Huperzine A Alleviates Oxidative Glutamate Toxicity in Hippocampal HT22 Cells via Activating BDNF/TrkB-Dependent PI3K/Akt/mTOR Signaling Pathway

Oxidative glutamate toxicity is involved in diverse neurological disorders including epilepsy and ischemic stroke. Our present work aimed to assess protective effects of huperzine A (HupA) against oxidative glutamate toxicity in a mouse-derived hippocampal HT22 cells and explore its potential mechanisms. Cell survival and cell injury were analyzed by MTT method and LDH release assay, respectively. The production of ROS was measured by detection kits. Protein expressions of BDNF, phosphor-TrkB (p-TrkB), TrkB, phosphor-Akt (p-Akt), Akt, phosphor-mTOR (p-mTOR), mTOR, phosphor-p70s6 (p-p70s6) kinase, p70s6 kinase, Bcl-2, Bax, and β-actin were assayed via Western blot analysis. Enzyme-linked immunosorbent assay was employed to measure the contents of nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Our findings illustrated 10 μM HupA for 24 h significantly protected HT22 from cellular damage and suppressed the generation of ROS. Additionally, after treating with LY294002 or wortmannin [the selective inhibitors of phosphatidylinositol 3 kinase (PI3K)], HupA dramatically prevented the down-regulations of p-Akt, p-mTOR, and p-p70s6 kinase in HT22 cells under oxidative toxicity. Furthermore, it was observed that the protein levels of BDNF and p-TrkB were evidently enhanced after co-treatment with HupA and glutamate in HT22 cells. The elevations of p-Akt and p-mTOR were abrogated under toxic conditions after blockade of TrkB by TrkB IgG. Cellular apoptosis was significantly suppressed (decreased caspase-3 activity and enhanced Bcl-2 protein level) after HupA treatment. It was concluded that HupA attenuated oxidative glutamate toxicity in murine hippocampal HT22 cells via activating BDNF/TrkB-dependent PI3K/Akt/mTOR signaling pathway.     

Apocynum venetum leaf extract protects rat cortical neurons from injury induced by oxygen and glucose deprivation in vitro

This study aimed to investigate the protective effect of Apocynum venetum leaf extract (AVLE) on an in vitro model of ischemia-reperfusion induced by oxygen and glucose deprivation (OGD) and further explored the possible mechanisms underlying protection. Cell injury was assessed by morphological examination using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) leakage; cell viability was measured by XTT reduction. Neuronal apoptosis was determined by flow cytometry, and electron microscopy was used to study morphological changes of neurons. Caspase-3,?-8, and?-9 activation and Bcl-2/Bax protein expression were determined by Western blot analysis. We report that treatment with AVLE (5 and 50?μg/mL) effectively reduced neuronal cell death and relieved cell injury induced by OGD. Moreover, AVLE decreased the percentage of apoptotic neurons, relieved neuronal morphological damage, suppressed overexpression of active caspase-3 and?-8 and Bax, and inhibited the reduction of Bcl-2 expression. These findings indicate that AVLE protects against OGD-induced injury by inhibiting apoptosis in rat cortical neurons by down-regulating caspase-3 activation and modulating the Bcl-2/Bax ratio.     

TAT-Mediated Protein Transduction of Nogo Extracellular Peptide 1-40 and its Biological Activity

