阿尔茨海默病与氧化应激

随着世界人口的老龄化，阿尔茨海默病（AD）已成为当前研究的热点之一，但目前其确切的发病机制还不完全清楚。本文主要针对AD中出现的氧化应激介体，阐明氧化应激在AD病理过程中可能的致病作用，并提出利用抗氧化途径来预防和治疗AD的可行性策略。     

Porcine reproductive and respiratory syndrome virus (PRRSV) causes apoptosis during its replication in fetal implantation sites

Reproductive failure due to porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by late-term abortions, early farrowing and an increase of dead and mummified fetuses and weak-born piglets. The mechanism of PRRSV-induced reproductive failure is poorly understood. Human pregnancies, complicated by some pathogens leading to reproductive disorders exhibit increased apoptosis in the fetal membranes. Because PRRSV-target cells are present in endometrium/fetal placentas from healthy sows and PRRSV-infected macrophages in other organs die by apoptosis, we hypothesized that PRRSV can replicate and induce apoptosis in the fetal implantation sites at the last stage of gestation. In the present study, identification, localization and quantification of the PRRSV-positive and apoptotic cells were performed in the fetal implantation sites. Three dams were inoculated intranasally with 10⁵ TCID₅₀ PRRSV 07V063 at 90 days of gestation and sampled at 10 days post-inoculation. Two non-inoculated dams that were euthanized at 100 days of gestation served as control animals. Inoculation of the dams resulted in a viremia that lasted until the end of the study. Transplacental PRRSV spread was detected in all inoculated dams. Using immunofluorescence staining, single PRRSV-positive cells were found in the endometrial connective tissues adjacent to both PRRSV-positive and PRRSV-negative fetuses. In the fetal placental mesenchyme of the PRRSV-positive fetuses, infected cells were more abundant and spread focally. Double staining showed that all PRRSV-positive cells in the fetal implantation sites were positive for sialoadhesin and CD163. Apoptotic cells (TUNEL+) were detected in endometrium and fetal placentas of both non- and PRRSV-inoculated dams. The number of apoptotic cells was significantly higher in PRRSV-positive endometrium/fetal placentas. PRRSV caused apoptosis in infected cells since 20-61% of PRRSV-positive cells were apoptotic and in surrounding cells since 43-91% of the apoptotic cells were virus-negative. The main conclusion obtained from the present study is that PRRSV replicates in the fetal implantation sites and causes apoptosis in infected macrophages and surrounding cells at the last stage of gestation. The possible mode of PRRSV replication in the fetal implantation sites and the events that might contribute to the reproductive disorders are discussed.     

Porcine arterivirus activates the NF-kappaB pathway through IkappaB degradation

Nuclear factor-kappaB (NF-kappaB) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-kappaB in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-kappaB activation was characterized by translocation of NF-kappaB from the cytoplasm to the nucleus, increased DNA binding activity, and NF-kappaB-regulated gene expression. NF-kappaB activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-kappaB activation. Degradation of IkappaB protein was detected late in PRRSV infection, and overexpression of the dominant negative form of IkappaBalpha (IkappaBalphaDN) significantly suppressed NF-kappaB activation induced by PRRSV. However, IkappaBalphaDN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-kappaB DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-kappaB was activated by PRRSV infection. Moreover, NF-kappaB-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-kappaB activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV.     

Low dose methotrexate induces apoptosis with reactive oxygen species involvement in T lymphocytic cell lines to a greater extent than in monocytic lines

Background: The mechanism by which low dose methotrexate (MTX) exerts its anti-inflammatory and immunosuppressive effect in rheumatoid arthritis (RA) patients is still debated. Recently it has been related to the induction of apoptosis.Objective: We investigated the degree of apoptotic induction by MTX in lymphocytic (Jurkat T, EL4 T, and Raji B) and monocytic cell lines (U937 and THP1) and its relation to reactive oxygen species (ROS) generation, as a possible mechanism underlying the apoptotic events.Methods: All cell types were incubated with a range of MTX concentrations (0.001, 0.01, 0.1, 1, and 10 μM) for up to 24 h. Cytotoxicity was assessed by Trypan Blue exclusion and MTT test; cell size and granularity by forward and side scatters (FSC, SSC). Apoptosis was measured by Annexin V test and FDA polarization; and mitochondrial ROS generation by DHR123 probe and by NAC inhibition.Results: MTX significantly reduced cell viability and proliferation in all cell lines, being most effective in the Jurkat T lymphocytic line. The MTX cytotoxicity (at the optimal concentrations corresponding to low dose MTX therapy) was attributed to apoptosis, as suggested by morphological changes (shrinkage, increased granularity) and confirmed by Annexin V binding and FDA hyperpolarization. The apoptotic induction and the ROS generation (statistically correlated to apoptosis) were most pronounced in the Jurkat and EL4 T cell lines, and were partially inhibited by the antioxidant N-acetyl L-cysteine (NAC).Conclusion: According to the present observations, MTX may most likely induce apoptosis through oxidative stress. The high susceptibility of T cell lines to MTX induced apoptosis may account for the beneficial effect of MTX treatment in rheumatoid arthritis, which is characterized by hyperproliferation of T cells.Received 29 June 2004; returned for revision 1 September 2004; returned for final revision 11 February 2005; accepted by M. J. Parnham 7 March 2005This research was supported by the Horowitz Foundation     

