BACKGROUND: Both apoptosis suppression gene Bcl-2 and apoptosis in duction gene Bax take parts in the apoptosis of neurons in ischemic penum bra. Whether would the polypeptide from chlamys farreri that is proved to be of anti-oxidation and anti-apoptosis in vitro protect the ischemic neurons from apoptosis? OBJECTIVE: To observe the effect of chlamys farreri on the Bcl-2 and Bax protein-associated apoptosis in penumbra and its role in neuron protection. DESIGN: A randomized trial.SETTING: Department of Anatomy, Medical College of Qingdao University.MATERIALS: The trial was conducted in Nerve Anatomy Laboratory of Medical College of Qingdao University from March to April 2000. The subjects were 32 adult Wistar rats that were randomly and averagely assigned into 4 groups: polypeptide chlamys farreri group, sterile water group, model control group and sham group. The chlamys farreri was provided by the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences.INTERVENTIONS: Model of brain ischemia and reperfusion was made in rats in polypeptide chlamys farreri, sterile water and model control group by occlusion of middle cerebral artery. The model was not established in rats in sham group. The rats in chlamys farreri group received intraperitoneal injection of chlamys farreri of volume fraction 0. 1 at the dose of 0. 1 mL/kg each day for 2 days prior to modeling and an extra injection 15 minutes just before modeling. And the rats in sterile water group received intraperitoneal injec tion of sterile water with the dose of 0. 1 mL/kg each for two days and an extra injection 15 minutes before modeling. Those in model control group and sham group were exposed to nothing. Then models were established in rats in chlamys farreri, sterile water and model control group by inserting 4-0 nylons sutures from external carotid artery through bifurcation of carotid artery,extracranial and intracranial segments of internal carotid artery till the initial part of middle cerebral artery to make acute ischemia of middle cerebral artery perfusion area. The model was considered successful by the presentation of Horner' s syndrome, adduction-flexion of right forearm when tail being lifted and right turning during walk of the rat. The rats in sham group underwent the same procedures as that in the other groups except for the occlusion of middle cerebral artery. Then brains of the rats were taken for immunohistochemical determination of Bcl-2 and Bax proteins. The protein expression was expressed by absorbance of the products of their immunological reaction.MAIN OUTCOME MEASURES: The expression differences betweenBcl-2 and Bax proteins in penumbras of the groups.RESULTS: There were 32 rats entered the stage of analysis after complement of subjects. ① Bcl-2 expression in penumbra: The absorbance in model control group and sterile water group were higher than that in sham group (0.453±0.048,0.510±0.061,0.211±0.023, F=683.78, q=21.13 to 24.74, P ＜ 0.01), and that in chlamys farreri group(0. 954 ±0. 059) was more than that in model control group and sterile water group( q = 38.08 to 41.69, P ＜ 0.01) . ② Bax expression in penumbra: The absorbance in model control group and sterile water group were higher than that in sham group (0. 834 ±0. 082, 0. 790 ±0. 102, 0. 125 ±0. 017, F=590.44, q =49.57 to 51.98, P ＜ 0.01 ) ] and that in chlamys farreri group (0.471 ± 0. 045 ) was suppressed as compared with that in model control group and sterile water group(q=23. 80 to 26. 23, P ＜0. 01).CONCLUSION: Chlamys farreri is capable of increasing Bcl-2 protein and decreasing Bax protein in cerebral penumbra to brake the initiation of neuron apoptosis after ischemia-reperfusion and preserve neuronic function in penumbra.

Aim To investigate the effect of α1-anti-trypsin Z variant (ATZ)overexpression on cell autoph-agy.Methods HEK 293T cells were transfected with pcDNA3.1 zeo+/ATM or pcDNA3.1 zeo+/ATZ,e-qual amount of empty vector was used as control.Cells were treated with NH4Cl for 4 hours and processed for detecting ATZ,LC3 and p62 by immunoblot.Mean-while ,expression and intracellular localization of ATZ, LC3 in 293 T cells were observed with double labeled immunofluorescence.The mRNA levels of autophagy-related genes were measured by real-time PCR.Immu-nohistochemistry was used to observe the morphology of ATZ-positive cells.Results Compared with the control,higher LC3Ⅱ levels and LC3 puncta were observed in ATZ transfected cells.Meanwhile,the levelsof p62 were decreased in ATZ transfected cells,andreversed by NH4 Cl (25 mmol·L -1 )treatment.Overexpression of ATZ increased the mRNA levels of Atg5and Atg12,but had no obvious influence on Beclin1.ATZoverexpressing cells presented abnormal morphologies.The nuclei became reduced,condensed,and even disappeared in ATZpositive cells.Conclusion ATZ overexpression increases autophagy activity whichmay be related to increasing Atg5 and Atg12 levels.

