雷公藤内酯醇对多发性骨髓瘤细胞凋亡及组蛋白H3K4甲基化的影响

目的：探讨雷公藤内酯醇（TPL）对多发性骨髓瘤RPMI8226细胞增殖、凋亡和组蛋白H3K4甲基化的影响。方法：以人多发性骨髓瘤细胞株RPMI8226为研究对象,在不同浓度（10、20、40、80、160 nmol/L） TPL中共培养不同时间（24 h、48 h、72 h）后,采用噻唑蓝（MTT）法检测细胞增殖活性;流式细胞术检测细胞凋亡和细胞周期;Western blot法检测组蛋白H3K4me2、H3K4me3的甲基化状态,实时荧光定量RT-PCR分析组蛋白甲基化酶SMYD3和组蛋白去甲基化酶LSD1的表达水平。结果：TPL对RPMI8226细胞有明显的增殖抑制作用,呈剂量和时间依赖性（P<0.05）;TPL对RPMI8226细胞有明显诱导凋亡的作用,并且随着TPL作用浓度的增加,细胞凋亡比例逐渐增加（P<0.05）;同时TPL还可以诱导RPMI8226细胞周期阻滞于G₂/M期;TPL以浓度依赖性降低组蛋白H3K4me2、H3K4me3的甲基化水平（P<0.05,P<0.01）,并抑制SMYD3和上调LSD1的表达（P<0.05）。结论：TPL可抑制RPMI8226细胞增殖、引起细胞周期阻滞于G₂/M期,并诱导其凋亡;通过抑制组蛋白甲基化酶SMYD3和增强组蛋白去甲基化酶LSD1的表达,降低组蛋白H3K4me3和H3K4me2的甲基化水平,这可能是TPL诱导多发性骨髓瘤细胞凋亡和抗肿瘤作用的机制之一。     

精氨酸缺乏对硼替佐米治疗多发性骨髓瘤的作用研究

摘要 目的：探讨精氨酸缺乏对硼替佐米(Bortezomib,BTZ) 治疗多发性骨髓瘤细胞的影响。方法：通过CCK8筛选BTZ对骨髓瘤细胞株(H929和RPMI 8226)治疗的最适药物浓度,比较在缺乏和富含精氨酸的两种培养基中的细胞增殖情况;通过使用PI染料标记细胞检测不同试验组细胞周期的分布,以及使用Annexin V/7AAD凋亡试剂盒检测BTZ对不同试验组细胞凋亡的影响。结果：BTZ降低了骨髓瘤细胞的存活率,并通过将细胞周期阻滞于G2/M、S期,抑制骨髓瘤细胞的增殖。缺乏精氨酸使细胞周期阻滞于S期,也抑制了骨髓瘤细胞的增殖。BTZ作用于缺乏精氨酸组的骨髓瘤细胞后,细胞凋亡百分比明显低于富含精氨酸组（H929细胞由约40%降至13.6%,RPMI8226凋亡百分比分别7.13%和19.27%）。结论：缺乏精氨酸和给予BTZ均阻滞细胞周期,抑制骨髓瘤细胞增殖;同时缺乏精氨酸降低了BTZ诱导骨髓瘤细胞的凋亡作用。     

脂多糖对多发性骨髓瘤细胞增殖的影响及其机制

目的:探讨脂多糖(lipopolysaccharide,LPS)对多发性骨髓瘤细胞增殖的影响及其作用机制。方法:采用LPS作用于多发性骨髓瘤细胞RPMI 8226后,通过MTT法检测细胞的增殖情况,流式细胞术检测细胞周期的变化,Western blot检测NF-κB信号通路中相关蛋白p65和IκB-α的表达情况。结果:随着LPS作用浓度(0-8μg/mL)的增加,RPMI 8226细胞增殖活力呈先上升后下降的趋势,在2μg/m L LPS作用下,RPMI8226细胞的增殖活力最显著(P0.05)。相同浓度的LPS诱导RPMI8226细胞不同时间,其细胞增殖呈时间依赖性。流式细胞术检测结果显示:不同浓度LPS(0.5、1、2μg/mL)作用RPMI 8226细胞后,G0/G1期细胞的比例明显低于对照组(P0.05),S期和G2/M期细胞比例明显增加(P0.05);Western blot检测结果显示:2μg/mL的LPS处理的RPMI8226细胞p65和IΚB-α蛋白的表达明显高于对照(P0.05)。结论:低浓度LPS能够诱导多发性骨髓瘤细胞RPMI 8226的增殖,其作用机制可能与上调信号通路NF-κB中相关蛋白p65和IκB-α有关。     