Oxidative stress: The core pathogenesis and mechanism of Alzheimer’s disease

As the number of patients with Alzheimer’s disease (AD) increases, it brings great suffering to their families and causes a heavy socioeconomic burden to society. A vast amount of funds and a mass of research have been devoted to elucidating the pathology of AD. However, the main pathogenesis is still elusive, and its mechanism is not completely clear. Research on the mechanisms of AD mainly focuses on the amyloid cascade, tau protein, neuroinflammation, metal ions, and oxidative stress hypotheses. Oxidative stress is as a bridge that connects the different hypotheses and mechanisms of AD. It is a process that causes neuronal damage and occurs in various pathways. Oxidative stress plays a critical role in AD and can even be considered a crucial central factor in the pathogenesis of AD. Previous reviews have also summarized the role of oxidative stress in AD, but these mainly review a specific signaling pathway. Taking oxidative stress as the central point, this review comprehensively expands on the roles of oxidative stress that are involved in the pathogenesis of AD. The vivid and easy-to-understand figures systematically clarify the connected roles of oxidative stress in AD and allow readers to further understand oxidative stress and AD.     

Caprine arthritis-encephalitis virus induces apoptosis in infected cells in vitro through the intrinsic pathway

Caprine arthritis-encephalitis virus (CAEV) is a lentivirus that causes natural inflammatory disease in goats, with chronic lesions in several different organs. CAEV infection of in vitro cultured cells is accompanied by apoptosis, but the involvement of the intrinsic and extrinsic pathways has not previously been elucidated. We have studied the activation of caspases-3, -8 and -9 by fluorescent assays in various goat cells infected in vitro by CAEV, and the effects of transfected dominant negative variants of theses caspases, to show that CAEV-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of mitochondrial pathway.     

Apoptotic signaling through reactive oxygen species in cancer cells

Reactive oxygen species(ROS) take part in diverse biological processes like cell growth,programmed cell death,cell senescence,and maintenance of the transformed state through regulation of signal transduction. Cancer cells adapt to new higher ROS circumstance. Sometimes,ROS induce cancer cell proliferation. Meanwhile,elevated ROS render cancer cells vulnerable to oxidative stress-induced cell death. However,this prominent character of cancer cells allows acquiring a resistance to oxidative stress conditions relative to normal cells. Activated signaling pathways that increase the level of intracellular ROS in cancer cells not only render up-regulation of several genes involved in cellular proliferation and evasion of apoptosis but also cause cancer cells and cancer stem cells to develop a high metabolic rate. In over the past several decades,many studies have indicated that ROS play a critical role as the secondary messenger of tumorigenesis and metastasis in cancer from both in vitro and in vivo. Here we summarize the role of ROS and anti-oxidants in contributing to or preventing cancer. In addition,we review the activated signaling pathways that make cancer cells susceptible to death.     

Rit GTPase regulates a p38 MAPK-dependent neuronal survival pathway

Rit, along with Rin and Drosophila Ric, comprises the Rit subfamily of Ras-related small GTPases. Although the cellular functions of many Ras family GTPases are well established, the physiological significance of Rit remains poorly understood. Loss of Rit sensitizes multiple mammalian cell lines and mouse embryonic fibroblasts (MEFs) derived from Rit^−/− mice to oxidative stress-mediated apoptosis. However, whether Rit-mediated pro-survival signaling extends to other cell types, particularly neurons, is presently unknown. Here, to examine these issues we generated a transgenic mouse overexpressing constitutively active Rit (Rit^Q79L) exclusively in neurons, under control of the Synapsin I promoter. Active Rit-expressing hippocampal neurons display a dramatic increase in oxidative stress resistance. Moreover, pharmacological inhibitor studies demonstrate that p38 MAPK, rather than a MEK/ERK signaling cascade, is required for Rit-mediated protection. Together, the present studies identify a critical role for the Rit-p38 MAPK signaling cascade in promoting hippocampal neuron survival following oxidative stress.     

