羟丁酸钠对大鼠原代培养皮层神经元缺氧复氧损伤的保护作用与GABAA受体的关系

目的 :探讨羟丁酸钠 (SO)对大鼠皮层神经元缺氧复氧损伤的保护作用与GABAA 受体的关系。方法 :采用原代培养大鼠皮层神经元建立缺氧复氧损伤模型 ,观察细胞的形态学 ,测定其LDH漏出率、MDA含量、SOD、GPx活力。结果 :缺氧复氧可引起神经元损伤 ,LDH漏出率增加 ,MDA含量升高 (P <0 .0 1) ,SOD、GPx活力降低 (P <0 .0 1)。SO能显著减少损伤神经元的LDH漏出率及MDA的生成 (P <0 .0 1) ,升高SOD、GPx(P <0 .0 1)的活力 ,这种作用可被GABAA 受体阻断剂seurinine减弱 (P <0 .0 1)。结论 :SO对缺氧复氧神经元损伤的保护作用可能与其激动GABAA 受体有关。     

L-肌肽对鱼藤素诱导SH-SY5Y细胞神经毒性的保护作用

目的研究L-肌肽是否能通过抗氧化活性降低鱼藤素导致的神经毒性。方法 SH-SY5Y细胞在L-肌肽(1、10、20、40、60、80、100 mmol·L~(-1))中培养24 h,CCK-8法测定细胞存活率。30 mmol·L~(-1) L-肌肽、20μmol·L~(-1)鱼藤素、20μmol·L~(-1)鱼藤素与30 mmol·L~(-1) L-肌肽作用于SH-SY5Y细胞24 h,AO/EB法检测细胞形态和凋亡情况。8μmol·L~(-1)鱼藤素、8μmol·L~(-1)鱼藤素与3 mmol·L~(-1) L-肌肽、8μmol·L~(-1)鱼藤素与30 mmol·L~(-1) L-肌肽、20μmol·L~(-1)鱼藤素、20μmol·L~(-1)鱼藤素与3 mmol·L~(-1) L-肌肽、20μmol·L~(-1)鱼藤素与30 mmol·L~(-1) L-肌肽、50μmol·L~(-1)鱼藤素、50μmol·L~(-1)鱼藤素与3 mmol·L~(-1) L-肌肽、50μmol·L~(-1)鱼藤素与30 mmol·L~(-1) L-肌肽作用于SH-SY5Y细胞24 h,CCK-8法检测细胞的增殖情况;双染流式细胞术检测细胞凋亡率;DCFH-DA流式细胞术检测细胞活性氧(ROS)水平。结果 20μmol·L~(-1)鱼藤素与30 mmol·L~(-1) L-肌肽共作用于细胞后,细胞抑制率降低9.07%,早期凋亡的细胞数减少,早期凋亡率降低9.35%,总凋亡率降低10.7%,ROS水平低于20μmol·L~(-1)鱼藤素组,结果差异具有统计学意义(P<0.05)。结论 L-肌肽能有效降低鱼藤素诱导SH-SY5Y细胞的神经毒性,保护细胞,其机制可能与降低氧化应激水平相关。     