Pravastatin激活自噬抑制糖皮质激素引起的髓核细胞凋亡

地塞米松注射可以缓解椎间盘退变引起的腰痛症状,但是具有一定的副作用。普伐他汀(Pravastatin)被发现可以缓解骨关节炎的炎症和症状,但是其对髓核细胞及椎间盘退变的作用及其机制尚不清楚。该文培养SD大鼠原代髓核细胞,用不同浓度地塞米松(dexamethasone,DXM)作用髓核细胞48 h后, DCFH-DA和MitoSOX Red染色分析细胞总活性氧(reactive oxygen species, ROS)和线粒体ROS水平, Annexin V/PI流式和DAPI染色分析细胞凋亡水平, N-acetyl-Lcysteine(NAC)抑制ROS水平。Western blot检测LC3-II、Beclin-1和P62等自噬相关蛋白质水平,ATG5 siRNA转染抑制自噬。结果显示,随着DXM处理浓度的增加,髓核细胞内总ROS和线粒体ROS水平及凋亡率升高(P0.05)。Pravastatin增加DXM处理下髓核细胞中LC3-II和Beclin-1蛋白质水平,降低P62蛋白质水平(P0.05)。Pravastatin可以抑制DXM诱导的髓核细胞中的ROS产生和细胞凋亡,而ATG5 siRNA抑制自噬后,显著逆转Pravastatin对细胞的保护作用(P0.05)。该研究结果提示, Pravastatin可能通过激活髓核细胞自噬抑制DXM诱导的ROS产生从而减少细胞凋亡。     

小豆蔻明调控自噬抑制卵巢癌SKOV3细胞增殖的研究

目的：卵巢癌为女性常见病,死亡率高,分子靶向治疗药物较少,研发高效低毒的靶向新药意义重大。细胞自噬（autophagy）维持着细胞内稳态和生长,是抗癌药物作用的关键靶部位。本课题旨在明确小豆蔻明（Cardamonin,CAR）调控自噬对卵巢癌SKOV3细胞增殖的影响。方法：体外培养卵巢癌SKOV3细胞,不同药物分组处理,荧光显微镜下观察单黄酰戊二胺（MDC）染色后细胞自噬囊泡,WesternBlot法检测细胞自噬相关蛋白LC3的表达,四氮唑蓝（MTT）法观察SKOV3细胞增殖情况,流式细胞术检测SKOV3细胞凋亡的变化。结果：自噬抑制剂3-MA显著性降低SKOV3细胞内MDC染色的荧光颗粒数目、LC3II蛋白表达,抑制细胞增殖;而CAR（高、中剂量）、雷帕霉素和AZD8055与3-MA联用后细胞内MDC荧光颗粒数目增多、LC3II蛋白表达增加、细胞增殖抑制率及凋亡率明显升高;且高剂量CAR（10^-6mol·L^-1）的作用比低剂量CAR（10^-6mol·L^-1）明显。结论：CAR能够抑制SKOV3细胞增殖,诱导细胞自噬,促进细胞凋亡。CAR有望成为卵巢癌药物治疗的先导化合物。本研究为进一步研发此类化合物提供了实验依据及一定的理论基础。     