Heterogeneity of swine lung macrophages inoculated by porcine reproductive and respiratory syndrome virus

The biological features of porcine alveolar macrophages (PAMs) and interstitial macrophages (IMs) were investigated, including morphology, nitric oxide (NO) secretion, cell viability and porcine reproductive and respiratory syndrome virus (PRRSV) mRNA expression post-inoculation with TJ-F10 or TJM-F92. Viability and NO secretion of PAMs and IMs were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Griess's assay, respectively. mRNA expression of PRRSV, inducible nitric oxide synthase (iNOS) and Arginase1 (Arg1) in PAMs and IMs were detected by quantitative real-time polymerase chain reaction technique. Our results show that PAMs were bigger and more granular than IMs and the Arg1/iNOS value was much higher in PAMs than in IMs. In addition, the vaccine strain TJM-F92 evoked higher NO production in PAMs and IMs compared with the wild type strain TJ-F10. In conclusion, our results indicate that the PAMs and IMs are heterogeneous in morphology, NO production and susceptibility to PRRSV.     

青蒿琥酯通过增加活性氧簇生成诱导人肝癌HepG2细胞凋亡

目的:探讨青蒿琥酯诱导人肝癌Hep G2细胞凋亡的机制及活性氧簇(ROS)在青蒿琥酯诱导Hep G2细胞凋亡中的作用。方法:采用MTT法观查青蒿琥酯对人肝癌Hep G2细胞存活的影响,Hoechst 33258荧光染色法观察细胞凋亡形态的变化,流式细胞术检测Hep G2细胞的凋亡率,DCFH-DA检测细胞凋亡过程中ROS的变化。Western blot检测细胞内凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3和细胞色素C(Cyt C)蛋白水平的变化。采用NADPH氧化酶抑制剂夹竹桃麻素(apocynin)预处理Hep G2细胞,Western blot检测NADPH氧化酶亚基p47~(phox)和p22~(phox)蛋白表达水平,流式细胞术检测ROS变化。结果:与对照组相比,青蒿琥酯作用于Hep G2细胞24 h后,细胞存活率明显减少(P0.05);细胞核呈致密浓染色,细胞凋亡比例升高(P0.05);ROS明显升高(P0.05);Western blot结果显示,青蒿琥酯作用后细胞内Bcl-2蛋白表达下调,Bax蛋白表达上调,Bax/Bcl-2蛋白表达比例升高,cleaved caspase-3和Cyt C蛋白水平升高。Apocynin预处理能降低青蒿琥酯给药组细胞内p47~(phox)和p22~(phox)蛋白表达及ROS的生成。结论:青蒿琥酯能诱导Hep G2细胞凋亡,其凋亡过程可能与ROS的生成增加相关。     

周期性张应变通过活性氧生成促进膝关节骨关节炎患者的软骨细胞发生凋亡

目的 探讨周期性张应变诱导体外培养的膝关节骨关节炎(osteoarthritis,OA)患者软骨细胞发生凋亡过程中的活性氧(reactive oxygen species,ROS)的作用机制.方法 对受力组软骨细胞施加频率0.5 Hz、强度20％延伸率的周期性张应变.对照组细胞的培养条件与受力组细胞一致,但不受力.N-乙酰半胱氨酸(NAC)和丁胱亚磺酰亚胺(BSO)干预组的软骨细胞在受力前分别使用NAC和BSO进行预处理.流式细胞术检测细胞凋亡率,DCFHDA为荧光探针检测细胞内ROS含量,分光光度法检测软骨细胞内caspase-9活性变化.结果 0.5 Hz、20％延伸率的周期性张应变可以刺激软骨细胞内ROS、caspase-9活性及细胞凋亡率显著升高(P＜0.05).使用NAC和BSO抑制及升高细胞内ROS含量后,可以明显抑制及促进周期性张应变诱导的软骨细胞的caspase-9活性及细胞凋亡率(P＜0.05).结论 周期性张应变通过促进ROS生成,进而激活caspase-9介导膝关节OA患者软骨细胞发生凋亡.抑制ROS的生成可以减轻周期性张应变导致的软骨细胞凋亡.     

