Quercetin in combating H2O2 induced early cell apoptosis and mitochondrial damage to normal human keratinocytes

Background Oxidative stress plays an important role in the pathogenesis of epidermal diseases. This study aimed to investigate the effects of quercetin on the anti-oxidative response and on mitochondrial protection in cultured normal human keratinocytes. Methods Cultured HaCaT cells were treated with different concentrations of H202 （0, 50, 100, 250, 500 pmol/L） for different periods of time （0.5, 1, 2, 4 hours） to establish an oxidative stress model. The cultured HaCaT cells were randomly assigned to control, H2O2, and quercetin＋H2O2 groups. For the quercetin groups, the cells were treated with different concentrations of quercetin （0, 10, 25, 50 umol/L） before exposure to H2O2. Morphological changes of the cells were observed under an inverted microscope and an electron microscope. The cell viability was detected by the MIF method. The cell apoptosis （AnnexinV/propidium iodide double stain） and mitochondrial membrane potential （△ψm） changes were detected by flow cytometry. Results An oxidative stress model of HaCaT cells was established under a suitable concentration （250 umol/L） and treated time of H2O2 （2 hours）. The cell viability and △ψm decreased in a concentration-dependent and time-dependent manner while the percentage of apoptotic cells significantly increased in the H2O2 groups compared with the control group （P 〈0.05）. The cell viability and △ψm of the quercetin treated group increased （P 〈0.05） and the percentage of apoptotic cells decreased at concentrations of 1-50 umol/L quercetin （P 〈0.01） compared with H2O2 treated group. Conclusion Quercetin can relieve the cell damage and apoptosis from H2O2 induced injury to HaCaT cells by anti-oxidation and mitochondrial protection.     

Protection of erythropoietin on experimental spinal cord injury by reducing the expression of thrombospondin-1 and transforming growth factor-β

Background Erythropoietin （EPO） functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production and neuron protection. This study aimed to determine the neuron protective effect of erythropoietin on experimental rats enduring spinal cord injury （SCI） by assessing thrombospondin-1 （TSP-1） level and transforming growth factor-β （TGF-β） in the development of a rat model of SCI. Methods Sixty Sprague-Dawley rats were randomly assigned to three groups： sham operation control group, SCI group and EPO treatment group. By using a weight-drop contusion SCI model, the rats in the SCI group and EPO treatment group were sacrificed at 24 hours and 7 days subsequently. The Basso, Beattie, and Bresnahan （BBB） scores were examined for locomotor function. Pathological changes were observed after HE staining. The expressions of thrombospondin-2 （TSP-1） and TGF-β were determined by immunohistochemical staining and Western blotting. Results Slighter locomotor dysfunction was discovered and it was recovered abruptly as higher BBB scores were found in the EPO treatment group than in the SCI group （P 〈0.01）. Pathologically, progressive disruption of the dorsal white matter and regeneration of a few neurons were also observed in SCI rats. TSP-1 and TGF-β expression increased at 24 hours and 7 days after SCI in the injured segment, and it was higher in the SCI group than in the EPO treatment group. Spinal cord samples from the animals demonstrated a TSP-1 optical density of 112.2±6.8 and TSP-1 positive cells of 5.7±1.3 respectively. After injury, the TSP-1 optical density and cell number increased to 287.2±14.3/mm^2 and 23.2±2.6/mm^2 at 24 hours and to 232.1±13.2/mm^2 and 15.2±2.3/mm^2 at 7 days respectively. When EPO treated rats compared with the SCI rats, the TSP-1 optical density and cell number decreased to 213.1 ±11.6/mm^2 and 11.9±1.6/mm^2 at 24 hours and to 189.9±10.5/mm^2 and 9.3±1.5/mm^2 at 7 days, respectively （P 〈0.01）. In the SCI rats, the TGF-β optical density and positive neuron number were 291.4±15.2/mm^2 and 28.8±4.9/mm^2 at 24 hours and 259.1±12.3/mm^2 and 23.9±4.1/mm^2 at 7 days respectively. They decreased in the EPO treated rats to 222.8±11.9/mm^2 and 13.7±2.1/mm^2 at 24 hours and to 196.5±9.7/mm^2 and 8.7±2.2/mm^2 at 7 days （P 〈0.01）. Conclusions Increased expression of TSP-1 and TGF-β can be found in the injured segment of the spinal cord at 24 hours and 7 days after injury. EPO treatment can effectively prevent pathological alterations from severe spinal cord injury by reduced expression of TSP-1 and TGF-β.     

