MAGED4B Promotes Glioma Progression via Inactivation of the TNF-α-induced Apoptotic Pathway by Down-regulating TRIM27 Expression / 神经科学通报·英文版

MAGED4B belongs to the melanoma-associated antigen family; originally found in melanoma, it is expressed in various types of cancer, and is especially enriched in glioblastoma. However, the functional role and molecular mechanisms of MAGED4B in glioma are still unclear. In this study, we found that the MAGED4B level was higher in glioma tissue than that in non-cancer tissue, and the level was positively correlated with glioma grade, tumor diameter, Ki-67 level, and patient age. The patients with higher levels had a worse prognosis than those with lower MAGED4B levels. In glioma cells, MAGED4B overexpression promoted proliferation, invasion, and migration, as well as decreasing apoptosis and the chemosensitivity to cisplatin and temozolomide. On the contrary, MAGED4B knockdown in glioma cells inhibited proliferation, invasion, and migration, as well as increasing apoptosis and the chemosensitivity to cisplatin and temozolomide. MAGED4B knockdown also inhibited the growth of gliomas implanted into the rat brain. The interaction between MAGED4B and tripartite motif-containing 27 (TRIM27) in glioma cells was detected by co-immunoprecipitation assay, which showed that MAGED4B was co-localized with TRIM27. In addition, MAGED4B overexpression down-regulated the TRIM27 protein level, and this was blocked by carbobenzoxyl-L-leucyl-L-leucyl-L-leucine (MG132), an inhibitor of the proteasome. On the contrary, MAGED4B knockdown up-regulated the TRIM27 level. Furthermore, MAGED4B overexpression increased TRIM27 ubiquitination in the presence of MG132. Accordingly, MAGED4B down-regulated the protein levels of genes downstream of ubiquitin-specific protease 7 (USP7) involved in the tumor necrosis factor-alpha (TNF-α)-induced apoptotic pathway. These findings indicate that MAGED4B promotes glioma growth via a TRIM27/USP7/receptor-interacting serine/threonine-protein kinase 1 (RIP1)-dependent TNF-α-induced apoptotic pathway, which suggests that MAGED4B is a potential target for glioma diagnosis and treatment.

MANF brakes TLR4 signaling by competitively binding S100A8 with S100A9 to regulate macrophage phenotypes in hepatic fibrosis

The mesencephalic astrocyte-derived neurotrophic factor (MANF) has been recently identified as a neurotrophic factor, but its role in hepatic fibrosis is unknown. Here, we found that MANF was upregulated in the fibrotic liver tissues of the patients with chronic liver diseases and of mice treated with CCl4. MANF deficiency in either hepatocytes or hepatic mono-macrophages, particularly in hepatic mono-macrophages, clearly exacerbated hepatic fibrosis. Myeloid-specific MANF knockout increased the population of hepatic Ly6Chigh macrophages and promoted HSCs activation. Furthermore, MANF-sufficient macrophages (from WT mice) transfusion ameliorated CCl4-induced hepatic fibrosis in myeloid cells-specific MANF knockout (MKO) mice. Mechanistically, MANF interacted with S100A8 to competitively block S100A8/A9 heterodimer formation and inhibited S100A8/A9-mediated TLR4-NF-κB signal activation. Pharmacologically, systemic administration of recombinant human MANF significantly alleviated CCl4-induced hepatic fibrosis in both WT and hepatocytes-specific MANF knockout (HKO) mice. This study reveals a mechanism by which MANF targets S100A8/A9-TLR4 as a "brake" on the upstream of NF-κB pathway, which exerts an impact on macrophage differentiation and shed light on hepatic fibrosis treatment.

Expression of RNF2 in breast carcinoma and its significance / 临床与实验病理学杂志

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.

Effects of dexmedetomidine on oxygen-glucose deprivation/reperfusion-induced neuronal apoptosis / 临床麻醉学杂志

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.

Autophagy alleviates neuronal toxicityinduced by abnormally phosphorylated tau protein / 中国药理学通报

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.

Effects of phosphocreatine postconditioning on cerebral ischemia-reperfusion injury in rats / 临床麻醉学杂志

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.

Effect of propofol on autophagy during oxygen-glucose deprivation/restoration in human liver cells / 中华麻醉学杂志

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.

Protective effects of MANF on oxygen-glucose deprivation/reperfusion-induced apoptotic ER stress in SH-SY5 Y neural cells / 中国药理学通报

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 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.

Effect of propofol preconditioning on endoplasmic reticulum stress induced by hypoxia-reoxygenation in HEPG2 cells / 中华麻醉学杂志

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.

Effect ofα1-antitrypsin Z variant overexpression on autophagy regulation / 中国药理学通报

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.

Effect of propofol pretreatment on endoplasmic reticulum stress-mediated apoptosis during focal cerebral ischemia-reperfusion in rats / 中华麻醉学杂志

Objective To evaluate the effect of propofol on the endoplasmic reticulum stress-mediated apoptosis during focal cerebral ischemia-reperfusion (I/R) in rats.Methods Eighty adult male Sprague-Dawley rats,weighing 240-280 g,were randomly divided into 4 groups (n =20 each):shame operation group (group S) ; focal cerebral I/R group (group FCIR); propofol pretreatment group (group P); intralipid pretreatment group (group Ⅰ).Focal cerebral I/R was induced by 4 h middle cerebral artery occlusion followed by reperfusion.Propofol was infused at a rate of 12 mg· kg-1 · h-1 starting from 30 min before ischemia until 15 min of ischemia in group P,while intralipid was given instead of propofol in group I.Neurological deficit scores (NDSs) were measured at 6 h of reperfusion in 10 rats chosen from each group and the rats were then sacrificed.Their left brains were removed for determination of brain water content.The left 10 rats were sacrificed and their brains were immediately removed for determination of the expression of C/EBP homologous protein (CHOP),Bcl-2,and activated caspase-3 in the left hippocampi by Western blot.Results Compared with group S,NDSs and brain water content were significantly increased,the expression of CHOP and activated caspase-3 was up-regulated,and the expression of Bcl-2 was down-regulated in group FCIR,NDSs was increased in group P (P ＜ 0.05).Compared with group FCIR,NDSs and brain water content were significantly decreased,the expression of CHOP and activated caspase-3 was down-regulated,and the expression of Bcl-2 was up-regulated in group P,and no significant change was found in each parameter in group Ⅰ (P ＞ 0.05).Conclusion Propofol can reduce focal cerebral I/R injury through inhibition of the endoplasmic reticulum stress-mediated apoptosis in rats.

Detection of Pathogens of Urogenital Infections and Their Drug-resistant Types by a DNA Chip / 中华皮肤科杂志

0.8). Conclusions This DNA chip combined with multiplex PCR is a rapid diagnostic assay with high specificity and sensitivity for the detection of Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma Urealyticum and their drug-resistance, and may be applied in the diagnosis of urogenital infections.