一氧化碳中毒迟发性脑病与脑细胞自噬的研究进展

一氧化碳中毒迟发性脑病(delayed encephalopathy after carbon monoxide poisoning,DEACMP)的发病率较高,对患者及家庭造成极大的痛苦和经济负担,其发病机制仍然不明。一氧化碳中毒时,一氧化碳可通过多种途径启动脑细胞自噬。自噬参与了DEACMP的发病,但自噬在疾病不同阶段的水平及发挥的作用,仍未得出一致的结论。     

Effect of age on the sensitivity of the rat thyroid gland to ionizing radiation

Exposure to ionizing radiation during childhood is a well-known risk factor for thyroid cancer. Our study evaluated the effect of age on the radiosensitivity of rat thyroid glands. Four-week-old (4W), 7 -week-old (7W), and 8-month-old (8M) male Wistar rats were exposed to 8 Gy of whole-body X-ray irradiation. Thyroids were removed 3–72 h after irradiation, and non-irradiated thyroids served as controls. Ki67-positivity and p53 binding protein 1 (53BP1) focus formation (a DNA damage response) were evaluated via immunohistochemistry. Amounts of proteins involved in DNA damage response (p53, p53 phosphorylated at serine 15, p21), apoptosis (cleaved caspase-3), and autophagy (LC3, p62) were determined via western blotting. mRNA levels of 84 key autophagy-related genes were quantified using polymerase chain reaction arrays. Ki67-positive cells in 4W (with high proliferative activity) and 7W thyroids significantly decreased in number post-irradiation. The number of 53BP1 foci and amount of p53 phosphorylated at serine 15 increased 3 h after irradiation, regardless of age. No increase in apoptosis or in the levels of p53, p21 or cleaved caspase-3 was detected for any ages. Levels of LC3-II and p62 increased in irradiated 4W but not 8M thyroids, whereas expression of several autophagy-related genes was higher in 4W than 8M irradiated thyroids. Irradiation increased the expression of genes encoding pro-apoptotic proteins in both 4W and 8M thyroids. In summary, no apoptosis or p53 accumulation was noted, despite the expression of some pro-apoptotic genes in immature and adult thyroids. Irradiation induced autophagy in immature, but not in adult, rat thyroids.     

异甘草素诱导人卵巢癌细胞株SKOV3凋亡及自噬的研究

目的：观察异甘草素（ISL）对人卵巢浆液性乳头状腺癌细胞株SKOV3体外增殖的抑制作用,并探讨其可能存在的分子机制。方法四甲基偶氮唑蓝（MTT）比色法观察ISL对SKOV3细胞生长的抑制作用;吖啶橙／溴化乙锭双荧光染色后观察细胞形态变化;流式细胞术AnnexinⅤ‐FITC／PI双染检测ISL对细胞凋亡的影响;Westernblot检测凋亡相关蛋白Bcl‐2、Bax及自噬相关蛋白Beclin1、LC3的表达水平。结果ISL能够抑制SKOV3细胞的增殖,呈时间、剂量依赖性;荧光染色后可见细胞核变圆,碎裂,染色质浓缩;流式细胞仪检测ISL（5、10、20μg／mL）作用于SKOV3细胞48h后,凋亡率明显高于ISL0μg／mL组,差异具有统计学意义（P＜0．05）;Westernblot结果显示ISL（5、10、20μg／mL）作用于SKOV3细胞48h后Bcl‐2蛋白的表达下调,Bax、Beclin1、LC3蛋白的表达上调,差异有统计学意义（P＜0．05）。结论ISL对人卵巢癌SKOV3细胞具有明显的抑制增殖和诱导凋亡的作用,其抗体外肿瘤机制可能与激活线粒体凋亡通路及自噬通路相关。     

The metabolic cross‐talk between epithelial cancer cells and stromal fibroblasts in ovarian cancer progression: Autophagy plays a role

Cancer and stromal cells, which include (cancer‐associated) fibroblasts, adipocytes, and immune cells, constitute a mixed cellular ecosystem that dynamically influences the behavior of each component, creating conditions that ultimately favor the emergence of malignant clones. Ovarian cancer cells release cytokines that recruit and activate stromal fibroblasts and immune cells, so perpetuating a state of inflammation in the stroma that hampers the immune response and facilitates cancer survival and propagation. Further, the stroma vasculature impacts the metabolism of the cells by providing or limiting the availability of oxygen and nutrients. Autophagy, a lysosomal catabolic process with homeostatic and prosurvival functions, influences the behavior of cancer cells, affecting a variety of processes such as the survival in metabolic harsh conditions, the invasive growth, the development of immune and chemo resistance, the maintenance of stem‐like properties, and dormancy. Further, autophagy is involved in the secretion and the signaling of promigratory cytokines. Cancer‐associated fibroblasts can influence the actual level of autophagy in ovarian cancer cells through the secretion of pro‐inflammatory cytokines and the release of autophagy‐derived metabolites and substrates. Interrupting the metabolic cross‐talk between cancer cells and cancer‐associated fibroblasts could be an effective therapeutic strategy to arrest the progression and prevent the relapse of ovarian cancer.     