Purpose To investigate the expression of RNF2 in breast disease tissues and cell lines,and to analyze the association between expression of RNF2 and clinicopathological characteristics in breast carcinoma.Methods Expression of RNF2 protein and mRNA levels was detected using immunohistochemistry of EnVision two-step and qRT-PCR in breast carcinoma and benign breast disease as well as in cell lines.Results RNF2 expression was sigmificantly higher in breast carcinoma tissue specimens compared with benign breast disease specimens (P ＜0.05).Besides,the expression of RNF2 protein was significantly associated to tumor size,lymph node status and TNM stage (P ＜ 0.05 for both),but was not related to age,histological grade,the expression of ER,PR and HER-2 (P ＞ 0.05 for both).Higher expression of RNF2 mRNA was detected in breast carcinoma cell lines compared with breast epithelial cell lines (P ＜ 0.05).Conclusion RNF2 is overexpressed in breast carcinoma and can be a potential therapeutic target for breast carcinoma.

Objective To investigate the effects of phosphocreatine postconditioning on cerebral ischemia-reperfusion(IR)injury in rats.Methods Thirty-six SD rats were randomly divided into three groups:groups Sham,IR (treated with normal saline)and PCr.IR was induced by intraluminal middle cerebral artery occlusion (MCAO).All treatments were given intravenously at the begining of reperfusion.Twenty-four hours after the reperfusion, neurological deficit score and magnetic resonance scan were performed.serum concentrations of malonaldehyde and 4-hydroxynonenal,cere-bral infarct volume and destruction of cerebral cortex were estimated.Neuronal apoptosis was further assessed by immunohistochemistry and immunofluorescent staining of caspase-3 and NeuN. Results Compared with group IR,phosphocreatine significantly decreased neurological deficit score, infarct volume,malonaldehyde and 4-hydroxynonenal levels(P < 0.05 ).Cortex structure was more complete,as well as neuronal apoptotic index was smaller in group PCr (P <0.05).Conclusion PCr can reduce cerebral infarct volume,thereby promote neurofunctional recovery.The mechanism of Pcr is related to reduced oxidative stress and inhibitted apopotosis during IR.

Objective To evaluate the effect of propofol on autophagy during oxygen-glucose deprivation and restoration (OGD/R) in human liver cells.Methods Human hepatic HL-7702 cells at the logarithmic growth phase were seeded into culture plates and randomly divided into 3 groups (n =12 each) using a random number table:control group (group C),OGD/R group,and propofol + OGD/R group (group P+OGD/R).The cells were cultured in normal culture medium in group C.In OGD/R and P+OGD/R groups,the cells were subjected to O2-glucose deprivation for 6 h followed by restoration of O2-glucose supply for 12 h.Propofol with a final concentration of 50 mmol/L was added at 10 min before oxygen-glucose deprivation.The cell viability was detected by MTT assay.The expression of autophagy-related proteins such as microtubule-associated protein light chain 3 (LC3) and Beclin-1 was evaluated by Western blot.Immunofluorescence was used to determine the number and distribution of autophagosomes.Results Compared with group C,the cell viability was significantly decreased,the expression of LC3 and Beclin-1 was significantly up-regulated (P＜0.05),and the number of autophagosomes was significantly increased in OGD/R and P+OGD/R groups.Compared with group OGD/R,the cell viability was significantly increased,the expression of LC3 and Beclin-1 was significantly down-regulated (P＜0.05),and the number of autophagosomes was significantly decreased in group P+OGD/R.Conclusion The mechanism by which propofol reduces OGD/R injury is probably related to inhibition of autophagy in human liver cells.