Aim Nogo extracellular peptide 1-40 (NEP1-40), a Nogo-66 antagonistic peptide, is one of the potential candidates for therapeutic intervention after central nervous system injury. This study is focused on the generation of TAT-NEP1-40 fusion protein and its transducible effects and biological activity. Methods TAT-NEP1-40 fusion protein was expressed in vitro. Transducible effects of TAT-NEP1-40 were analyzed by using immunofluorescence staining or Western blot in vitro and in vivo. The biological activity of TAT-NEP1-40 was assessed by its effects against oxygen and glucose deprivation (OGD)-induced PC12 cell damages. Results Our results showed that the TAT-NEP1-40 fusion protein was successfully expressed, purified, and refolded. Western blot analysis and immunofluorescence staining confirmed the delivery of TAT-NEP1-40 protein into PC12 cells and rat brains. OGD caused cell apoptosis or death, decreased cell viability, increased lactate dehydrogenase release in medium and the Bax/Bcl-2 ratio, all of which were prevented by the TAT-NEP1-40 fusion proteins when added exogenously to culture medium. In addition, TAT-NEP1-40 promoted neurite outgrowth of PC12 cells exposed to OGD. Conclusion These results demonstrate that the TAT-NEP1-40 can be successfully generated and efficiently transduced into PC12 cells and rat brains. The TAT-NEP1-40 can protect PC12 cells against OGD and promote neurite outgrowth. This finding suggests that the transducible TAT-NEP1-40 fusion protein offers a possibility of the development of novel therapy for cerebral injuries via delivery of the biologically active TAT-NEP1-40 fusion protein into injured sites. Qiang Wang and Xingchun Gou contributed equally to this article.     

生长分化因子11对甲醛诱导的海马神经细胞凋亡的影响

目的探讨生长分化因子11（GDF11）对甲醛诱导的海马神经（HT22）细胞毒性的影响。 方法把HT22细胞分为对照组（细胞未做任何处理）、甲醛组（50、100、200 μmol/ L甲醛处理细胞）和GDF11+甲醛组（GDF11转染细胞后用100 μmol/L甲醛处理）。细胞计数试剂盒（CCK8）法检测HT22细胞的活力;蛋白免疫印迹法检测HT22细胞凋亡相关蛋白Bax以及Bcl-2的变化;caspase-3活性检测试剂盒检测HT22细胞内caspase-3活性;DCFDA染色流式细胞仪检测HT22细胞中活性氧（ROS）水平。三组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。 结果与对照组比较,甲醛组HT22细胞活力（92.23±0.20比56.12±0.61）和Bcl-2蛋白表达（220.32±2.21比150.25±0.31）水平均降低,差异具有统计学意义（P均< 0.05）;而caspase-3活性（95.36±1.74比190.17±2.14）、Bax蛋白表达（132.19±1.21比150.17±1.06）和ROS水平（1099.32±75.47比2802.17±126.49）均升高,差异具有统计学意义（P均< 0.05）。GDF11转染HT22细胞后,与甲醛组比较,GDF11+甲醛组HT22细胞活力升高（56.12±0.61比83.11±1.64）,Bax蛋白表达（270.03±0.17比150.17±1.06）降低,Bcl-2蛋白表达（150.25±0.31比187.34±1.52）升高,caspase-3活性降低（190.17±2.14比105.31±4.12）和ROS水平降低（2802.17±126.49比1305.36±68.45）,差异具有统计学意义（P均< 0.05）。 结论GDF11能够逆转甲醛对HT22细胞凋亡的诱导作用以及降低甲醛对HT22细胞ROS水平的增加作用,此机制对防治甲醛的神经毒性具有重要意义。     

Inhibition of NADPH oxidase reduces myocardial oxidative stress and apoptosis and improves cardiac function in heart failure after myocardial infarction

