基于TMT技术的激素性股骨头坏死硬化带修复模式探索及临床验证

背景:股骨头内硬化带是激素性股骨头坏死疾病发展过程中重要的影像学特征并与预后相关。过氧化物酶体增殖物活化受体γ协同刺激因子1α(peroxisome proliferator-activated receptor gamma coactivator 1α,PGC-1α)被证明具有成骨、促血管形成、抗线粒体凋亡等生物活性,与激素性股骨头坏死的骨修复可能存在紧密关联。目的:筛选激素性股骨头坏死硬化带区与正常区的差异蛋白,筛选出硬化带的关键蛋白,并验证关键蛋白在激素性股骨头坏死股骨头标本中的差异表达;探索激素性股骨头坏死硬化带修复模式。方法:取激素性股骨头坏死行人工全髋关节置换术取出的股骨头标本,通过串联质谱标签技术筛选硬化带区与正常区的差异表达基因,并进行GO与KEGG信号通路分析,构建关键靶点的蛋白互作网络、筛选关键基因。观察关键蛋白在激素性股骨头坏死硬化带中的表达,通过Western blotting、免疫组化验证关键蛋白在硬化带中的表达。结果与结论:①通过串联质谱标签定量蛋白质谱检测发现:相比于正常区骨组织,股骨头硬化带骨组织中具有显著差异表达(Log_(2)FC>1.20、Log_(2)FC<0.84和P<0.05)的蛋白质有609个,其中发生上调的蛋白质290个,发生下调的蛋白质319个;②通过GO与KEGG通路富集分析,发现在前10位的富集通路中,Wnt信号通路与生命周期调控通路与骨修复关系密切;在生命周期调控通道中,PGC-1α是重要的蛋白之一;③与激素性股骨头坏死标本正常区相比,Western blotting验证了PGC-1α、NRF-1在硬化带中低表达,Cleaved Caspase-3在硬化带中高表达;④光镜下免疫组化结果显示,股骨头组织标本中硬化区及正常区的PGC-1α、NRF1及Cleaved Caspase-3阳性染色分布,可以发现骨小梁、成骨细胞和骨髓均有表达存在;硬化带区Cleaved Caspase-3表达更为明显;⑤结论:激素性股骨头坏死硬化带中存在包含Wnt等成骨相关通路活跃、以PGC-1α低表达为特征的氧化凋亡活跃等生物学行为;PGC-1α在激素性股骨头坏死硬化带中低表达可能与氧化凋亡活跃相关。

Redox status of thioredoxin-1 （TRX1） determines the sensitivity of human liver carcinoma cells （HepG2） to arsenic trioxide-induced cell death

lntracellular redox homeostasis plays a critical role in determining tumor cells＇ sensitivity to drug-induced apoptosis. Here we investigated the role of thioredoxin-1 （TRX1）, a key component of redox regulation, in arsenic trioxide （AS2O3）-induced apoptosis. Over-expression of wild-type TRX1 in HepG2 cells led to the inhibition of As2O3-induced cytochrome c （cyto c） release, caspase activation and apoptosis, and down-regulation of TRX1 expression by RNAi sensitized HepG2 cells to As2O3-induced apoptosis. Interestingly, mutation of the active site of TRX1 from Cys^32/35 to Ser^32/35 converted this molecule from an apoptotic protector to an apoptotic promoter. In an effort to understand the mechanisms of this conversion, we used isolated mitochondria from mouse liver and found that recombinant wild-type TRX1 could protect mitochondria from the apoptotic changes. In contrast, the mutant form of TRX1 alone elicited mitochondria-related apoptotic changes, including the mitochondrial permeability transition pore （mPTP） opening, loss of mitochondrial membrane potential, and cyto c release from mitochondria. These apoptotic effects were inhibited by cyclosporine A （CsA）, indicating that mutant TRX1 targeted to mPTP. Alteration of TRX1 from its reduced form to oxidized form in vivo by 2,4-dinitrochlorobenzene （DNCB）, a specific inhibitor ofTRX reductase, also sensitized HepG2 cells to As203-induced apoptosis. These data suggest that TRX1 plays a central role in regulating apoptosis by blocking cyto c release, and inactivation of TRX1 by either mutation or oxidization of the active site cysteines may sensitize tumor cells to As2O3-induced apoptosis.