新合成的7-氮杂异靛蓝抑制A549细胞增殖的作用及可能机制

研究新合成的N~1-(正-丁基)-7-氮杂异靛蓝(N~1-(n-butyl)-7-azaisoindigo,7-AI-b)抑制人非小型肺癌细胞A549的增殖,初步探索其抗肿瘤的机制。以不同浓度的7-AI-b作用于A549,MTT检测与相差显微镜观察相结合,分析7-AI-b对细胞增殖的影响;MDC染色和Western印迹检测自噬标志蛋白(LC3)的表达,研究药物处理后细胞是否发生了自噬。PI单染,流式检测细胞周期的变化;Fluo-3-AM荧光探针检测细胞中Ca~(2+)的含量。结果发现,7-AI-b以时间和剂量依赖性方式抑制细胞的增殖;MDC染色后,自噬小泡增多,LC3的总表达量增加,且由LC3-Ⅰ向LC3-Ⅱ的转变量升高。流式细胞检测发现细胞被阻滞在G_0/G_1期;同时,药物处理后,细胞内Ca~(2+)出现了超载。由此,新合成的7-AI-b有很好的抑制肿瘤细胞增殖的作用,其机制与引起细胞周期阻滞、诱导细胞自噬有关,胞内Ca~(2+)超载也参与了该作用。     

小豆蔻明调控自噬抑制卵巢癌SKOV3 细胞增殖的研究

目的：卵巢癌为女性常见病,死亡率高,分子靶向治疗药物较少,研发高效低毒的靶向新药意义重大。细胞自噬（autophagy） 维持着细胞内稳态和生长,是抗癌药物作用的关键靶部位。本课题旨在明确小豆蔻明（Cardamonin,CAR）调控自噬对卵巢癌 SKOV3 细胞增殖的影响。方法：体外培养卵巢癌SKOV3 细胞,不同药物分组处理,荧光显微镜下观察单黄酰戊二胺（MDC）染色 后细胞自噬囊泡,Western Blot 法检测细胞自噬相关蛋白LC3的表达,四氮唑蓝（MTT）法观察SKOV3 细胞增殖情况,流式细胞 术检测SKOV3 细胞凋亡的变化。结果：自噬抑制剂3-MA 显著性降低SKOV3 细胞内MDC染色的荧光颗粒数目、LC3Ⅱ蛋白表 达,抑制细胞增殖;而CAR（高、中剂量）、雷帕霉素和AZD8055 与3-MA 联用后细胞内MDC荧光颗粒数目增多、LC3Ⅱ蛋白表达 增加、细胞增殖抑制率及凋亡率明显升高;且高剂量CAR（10-5mol·L-1）的作用比低剂量CAR（10-6mol·L-1）明显。结论：CAR能够 抑制SKOV3 细胞增殖,诱导细胞自噬,促进细胞凋亡。CAR有望成为卵巢癌药物治疗的先导化合物。本研究为进一步研发此类 化合物提供了实验依据及一定的理论基础。     

Genistein对卵巢癌铂类耐药细胞株CP70增殖凋亡的影响及与细胞内ROS的相关性

目的:探讨Genistein对卵巢癌铂类耐药细胞CP70增殖、凋亡的影响及与细胞内活性氧水平的关系。方法:采用MTT法检测Genistein对CP70细胞增殖的影响;流式细胞仪分析不同药物处理后对细胞凋亡的影响,线粒体膜电位及细胞内ROS水平的变化情况。结果:Genistein对CP70细胞增殖表现出剂量和时间依赖性的抑制作用,并能诱导其凋亡;Genistein作用于CP70细胞后,可使其线粒体膜电位降低,并引发了细胞内ROS水平的显著升高;ROS抑制剂NAC预处理CP70细胞后,有效抑制了ROS的产生,并降低了细胞凋亡率,与未加NAC组相比差异有显著性(P0.05)。结论:Genistein能抑制铂类耐药卵巢癌细胞CP70的增殖,并促进其凋亡,这与细胞内ROS水平的升高有关,可能是Genistein抗肿瘤诱导细胞凋亡的机制之一。     