红景天苷衍生物S01对谷氨酸及缺氧缺糖所致SH-SY5Y细胞损伤的保护作用

目的探讨红景天苷衍生物S01(3′,4′,5′-三甲氧基苄基-1-硫代-β-D-吡喃葡萄糖苷)对神经细胞的保护作用,并初步探讨其作用机制。方法用不同浓度S01(2,10和50mg.L-1)预处理SH-SY5Y细胞,12h后用谷氨酸或连二亚硫酸钠(Na2S2O4)诱导SH-SY5Y细胞损伤,用MTT法检测细胞存活率,比色法检测乳酸脱氢酶(LDH)释放、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,荧光探针负载法检测细胞内活性氧(ROS)水平和细胞内游离钙离子浓度([Ca2+]i),化学发光法检测细胞中腺苷三磷酸(ATP)水平。结果在谷氨酸所致SH-SY5Y细胞损伤模型中,与对照组比较,模型组细胞存活率、SOD活性和ATP水平降低,LDH释放、MDA含量和细胞内ROS水平增加。与模型组比较,S01(2,10和50mg.L-1)可提高细胞存活率,减少谷氨酸引起的LDH释放,拮抗谷氨酸引起的SOD活性和ATP水平降低,并拮抗MDA和ROS水平升高。在Na2S2O4所致SH-SY5Y细胞缺氧缺糖损伤模型中,与对照组比较,模型组细胞存活率下降,LDH释放增多,细胞[Ca2+]i升高。与模型组比较,S01(2,10和50mg.L-1)可提高细胞存活率,减少缺氧缺糖引起的LDH释放和细胞内钙超载。结论S01对谷氨酸和缺氧缺糖所致神经细胞损伤具有保护作用,提示S01可能对脑缺血具有治疗作用。     

鹅不食草通过激活ERK/Nrf2信号通路发挥神经保护的作用机制

目的氧化应激与神经退行性疾病的发生发展密切相关,传统中草药鹅不食草具有抗氧化特性。本课题利用鹅不食草乙醇提取物(ECM)深入研究鹅不食草的抗氧化效果以及分子机制,并探讨鹅不食草通过抗氧化发挥神经保护从而改善小鼠学习记忆的作用效果。方法采用醇提法提取ECM,并运用高效液相色谱技术从ECM中分离纯化倍半萜类化合物,利用核磁共振对其进行结构鉴定。体外实验利用叔丁基过氧化氢(t BHP)和谷氨酸盐在神经母细胞瘤细胞(SH-SY5Y)和小鼠嗜铬细胞瘤细胞(PC12)中构建氧化损伤模型;在体内实验中,选用昆明小鼠皮下注射D-半乳糖(D-Gal,每天120 mg·kg~(-1))建立亚急性衰老模型,并在造模2个月后用ECM(每天100,200和400 mg·kg~(-1))继续灌胃1个月检测对小鼠的体内抗氧化效果。通过DCFH-DA荧光探针标记检测活性氧(ROS)含量;利用相关试剂盒检测细胞脂质过氧化产物丙二醛(MDA)和抗氧化小分子GSH的含量;用免疫印迹检测各种抗氧化蛋白的含量;采用Nissl染色观察鹅不食草对海马神经元的保护作用;利用Morris水迷宫检测鹅不食草改善小鼠学习记忆的能力。结果首先我们检测鹅不食草在细胞水平的抗氧化效果和分子机制。谷氨酸盐和t BHP可以显著升高细胞内ROS含量,促进SH-SY5Y和PC12细胞死亡,并降低线粒体膜电位,下调GSH的含量,抑制SOD活性,同时升高MDA水平。而ECM(0.5～2.0 mg·L-1)预处理可以显著降低细胞内ROS含量,抑制氧化诱导的细胞死亡,并回复线粒体膜电位,降低MDA含量,同时GSH含量和SOD活性也回复至正常水平。ECM可以显著降低t BHP诱导的p38 MAPK和JNK磷酸化水平,同时维持了ERK的磷酸化水平。进一步发现ECM可以诱导Nrf2的核转位进而促进下游抗氧化相关蛋白的表达,包括血红素加氧酶-1(HO-1)、超氧化物歧化酶2(SOD2)和NAD(P)H:醌氧化还原酶-1(NQO-1),表明ECM可以显著活化细胞内Nrf2信号通路;而ERK抑制剂U0216则显著抑制ECM诱导的Nrf2通路活化,表明ECM通过激活ERK活化Nrf2通路从而发挥抗氧化作用。在D-Gal诱导的亚急性衰老小鼠模型中,ECM同样可以显著降低海马和皮质内MDA水平,增加GSH含量;在海马组织中,ECM可以抑制p38 MAPK和JNK的活化,增强ERK磷酸化水平,激活Nrf2信号通路,恢复小鼠海马中HO-1,NQO-1和SOD2的表达水平,并维持海马神经元突触蛋白的表达水平。在Morris水迷宫定位航行试验中,ECM处理组小鼠的逃避潜伏期较模型组明显缩短,并在空间探索实验中增加了小鼠的穿越平台次数,从而表明鹅不食草可以显著抑制氧化应激导致的神经损伤并改善学习记忆。为了探索鹅不食草发挥抗氧化作用的主要活性成分,从ECM中分离纯化出了4种倍半萜类化合物,经鉴定确定为6-O-angeloylprenolin、arnicolide D、arnicolide C和microhelenin C4种单体化合物;实验发现6-O-angeloylprenolin和arnicolide D在低剂量下(0.5～2μmol·L-1)可以非常显著的抑制ROS产生,并保护t BHP对SH-SY5Y和PC12造成的氧化损伤,同时2种单体化合物可以显著活化Nrf2信号通路。从而明确了鹅不食草发挥抗氧化作用的主要活动单体成分。结论 ECM能够通过激活ERK/Nrf2信号通路保护神经元细胞免受氧化应激诱导的损伤,并显著改善小鼠的学习记忆能力;其中6-O-angeloylplenolin和arnicolide D2种单体化合物作为主要活性成分发挥抗氧化特性。提示鹅不食草具有显著抗氧化作用并可以作为抗氧化化合物的来源,为治疗神经退行性疾病药物研发提供了新的选择。     