Different suppressive effect of lidocaine on persistent Na^＋ current and transient Na^＋ current in dorsal root ganglion neurons

Objective ：To investigate the different suppressive effect of lidocaine on persistent Na^＋ current and transient Na^＋ current in injured or uninjured dorsal root ganglion neurons. Methods： Totally 23 SD rats were randomly divided into 2 groups： control group （n： 10） and chronically compressed DRG （dorsal root ganglion） group （CCD group, n= 13）. Rats were anesthetized and DRG was isolated. Single DRG neuron was isolated by enzymatic disassociation method. Persistent Na^＋ current （INap） and transient Na^＋ current （INaT） were elicited in voltage clamp mode. Results： The presence of INap was testified in most DRG neurons （38/46 neurons in CCD group and 31/39 neurons in control group, P〉0. 05）. However, the cur- rent density of INap in CCD group （4. 6±0. 6 pA/pF, n=38 neurons） was greater than that in control group （2.5±0.4 pA/pF, n=31 neurons） （P〈0. 05）. The characteristics of INap was observed and found that INap could he blocked by 0.2 μmol/L tetrodotoxin easily. Furthermore, the does-effect relationship of lidocaine on INaP and IN.T were also examined. INaP and IN.T were suppressed by different concentrations of li- docaine, the range for INap was 5-20 μmol/L and for INaT was 0. 05-2 mmol/L. Conclusion： INap and INaT were suppressed by different concentrations of lidocaine. INap was suppressed by very low concentration of lidocaine （5-20 μmol/L）. However, INaT could only be blocked by high concentration of lidocaine （0.05-2 mmol/L）.     

Effects of Livin gene RNA interference on apoptosis of cervical cancer Hela cells and enhanced sensitivity to cisplatin

The recombinant plasmids pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into Hela cells and cisplatin was added with different concentrations in order to study the inhibitory effects of Livin gene, increase the apoptosis induced by cisplatin, and detect the expression ofBcl-2, Bax, caspase-3, and survivin genes. The pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into Hela cells, and the expression levels of Livin, Bcl-2, Bax, caspase-3, and survivin genes were detected by using fluorescence quantitative real-time PCR. Then cisplatin at different concentrations （3.0, 6.0 and 9.9 μg/mL） was added into the transfected Hela cells, and 24, and 48 h later, the apoptosis rate was measured by flow cytometry. After transfection of pGenesil-l-BIRC71 and pGenesil-1-BIRC72 into Hela cells, the expression level of Livin gene was obviously reduced, and the apoptosis rate was significantly increased in transfection group as compared with control group （P〈0.05）. Cisplatin could increase the apoptosis rate in a dose- and time-dependent manner. After cisplatin was added, the expression levels of Bcl-2 mRNA were reduced, and those of Bax, caspase-3, and survivin mRNA were increased in transfection group as compared with those in control group （P〈0.05）. It was concluded that shRNA expression vector targeting Livin gene could inhibit the expression of Livin gene in Hela cells and enhance the apoptosis induced by cisplatin, which was related to the decreased expression of Bcl-2 and activation of Bax and caspase-3. Survivin might play an important role as an antagonist in the process of apoptosis induction.     