CHOP双重调控衣霉素诱导的DU-145细胞凋亡及自噬的研究

目的研究内质网应激分子CHOP调控细胞凋亡与自噬的作用和机制。 方法利用衣霉素诱导DU-145细胞产生内质网应激,Western Blot法检测内质网应激相关分子Grp78、Grp94、p-eIF2α和CHOP及自噬蛋白LC3Ⅱ、Atg5和Beclin1的表达;用流式细胞术检测细胞凋亡水平;沉默CHOP基因,用Western Blot法检测凋亡蛋白PARP、Caspase3的表达,流式细胞术检测细胞凋亡;并利用免疫荧光检测自噬标志性蛋白LC3B的表达。 结果衣霉素诱导DU-145细胞内质网应激能诱导一定程度的细胞凋亡,衣霉素处理8、12、24?h的细胞凋亡率分别为3.27﹪±1.02﹪,8.97﹪±0.71﹪和11.67﹪±1.41﹪,处理12?h及24?h的细胞凋亡率与对照组相比差异具有统计学意义（P < 0.01）。同时也能通过抑制PI3K/AKt/mTOR信号通路激活DU-145细胞自噬。CHOP基因沉默抑制细胞凋亡,shCtrl组细胞凋亡率为32.17﹪±3.93﹪,shCHOP-1组细胞凋亡率为23.53﹪±3.41﹪,两组相比差异具有统计学意义（P < 0.05）。且CHOP基因沉默能促进细胞自噬分子LC3B的表达。 结论衣霉素诱导DU-145细胞内质网应激状态下,CHOP在细胞凋亡与自噬之间发挥双重调控作用。     

硼替佐米增强P16蛋白表达诱导骨髓瘤细胞衰老

目的：研究蛋白酶体抑制剂硼替佐米诱导骨髓瘤RPMI8226、MMH929细胞衰老作用,并进一步探讨其作用机制。方法：硼替佐米0.1-100nmol/L处理骨髓瘤RPMI8226、MMH929细胞48、72h,MTT法检测细胞存活率、药物IC50值。选择药物IC50值1/10剂量处理骨髓瘤RPMI8226、MMH929细胞0、24、48H后检测衰老相关β-半乳糖苷酶染色率。流式细胞术检测细胞周期情况及凋亡率。Western-blot检测相关蛋白表达。结果：硼替佐米处理骨髓瘤细胞RPMI8226、MMH929后48小时IC50值：RPMI8226：19.05 nmol/L,MMH929：18.45nmol/L。以硼替佐米2 nmol/L处理骨髓瘤RPMI8226、MMH929细胞0、24、48H后发现β-半乳糖苷酶染色率、细胞G0/G1期比例明显上升与药物作用时间呈正相关,Western-blot检测细胞周期调控蛋白发现P53、PTEN蛋白无变化,P16蛋白与药物作用时间正相关。结论：硼替佐米通过增强P16蛋白表达诱导骨髓瘤细胞RPMI8226、H929衰老。     

Gadd45b prevents autophagy and apoptosis against rat cerebral neuron oxygen-glucose deprivation/reperfusion injury

Autophagic (type II) cell death has been suggested to play pathogenetic roles in cerebral ischemia. Growth arrest and DNA damage response 45b (Gadd45b) has been shown to protect against rat brain ischemia injury through inhibiting apoptosis. However, the relationship between Gadd45b and autophagy in cerebral ischemia/reperfusion (I/R) injury remains uncertain. The aim of this study is to investigate the effect of Gadd45b on autophagy. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R) model of rat primary cortex neurons, and lentivirus interference used to silence Gadd45b expression. Cell viability and injury assay were performed using CCK-8 and LDH kit. Autophagy activation was monitored by expression of ATG5, LC3, Beclin-1, ATG7 and ATG3. Neuron apoptosis was monitored by expression of Bcl-2, Bax, cleaved caspase3, p53 and TUNEL assay. Neuron neurites were assayed by double immunofluorescent labeling with Tuj1 and LC3B. Here, we demonstrated that the expression of Gadd45b was strongly up-regulated at 24 h after 3 h OGD treatment. ShRNA-Gadd45b increased the expression of autophagy related proteins, aggravated OGD/R-induced neuron cell apoptosis and neurites injury. ShRNA-Gadd45b co-treatment with autophagy inhibitor 3-methyladenine (3-MA) or Wortmannin partly inhibited the ratio of LC3II/LC3I, and slightly ameliorated neuron cell apoptosis under OGD/R. Furthermore, shRNA-Gadd45b inhibited the p-p38 level involved in autophagy, but increased the p-JNK level involved in apoptosis. ShRNA-Gadd45b co-treatment with p38 inhibitor obviously induced autophagy. ShRNA-Gadd45b co-treatment with JNK inhibitor alleviated neuron cell apoptosis. In conclusion, our data suggested that Gadd45b inhibited autophagy and apoptosis under OGD/R. Gadd45b may be a common regulatory protein to control autophagy and apoptosis.     