The relationship between Cd‐induced autophagy and lysosomal activation in WRL‐68 cells

This study shows that Cd induces autophagy in the human's embryonic normal liver cell line (WRL‐68). The expression of LC3B‐II and the mature cathepsin L were analyzed by Western blotting. The autophagosomes and lysosomes were directly visualized by electron microscopy and confocal microscopy analysis in Cd‐exposed WRL‐68 cells. In this study, we first found that autophagy induced the activation of lysosomal function in WRL‐68 cells. The lysosomal activation was markedly decreased when the cells were co‐treated with 3‐MA (an inhibitor of autophagy). Secondly, we provided the evidence that the activation of lysosomal function depended on autophagosome–lysosome fusion. The colocalization of lysosome‐associated membrane protein‐2 (LAMP2) and GFP‐LC3 was significantly reduced, when they were treated with thapsigargin (an inhibitor of autophagosome–lysosome fusion). We demonstrated that deletion or blockage of the autophagosome–lysosome fusion process effectively diminished lysosomal activation, which suggests that lysosomal activation occurring in the course of autophagy is dependent on autophagosome–lysosome fusion. Thirdly, we provided evidence that the activation of lysosomal function was associated with lysosomal acid. We investigated the relationship between autophagosome–lysosome fusion and pH in acidic compartments by visualizing fusion process in WRL‐68 cells. This suggests that increasing pH in acidic compartments in WRL‐68 cells inhibits the autophagosome–lysosome fusion. Finally, we found that the activation of lysosomal function was associated with Ca²⁺ stores and the intracellular Ca²⁺ channels or pumps were possibly pH‐dependent. Copyright © 2015 John Wiley & Sons, Ltd.     

Carnosic acid induces autophagic cell death through inhibition of the Akt/mTOR pathway in human hepatoma cells

The therapeutic goal of cancer treatment is now geared towards triggering tumour‐selective cell death with autophagic cell death being required for the chemotherapy of apoptosis‐resistant cancer. In this study, Carnosic acid (CA), a polyphenolic diterpene isolated from Rosemary (Rosemarinus officinalis), significantly induced autophagic cell death in HepG2 cells. Ca treatment caused the formation of autophagic vacuoles produced an increasing ratio of LC3‐II to LC3‐I in a time‐ and dose‐dependent manner but had no effect on the levels of autophagy‐related protein ATG6 and ATG13 expression. Autophagy inhibitors, 3‐methyladenine (3‐MA), chloroquine and bafilomycin A1, or ATG genes silencing in HepG2 cells significantly inhibited CA‐induced autophagic cell death. The CA treatment decreased the levels of phosphorylated Akt and mTOR without any effects on PI3K or PTEN. Most importantly, overexpression of Akt and knockdown of PTEN attenuated autophagy induction in CA‐treated cells. Taken together, our results indicated that CA induced autophagic cell death through inhibition of the Akt/mTOR pathway in human hepatoma cells. These findings suggest that CA has a great potential for the treatment of hepatoma via autophagic induction. Copyright © 2014 John Wiley & Sons, Ltd.     

β‐asarone and levodopa co‐administration protects against 6‐hydroxydopamine‐induced damage in parkinsonian rat mesencephalon by regulating autophagy: down‐expression Beclin‐1 and light chain 3B and up‐expression P62