Suppressive effects of genomic imprinted gene PEG10 on hydrogen peroxide-induced apoptosis in L0<Subscript>2</Subscript> cells

The effects of PEG10 on hydrogen peroxide （H2O2）-induced apoptosis in human normal liver cell line L02 were investigated. The PEG10 gene was transfected into L02 cells by lipofectamine, the positive clone was screened by G418 and defined as L02/PEG10, while the cell transfected with empty expression vector （pEGFP-N1） was defined as L02/vector. L02/vector and parental L02 cells served as control. RT-PCR and Western blotting were employed to detect the expression of target genes. H2O2 （50–400 mmol/L） was administered to induce the apoptosis of L02 cells. Cells viability was measured by MTT and the morphological changes of apoptotic cells were determined by fluorescence microscopy using hoechst33342 nuclei staining. DNA fragmentation was observed by agarose gel electrophoresis. PEG10 mRNA and protein levels in L02/PEG10 cells were significantly increased as compared with those in the control cells. After treatment with 400 mmol/L H2O2 for 24 h, the cellular growth inhibition rate of L02/PEG10 cells was significantly lower （58.2%） than that of L02 （92.5%） and L02/vector （88%）. Distinct morphological changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were not observed in L02/PEG10. Ladder-like DNA fragmentation in a dose-dependent manner was observed in both L02 and L02/vector cell lines, but not in L02/PEG10. PEG10 over-expression significantly inhibited cytotoxicity induced by H2O2 on human normal liver cell line L02 by antagonizing H2O2-induced apoptosis.     

Protection of INS-1 Cells from Free Fatty Acid-induced Apoptosis by Inhibiting the Glycogen Synthase Kinase-3

To examine the role of glycogen synthase kinase 3 （GSK-3） in the apoptosis of pancreatic β-cells to better understand the pathogenesis and to find new approach to the treatment of type 2 diabetes, apoptosis was induced by oleic acid （OA） in INS-1 cells and the activity of GSK-3 was inhibited by LiCl. The PI staining and flow cytometry were employed for the evaluation of apoptosis. The phosphorylation level of GSK-3 was detected by Western blotting. The results showed that OA at 0.4 mmol/L could cause conspicuous apoptosis of INS- 1 cells and the activity of GSK-3 was significantly increased. After the treatment with 24 mmolFL of LiCl, a inhibitor of GSK-3, the OA-induced apoptosis of INS-1 cells was lessened and the phosphorylation of GSK-3 was increased remarkably. It is concluded that GSK-3 activation plays an important role in OA-induced apoptosis in pancreatic β-cells and inhibition of the GSK-3 activity can effectively protect INS-1 cells from the OA-induced apoptosis. Our study provides a new experimental basis and target for the clinical treatment of type-2 diabetes.     

Effects of 3-aminobenzamide on expressions of poly（ADP ribose） polymerase and apoptosis inducing factor in cardiomyocytes of rats with acute myocardial infarction

Background Poly（ADP-ribose） polymerase （PARP） plays an important role in cell survival and death. However, the mechanisms involved are not fully understood. Therefore, we investigated the effect of inhibition of PARP on acute myocardial infarction （AMI） at different time points in rats. Methods AMI was induced in rats by ligating the left anterior descending coronary artery. One group received 3-aminobenzamide （3-AB, a kind of PARP inhibitor） （30 mg/kg） by intraperitoneal injection. The changes of ultramicrostructure of cardiocytes in infarction region were noted, PARP cleavage was measured by Western blotting, and expressions of protein of PARP and apoptosis inducing factor （AIF） were measured by immunohistochemical staining after treatment with 3-AB for 2 hours, 4 hours, 6 hours, 1 week, 4 weeks and 8 weeks. Results Few damages to the ultramicrostructure of cardiocytes were observed after treatment with 3-AB. PARP cleavage was detected as early as 4 hours and markedly increased by 6 hours following AMI without 3-AB, but was not found until 6 hours following AMI treated with 3-AB. There were significant differences between 3-AB and AMI groups at the same time points. The expression of PARP was observed gradually increased, but that of AIF was suppressed for 6 hours after treatment of 3-AB, compared with AMI groups in positive cells at the same time points. There was significantly less cleavage of PARP and more PARP expression in 3-AB treated group compared with AMI and control groups at all matched time points. Conclusions Our results suggest that 3-AB inhibits degradation of PARP, increases the expression of PARP protein, and suppresses the expression of AIF protein. Inhibition of PARP activity may protect cardiocytes in rats with AMI and reduce apoptosis.     

Caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epithelial cell apoptosis induced by hydrogen in vitro

Objective To investigate the role of caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epitheli alcell apoptosis induced by hydrogen peroxide (H2O2 ) in vitro.Methods Rat lenses were incubated in modified Eagle‘s medium containing 2 mmol/L H2O2 to induce apoptosis in vitro. Apoptosis in lens epithelial cells was assessed by transmission electron microscopy and annexin V-propidium iodide (PI) double staining flow cytometry after 12, 24 and 48 h of incubation. The activity of caspase-3 was analyzed by western blotting.Results Observations under transmission electron microscopy revealed that 2 mmol/L H2O2 could effectively induce lens epithelial cell apoptosis in vitro. Caspase-3 activity increased during cell apoptosis and the peak measurement occurred at 24 h after treatment with H2O2. Cell apoptosis was blocked by caspase-3 inhibitor Ac-DEVD-CHO.Conclusions The activation of caspase-3 plays an important role in executing apoptosis in H2O2-treated lens epithelial cells and in the formation of cataract. The caspase-3 inhibitor Ac-DEVD-CHO may effectively prevent lens epithelial cell apoptosis caused by oxidative injury.     

神经生长因子及抑制因子对鸡胚背根神经节轴突生长的影响

研究神经生长因子和神经生长抑制因子鸡胚背根神经节轴突生长的影响。方法分离培养鸡胚ＤＲＧ,加入ＮＧＦ、ＮＧＩ并观察它们对轴突生长的影响。结果ＤＲＧ轴突在含ＮＧＦ的培养液中迅速生长,加入ＮＧＩ后这种生长被抑制。结论ＮＧＦ可促进ＤＲＧ轴突的生长,而ＮＧＩ抑制它的生长。     

二苯乙烯苷上调Bcl-2抑制内皮细胞凋亡

目的研究二苯乙烯苷（TSG）对过氧化氢（H2O2）诱导人脐静脉内皮细胞凋亡的作用,以及对抗凋亡基因Bcl-2表达的影响。方法体外培养人脐静脉内皮细胞,筛选造模内皮细胞凋亡的H2O2浓度,以不同浓度二苯乙烯苷作用24 h。采用MTT法检测细胞生长活力,Hoechst33258染色观察细胞形态,流式细胞仪检测细胞凋亡率,Western-blot检测Bcl-2蛋白的含量。结果过氧化氢能引起内皮细胞损伤,抑制细胞增殖,并且随着浓度增加,细胞生存率逐渐降低。其中300μmol/L H2O2作用后细胞增殖明显受到抑制,Hoechst33258染色可见大量凋亡细胞,流式细胞仪检测出明显的凋亡峰,Bcl-2蛋白的表达量显著减少;加入二苯乙烯苷作用后,随着TSG浓度增加,细胞生存率增加,凋亡率逐渐降低;与H2O2造模组比较,10μmol/L的二苯乙烯苷预处理能够显著提高细胞生存率,抑制细胞的凋亡,并且使Bcl-2表达增加（P〈0.05）。结论二苯乙烯苷能抑制过氧化氢诱导的内皮细胞凋亡,该作用与上调Bcl-2表达有关。     

幽门螺杆菌及其相关细胞因子对胃上皮细胞凋亡的调控及相关机制

目的研究幽门螺杆菌（Hp）及其相关细胞因子对胃上皮细胞凋亡的影响,以探讨Hp的致病机理。方法以人胃永生化上皮细胞株（GES-）和人胃癌细胞株（AGS）作为研究对象。流式细胞术检测Hp毒力菌株（NCTC11637）单独及分别联合Hp相关细胞因子[白细胞介素（IL）-1β、IL-8及IL-10]对两种细胞系凋亡的影响;检测IL-1β、IL-8及IL-10对外源性Fas配体（FasL）诱导的两种细胞系凋亡的影响。逆转录-聚合酶链反应检测Hp及其相关细胞因子（IL-1β、IL-8及IL-10）对两种细胞系FasmRNA基础表达水平的影响。结果（1）Hp能诱导GES-1和AGS细胞凋亡增加113．0％和47．0％（均P〈0．05）;IL-1β、IL-10能抑制Hp诱导的GES-1（29．6％、25．8％）和AGS（19．7％、21．1％）细胞凋亡（均P〈0．05）;IL-8能增强Hp诱导的GES-1细胞凋亡（19．9％,P〈0．05）。（2）Hp能上调GES-1和AGS细胞FasmRNA基础表达水平（89．0％和36．0％,P〈0．05）;IL-1β、IL-10对两种细胞FasmRNA基础表达无显著影响（P〉0．05）;IL-8能上调GES-1（33．0％）细胞FasmRNA基础表达（P〈0．05）。（3）IL-1β、IL-10能抑制FasL诱导的GES-1（30．3％、32．5％）和AGS（44．2％、51．5％）细胞凋亡（P〈0．05）;IL-8能增强FasL诱导的GES。1细胞凋亡（100％,P〈0．05）。结论Hp可通过上调Fas受体表达直接介导胃上皮细胞凋亡。Hp相关细胞因子IL-8可能通过上调Fas受体表达来增强Hp诱导的胃上皮细胞凋亡;IL—1β和IL-10可能通过其他机制来抑制Hp诱导的胃上皮细胞凋亡。     