mTOR信号通路诱导的自噬在心肌细胞缺氧损伤中的作用研究

目的:探讨自噬在心肌细胞缺氧损伤中的作用及分子机制。方法:体外分离培养乳鼠心肌细胞,体外建立缺氧/去血清(H/SD)模型以模拟在体的缺血环境。分别给予自噬抑制剂3-甲基腺嘌呤(3MA,5 mM)和mTOR抑制剂雷帕霉素(1.0μg/L)调节心肌细胞自噬水平。分别采用TUNEL染色检测心肌细胞凋亡,Western blot方法检测心肌细胞蛋白表达水平。结果:H/SD损伤可以显著诱导心肌细胞自噬水平(P0.05),并且细胞自噬水平可以被3-MA及雷帕霉素调节。同时,H/SD可以显著增加心肌细胞凋亡(P0.05),而给予3-MA抑制自噬水平可以减少细胞凋亡(P0.05)。相反,雷帕霉素增加自噬同样可以加重缺氧导致的心肌细胞凋亡(P0.05)。H/SD损伤过程中,心肌细胞mTOR信号通路被激活,而自噬抑制剂3-MA可以显著提高缺氧条件下心肌细胞中p-mTOR(Ser2448)的表达水平(P0.05),并增加mTOR下游分子p-p70S6k(P0.05)和p-S6(P0.05)的表达。结论:mTOR信号通路诱导的细胞自噬可能参与了缺氧损伤诱导的心肌细胞凋亡。     

Ulk1/FUNDC1 Prevents Nerve Cells from Hypoxia-Induced Apoptosis by Promoting Cell Autophagy

Cell autophagy and cell apoptosis are both observed in the process of hypoxia-induced ischemic cerebral infarction (ICI). Unc-51 like autophagy activating kinase 1 (Ulk1) and FUN14 Domain-containing Protein 1 (FUNDC1) are both involved in the regulation of cell autophagy. This study aimed to investigate the regulatory effects of Ulk1 and FUNDC1 on hypoxia-induced nerve cell autophagy and apoptosis. Cell viability was measured using cell counting kit-8 (CCK-8) assay. Cell apoptosis was detected using Annexin V-PE/7-ADD staining assay. qRT-PCR was used to quantify the mRNA levels of Ulk1 and FUNDC1 in PC-12 cells. Cell transfection was performed to up-regulate the expression of Ulk1. 3-Methyladenine (3-MA) was used as autophagy inhibitor and rapamycin was used as autophagy activator in our experiments. SP600125 was used as c-Jun N-terminal kinase (JNK) inhibitor. Western blotting was performed to analyze the expression levels of key factors that are related to cell autophagy, apoptosis and JNK pathway. We found that hypoxia simultaneously induced apoptosis and autophagy of PC-12 cells. The activation of Ulk1 and FUNDC1 were also found in PC-12 cells after hypoxia induction. Overexpression of Ulk1 promoted the activation of FUNDC1 and prevented PC-12 cells from hypoxia-induced apoptosis. Suppression of Ulk1 had opposite effects. Furthermore, we also found that JNK pathway participated in the effects of Ulk1 overexpression on PC-12 cell apoptosis reduction. To conclude, Ulk1/FUNDC1 played critical regulatory roles in hypoxia-induced nerve cell autophagy and apoptosis. Overexpression of Ulk1 prevented nerve cells from hypoxia-induced apoptosis by promoting cell autophagy.     