Increases in NADPH oxidase activity, oxidative stress, and myocyte apoptosis coexist in failing hearts. In cardiac myocytes in vitro inhibition of NADPH oxidase reduces apoptosis. In this study, we tested the hypothesis that NADPH oxidase inhibition reduces myocyte apoptosis and improves cardiac function in heart failure after myocardial infarction (MI). Rabbits with heart failure induced by MI and sham-operated animals were randomized to orally receive apocynin, an inhibitor of NADPH oxidase (15 mg per day) or placebo for 4 weeks. Left ventricular (LV) dimension and function were assessed by echocardiography and hemodynamics. Myocardial NADPH oxidase activity was measured by superoxide dismutase-inhibitable cytochrome c reduction assay, NADPH oxidase subunit p47phox expression by Western blot and immunofluorescence analysis, myocardial oxidative stress evaluated by 8-hydroxydeoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE) using immunohistochemistry, and myocyte apoptosis by TUNEL assay. MI rabbits exhibited LV dilatation and systolic dysfunction measured by LV fractional shortening and the maximal rate of LV pressure rise (dP/dt). These changes were associated with increases in NADPH oxidase activity, p47phox protein expression, 8-OHdG expression, 4-HNE expression, myocyte apoptosis, and Bax protein and a decrease in Bcl-2 protein. Apocynin reduced NADPH oxidase activity, p47phox protein, oxidative stress, myocyte apoptosis, and Bax protein, increased Bcl-2 protein, and ameliorated LV dilatation and dysfunction after MI. The results suggest that inhibition of NADPH oxidase may represent an attractive therapeutic approach to treat heart failure.     

SOD2在姜黄素减轻氧糖剥夺所致神经元损伤中的作用

目的:探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了姜黄素(Curcumin,Cur)对氧糖剥夺模型(Oxygen-Glucose Deprivation,OGD)损伤神经元的保护作用。方法:本研究采用HT22神经元细胞暴露于OGD环境中3 h模拟神经元缺血缺氧损伤,SOD2-si RNA抑制神经元SOD2蛋白表达后,通过噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,比色法测量培养基乳酸脱氢酶(Lactic Dehydrogenase LDH)水平,流式细胞仪计算细胞凋亡率,Western blot测定凋亡蛋白Cleaved Caspase-3表达,并观察细胞形态和线粒体功能。结果:与正常培养的Control组相比,OGD组细胞活力显著降低,LDH释放明显增加,细胞凋亡率和Cleaved Caspase-3表达显著上升,细胞形态破坏并降低线粒体膜电位(MMP)和线粒体复合物1(Mitochondrial Complex 1 Activity)的活力(P0.05),100 ng/ml的Cur可显著减轻OGD诱导的神经元细胞的上述损伤性改变(P0.05)。而SOD2-si RNA显著逆转Cur对OGD诱导的神经元细胞损伤的保护作用(P0.05),SC-si RNA则未对Cur产生的神经保护作用造成显著干扰(P0.05)。结论:Cur可能通过上调SOD2的表达,减轻OGD对神经元细胞的损伤。     

NADPH oxidase is involved in angiotensin II-induced apoptosis in H9C2 cardiac muscle cells: effects of apocynin

Angiotensin II stimulates NADPH oxidase activity in vascular cells. However, it is not fully understood whether angiotensin II, which plays an important role in heart failure, stimulates NADPH oxidase activation and expression in cardiac myocytes. Previous studies have shown that angiotensin II induces myocyte apoptosis, but whether the change is mediated via NADPH oxidase remains to be elucidated. In this study we proposed to determine whether angiotensin II stimulated NADPH oxidase activation and NADPH oxidase subunit p47-phox expression in H9C2 cardiac muscle cells. If so, we would determine whether the NADPH oxidase inhibitor apocynin prevented angiotensin II-induced apoptosis. The results showed that angiotensin II increased NADPH oxidase activity, p47-phox protein and mRNA expression, intracellular reactive oxygen species, and apoptosis in H9C2 cells. Angiotensin II elevated p38 mitogen-activated protein kinase (MAPK) activity, decreased Bcl-2 protein, and increased Bax protein and caspase-3 activity. Apocynin treatment inhibited angiotensin II-induced NADPH oxidase activation and increases in p47-phox expression, intracellular reactive oxygen species, and apoptosis. The effect of apocynin on apoptosis was associated with reduced p38 MAPK activity, increased Bcl-2 protein, and decreased Bax protein and caspase-3 activity. These results suggest that angiotensin II-induced apoptosis is mediated via NADPH oxidase activation probably through p38 MAPK activation, a decrease in Bcl-2 protein, and caspase activation.     

