively for 60 min.The neurons in the antagonist groups were pre-incubated for 10 min with different type of receptor antagonists(10-8 mol/L),and then stimulated with CCK8(10-7 mol/L)for 60 min.Reactions were terminated by freezing neurons in liquid nitrogen,and then neuron proteins were collected.The [32P]-labeled lysates isoelectrically focused on IPG Drystrip pH4-7 linear gels and subsequently separated by second-dimensional SDS-PAGE.The 2-DE patterns of phosphoproteins were imaged with autoradiography for 7 days at-70 ℃.Then the phosphoprotein spots were inquired in Swiss-Prot and PPDB protein databases,and analyzed by PDQuest 2D software.RESULTS:Autoradiograph of the 2-DE maps showed that phosphorylation modifications of 46 proteins were involved in CCK signaling transduction in neuron,including protein kinase such as MK07,PI3K,PKC(?,?,?),PKA?,PKG?,AKT3 etc.Intracellular signal molecular such as RB17,GBO?,transcription factor,receptor for growth factor and nuclear protein were also observed.When the neurons were treated with L364 718,the phosphorylation of PKC? and P55G declined obviously,while the phosphorylation of PKC?,PKG?,OGFR and EGFR reduced when the CCK_B specific antagonist L365 260 was used.CONCLUSION:Both CCKA and CCK_B lead to the signaling transduction of CCK8 in neuron,but CCK_B probably play a more important role.The results suggest that cAMP-PKA,MAPK,JNK,PI3K-PKB and cGMP-PKG pathways are involved in CCK_B mediated signaling transduction.