表没食子儿茶素促进Nrf2的核转位减轻Aβ_(25-35)对SH-SY5Y细胞的损伤

目的研究表没食子儿茶素(epigallocatechin,EGC)对Aβ_(25-35)损伤SH-SY5Y细胞的保护作用及机制。方法采用Aβ_(25-35)损伤SH-SY5Y细胞,MTT和LDH法,考察EGC对SH-SY5Y细胞活力的影响。应用DCFH-DA荧光探针检测细胞内ROS水平,通过测定细胞内SOD、GSH-Px和MDA活性,考察细胞氧化应激水平。Western blot测定细胞中Nrf2、NQO1、HO-1、Prdx6和Trx1蛋白含量。结果 Aβ_(25-35)损伤SH-SY5Y细胞,导致细胞存活率明显降低。5、10、20μmol·L~(-1) EGC能够减轻Aβ_(25-35)诱导的SH-SY5Y细胞损伤及LDH释放。而且EGC能够减少Aβ_(25-35)诱导的细胞内ROS水平的增加,减轻氧化应激损伤。同时,EGC能够明显增强Aβ_(25-35)损伤后Nrf2、NQO1、HO-1、Prdx6和Trx1的表达。结论EGC能够抑制Aβ_(25-35)诱导的SH-SY5Y细胞损伤,通过促进Nrf2核转位,提高抗氧化水平,降低Aβ_(25-35)神经毒性。     

基于CREB通路研究3,6’-二芥子酰基蔗糖的神经保护分子机制

目的 3,6’-二芥子酰基蔗糖(DISS)是远志中的寡糖酯类成分,通过研究DISS对人神经母细胞瘤细胞SH-SY5Y的增殖作用和对环磷酸腺苷反应元件结合蛋白(CREB)通路的影响,探究其发挥神经保护作用的分子机制。方法采用四甲基噻唑蓝(MTT)法测定SH-SY5Y细胞生长曲线,筛选最佳种板密度和给药时间,并检测不同浓度DISS对细胞增殖的影响;采用Western blotting法检测细胞给予DISS后不同时间点CREB、磷酸化CREB(p-CREB)和脑源性神经生长因子(BDNF)的表达水平;给予调节CREB活性的相关上游通路拮抗剂U0126(MEK拮抗剂)、H89(PKA拮抗剂)、KN93(CaMK拮抗剂)拮抗CREB上游通路,并给予DISS保护,用Western blotting法检测DISS对拮抗前后细胞内CREB,p-CREB,BDNF蛋白表达的影响。结果 SH-SY5Y最佳中板密度为2×108L-1,DISS(60μmol·L-1,30μmol·L-1)对SH-SY5Y细胞具有明显的促增殖作用(P<0.05);与正常细胞相比,DISS给药15 min时可显著增加细胞内p-CREB,BDNF的表达(P<0.05);DISS可明显增加拮抗后SH-SY5Y细胞内CREB,p-CREB,BDNF的表达水平(P<0.05)。结论 DISS可以调节CREB活性并促进下游BDNF等相关因子的表达,其神经保护作用的机制可能与活化Ca2+/CaM/CaMK-CREB-BDNF信号通路有关。     