Neuroprotective Effects of <Emphasis Type="Italic">Sigesbeckia pubescens</Emphasis> Extract on Glutamate-Induced Oxidative Stress in HT22 Cells via Downregulation of MAPK/caspase-3 Pathways

Sigesbeckia pubescens (SP) is a traditional Chinese medicine, possessing antioxidant and anti-inflammatory activities. In this study, we evaluate the neuroprotective activities of SP extract on glutamate-induced oxidative stress in HT22 cells and the molecular mechanism underlying neuroprotection. We applied 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), crystal violet, reactive oxygen species (ROS), lactate dehydrogenase (LDH), quantitative real-time polymerase chain reaction (qPCR), and western blot analyses for assessing the neuroprotective effects of SP extract. The experimental study revealed that SP considerably increased the cell viability, and reduced the oxidative stress promoted ROS and LDH generation in HT22 cells in a dose-dependent manner. Additionally, the morphology of HT22 cells was effectively improved by SP. Upregulated gene expressions of mitogen-activated protein kinase (MAPK) were markedly attenuated by SP. Similarly, SP notably suppressed the ROS-mediated phosphorylation of MAPK (pERK1/2, pJNK, and pp38) cascades and activation of apoptotic factor caspase-3 signaling pathway that overall contributed to the neuroprotection. Taken together, SP may exert neuroprotective effects via alteration of MAPK and caspase-3 pathways under oxidative stress condition. Therefore, SP is a potential agent for preventing oxidative stress-mediated neuronal cell death.     

Ischemia-activated microglia induces neuronal injury via activation of gp91phox NADPH oxidase

Although glial cells play a major role in the pathogenesis of many neurological diseases by exacerbating neuronal and non-neuronal cell death, the mechanisms involved are unclear. We examined the effects of microglia-(MCM) or astrocyte-(ACM) conditioned media obtained by chemical ischemia on the neuronal injury in SH-SY5Y cells. Chemical ischemia was induced by the treatment with NaN₃ and 2-deoxy-d-glucose for 2 h. MCM-treated SH-SY5Y cells showed reduced the viability, increased caspase-3 activity, decreased Bcl-2/Bax ratio, and increased cytochrome c release, increased inflammatory cytokines, and increased reactive oxygen species (ROS) generation. MCM also increased gp91phox nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which was inhibited by NADPH oxidase inhibitor, apocynin, and gp91phox siRNA. However, ACM did not show any significant changes. The results suggest that microglia activated by ischemic insult may increase reactive oxygen species generation via activation of gp91phox NADPH oxidase, resulting in neuronal injury.     

Downregualtion of dynamin-related protein 1 attenuates glutamate-induced excitotoxicity via regulating mitochondrial function in a calcium dependent manner in HT22 cells

Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Dynamin related protein 1 (Drp-1), one of the GTPase family of proteins that regulate mitochondrial fission and fusion balance, is associated with apoptotic cell death in cancer and neurodegenerative diseases. Here we investigated the effect of downregulating Drp-1 on glutamate excitotoxicity-induced neuronal injury in HT22 cells. We found that downregulation of Drp-1 with specific small interfering RNA (siRNA) increased cell viability and inhibited lactate dehydrogenase (LDH) release after glutamate treatment. Downregulation of Drp-1 also inhibited an increase in the Bax/Bcl-2 ratio and cleavage of caspase-9 and caspase-3. Drp-1 siRNA transfection preserved the mitochondrial membrane potential (MMP), reduced cytochrome c release, enhanced ATP production, and partly prevented mitochondrial swelling. In addition, Drp-1 knockdown attenuated glutamate-induced increases of cytoplasmic and mitochondrial Ca²⁺, and preserved the mitochondrial Ca²⁺ buffering capacity after excitotoxicity. Taken together, these results suggest that downregulation of Drp-1 protects HT22 cells against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the preservation of mitochondrial function through regulating intracellular calcium homeostasis.     