Inhaled hydrogen gas therapy for prevention of noise-induced hearing loss through reducing reactive oxygen species

Reactive oxygen species (ROS) that form in the inner ear play an important role in noise-induced hearing loss (NIHL). Recent studies have revealed that molecular hydrogen (H₂) has great potential for reducing ROS. In this study, we examined the potential of hydrogen gas to protect against NIHL. We tested this hypothesis in guinea pigs with 0.5%, 1.0% and 1.5% H₂ inhalation in air for 5 h a day after noise exposure, for five consecutive days. All animals underwent measurements for auditory brainstem response after the noise exposure; the results revealed that there was a better improvement in the threshold shift for the 1.0% and 1.5% H₂-treated groups than the non-treated group. Furthermore, outer hair cell (OHC) loss was examined 7 days after noise exposure. A significantly higher survival rate of OHCs was observed in the 1.0% and 1.5% H₂-treated group as compared to that of the non-treated group in the basal turn. Immunohistochemical analyses for 8-hydroxy-2′-deoxyguanosine (8-OHdG) were performed to examine the amount of oxidative DNA damage. While strong immunoreactivities against 8-OHdG were observed of the non-treated group, the H₂-treated group showed decreased immunoreactivity for 8-OHdG. These findings strongly suggest that inhaled hydrogen gas protects against NIHL.     

Porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages

Different viruses have evolved strategies that inhibit apoptosis of the host cell early in infection and/or induce apoptosis in the host cell late in infection. In this study, it was investigated if and when porcine reproductive and respiratory syndrome virus (PRRSV) modulates apoptosis in PRRSV-infected macrophages. The PRRSV replication cycle in macrophages was completed within 12 h post-inoculation (hpi). PRRSV-infected macrophages, treated with staurosporine at 4, 5, 6 and 8 hpi, were significantly protected against staurosporine-induced apoptosis, but PRRSV-infected macrophages, treated with staurosporine at 12 hpi, were not. In contrast, starting from 12 hpi, all PRRSV-infected macrophages died by caspase-dependent apoptosis, which culminated in secondary necrosis. Treatment of PRRSV-infected macrophages with Z-Val-DL-Asp-fluoromethylketone indicated that apoptosis late in infection was not essential for efficient virus release. Anti- and pro-apoptotic activities were also observed in PRRSV-infected Marc-145 cells. In conclusion, this study shows that PRRSV stimulates anti-apoptotic pathways in macrophages early in infection and that PRRSV-infected macrophages die by apoptosis late in infection.     

Suppression of porcine reproductive and respiratory syndrome virus replication in MARC-145 cells by shRNA targeting ORF1 region

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine producing area. The current vaccine strategies cannot provide complete protection against PRRSV. The objective of this study was to determine if specific short-hairpin RNA (shRNA) directed against different genomic regions of ORF1b of PRRSV could be utilized to inhibit virus replication in MARC-145 cells. Two shRNA expression vectors targeting ORF1b gene of PRRSV were constructed and delivered into MARC-145 cells, and then infected with PRRSV. The results showed that PRRSV-specific cytopathic effect (CPE) could be inhibited in the cells transfected with pSUPER-P2 and pSUPER-P3, and the virus titers in the cells transfected with pSUPER-P2 and pSUPER-P3 were lower than those control cells by approximately 100 fold. Moreover, the expression of ORF1 of PRRSV in the cells was reduced both at RNA and protein levels comparing to the controls. It indicated that vector-based shRNA targeting ORF1 region could effectively inhibit PRRSV replication in MARC-145 cells. Guanming Li and Juan Huang contributed equally to this work.     

活性氧在淋巴细胞信号转导中的作用

确切证据表明,活性氧(ROS)参与了淋巴细胞内一系列生理或病理活动的调节。尽管对ROS调节免疫功能的分子机制仅有初步认识,抑制性信号分子如酪氨酸磷酸酶或NF-κB抑制分子I-κB的氧化失活,可能是淋巴细胞内氧化还原信号调节的关键点。根据细胞类型、氧化应激程度及自由基产生的胞内区域的不同,ROS发挥不同生物学效应。本文综述了ROS在淋巴细胞信号转导中的作用,并分析了其在正常和病理免疫应答中可能扮演的角色。