鱼藤素对SH-SY5Y神经母细胞瘤细胞凋亡的诱导作用

目的研究鱼藤素对SH-SY5Y细胞凋亡的诱导作用。方法鱼藤素(0、0.625、1.25、2.5、5、10、20μmol·L~(-1))处理SH-SY5Y细胞24、48、72 h后,CCK-8法测定细胞存活率。鱼藤素(0、8、20、50μmol·L~(-1))处理SH-SY5Y细胞24 h,光镜下及AO/EB双染分别观察细胞形态和凋亡形态,流式细胞术检测细胞凋亡率,DCFH-DA荧光探针法检测细胞活性氧水平,分光光度法检测caspase-3活化程度。结果鱼藤素对SH-SY5Y细胞存活率呈时间和浓度依赖性抑制作用,作用24、48、72 h的IC50值分别为(26.07±2.18)、(18.33±0.94)、(12.5±1.49)μmol·L~(-1)。鱼藤素处理24 h后,细胞凋亡率、细胞活性氧水平明显上升(P<0.05),caspase-3活化程度升高,且三者均有浓度-效应特征。结论鱼藤素可抑制SH-SY5Y细胞存活,诱导细胞凋亡,其机制可能与升高活性氧水平和活化caspase-3相关。     

脑CYP2E1参与脂多糖诱导的神经元损伤

目的研究脂多糖(LPS)所致炎症与脑细胞色素P4502E1和CYP2E1之间的相互影响。方法采用具有胆碱能神经元特征的IMR-32人神经母细胞瘤细胞系,分别给予低剂量(0.1 mg·L~(-1))和高剂量(1.0 mg·L~(-1))LPS处理24 h,检测LDH和SOD活性。在LPS处理前45 min,分别加入p38抑制剂SB203580和ERK抑制剂U0126处理IMR-32细胞,观察MAPK信号系统对神经元CYP2E1表达的影响。采用具有多巴胺能神经元特征的SH-SY5Y人神经母细胞瘤细胞系建立高表达CYP2E1细胞系,并与正常SH-SY5Y细胞同时给予低剂量(0.1 mg·L~(-1))和高剂量(1.0 mg·L~(-1))LPS处理24 h后检测LDH和SOD活性。结果与对照组相比,高剂量LPS处理IMR-32细胞,SOD的活力下降15.0%(P<0.01),LDH上升1.38倍(P<0.01),CYP2E1 mRNA升高1.25倍(P<0.01),蛋白水平升高1.19倍(P<0.05)。p38和ERK抑制剂可拮抗高剂量LPS对CYP2E1的诱导作用。低剂量LPS处理CYP2E1高表达SH-SY5Y细胞,LDH的升高幅度较非高表达的对照组上升了1.28倍(P<0.01),SOD活力下降幅度增加了3.53倍(P<0.01);高剂量LPS使得CYP2E1高表达SH-SY5Y细胞LDH的升高幅度较非高表达的对照组上升了1.54倍(P<0.01),SOD活力下降幅度增加了2.17倍(P<0.01)。结论 LPS可上调神经元所表达的CYP2E1水平,其调控作用可能与ERK和p38信号传导通路相关。高表达CYP2E1加剧LPS对神经元的损伤,提示CYP2E1参与了炎症所致神经细胞损伤的病理过程。     