Pretreatment with 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside Attenuates Cerebral Ischemia/Reperfusion-Induced Injury In Vitro and In Vivo

Salidroside, extracted from the root of Rhodiola rosea L, is known for its pharmacological properties, in particular its neuroprotective effects. 2-(4-Methoxyphenyl) ethyl-2-acetamido-2-deoxy-β-D- pyranoside (GlcNAc-Sal), an analog of salidroside, was recently synthesized and shown to possess neuroprotective properties. The purpose of the current study was to investigate the neuroprotective effects of GlcNAc-Sal against oxygen–glucose deprivation-reperfusion (OGD-R)-induced neurotoxicity in vitro and global cerebral ischemia-reperfusion (GCI-R) injury in vivo. Cell viability tests and Hoechst 33342 staining confirmed that GlcNAc-Sal pretreatment markedly attenuated OGD-R induced apoptotic cell death in immortalized mouse hippocampal HT22 cells. Western blot, immunofluorescence and PCR analyses revealed that GlcNAc-Sal pretreatment restored the balance of pro- and anti-apoptotic proteins and inhibited the activation of caspase-3 and PARP induced by OGD-R treatment. Further analyses showed that GlcNAc-Sal pretreatment antagonized reactive oxygen species (ROS) generation, iNOS-derived NO production and NO-related apoptotic cell death during OGD-R stimulation. GCI-R was induced by bilateral common carotid artery occlusion (BCCAO) and reperfusion in mice in vivo. Western blot analysis showed that GlcNAc-Sal pretreatment decreased the expression of caspase-3 and increased the expression of Bcl-2 (B-cell lymphoma 2)/Bax (Bcl-2-associated X protein) induced by GCI-R treatment. Our findings suggest that GlcNAc-Sal pretreatment prevents brain ischemia reperfusion injury by the direct or indirect suppression of cell apoptosis and GlcNAc-Sal could be developed as a broad-spectrum agent for the prevention and/or treatment of cerebral ischemic injury.     

PML/RARalpha fusion protein mediates the unique sensitivity to arsenic cytotoxicity in acute promyelocytic leukemia cells: Mechanisms involve the impairment of cAMP signaling and the aberrant regulation of NADPH oxidase

Acute promyelocytic leukemia (APL) cells are characterized by PML/RARalpha fusion protein, high responsiveness to arsenic trioxide (ATO)-induced cytotoxicity and an abundant generation of reactive oxygen species (ROS). In this study we investigated the association among these three features in APL-derived NB4 cells. We found that NADPH oxidase-derived ROS generation was more abundant in NB4 cells compared with monocytic leukemia U937 cells. By using PR9, a sub-line of U937 stably transduced with the inducible PML/RARalpha expression vectors, we attributed disparities on ROS generation and ATO sensitivity to the occurrence of PML/RARalpha fusion protein, since PML/RARalpha-expressing cells appeared higher NADPH oxidase activity, higher ROS level and higher sensitivity to ATO. On the other hand, the basal intensity of cAMP signaling pathway was compared between NB4 and U937 as well as between PR9 cells with or without PML/RARalpha, demonstrating that PML/RARalpha-expressing cells had an impaired cAMP signaling pathway which relieved its inhibitory effect on NADPH oxidase derived ROS generation. In summary, the present study demonstrated the correlation of PML/RARalpha with cAMP signaling pathway, NADPH oxidase and ROS generation in APL cells. PML/RARalpha that bestows NB4 cells various pathological features, paradoxically also endows these cells with the basis for susceptibility to ATO-induced cytotoxcity.     