Objective To evaluate the effect of propofol preconditioning on endoplasmic reticulum stress induced by hypoxia-reoxygenation (H/R) in HEPG2 cells.Methods HEPG2 cells were randomly divided into 4 groups using a random number table:control group (group C),propofol group (group P),H/R group and H/R + propofol preconditioning group (group PP).In group C,the cells were cultured routinely for 42 h.In group H/R,after being cultured routinely for 6 h,the cells were exposed to 1％ O2 + 5％ CO2 + 94％ N2 for 12 h followed by 12 h reoxygenation.In group PP,the cells were cultured for 6 h in the culture medium containing propofol 10 μmol/L (final concentration),and then H/R was induced.The cell viability was detected by MTT assay.The expression of immunoglobulin heavy chain-binding protein (BIP),C/EBP homologous protein (CHOP) and activated caspase-3 was determined by Western blot.The expression of BIP,CHOP and caspase-3 mRNA was determined by RT-PCR.Results Compared with group C,the cell viability was significantly decreased,and the expression of BIP,CHOP and activated caspase-3 protein and mRNA was up-regnlated in H/R and PP groups,and no significant changes were found in the parameters mentioned above in group P.Compared with group H/R,the cell viability was significantly increased,and the expression of BIP,CHOP and activated caspase-3 protein and mRNA was down-regulated in PP group.Conclusion Propofol preconditioning can promote the cell proliferation and attenuate H/R injury to HEPG2 cells through inhibiting endoplasmic reticulum stress.

Aim To investigate the protective effects of MANF on human neuroblastoma SH-SY5 Y cells suf-fering from oxygen-glucose deprivation/reperfusion ( OGD/R) and the underlying mechanism. Methods SH-SY5Y cells were treated with OGD for 6 h, fol-lowed by reperfusion for 12 h. Meanwhile, the cells were incubated with 2 μmol · L-1 recombinant human protein MANF for 12 h during reperfusion. The cell morphology was observed under an optical microscope. The cell viability was determined by MTT assay. PI
staining was performed to detect the number of dead cells. Western blot was performed to determine the protein levels of endogenous MANF, glucose-related protein 78 ( GRP78/BiP) , phosphorylated inositol re-quiring enzyme 1 ( p-IRE1 ) , phosphorylated eukaryot-ic translation initiator factor 2α ( p-eIF2α) , cleaved caspase-3, and C/EBP-homologous protein (CHOP). Results The cells exposed to OGD/R became smaller and round, and the neurites of the cells were shortened or disappeared . Recombinant human protein MANF
improved the survival rate ( P <0. 05 ) and decreased the death rate ( P <0. 05 ) of SH-SY5 Y cells treated with by OGD/R. Western blot assay showed that the endoplasmic reticulum ( ER) stress-associated proteins GRP78/BiP, p-IRE1, p-eIF2α, and MANF were in-creased significantly after OGD/R treatment, compared with the untreated controls. However, the increases of secretion levels of apoptosis-associated proteins CHOP
and cleaved caspase-3 in SH-SY5 Y cells induced by OGD/R were significantly suppressed by MANF. Con-clusion OGD/R up-regulates the ER stress-associated proteins and causes apoptosis. MANF inhibits OGD/R-induced cell death, which may be related to attenua-ting ER stress-induced apoptosis.

Aim To observe the effects of rapamycin (Rapa) and starvation-induced autophagy on the morphology of neuronal cells, tau protein aggregation and expression of phosphorylated tau protein, to explore the possible mechanism of cytoprotective effect of these two classical autophagy inducers on phosphorylated tau expressing cells.Methods N2a cells were transfected with GFP-tau plasmid, and equal amount of empty vector was used as control.Then cells were incubated with or without okadaic acid(OA) for 12 h, followed by treatment with autophagy inducers rapamycin(Rapa) and EBSS, autophagy inhibitor Bafilomycin A1(Baf A1) for 6 h.DAB was used to observe tau expression and cell morphology.Confocal microscopy was used to observe the intracellular tau aggregation.TUNEL assay and cleaved caspase-3 level were used to detect cell apoptosis.Immunoblot was used to detect the expression of phosphorylated tau and autophagy-related proteins.Results Our study showed that the N2a cells treated with OA exhibited small cell body, retracted processes and increased tau aggregation, compared with only tau-expressing cells.Rapa and EBSS treatment significantly improved cell morphology, decreased tau aggregation and reduced cell apoptosis.On the contrary, Baf A1 treatment induced aberrant cell shape and increased tau aggregation and cell apoptosis.In addition, Rapa significantly decreased the high molecular weight, phosphorylated tau whereas EBSS especially decreased the low molecular weight phosphorylated tau.Conclusions Rapa and EBSS is alleviate hyperphosphorylated tau-induced cytotoxicity through different mechanism.Rapamycin mainly decreases phosphorylated tau oligomers, while EBSS liable to decrease the soluble phosphorylated tau.