20-羟基蜕皮甾酮对H_2O_2诱导SH-SY5Y细胞损伤的保护作用

本文旨在研究一种类固醇激素即20-羟基蜕皮甾酮对H2O2诱导的SH-SY5Y细胞损伤的保护作用及其作用机制。预孵育20-羟基蜕皮甾酮可以明显提高H2O2诱导的SH-SY5Y细胞的生存率,减少乳酸脱氢酶（LDH）漏出率,抑制脂质过氧化水平（MDA）升高,提高超氧化物歧化酶（SOD）活性。此外,20-羟基蜕皮甾酮可以通过降低Bax/Bcl-2的比值,延缓caspase-3的活化来抑制H2O2诱导的细胞凋亡。本研究表明20-羟基蜕皮甾酮对H2O2诱导的SH-SY5Y细胞具有保护作用,有可能用于由氧化应激和凋亡导致的神经退行性疾病的治疗。     

基于Nrf2信号通路的三七总皂苷对Aβ_(25-35)诱导PC12细胞凋亡的保护作用机制研究

目的探讨三七总皂苷对Aβ_(25-35)诱导的PC12细胞凋亡的保护作用及分子机制。方法利用Aβ_(25-35)诱导PC12细胞损伤模型,MTT法检测PC12细胞活力,试剂盒测定LDH漏出量、ROS水平、MDA含量、Caspase-3活力以及抗氧化酶SOD、CAT和GHS-Px活力,TUNEL染色检测细胞凋亡,JC-1染色观察线粒体膜电位,Western blot检测相关蛋白HO-1、Lamin-B、细胞核内Nrf2及总Nrf2的蛋白表达。结果三七总皂苷能够明显抑制Aβ_(25-35)诱导的PC12细胞活力下降(P<0.01)和LDH漏出(P<0.01),减少ROS和MDA的产生(P<0.01),增加SOD、CAT和GSH-Px活力(P<0.01),稳定线粒体膜电位,抑制caspase-3的活化(P<0.01)。而且,三七总皂苷还能够上调PC12细胞细胞核Nrf2、总Nrf2和细胞质中HO-1的蛋白表达。HO-1抑制剂Sn PP能够抑制三七总皂苷的神经保护作用。结论三七总皂苷可能通过上调Nrf2/HO-1信号通路,从而抑制Aβ25-35诱导PC12细胞氧化应激和凋亡。     

MCI-186对β-淀粉样肽诱导SH-SY5Y细胞凋亡及p38MAPK蛋白表达的影响

目的研究依达拉奉(MCI-186)对阿尔茨海默病(AD)细胞损伤的保护作用及其机制。方法将人神经母细胞瘤SH-SY5Y细胞分为五组:空白对照组、溶剂对照组、β淀粉样肽(Aβ)组、Aβ+溶剂对照组和Aβ+药物处理组。用Aβ25-35处理SH-SY5Y细胞,建立AD细胞模型。采用DAPI荧光染色法计数凋亡细胞,Western blot法检测磷酸化的p38丝裂原活化蛋白激酶(p-p38MAPK)表达。结果 Aβ25-35引起时间和剂量依赖性的SH-SY5Y细胞凋亡,并使p-p38MAPK表达增加(P<0.05)。MCI-186与p38MAPK抑制剂SB239063均抑制Aβ25-35诱导的SH-SY5Y细胞凋亡及p-p38MAPK表达(P<0.05)。结论 MCI-186通过抑制p38MAPK磷酸化,对Aβ25-35诱导的SH-SY5Y细胞凋亡起到保护作用。     

Monocyte-mediated rotenone neurotoxicity towards human neuroblastoma SH-SY5Y: role of mitogen-activated protein kinases