Effects of Estrogen and Phytoestrogen Treatment on an In Vitro Model of Recurrent Stroke on HT22 Neuronal Cell Line

An increase of stroke incidence occurs in women with the decline of estrogen levels following menopause. This ischemic damage may recur, especially soon after the first insult has occurred. We evaluated the effects of estrogen and phytoestrogen treatment on an in vitro recurrent stroke model using the HT22 neuronal cell line. HT22 cells were treated with 17β-estradiol or genistein 1 h after the beginning of the first of two oxygen and glucose deprivation/reoxygenation (OGD/R) cycles. During the second OGD, there was a deterioration of some components of the electron transport chain, such as cytochrome c oxidase subunit 1 with a subsequent increase of reactive oxygen species (ROS) production. Accordingly, there was also an increase of apoptotic phenomena demonstrated by poly(ADP-ribose) polymerase 1 cleavage, Caspase-3 activity, and Annexin V levels. The recurrent ischemic injury also raised the hypoxia-inducible factor 1α and glucose transporter 1 levels, as well as the ratio between the lipidated and cytosolic forms of microtubule-associated protein 1A/1B-light chain 3 (LC3-II/LC3-I). We found a positive effect of estradiol and genistein treatment by partially preserving the impaired cell viability after the recurrent ischemic injury; however, this positive effect does not seem to be mediated neither by blocking apoptosis processes nor by decreasing ROS production. This work contribute to the better understanding of the molecular mechanisms triggered by recurrent ischemic damage in neuronal cells and, therefore, could help with the development of an effective treatment to minimize the consequences of this pathology.     

Ginkgolides and bilobalide protect BV2 microglia cells against OGD/reoxygenation injury by inhibiting TLR2/4 signaling pathways

Ginkgolide and bilobalide are major trilactone constituent of Ginkgo biloba leaves and have been shown to exert powerful neuroprotective properties. The aims of this study were to observe the inhibitory effects of ginkgolide and bilobalide on the activation of microglial cells induced by oxygen–glucose deprivation and reoxygenation (OGD/R) and the specific mechanisms by which these effects are mediated. For detecting whether ginkgolide and bilobalide increased cell viability in a dose-dependent manner, BV2 cells were subjected to oxygen–glucose deprivation for 4 h followed by 3 h reoxygenation with various concentrations of drugs (6.25, 12.5, 25, 50, and 100 μg/ml). The extent of apoptosis effect of OGD/R with or without ginkgolide and bilobalide treatment were also measured by Annexin V-FITC/PI staining. Similarly, the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and IL-10 were detected using a specific Bio-Plex Pro? Reagent Kit. The effects of ginkgolide and bilobalide on protein levels of TLR2/4, MyD88, p-TAK1, p-IKKβ, p-IkBα, NF-κB p65, Bcl-2, Bax, Bak, RIP3, cleaved-Caspase-3, cleaved PARP-1 and cellular localization of NF-κB p65 were evaluated by Western blot and double-labeled immunofluorescence staining, respectively. OGD/R significantly decreased the cell viability and increased the release of IL-1β, IL-6, IL-8, IL-10, TNF-α in BV2 microglia cells; these effects were suppressed by ginkgolide and bilobalide. Meanwhile, ginkgolide and bilobalide also attenuated the OGD/R-induced increases in TLR2, TLR4, MyD88, Bak, RIP3 levels and reversed cleaved caspase-3/caspase-3, Bax/Bcl-2 and cleaved PARP-1/PARP-1 ratio. Furthermore, ginkgolide and bilobalide also downregulated p-TAK1, p-IkBα, and p-IKKβ and inhibited the OGD/R-induced transfer of NF-κB p65 from cytoplasm to nucleus in BV2 microglia cells. The results showed that ginkgolide and bilobalide can inhibit OGD/R-induced production of inflammatory factors in BV2 microglia cells by regulating the TLRs/MyD88/NF-κB signaling pathways and attenuating inflammatory response. The possible mechanism of anti-inflammatory and neuroprotective effects of ginkgolides results from the synergistic reaction among each monomer constituents.     