Objective To investigate protective effects of dexmedetomidine on oxygen-glucose deprivation/reperfusion(OGD/R)-induced neuronal apoptosis.Methods SH-SY5Y cells were differentiated to neurons with ATRA and followed by TPA.According to the results of preliminary experiment, OGD/R modle was constructed by oxygen-glucose deprivation(OGD) for 12 h and reperfusion(R) for another 12 h.During the start of the OGD, neurons were immediately divided into six groups: group D0(0 μmol/L dexmedetomidine), group D1(0.1 μmol/L dexmedetomidine), group D2 (1 μmol/L dexmedetomidine), group D3 (10 μmol/L dexmedetomidine), group D4(100 μmol/L dexmedetomidine), group D5 (1 000 μmol/L dexmedetomidine).After reperfusion 12 h, the cell viability was evaluated by the method of MTT.The cellular apoptosis was observed by flow cytometry method.The protective effects of different concentration dexmedetomidine on OGD/R-induced neuronal apoptosis were investigated.Then in chosen the exact group having protective effects, endoplasmic reticulum stress specific protein mesencephalic astrocyte-derived neurotrophic factor (MANF) and pro-apoptotic protein Caspase-3 and CHOP were detected by Westernblot method.Results Compared with group D0, there was no difference on the cell viability and cellular apoptosis induced by OGD/R in groups D1 and D2, but a significant decrease and increase in groups D4 and D5 (P<0.01 or P<0.05).And only group D3 had a neuroprotective effect, significantly increased the cell viability and inhibited the apoptosis (P<0.01).Further studys found that group D3 significantly up-regulated ER stress specific protein MANF (P<0.01) and inhibited up-regulation of Caspase-3 and CHOP (P<0.01).Conclusion These data suggest that 10 μmol/L dexmedetomidine had neuroprotective effect on OGD/R-induced neuronal apoptosis and significantly increased cell viability.Our results also indicate that up-regulation of ER stress specific protein MANF and inhibition of CHOP and Caspase-3 by MANF are involved in the neuroprotective effects of Dexmedetomidine.

Objective To conduct the comparative comprehensive evaluation on the actual healthy effects and safety of two kinds of healthy foods capsule A and B made of Chinese medicinal herbs on sale through the low-nutritional sub-health mice model com-bined with the benefit-damage index-general score(BDI-GS) approach ,and to perform the discussion on the relevant problems a-round healthy foods .Methods The experimental healthy ICR male mice during growth period were fed with maize low-nutritional feed and the mixed feed with 3 doses of 0 .25% ,0 .5% ,0 .75% healthy foods for 12 d and the mice body masses were recorded .Af-ter dissection ,9 items of the organ index and their BDI ,GS and serum biochemical indicators were performed the statistics .Results In the capsule A ,the medium and high dose groups manifested certain health-promoting effect ,while the slight negative effect exis-ted in the low dose group ,which was expressed in the GS values ;but in the capsule B ,3 doses all caused the damage to main internal organs in different degrees ,which was expressed in BDI<1 .0 and GS<9 .0 .Conclusion At present ,despite of possessing similar ingredients ,Chinese medicinal healthy foods in market are of greater differences in intrinsic qualities ,and even partial products have some adverse effect ,the healthy functions and safety are not enough to be fully ensured .Through the systematic evaluation of the BDI-GS system ,the criteria of marketing threshold for healthy foods will be increased so as to enhance their effects and safety level .