Increasing evidence has suggested an important role for rotenone in the pathogenesis of Parkinson's disease (PD). In this report, sequential linking of two culture systems, monocytic THP-1 cell line and SH-SY5Y neuroblastoma, was utilized. The supernatant from rotenone-stimulated THP-1 cells was used as the incubating medium for the second culture which adopted cells of the SH-SY5Y neuroblastoma. At 6.25-50 nM, concentrations that were nontoxic to SH-SY5Y directly, rotenone induced dose-dependent cell death on SH-SY5Y through stimulating monocyte THP-1 within a period of 48 h. Cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Hoechst 33258 staining revealed that the treatment of SH-SY5Y with rotenone-stimulated THP-1 supernatant resulted in condensed nuclei and a decrease in cell size. Apoptotic rate measured by flow cytometric analysis indicated that at 25 and 50 nM, the percentage of apoptotic SH-SY5Y cells accumulated to 31.5% and 37.0% respectively. We further investigated whether rotenone (50 nM) activated mitogen-activated protein kinase (MAPK) cascades, and found it had effect on p38 MAPK and ERK in THP-1 cells, but not JNK. Pretreatment of THP-1 cells with the MAPK kinase inhibitor, PD98059, inhibited THP-1 cell-mediated rotenone neurotoxicity towards SH-SY5Y, whereas the p38 MEK inhibitor, SB203580, had no effect. These results suggested that activation of microglia intracellular signaling pathway may also involve in microglia-enhanced rotenone neurotoxicity.     

Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells: role of ERK1/2 in H2O2-induced cell death

Reactive oxygen species including H(2)O(2) activate an array of intracellular signalling cascades that are closely associated with cell death and cell survival pathways. The human neuroblastoma SH-SY5Y cell line is widely used as model cell system for studying neuronal cell death induced by oxidative stress. However, at present very little is known about the signalling pathways activated by H(2)O(2) in SH-SY5Y cells. Therefore, in this study we have investigated the effect of H(2)O(2) on extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (PKB) activation in undifferentiated and differentiated SH-SY5Y cells. H(2)O(2) stimulated time and concentration increases in ERK1/2, JNK and PKB phosphorylation in undifferentiated and differentiated SH-SY5Y cells. No increases in p38 MAPK phosphorylation were observed following H(2)O(2) treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 ((2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited H(2)O(2)-induced increases in ERK1/2 and PKB phosphorylation. Furthermore, H(2)O(2)-mediated increases in ERK1/2 activation were sensitive to the MAPK kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK responses were blocked by the JNK inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Treatment of SH-SY5Y cells with H(2)O(2) (1 mM; 16 h) significantly increased the release of lactate dehydrogenase (LDH) into the culture medium indicative of a decrease in cell viability. Pre-treatment with wortmannin, SP 600125 or SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; p38 MAPK inhibitor) had no effect on H(2)O(2)-induced LDH release from undifferentiated or differentiated SH-SY5Y cells. In contrast, PD 98059 and LY 294002 significantly decreased H(2)O(2)-induced cell death in both undifferentiated and differentiated SH-SY5Y cells. In conclusion, we have shown that H(2)O(2) stimulates robust increases in ERK1/2, JNK and PKB in undifferentiated and differentiated SH-SY5Y cells. Furthermore, the data presented clearly suggest that inhibition of the ERK1/2 pathway protects SH-SY5Y cells from H(2)O(2)-induced cell death.     

不同来源石斛对SH-SY5Y细胞缺血性损伤保护作用的比较

目的研究市售种植铁皮枫斗和组培铁皮石斛对谷氨酸损伤的SH—SY5Y细胞的保护作用。方法利用血清药理学方法制备含药血清,用SH—SY5Y细胞建立谷氨酸损伤模型,观察72h损伤细胞增殖情况,MTT实验测定细胞活性,乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）和丙二醛（MDA）的测定研究两种来源石斛对神经细胞损伤保护作用。结果种植和组培石斛均可提高SH-SY5Y细胞谷氨酸损伤的存活率,降低培养液中LDH含量和细胞内MDA含量,提高SOD活力,其中铁皮石斛胚芽的抗氧化能力优于市售铁皮枫斗。结论石斛具有对SH—SY5Y细胞谷氨酸损伤的保护作用;组培铁皮石斛较市售铁皮枫斗保护作用更加显著,初步证明经过组培繁殖的铁皮石斛作用效果优于市售石斛属药用植物。     

