Neuroprotective and antioxidant properties of FKBP-binding immunophilin ligands are independent on the FKBP12 pathway in human cells

We focused on immunophilin isoforms in order to clarify the neuroimmunophilins which were identified as targets for the immunophilin ligands to elicit a neuroprotective effect. Although the expressions of five FK506-binding protein (FKBP) mRNAs were detected in both SH-SY5Y (human neuroblastoma) and U251 (human glioma) cell lysates, the FKBP12 mRNA expression was detected in only the SH-SY5Y cells, and not the U251 cells. However, we found that the SH-SY5Y and the U251 cells were equipotent in the intensity of cellular protection of FK506 (an immunosuppressive immunophilin ligand) and GPI1046 (a non-immunosuppressive FK506 analog), indicating that the protective effect and glutathione activation of FK506 and GPI1046 had little need to bind FKBP12. Therefore, we conclude that the neuroprotective and antioxidant properties of immunophilin ligands are independent on the FKBP12 pathway.     

Inhibitory effect of the immunosuppressant FK506 on apoptotic cell death induced by HIV-1 gp120

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 may play a central role in inducing immunoregulatory disorders after HIV infection. The apoptotic death of normal human peripheral blood mononuclear cells was induced by priming with gp120 followed by stimulation with an anti-T cell receptor (TCR) antibody. Tumor necrosis factor- produced by gp120-binding macrophages may be important to induce this cell death. Treatment of gp120-primed cells with an immunosuppressant (FK506) before TCR signaling inhibited apoptotic cell death, and this blocking effect of FK506 was concentration dependent. FK506 did not have any influence on cell growth and viability over the range of concentrations tested. These findings suggest that FK506 is a potentially useful drug in delaying the onset of AIDS after HIV infection.     

Alginate oligosaccharide protects against endoplasmic reticulum- and mitochondrial-mediated apoptotic cell death and oxidative stress

Oxidative stress is a major component of harmful cascades activated in neurodegenerative disorders. We sought to elucidate possible effects of alginate oligosaccharide (AOS) on H(2)O(2)-induced cell death and to determine the underlying molecular mechanisms in neuron-like PC12 cells. We found that AOS treatment protected PC12 cells against H(2)O(2)-induced endoplasmic reticulum (ER) and mitochondrial-dependent apoptotic cell death. AOS promoted Bcl-2 expression, while blocked Bax expression and inhibited H(2)O(2)-induced caspase-3 activation. It also blocked PARP cleavage. AOS acted on key molecules in apoptotic cell death pathway and reduced p53, p38, c-June NH2-terminal kinase phosphorylations, inhibited NFkB, and enhanced Nrf2 activation. These results suggest that treatment of PC12 cells with AOS can block H(2)O(2)-induced oxidative stress and caspase-dependent apoptotic cascades originating from both ER and mitochondria. Our in?vivo experiments further confirm the neuroprotective potential of AOS against Aβ-induced neural damage. According to our data, the involvement of caspase-independent pathway in AOS-induced protection appears to be unlikely.     

Effect of FK506 on the interleukin 15-driven proliferation and apoptosis of anti-CD3-activated umbilical cord blood T cells.