Protection of Acanthopanax Senticosus Saponin on Free Radical Injury Induced Aging of Nerve Cell

Ｉｔｉｓｒｅｃｅｎｔｌｙｈｅｌｄｔｈａｔｎｅｒｖｅｃｅｌｌａｇｉｎｇｉｓｃｌｏｓｅｌｙｒｅｌａｔｅｄｔｏｔｈｅｆｒｅｅｒａｄｉｃａｌａｎｄａｎｔｉ ｏｘｉｄａｓｅａｃｔｉｖｉｔｙ，ｔｈｅＡｃａｎｔｈｏｐａｎａｘ（ＡＰ）ｉｎｊｅｃｔｉｏｎｃａｎｅｌｅｖａｔｅ     

神经生长因子诱导背根节感觉神经元细胞iNOS和P物质的表达及干扰素调节因子-1的作用

目的:研究神经生长因子(nerve growth factor,NGF)对脊髓C7-T5背根节感觉神经元细胞内诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和P物质表达的影响及干扰素调节因子-1(interferon regulatory factor-1,IRF-1)的...     

Neurotrophin 3对本体感觉神经作用的研究进展

Neurotrophin 3（NT-3）是本体感觉神经存活和发挥生理功能所必需的营养因子.在胚胎期缺失NT-3,可直接导致本体感觉神经元凋亡,进而导致本体感受器（肌梭）不能分化发育.同时,NT-3在本体感觉神经投射和形成突触联系中起关键的诱导作用.成年后缺失NT-3,也同样可导致本体感觉神经功能下降.在病理状态下NT-3可以保护本体感觉神经,并促进其恢复正常生理功能.     

N-乙酰-L-半胱氨酸对H2O2引起的血管内皮细胞损伤的拮抗效应

目的 观察N-乙酰-L-半胱氨酸(NAC，痰易净)对H2O2引起的血管内皮细胞损伤的影响。方法 通过测定乳酸脱氢酶(LDH)释放率和脂质过氧化物(LPO)值，MTT法测定细胞增殖活性，流式细胞术测细胞凋亡率，研究体外培养的人脐静脉内皮细胞在不同浓度H2O2作用下的细胞损伤及凋亡，以及NAC对抗细胞损伤及凋亡的效应。结果 随着H2O2浓度增加，细胞凋亡率明显增高，细胞增殖活力降低，细胞脂质过氧化物生成增加，LDH释放率增加，并在一定范围内呈剂量-效应关系。在H2O2中同时加入0．5mmol／L NAC，可使细胞活力增强，凋亡率降低，细胞脂质过氧化物生成显减少，LDH释放率降低。结论 NAC可以对抗H2O2引起的血管内皮细胞损伤。     