Oxidative stress in ovariectomy menopause and role of chondroitin sulfate

Oxidative stress due to reactive oxygen species (ROS) can cause oxidative damage to cells. Cells have a number of defense mechanisms to protect themselves from the toxicity of ROS. Mitochondria are especially important in the oxidative stress as ROS have been found to be constantly generated as an endogen threat. Mitochondrial defense depends mainly on superoxide dismutase (SOD) and glutathione peroxidase (GPx), whereas microsomal defense depends on catalase (CAT), which is an enzyme abundant in microsomes. SOD removes superoxide anions by converting them to H2O2, which can be rapidly converted to water by CAT and GPx. Also, GPx converts hydroperoxide (ROOH) into oxidized-glutathione (GSSG). Ovariectomized (OVX) rats are used as an oxidative stress model. An ovariectomy increased the levels of MDA, one of the end-products in the lipid peroxidative process, and decreased levels of the antioxidative enzymes; SOD, CAT and GPx. However, Chondroitin sulfate (CS) decreased the levels of MDA, but increased the levels of SOD, CAT and GPx in a dose-dependent manner. Moreover, inflammation and cirrhosis of liver tissue in CS- treated rats were significantly decreased. These results suggest that CS might be a potential candidate as an antioxidative reagent.     

Lonicera japonica THUNB. protects 6-hydroxydopamine-induced neurotoxicity by inhibiting activation of MAPKs, PI3K/Akt, and NF-κB in SH-SY5Y cells

In this study, we investigated the neuroprotective effects of Lonicera japonica THUNB. extract (LJ) on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells. We found that LJ significantly increased cell viability decrease, lactate dehydrogenase release (LDH), morphological changes, nuclear condensation, fragmentation, and reactive oxygen species (ROS) production induced by 6-OHDA in SH-SY5Y cells. The cytoprotection afforded by pretreatment with LJ was associated with increases of the glutathione (GSH) level, superoxide dismutase (SOD) activity, and catalase (CAT) activity in 6-OHDA-induced SH-SY5Y cells. In addition, LJ strikingly inhibited 6-OHDA-induced mitochondrial dysfunctions including reduction of mitochondria membrane potential (MMP) and activation of cleaved poly-ADP-ribose polymerase (PARP), cleaved caspase-3, cleaved caspase-9, increased Bax, as well as decreased Bcl-2 and Bcl-xL. Additionally, LJ dramatically attenuated 6-OHDA-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK 1/2), and phosphoinositide 3-kinase (PI3K)/Akt. Meanwhile, LJ counteracted nuclear factor-κB (NF-κB) activation by blocking its translocation to the nucleus. These findings suggest that LJ has a potent anti-parkinsonism; this effect was mediated, at least in part, by inhibition of neurotoxicity, apoptotic cascade events, and oxidative stress via activation of MAPKs, PI3K/Akt, and NF-κB.     

PTD-SOD对Na2S2O4诱导的SH-SY5Y细胞损伤的保护和修复作用研究

目的研究PTD-SOD对Na2S2O4诱导的SH-SY5Y细胞损伤的保护和修复作用。方法建立Na2S2O4诱导的SH-SY5Y细胞缺氧损伤模型,采用MTT法检测融合蛋白PTD-SOD对细胞存活率的影响,LDH释放分析检测PTD-SOD对细胞损伤后的保护作用及损伤后细胞内氧化与抗氧化水平的变化。结果对Na2S2O4诱导的SH-SY5Y细胞缺氧损伤,MTT活性检测、LDH分析及氧化与抗氧化水平分析都表明PTD-SOD能明显提高细胞存活率和细胞内抗氧化能力。结论PTD-SOD融合蛋白能保护Na2S2O4诱导缺氧损伤的SH-SY5Y细胞,具有抑制细胞死亡,改善损伤细胞内抗氧化能力。     