BACKGROUND: Increased susceptibility of umbilical cord blood (CB) T cells to FK506 immunosuppression may contribute to the lessened severity of graft-vs-host disease in CB transplantation. OBJECTIVE: To investigate the FK506 sensitivity of interleukin 15 (IL-15)- and IL-2-driven proliferation and apoptosis of anti-CD3-stimulated CB T cells compared with adult peripheral blood (APB) T cells. METHODS: Surface flow cytometric analysis (CD25 and CD95), carboxyfluorescein diacetae succinimidyl ester staining to track CD3+ T-cell division, and flow cytometric analysis of apoptotic cell death using Annexin V were performed to determine the effect of FK506 on CD3+ T-cell activation and apoptosis after anti-CD3 stimulation in the presence of IL-15 or IL-2. RESULTS: IL-15 is superior to IL-2 in promoting CD25 expression and proliferation of anti-CD3-stimulated CB and APB T cells. Although IL-15-driven proliferation evaluated by carboxyfluorescein diacetae succinimidyl ester staining revealed comparable sensitivity to FK506 in anti-CD3-stimulated CB and APB T cells, IL-15-driven CD25 up-regulation in CB T cells was more sensitive to FK506 inhibition than APB T cells. FK506 down-regulated anti-CD3-induced apoptosis in CB and APB T cells (P < .01). However, the FK506 sensitivity of anti-CD3-induced T-cell apoptosis was lost in IL-15-supplemented CB cultures (P = .51) but not in corresponding APB cultures (P = .002). The IL-15-enhanced Fas expression on CB T cells (CD95) was decreased by FK506, similar to that observed with adults. CONCLUSIONS: We observed differential FK506 sensitivity of IL-15-driven CD25 up-regulation and apoptotic response comparing CB and APB T cells. This finding suggests the potential therapeutic benefit of FK506 in ameliorating graft-vs-host disease by decreasing IL-15-driven donor T-cell proliferation without inhibiting associated activation-induced apoptosis during CB transplantation.     

Cyclosporin a and tacrolimus (FK506) differentially alter T-cell receptor expression in vivo

Cyclosporin A (CSA) and tacrolimus (FK506) are two common immunosuppressive agents used post blood and marrow transplantation. Despite similarity in their accepted modes of action, we observed polarized effects of CSA and FK506 on the in vivo human T cell repertoire. To determine the possible mechanism for this difference, the effects of CSA and FK506 on cell viability, cell proliferation, interleukin-2 production, and calcineurin inhibition were determined in vitro. Our data suggest that a secondary mechanism of action exists for the different T-cell repertoire induced by exposure to CSA and FK506.     

Nicotine switches the form of H(2)O(2)-induced cell death from apoptosis to necrosis in U937 cells

Cigarette smoking is a major risk factor for lung diseases and oral diseases. The present study examined the effect of nicotine on H(2)O(2)-induced apoptosis in U937 cells. In the presence of 1 microg/ml nicotine, cell viability was not affected, but the cells were unable to undergo H(2)O(2)-induced apoptosis and instead died as a result of necrosis. Nicotine reduced the intracellular level of ATP, which is an important determinant of the form of cell death. These results suggest that nicotine can alter the cell death pathway by reducing the level of intracellular ATP, and that this effect may contribute to cigarette smoke-induced tissue destruction.     

硫化氢对抗过氧化氢对PC12细胞的损伤作用

目的探讨硫化氢(hydrogen sulfide,H2S)对抗过氧化氢(hydrogen peroxide,H2O2)对PC12细胞的损伤作用及有关机制。方法应用H2O2在PC12细胞建立氧化应激损伤的实验模型;应用甲氮甲唑蓝(MTT)法检测细胞存活率,碘化丙啶(PI)染色流式细胞技术(FCM)检测细胞凋亡率,罗丹明123(Rhodamine 123,Rh123)染色FCM检测细胞线粒体膜电位(mitochondrial membrane potential,MMP),双氢罗丹明123染色FCM检测细胞内活性氧(reactive oxygen species,ROS)的含量。应用硫化氢钠(sodium hydrosulfide,NaHS)作为H2S的供体。结果200μmol和400μmolH2O2作用PC12细胞24h均使细胞的存活率明显降低及凋亡率显著增加,200μmolH2O2引起PC12细胞的MMP明显降低及ROS生成显著增多。当NaHS与H2O2(200或400μmol/L)共同作用于PC12细胞时,NaHS(100～400μmol/L)浓度依赖性的阻断H2O2引起PC12细胞的存活率降低及细胞凋亡率增加。400μmolNaHS明显地阻断200μmolH2O2引起PC12细胞的MMP降低及ROS增多。结论H2S能明显地保护PC12细胞对抗H2O2引起的损伤,阻断MMP降低及ROS生成可能是H2S的细胞保护机制之一。     

Cyclosporin A and Tacrolimus (FK506) Differentially Alter T-cell Receptor Expression In Vivo

Cyclosporin A (CSA) and tacrolimus (FK506) are two common immunosuppressive agents used post blood and marrow transplantation. Despite similarity in their accepted modes of action, we observed polarized effects of CSA and FK506 on the in vivo human T cell repertoire. To determine the possible mechanism for this difference, the effects of CSA and FK506 on cell viability, cell proliferation, interleukin-2 production, and calcineurin inhibition were determined in vitro. Our data suggest that a secondary mechanism of action exists for the different T-cell repertoire induced by exposure to CSA and FK506.     