梓醇对氧糖剥夺诱导PC1 2 细胞凋亡的保护作用

目的：观察梓醇对氧糖剥夺（OGD）诱导PC12细胞凋亡的保护作用。方法：采用Hoechst 33258 DNA染色法,四甲基偶氮唑盐（MTT）检测细胞活性;化学比色法测定乳酸脱氢酶（LDH）的释放量,用流式细胞技术检测细胞凋亡比例以及P53和Bcl-2蛋白。结果：OGD可导致PC12细胞活力明显下降,LDH释放量增加、P53蛋白表达上升,Bcl-2蛋白表达下降。梓醇可明显改善细胞形态结构,显著降低LDH释放量、降低P53蛋白的表达,提高Bcl-2蛋白的表达,降低细胞凋亡率。结论：梓醇通过调节细胞凋亡相关基因的表达而抑制细胞凋亡。     

L-茶氨酸对H2O2致L02细胞损伤的保护作用及其机制研究

研究L-茶氨酸对肝细胞损伤的保护作用及其机制。利用H2O2诱导的LO2肝细胞损伤模型,分别用MTT法检测细胞存活率、测定LDH、流式细胞术检测细胞凋亡率、Western blot法检测Caspase-3和PARP蛋白表达7LBax／Bcl-2比值的变化,评价L-茶氨酸是否能保护H2O2诱导的肝细胞损伤。结果表明,L-茶氨酸能提高H2O2损伤的L02细胞存活率,减少LDH的渗漏,降低肝细胞凋亡,且L-茶氨酸通过抑制caspase-3的激活和PARP的切割及Bax／Bcl-2比值的升高而发挥抗凋亡的作用。L-茶氨酸对肝细胞损伤有一定的治疗和保护作用。     

miR-20b-5p靶向MAPK1调控脑出血过程中脑微血管内皮细胞铁死亡

摘要 目的：探讨miR-20b-5p对氧糖剥夺（OGD）/Hemin处理的脑微血管内皮细胞（BMVEC）功能的影响及机制。方法：将BMVEC分为Control组、agomir-NC组、agomir-miR-20b-5p组、antagomir-NC组和antagomir-miR-20b-5p组。使用Lipofectamine 2000试剂对细胞进行相应的转染处理。BMVEC转染后，将BMVEC再分为Control组、OGD/Hemin组（O/H组）、OGD/Hemin+agomir-NC组（O/H+agomir-NC组）、OGD/Hemin+agomir-miR-20b-5p组（O/H+agomir-miR-20b-5p组）、OGD/Hemin+antagomir-NC组（O/H+antagomir-NC组）和OGD/Hemin+antagomir-miR-20b-5p组（O/H+antagomir-miR-20b-5p组）。Control组BMVEC正常培养，其他组BMVEC进行OGD/Hemin处理。MTT法检测BMVEC增殖，TUNEL染色检测BMVEC凋亡，Transwell检测BMVEC迁移。使用试剂盒检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和丙二醛（MDA）水平。使用Iron Assay试剂盒检测Fe²⁺含量。通过qRT-PCR检测miR-20b-5p和MAPK1 mRNA水平。通过Western blot检测MAPK1、Bax、Bcl-2、谷胱甘肽过氧化物酶4（GPX4）和前列腺素内过氧化物合酶2（PTGS2）蛋白表达水平。通过免疫荧光染色检测MAPK1的荧光强度水平。结果：与Control组和agomir-NC组比较，agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高（P<0.05）。与Control组和antagomir-NC组比较，antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低（P<0.05）。与Control组比较，O/H组BMVEC中的miR-20b-5p水平降低，细胞活力降低，TUNEL阳性率和Bax蛋白表达水平升高，Bcl-2蛋白表达水平降低，迁移数量降低，SOD和GSH-Px活性降低，MDA含量升高，Fe²⁺含量和PTGS2的蛋白表达水平升高，GPX4的蛋白表达水平降低，MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。与O/H组和O/H+agomir-NC组比较，O/H+agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高，细胞活力升高，TUNEL阳性率和Bax蛋白表达水平降低，Bcl-2蛋白表达水平升高，迁移数量升高，SOD和GSH-Px活性升高，MDA含量降低，Fe²⁺含量和PTGS2的蛋白表达水平降低，GPX4的蛋白表达水平升高，MAPK1的mRNA和蛋白表达水平以及相对荧光强度降低（P<0.05）。与O/H组和O/H+antagomir-NC组比较，O/H+antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低，细胞活力降低，TUNEL阳性率和Bax蛋白表达水平升高，Bcl-2蛋白表达水平降低，迁移数量降低，SOD和GSH-Px活性降低，MDA含量升高，Fe²⁺含量和PTGS2的蛋白表达水平升高，GPX4的蛋白表达水平降低，MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。结论：本研究表明上调miR-20b-5p通过抑制OGD/Hemin处理的BMVEC中MAPK1的表达从而抑制了铁死亡途径。     