自噬对高糖诱导的人冠状动脉内皮细胞凋亡的保护作用

目的:研究自噬对高糖诱导的人冠状动脉内皮细胞凋亡的影响。方法:将人冠状动脉内皮细胞,分别用常规培养基(正常对照组)、含30 mmol/L D-葡萄糖的高糖培养基(高糖组)、高糖培养基合并雷帕霉素(Rapamycin,RAPA;100 nmol/L)干预(RAPA组)和高糖培养基合并3-甲基腺嘌呤(3-Methyladenine,3-MA,5 mmol/L)干预(3-MA组)培养。利用CCK-8法检测细胞生长活力,使用流式细胞术检测细胞凋亡水平,western blot检测细胞自噬标记蛋白(Beclin1)的表达水平。结果:(1)高糖溶液刺激内皮细胞24 h后,细胞生长活力为正常组的55.0%(P0.01),自噬标记蛋白Beclin1的表达水平明显增加,凋亡水平为正常组的2.0倍;(2)与高糖组相比,RAPA组细胞生长活力明显增加,Beclin1的表达明显升高(P0.01),凋亡水平为高糖组的70.1%;(3)与高糖组相比,3-MA组细胞生长活力明显减少,Beclin1的表达明显降低(P0.01),凋亡水平为高糖组的1.42倍。结论:细胞自噬可能对高糖诱导的人冠状动脉内皮细胞具有凋亡保护作用。     

Sirtuin 6 protects human retinal pigment epithelium cells from LPS-induced inflammation and apoptosis partly by regulating autophagy

ABSTRACT

Lipopolysaccharides (LPS)-induced retinal inflammation is an important factor in retinal diseases. This study was aimed to investigate the effect of Sirt6 on LPS-induced retinal injury. ARPE-19 cells were incubated with LPS to induce inflammation. The cell viability was determined using CCK-8 assay. The mRNA level and protein expression of corresponding genes was detected using qRT-PCR and western blot, respectively. The production of inflammatory cytokines was measured using ELISA kit. The levels of oxidative stress-related factors were measured using their detection kits. Cell apoptosis was observed using TUNEL assay. The results showed that Sirt6 was downregulated after LPS treatment. Sirt6 strengthened LPS-induced autophagy by promoting the expression of LC3II/I, beclin1 and ATG5. Sirt6 treatment significantly inhibited LPS-induced inflammation, oxidative stress and cell apoptosis, which was then partly abolished by 3 MA. These results suggest Sirt6 to be an important regulator for LPS-induced inflammation, oxidative stress, and apoptosis partly by regulating cell autophagy.     

Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER–ERK–mTOR pathway

Estradiol could protect osteoblast against apoptosis, and apoptosis and autophagy were extensively and intimately connected. The aim of the present study was to test the hypothesis that autophagy was present in osteoblasts under serum deprivation and estrogen protected against osteoblast apoptosis via promotion of autophagy. MC3T3-E1 osteoblastic cells were cultured in a serum-free and phenol red-free minimal essential medium (α-MEM). Ultrastructural analysis, lysosomal activity assessment and monodansycadaverine (MDC) staining were employed to determine the presence of autophagy, and real time PCR was used to evaluate the expression of autophagic markers. Meanwhile, the osteoblasts were transferred in a serum-free and phenol red-free α-MEM containing either vehicle or estradiol. Apoptosis and autophagy was assessed by using the techniques of real-time PCR, Western blot, immunofluorescence assay, and flow cytometry. The possible pathway through which estrogen promoted autophagy in the serum-deprived osteoblasts was also investigated. Real-time PCR demonstrated the expression of LC3, beclin1 and ULK1 genes in osteoblasts under serum deprivation, and immunofluorescence assay verified high expression of proteins of these three autophagic bio-markers. Lysosomes and autolysosomes accumulated in the cytoplasm of osteoblasts were also detected under transmission electron microscopy, MDC staining and lysosomal activity assessment. Meanwhile, estradiol significantly decreased the expression of proteins of the bio-markers of apoptosis, and at the same time increased the expression of proteins of the bio-markers of autophagy in the serum-deprived osteoblasts. Furthermore, the estradiol-promoted autophagy in serum-deprived osteoblasts could be blocked by estrogen receptor (ER) antagonist (ICI 182780), and estradiol failed to rescue the cells pretreated with an inhibitor of vacuolar ATPase (bafilomycin A) from apoptosis. Serum deprivation resulted in apoptosis through activation of Caspase-3 and induced autophagy through inhibition of phospho-mammalian target of rapamycin (p-mTOR). Both 3-methyladenine (3MA) and U0126 led to increase of apoptosis in osteoblasts with serum deprivation. Estradiol failed to over-ride the inhibitory effect of 3MA on phosphorylation of AKT but directly led to dephosphorylation of mTOR and upregulation of LC3 protein expression. However, the estradiol-enhanced LC3 protein expression was significantly suppressed by U0126 through inhibition of phosphorylation of extracellular signal-regulated kinase (ERK). Estradiol rescued osteoblast apoptosis via promotion of autophagy through the ER–ERK–mTOR pathway.     