Suppression of B cell activation by cyclosporin A, FK506 and rapamycin.

The effects of the immunosuppressants cyclosporin A (CsA), FK506 and rapamycin have been compared using murine B cells activated with a variety of mitogens. FK506 is a macrolide antibiotic that has been recently shown to inhibit T cell activation by a mechanism that appears similar to that of CsA. Rapamycin is a macrolide structurally related to FK506 whose mechanism of T cell suppression appears to be distinct from that of FK506 and CsA. While CsA and FK506 were found to preferentially inhibit B cell activation caused by stimuli which induce a rise in intracellular calcium, rapamycin partially inhibited activation by all stimuli tested, including those which are not associated with a calcium flux. All three compounds were found to inhibit cell cycle progression within the G1 phase; however, the rapamycin-sensitive event within G1 was completed earlier than the G1 events inhibited by CsA and FK506. In addition, inhibition of anti-IgM-activated B cells with CsA and FK506, but not with rapamycin, resulted in cell death. These data suggest that although CsA, FK506 and rapamycin are all inhibitors of B cell activation, the inhibitory activity of rapamycin can be clearly distinguished from that of CsA and FK506. Although the suppressive effects of CsA and FK506 on B cell proliferation were nearly identical in this study, their biological activities were distinguishable since FK506, but not CsA, could antagonize rapamycin-mediated suppression.     

Neuroprotective effects of FK506 against excitotoxicity in organotypic hippocampal slice culture

FK506 has been originally classified as an immunosuppressant and is known to exhibit neurotrophic actions in vitro and protective effects on some neurological conditions. We investigated the neuroprotective effects of FK506 on kainic acid (KA)-induced neuronal death in organotypic hippocampal slice cultures (OHSCs). After an 18 h KA (5 μM) treatment, significantly neuronal death was detected in the CA3 region using propidium iodide staining. However, neuronal death was significantly prevented at 24 and 48 h after treatment with 0.1 μM FK506. Using cresyl violet staining, we also observed that an increased number of CA3 neurons survived in the 0.1 μM FK506 group compared to the KA only group. Based on the results of the Western blot analysis, the expressions of 5-lipoxygenase and caspase-3 were reduced 24 h after 0.1 μM FK506 treatment. The levels of superoxide dismutase (SOD) and phospho-Akt expression were increased by treatment with 0.1 μM FK506. These results suggest that FK506 may have a positive role in protecting neurons against cell death in the KA injury model of OHSCs.     

NOD8对H2O2诱导的人肝细胞凋亡的影响

 目的:探讨NOD8对H2O2诱导的人肝细胞L02凋亡的影响。 方法: pEGFP-C2 及pEGFP-NOD8重组质粒经JetPRIME介导转染L02细胞;用H2O2诱导细胞凋亡。实验分为pEGFP-C2组、pEGFP-C2+H2O2组和pEGFP-NOD8+H2O2组。采用MTT法检测细胞活性,Western blotting检测细胞NOD8的蛋白表达,Hoechst 33342染色检测细胞凋亡情况,流式细胞术检测细胞凋亡率,比色法检测细胞caspase-3活性。 结果: 通过MTT检测不同浓度（0.2～2 mmol/L）H2O2刺激6 h后的细胞活性,确定1 mmol/L H2O2为诱导细胞凋亡的剂量。Western blotting检测结果显示,转染pEGFP-NOD8质粒的细胞NOD8蛋白表达明显增加。Hoechst 33342染色法观察发现,pEGFP-C2+H2O2组有较多细胞出现强蓝色荧光细胞核,细胞凋亡较多,而pEGFP-NOD8+H2O2组细胞凋亡明显减少。流式细胞术分析显示,pEGFP-C2+H2O2组的细胞凋亡率明显升高,pEGFP-NOD8+H2O2组的细胞凋亡率则显著下降。pEGFP-C2+H2O2组细胞的caspase-3活性明显升高,而pEGFP-NOD8+H2O2组细胞的caspase-3活性显著下降。 结论: NOD8可抑制H2O2诱导的L02细胞凋亡,其作用机制可能与NOD8抑制细胞的caspase-3活性有关。     