SIRT3在氧糖剥夺再灌注损伤小鼠神经元中的表达及意义

目的:通过建立体外脑缺血模型,探讨沉默信息因子3(SIRT3)在小鼠皮层神经元氧糖剥夺再灌注(OGD/R)损伤后的表达和意义。方法:C57BL/6J小鼠皮层神经元原代培养7天后,以氧糖剥夺不同时长(2 h、4 h、6 h、8 h)再灌注24 h作为观察时间点,利用细胞增殖-毒性检测试剂盒(Cell Counting Kit-8,CCK-8)检测细胞活力;小鼠乳酸脱氢酶(LDH)试剂盒检测LDH释放;蛋白印迹法(Western blot WB)观察微管相关蛋白1轻链3(LC3-Ⅱ)、活化凋亡蛋白3(Cleaved caspase-3)、以及SIRT3的表达变化;免疫荧光下进一步观察LC3-II、SIRT3表达。结果:与正常组比,随着氧糖剥夺时间的延长,LDH释放量呈台阶式升高(P0.01),而神经元活性进展性下降(P0.01);蛋白印迹结果发现在缺血损伤后LC3-Ⅱ整体上调,并于OGD 4h达峰值,SIRT3分子表达趋势与LC3-Ⅱ相似均呈抛物线状,而Cleaved caspase-3整体上调;相应的,细胞免疫荧光结果显示缺血损伤后神经元胞体和突起中LC3呈点状高表达,与此同时SIRT3荧光强度亦增高。结论:神经元缺血时间越长损伤越重;LC3-Ⅱ和SIRT3表达呈现相似性;SIRT3可能通过调控线粒体自噬参与了拮抗神经元缺血损伤的作用。     

NOX2 deficiency ameliorates cerebral injury through reduction of complexin II-mediated glutamate excitotoxicity in experimental stroke

Although NADPH oxidase (NOX)-mediated oxidative stress is considered one of the major mechanisms triggering the pathogenic actions of ischemic stroke and very recent studies have indicated that NADPH oxidase is a major source of reactive oxygen species (ROS) production controlling glutamate release, how neuronal NADPH oxidase activation is coupled to glutamate release is not well understood. Therefore, in this study, we used an in vivo transient middle cerebral artery occlusion model and in vitro primary cell cultures to test whether complexins, the regulators of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion, are associated with NOX2-derived ROS and contribute to glutamate-mediated excitotoxicity in ischemic stroke. In this study, we first identified the upregulation of complexin II in the ischemic brain and evaluated its potential role in ischemic stroke showing that gene silencing of complexin II ameliorated cerebral injury as evidenced by reduced infarction volume, neurological deficit, and neuron necrosis accompanied by decreased glutamate levels, consistent with the results from NOX2^−/− mice with ischemic stroke. We further demonstrated that complexin II expression was mediated by NOX2 in primary cultured neurons subjected to oxygen–glucose deprivation (OGD) and contributed to OGD-induced glutamate release and neuron necrosis via SNARE signaling. Taken together, these findings for the first time provide evidence that complexin II is a central target molecule that links NADPH oxidase-derived ROS to glutamate-mediated neuronal excitotoxicity in ischemic stroke.