不同潮气量机械通气对ALI犬肾脏的影响

目的观察不同潮气量(Vt)机械通气(MV)对油酸所致急性肺损伤犬肾脏的影响。方法用静脉注射油酸法制备犬急性肺损伤模型,造模成功后随机分为两组,分别接受不同Vt的MV(通气时间均为6h):小Vt组(L组,Vt=6ml/kg,PEEP=10cmH2O);大Vt组(H组,Vt=20ml/kg,PEEP=10 cmH2O)。动态观察血肌酐、尿素氮变化。在MV6 h后放血处死动物,开腹取肾组织,行病理学检查及TUNEL法观察细胞凋亡情况。结果急性肺损伤犬血肌酐、尿素氮呈上升趋势,L组与H组比较差异有显著性(P<0.01);肾小管组织出现不同程度的坏死、凋亡,肾脏组织学评分、细胞凋亡率两组间比较差异有显著性(P<0.01)。结论小Vt MV在一定程度上可降低不当MV导致的肾损伤,保护肾功能,避免出现急性肾功能不全。     

H2O2预处理对骨髓间质干细胞凋亡适应性保护作用的研究

 【目的】 探讨低浓度H2O2预处理对高浓度H2O2引起骨髓间质干细胞凋亡的影响.【方法】 大鼠骨髓间质干细胞(BMSC)经体外培养扩增、纯化、鉴定.BMSC分别被暴露于0,50,100,200,300,400,500 μmol/L H2O2,流式细胞仪(FCM)检测不同浓度H2O2处理对BMSC细胞凋亡的影响.然后分别用20 μmol/L ,50 μmol/L,100 μmol/L H2O2预处理24 h,用FCM和Hoechst33324染色观察低浓度H2O2预处理对高浓度H2O2引起骨髓间质干细胞凋亡的影响, RT-PCR检测不同浓度H2O2预处理组BMSC Caspase-3和Bcl-2基因的表达.【结果】 BMSC经体外培养扩增、流式细胞仪鉴定,符合其表面标志,FCM检测凋亡显示:H2O2呈浓度依赖性诱导BMSC凋亡,且50 μmol/L H2O2预处理可降低500 μmol/L H2O2诱导的BMSC凋亡率;Hoechst33324染色结果显示:对照组BMSC染色质分布均匀,为弥散均匀的低强度荧光;500 μmol/L H2O2组BMSC胞核呈浓染致密的固缩形态或颗粒状荧光;50 μmol/L H2O2预处理组所致的凋亡细胞数明显少于500 μmol/L H2O2处理组所引起的细胞凋亡数,100 μmol/L H2O2预处理组所致的凋亡细胞数与500 μmol/L H2O2处理组比较无明显差异、RT-PCR结果显示50 μmol/L H2O2预处理组与单独使用500 μmol/L H2O2处理组比较,其BMSC的Bcl-2表达增加和Caspase-3基因表达降低(P < 0.05),100 μmol/L H2O2预处理组与单独使用500 μmol/L H2O2处理组比较,其BMSC的Bcl-2、Caspase-3表达无明显差异(P > 0.05)。 【结论】 低浓度H2O2预处理对BMSC凋亡具有适应性保护作用,其机制可能与H2O2预处理使BMSC Bcl-2基因表达增加和Caspase-3基因表达降低有关。     

高渗糖溶液鞘内注射对大鼠神经病理和生理的影响

目的 观测高渗糖溶液鞘内用药对大鼠行为学以及对大鼠背根神经节病理结构的影响.方法 70只成年SD大鼠,制作鞘内置管模型,72h后行为学观察无神经损伤者随机分成3组,分别鞘内注射NS (Normal saline),5％ GS(Glucose solution,),10％GS.用平行学实验和体重变化观察高渗糖溶液对大鼠的行为学影响；每组大鼠按鞘内注药后2h、24h、48h和72h分别处死并分离L4-5节段背根神经节,光镜及电镜下观察细胞结构随着时间变化和糖浓度变化的改变.结果 63只大鼠进入最终结果分析.给药后所有大鼠均未出现行为学方面的神经损伤表现.背根神经节损伤以鞘内注射葡萄糖溶液后2h-24h表现最明显,符合细胞凋亡的特点,且10％GS组的神经损伤重于5％GS组.48h-72h后神经损伤的病理表现有所减轻,甚至消失.结论 高渗葡萄糖溶液有诱发大鼠背根神经节细胞凋亡、脱髓鞘的作用,并且随着葡萄糖浓度的增加,其损伤程度越显著.但未对大鼠的行为能力造成影响.