左卡尼汀通过抑制钙/钙调素依赖蛋白激酶Ⅱ信号通路抑制过氧化氢诱导的大鼠心肌细胞凋亡

 目的： 观察左卡尼汀对过氧化氢（H2O2）诱导的大鼠心肌细胞凋亡的保护作用及其机制。方法：利用200 μmol/L H2O2刺激12 h,建立体外原代培养新生乳鼠心肌细胞凋亡模型。Ca2+螯合剂1,2-双(2-氨基苯氧基)乙烷-N, N, N′, N′-四乙酸(BAPTA)、钙调素依赖蛋白激酶II (CaMKII)特异性抑制剂KN93及左卡尼汀分别于加入H2O2前30 min或1 h加入,以检测这3种药物对H2O2刺激下心肌细胞活力、细胞凋亡、细胞内静息钙浓度（［Ca2+］i）及磷酸化CaMKII (p-CaMKII)表达的影响。利用MTT比色法检测心肌细胞活力;流式细胞仪检测细胞凋亡率;利用激光共聚焦扫描检测［Ca2+］i;蛋白质免疫印迹法检测cleaved caspase-3及p-CaMKII的表达。结果：模型组经200 μmol/L H2O2作用12 h后,细胞活力显著下降,细胞凋亡率显著增加。BAPTA、KN93及左卡尼汀预处理显著抑制上述细胞损伤。进一步研究发现,H2O2 诱导的 ［Ca2+］i水平升高、cleaved caspase-3及p-CaMKII的表达增加均可被上述3种药物不同程度地抑制。结论：左卡尼汀可抑制H2O2所致的心肌细胞凋亡,该心肌保护作用可能与其抑制Ca2+/CaMKⅡ信号通路有关。     

雌激素对H2O2诱导的氧化应激的保护作用及机制

为了探讨雌激素17β-estradiol对H2O2诱导的氧化应激的影响及其可能机制。本研究将H2O2作用于大鼠皮层神经元,利用四甲基偶氮唑盐(MTT)检测单独H2O2或者17β-estradiol存在时其对细胞活力和乳酸脱氢酶(LDH)释放的影响;检测单独H2O2或者17β-estradiol存在时其对糖原合成激酶-3β(GSK-3β)活性的影响及其对磷酸化和非磷酸化GSK-3β表达的影响;检测GSK-3β抑制剂LiCl对H2O2诱导的细胞活力的影响;检测雌激素受体抑制剂ICI-182780存在时17β-estradiol对GSK-3β表达的影响。结果显示:(1)H2O2作用于大鼠皮层神经元后显著降低细胞的活力,各种浓度的17β-estradiol预处理细胞后均能部分阻断H2O2对细胞活力的影响;(2)H2O2作用后显著增加细胞乳酸脱氢酶的释放,而17β-estradiol则部分拮抗此种作用;(3)H2O2增加了GSK-3β的活性,而提前加入17β-estradiol则可以降低H2O2诱导的GSK-3β活性增加;(4)H2O2降低了GSK-3β的磷酸化,而17β-estradiol则部分拮抗了这种作用;(5)GSK-3β抑制剂LiCl也可以拮抗H2O2诱导的细胞活力下降;(6)与对照组相比,17β-estradiol本身亦可增加GSK-3β的磷酸化,此作用可被雌激素受体抑制剂ICI-182780部分拮抗,而ICI-182780本身对GSK-3β的磷酸化无显著影响。以上结果提示,17β-estradiol可以拮抗H2O2诱导的氧化应激作用,17β-estradiol通过与其受体结合而抑制GSK-3β的活性可能是其发挥保护作用的机制之一。