In this study, we investigated Beclin‐1, light chain (LC)3B, and p62 expression in 6‐hydroxydopamine (6‐OHDA)‐induced parkinsonian rats after β‐asarone and levodopa (l ‐dopa) co‐administration. Unilateral 6‐OHDA injection into the medial forebrain bundle was used to create the models, except in sham‐operated rats. Rats were divided into eight groups: sham‐operated group; 6‐OHDA model group; madopar group (75 mg/kg, per os (p.o.)); l ‐dopa group (60 mg/kg, p.o.); β‐asarone group (15 mg/kg, p.o.); β‐asarone + l ‐dopa co‐administered group (15 mg/kg + 60 mg/kg, p.o.); 3‐methyladenine group (500 nmol, intraperitoneal injection); and rapamycin group (1 mg/kg, intraperitoneal injection). Then, Beclin‐1, LC3B, and p62 expression in the mesencephalon were detected. The mesencephalon was also observed by transmission electron microscope. The results showed that Beclin‐1 and LC3B expression decreased and that p62 expression increased significantly in the madopar, l ‐dopa, β‐asarone, and co‐administered groups when compared with the 6‐OHDA model. Beclin‐1 and LC3B expression in the β‐asarone and co‐administered groups were less than in the madopar or l ‐dopa groups, whereas p62 expression in the β‐asarone and co‐administered groups was higher than in the madopar or l ‐dopa groups. In addition, a significant decrease in autophagosome was exhibited in the β‐asarone and co‐administered groups when compared with the 6‐OHDA group. Our findings indicate that Beclin‐1 and LC3B expression decreased, whereas p62 expression increased after co‐administration treatment. In sum, all data suggest that the co‐administration of β‐asarone and l ‐dopa may contribute to the treatment of 6‐OHDA‐induced damage in rats by inhibiting autophagy activity.     

黄芪多糖对大鼠缺氧/复氧诱导的心肌细胞自噬及凋亡抑制作用的机制探讨

目的：探究黄芪多糖（APS）通过调控高迁移率族蛋白1/Toll样受体4/核转录因子-κB(HMGB1/TLR4/NF-κB)信号通路对大鼠缺氧/复氧（H/R）诱导的心肌细胞自噬及凋亡的抑制作用。方法：建立H9C2心肌细胞H/R损伤模型并分为4组：对照组、H/R组、APS组和HMGB1抑制剂组。CCK-8法和EdU染色法检测细胞增殖能力;酶联免疫吸附试验检测细胞肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-6含量;透射电镜观察细胞自噬小体的形成;膜联蛋白V-异硫氰酸荧光素/碘化丙啶（AnnexinV-FITC/PI）双染法检测细胞凋亡;实时定量PCR检测细胞HMGB1、TLR4、NF-κB p65 mRNA表达水平;蛋白免疫印迹法检测细胞HMGB1、TLR4、NF-κB p65、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白（Bax）、含半胱氨酸的天冬氨酸蛋白水解酶-3(caspase-3)、P62、微管相关蛋白1A/1B-轻链3(MAP1LC3,缩写为LC3)-Ⅱ蛋白表达水平。结果：与对照组相比,H/R组细胞增殖能力明显减弱,凋亡及自噬水平明显增加,细胞内可见大量自...     

Rapamycin attenuates articular cartilage degeneration by inhibiting β-catenin in a murine model of osteoarthritis

Purpose: To investigate whether systemic injection of rapamycin attenuates articular cartilage degeneration by inhibiting β-catenin in a murine model of osteoarthritis (OA).

Materials and methods: Ten-week-old male C57BL/6j wild-type (WT) mice and SOST-knockout (SOST^?/?) mice were randomized to a sham group, a vehicle-treated group, and a rapamycin-treated group. Mice in the vehicle-treated group underwent destabilizing of the medial meniscus (DMM) in the right knee, and were then treated with vehicle. Mice in the rapamycin treatment group underwent DMM and were treated with rapamycin. Safranin O-Fast green staining and Osteoarthritis Research Society International (OARSI) modified Mankin score were used to evaluate the histopathological features of the articular cartilage in the knee. The expression of light chain 3 (LC3) was evaluated by immunofluorescence, whereas the expression of ATG5, matrix metallopeptidase 13 (MMP-13), vascular endothelial growth factor (VEGF), sclerostin, and β-catenin were evaluated by immunohistochemistry. TUNEL staining was used to determine apoptosis of chondrocytes.

Results: In vehicle-treated mice when compared with mice in the sham group, the OARSI scores, expression of MMP-13, VEGF, sclerostin, β-catenin, and chondrocyte apoptosis were significantly increased, whereas the expression of LC3 and ATG5 were significantly decreased. A systemic injection of rapamycin activated chondrocyte autophagy, which increased the expression of LC3 and ATG-5, and reduced OARSI scores, the expression of β-catenin, MMP-13, and VEGF, and chondrocyte apoptosis in rapamycin treated mice when compared with vehicle-treated mice.

Conclusions: Systemic injection of rapamycin attenuated articular cartilage degeneration by inhibiting β-catenin in a murine model of OA.