The study on neuroprotective mechanism of water extract of Fomitopsis Pinicola on mesencephala dopaminergic neurons induced by MPP+

Objective: To observe the neuroprotective mechanism of water extract of Fomito^p^is Pinicola on MPP+ induced apoptosis of mesencephala dopaminergic cells in vitro. Methods: The antioxidant activity of fungi was determined by FRAP method. The anti-inflammatory activity of the fungi was detected by LPS-induced NO release method. Mesencephalic dopaminergic neurons were labeled by TH staining to observe the survival of THir neurons. Results: In the anti-oxidant activity assay, the Trolox equivalent anti-oxidant capacity (TEAC) of water extract of Fomitopsis Pinicola was determined to be ( 165.80±7.13 )μmol Trolox/g extract. Water extracts o f Fomitopsis Pinicola treatment(100, 5 0 ,2 5 , 12.5^g/ml) decreased NO formation significantly. MPP+ induced significant chromatin condensation in the nuclei of mesencephala dopaminergic neurons with nuclear lysis, the mitochondrial membrane potential decreased remarkably, and ROS production increased significantly. Compared with the MPP+ control group, the morphological changes of cell nuclei after apoptosis was reversed by water extract of Fomitopsis Pinicola. Water extract of Fomitopsis Pinicola treatment (50,25,12.5^g/ml) dramatically increased relative mitochondrial membrane potential compared with MPP+ control respectively. Compared with the MPP+ control, water extract of Fomitopsis Pinicola treatment (50, 25^g/ml) significantly decreased relative ROS formation respectively. Conclusions: Water extract of Fomitopsis Pinicola showed significant neuroprotective effect on mesencephala dopaminergic cells induced by MPP+. The water extract of Fomitopsis Pinicola showed antioxidant and anti-inflammatory activities. The mechanism of neuroprotective effect of water extract of Fomitopsis Pinicola may be related to inhibitory on mitochondrial oxidative stress.

Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing

Resveratrol(3,5,4’-trihydroxystilbene,RSV) has been widely used in mammalian cells,but whether it can be used during freezing boar semen is still unknown.The effects of RSV treatment during boar semen freezing on its anti-freezing ability,apoptosis,and possible apoptotic pathways were observed in this study.Sperm motility,mitochondrial membrane potential(ΔΨm),adenosine triphosphate(ATP) content,terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL)-positive apoptotic state,and messenger RNA(mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested.The results showed that:(1) Compared with fresh sperm,the motility,normal acrosome rate,and plasma membrane integrity rate of frozen boar sperm decreased significantly(P<0.05),and RSV did not significantly increase the sperm motility(0.44 vs.0.40,P>0.05),but it did significantly improve the normal acrosome rate(57.65% vs.47.00%,P<0.05) and plasma membrane integrity rate(46.67% vs.38.85%,P<0.05).(2) After freezing,most boar sperm showed low mitochondrial ΔΨm.RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm.(3) RSV treatment significantly decreased reactive oxygen species(ROS) levels(58.65% vs.88.41%,P<0.05)and increased the ATP content(0.49 μmol/L vs.0.25 μmol/L,P<0.05) of boar sperm during freezing.(4) The apoptotic rate of the freezing group(80.41%) with TUNEL detection increased significantly compared to the fresh group(9.70%,P<0.05),and RSV treatment greatly decreased the apoptotic rate(68.32%,P<0.05).(5) Real-time polymerase chain reaction(RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway(tumor necrosis factor-α(TNF-α),Fas ligand(FasL),and Caspase-8),but also the genes from the mitochondria-mediated apoptotic pathway(manganese superoxide dismutase(MnSOD),B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),and Caspase-9) were both significantly changed after freezing.RSV treatment during freezing greatly changed their expression levels.Although RSV treatment during boar semen freezing did not significantly increase motility after thawing,it still played an efficient antioxidant role,which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor-and mitochondria-mediated apoptotic pathways.