Rottlerin induces autophagy and apoptotic cell death through a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells: the protective role of autophagy in apoptosis

Rottlerin is widely used as a protein kinase C-delta inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8 and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-delta by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-delta overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-delta-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.     

细胞自噬对酒精诱导的肝细胞毒性的保护作用

目的：研究细胞自噬对酒精诱导的人肝细胞系（CL-1）的保护作用。方法：培养正常肝细胞系CL-1细胞,80mmol／L酒精常规处理24小时,采用CCK-8法观察酒精对细胞活力的影响;流式细胞技术观察酒精对细胞凋亡的影响;免疫蛋白印迹及转染GFP-LC3法检测细胞自噬水平;选用rapamycin和3-MA调节细胞自噬,观察酒精处理后细胞活力及凋亡的变化。结果：酒精处理体外培养的CL-1细胞,实验组较对照组细胞活力下降（P〈0．05）;实验组细胞46．2％发生凋亡,显著高于对照组8．4％;LC3II及Beclinl水平显著高于对照组;GFP-LC3荧光数显著高于对照组（P〈0．05）;调节细胞自噬水平,rapamycin组细胞活性增加（P〈0．01）,31．1％（46．2％）细胞发生凋亡;3-MA组细胞活性降低（P〈0．05）,54．1％（46．2％）细胞发生凋亡。结论：酒精处理降低CL-1细胞活性,促进凋亡,提高自噬水平;提高或降低细胞自噬水平,细胞凋亡及活力随之降低和增加;细胞自噬能够对抗酒精诱导的肝细胞凋亡。     

Ischemic conditioning by short periods of reperfusion attenuates renal ischemia/reperfusion induced apoptosis and autophagy in the rat

Prolonged ischemia amplified iscehemia/reperfusion (IR) induced renal apoptosis and autophagy. We hypothesize that ischemic conditioning (IC) by a briefly intermittent reperfusion during a prolonged ischemic phase may ameliorate IR induced renal dysfunction. We evaluated the antioxidant/oxidant mechanism, autophagy and apoptosis in the uninephrectomized Wistar rats subjected to sham control, 4 stages of 15-min IC (I15 × 4), 2 stages of 30-min IC (I30 × 2), and total 60-min ischema (I60) in the kidney followed by 4 or 24 hours of reperfusion. By use of ATP assay, monitoring O₂ ^-. amounts, autophagy and apoptosis analysis of rat kidneys, I60 followed by 4 hours of reperfusion decreased renal ATP and enhanced reactive oxygen species (ROS) level and proapoptotic and autophagic mechanisms, including enhanced Bax/Bcl-2 ratio, cytochrome C release, active caspase 3, poly-(ADP-ribose)-polymerase (PARP) degradation fragments, microtubule-associated protein light chain 3 (LC3) and Beclin-1 expression and subsequently tubular apoptosis and autophagy associated with elevated blood urea nitrogen and creatinine level. I30 × 2, not I15 × 4 decreased ROS production and cytochrome C release, increased Manganese superoxide dismutase (MnSOD), Copper-Zn superoxide dismutase (CuZnSOD) and catalase expression and provided a more efficient protection than I60 against IR induced tubular apoptosis and autophagy and blood urea nitrogen and creatinine level. We conclude that 60-min renal ischemia enhanced renal tubular oxidative stress, proapoptosis and autophagy in the rat kidneys. Two stages of 30-min ischemia with 3-min reperfusion significantly preserved renal ATP content, increased antioxidant defense mechanisms and decreased ischemia/reperfusion enhanced renal tubular oxidative stress, cytosolic cytochrome C release, proapoptosis and autophagy in rat kidneys.