DL0410 attenuates oxidative stress and neuroinflammation via BDNF/TrkB/ERK/CREB and Nrf2/HO-1 activation

Oxidative stress and neuroinflammation have been deeply associated with Alzheimer’s disease. DL0410 is a novel acetylcholinesterase inhibitor with potential anti-oxidative effects in AD-related animal models, while the specific mechanism has not been fully clarified. In this study, DL0410 was predicted to be related to the modification of cell apoptosis, oxidation-reduction process, inflammatory response and ERK1/ERK2 cascade by in silico target fishing and GO enrichment analysis. Then the possible protective effects of DL0410 were evaluated by hydrogen peroxide (H₂O₂)-induced oxidative stress model and lipopolysaccharides (LPS)-induced neuroinflammation model H₂O₂ decreased the viability of SH-SY5Y cells, induced malondialdehyde (MDA) accumulation, mitochondrial membrane potential (Δψm) loss and cell apoptosis, which could be reversed by DL0410 dose-dependently, indicating that DL0410 protected SH-SY5Y cells against H₂O₂-mediated oxidative stress. Western blot analysis showed that DL0410 increased the H₂O₂-triggered down-regulated TrkB, ERK and CREB phosphorylation and the expression of BDNF. In addition, TrkB inhibitor ANA-12, ERK inhibitor SCH772984 and CREB inhibitor 666-15 eliminated the inhibition of DL0410 on MDA accumulation and Δψm loss. Furthermore, DL0410 attenuates inflammatory responses and ROS production in LPS-treated BV2 cells, which is responsible for Nrf2 and HO-1 up-regulation. The present study demonstrates that DL0410 is a potential activator of the BDNF/TrkB/ERK/CREB and Nrf2/HO-1 pathway and may be a potential candidate for regulating oxidative stress and neuroinflammatory response in the brain. Together, the results showed that DL0410 is a promising drug candidate for treating AD and possibly other nervous system diseases associated with oxidative stress and neuroinflammation.     

2,3-吲哚醌对肿瘤凋亡抑制因子survivin的影响及作用机制

目的研究2,3-吲哚醌(2,3-dioxoindoline,isatin,ISA)在体外对人神经母瘤细胞(SH-SY5Y)分泌肿瘤凋亡抑制因子survivin的影响,并探讨其作用机制。方法体外培养人神经母细胞瘤细胞SH-SY5Y细胞,将SH-SY5Y细胞随机分为对照组和ISA不同浓度(100、200、400μmol.L-1)处理组,采用荧光染色、DNA Ladder分析细胞凋亡,RT-PCR及Westernblot等方法检测不同浓度ISA作用后survivin及p53在mRNA和蛋白水平的变化,流式细胞术(flow cytometry,FCM)检测Caspase-3蛋白水平的变化。结果ISA(100、200、400μmol.L-1)作用SH-SY5Y细胞48h后,在荧光显微镜下观察到400μmol.L-1ISA组细胞出现典型的凋亡形态学改变:核染色质聚集、核碎裂、胞质浓缩;琼脂糖凝胶电泳显示400μmol.L-1ISA处理组细胞出现明显的梯形条带;RT-PCR及Western blot显示:随着ISA药物浓度的增加,survivin mRNA及蛋白表达逐渐减少(P<0.05),野生型p53mRNA及蛋白表达水平逐渐增加,各用药组与对照组比较差异均有统计学意义(P<0.05),并呈现浓度依赖关系;FCM结果显示随着ISA药物浓度的增加,活化Caspase-3的表达率分别为18.38%、26.93%、35.66%,明显高于对照组(11.23%,P<0.05)。结论ISA能明显诱导SH-SY5Y细胞凋亡,可能机制是抑制凋亡抑制蛋白(IAP)家族中的生存蛋白(survivin)表达继而增加活化Caspase-3水平来实现的,而这一过程可能与ISA上调野生型p53的表达有关。