Outer membrane protein A of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in RAW264.7 cells

AIM:To analyze the effects of outer membrane protein A (OmpA) from Acinetobacter baumannii ATCC 19606 on the autophagy of RAW264.7 cells. METHODS:The RAW264.7 cell model stimulated by OmpA was established. The effects of OmpA on the autophagy of RAW264.7 cells were detected by immunofluorescence, Western blot and transmission electron microscopy. RESULTS:The OmpA increased the expression of LC3B-Ⅱ and reduced the phosphorylation levels of Akt, mTOR and p70S6K. Rapamycin further reduced the phosphorylation levels of mTOR and p-70S6K, and increased the expression of LC3B-Ⅱ induced by OmpA. CONCLUSION:The OmpA of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in the RAW264.7 cells. This work provides a basis for further research on the molecular mechanism of autophagy induced by Acinetobacter baumannii to find a new method against the infection of Acinetobacter baumannii.     

SNAT2介导蛋氨酸对牛乳腺上皮细胞增殖与自噬的调节作用

钠离子依赖性中性氨基酸转运体2（SNAT2）是一种氨基酸转运蛋白，可转运中性氨基酸，广泛分布于多种细胞中。氨基酸既可作为蛋白质合成的底物，也是调节细胞新陈代谢的关键信号分子，但SNAT2是否介导氨基酸调节BMECs增殖和自噬尚未见报道。本研究利用CASY细胞计数和Western blotting技术检测SNAT2过表达和siRNA干扰后牛乳腺上皮细胞（BMECs）增殖情况以及SNAT2对自噬标志蛋白LC3-Ⅰ/Ⅱ表达量的影响，并利用免疫荧光检测细胞自噬斑点（LC3-Ⅱ）变化。结果显示，SNAT2过表达时，p-PI3K、p-mTOR和Cyclin D1表达量增加，反之，p-PI3K、p-mTOR和Cyclin D1表达量下降。SNAT2抑制时，LC3-Ⅱ表达量增加，免疫荧光检测自噬斑点增多。添加自噬增强剂海藻糖（trehalose，Tre）和蛋氨酸（methionine，Met）后，与单一添加Tre组相比，Met+Tre组p-mTOR表达量增加，LC3-Ⅱ表达量降低，胞浆内绿色自噬斑点减少；添加Tre和Met并抑制SNAT2时，p-mTOR表达量下降，LC3-Ⅱ表达量增多，胞浆内绿色自噬斑点增加。以上结果表明，SNAT2可介导Met通过调控PI3K-mTOR/Cyclin D1信号通路调节BMECs的增殖与自噬。     

Cepharanthine induces autophagy via PI3K/AKT/mTOR signaling pathway in ovarian cancer SKOV3 cells

AIM: To investigate the autophagy of human ovarian cancer SKOV3 cells induced by cepharanthine and to explore its mechanism. METHODS: The effect of cepharanthine on the viability of ovarian cancer SKOV3 cells was measured by CCK-8 assay. The SKOV3 cells were treated with cepharanthine, and then the formation of autophagosome was observed with acridine orange staining under fluorescence microscope. The protein levels of LC3, AKT, p-AKT, mTOR, p-mTOR and GAPDH in the SKOV3 cells treated with cepharanthine were determined by Western blot.RESULTS: Cepharanthine significantly inhibited the viability of ovarian cancer SKOV3 cells in a dose-dependent manner (P<0.05). The number of the intracellular acidic autophagosomes with bright red fluorescence was significantly increased after cepharanthine treatment in the SKOV3 cells. The expression of LC3-Ⅱ in SKOV3 cells was significantly enhanced after cepharanthine treatment. Furthermore, treatment with cepharanthine in the SKOV3 cells also resulted in a significant down-regulation of phosphorylated form of AKT and mTOR (P<0.01), while the total protein level was not changed. Combination of cepharanthine and 3-methyladenine resulted in a substantial decrease in the cell viability compared with using cepharanthine alone.CONCLUSION: Cepharanthine significantly inhibits the growth of human ovarian cancer SKOV3 cells and induces the autophagy, which may be correlated with down-regulation of PI3K/AKT/mTOR signaling pathway.     

Nutrient starvation affects expression of LC3 family at the feto-maternal interface during murine placentation

LC3 − the mammalian homolog of Atg8 − was found as autophagosome membrane binding protein in mammals and widely used as an autophagosomal marker. LC3A, B and C show different expression patterns in each tissue. The aim of this study was to reveal the differences of expression patterns among LC3 families in mouse placenta under normal condition and nutrient starving condition. LC3A and B were highly expressed in decidual cells. LC3A and B were increased in D14 compared with D12 and D16 in mouse placenta, while LC3C was decreased. Starvation induced increase in LC3B expression specifically. Immunohistochemistry showed different expression patterns among LC3A, B and C. LC3A expression in syncytiotrophoblast was vanished by starvation. The results of real time RT-PCR suggested differences between D12 and D16 in autophagic cascade induced by starvation. Taken together, this study suggests that autophagy could play a role in placental invasion system and that nutrient starvation affects LC3B expression.     

Research progress on autophagy in treatment of glioma

Autophagy is a transport pathway from the cytoplasm to the lysosome, which is a major intracellular degradation/recycling system ubiquitous in eukaryotic cells. Autophagy regulation has achieved some gratifying results in the treatment of glioma. It is currently an exciting field of clinical development. In chemotherapy or radiotherapy, autophagy-related drugs are currently used in vitro and in vivo for treating tumors with significant effects. Autophagy inducers and inhibitors may potentially block tumor formation and enhance the anti-cancer immune response. A more comprehensive understanding of the role of autophagy in different stages of glioma development may guide the development of new therapeutic strategies.     

Notch1 promotes renal tubulointerstitial fibrosis in renal tissues of diabetic mice by inhibiting autophagy through Akt/mTOR signaling pathway

AIM: To observe the changes of Notch1 expression and autophagy in the renal tissues of diabetic mice, and to explore the regulatory effect of Notch1 on tubulointerstitial fibrosis by inhibiting autophagy in diabetic nephro-pathy. METHODS: The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice), with 8 rats in each group. After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. The protein expression of Notch1 in the renal tubular epithelial cells was observed by immunohistochemical staining. The protein levels of Notch1, PTEN, p-Akt (Thr308), Akt, p-mTOR (Ser2448), mTOR, LC3, P62, collagen type Ⅰ (Col-Ⅰ) and collagen type Ⅲ (Col-Ⅲ) were determined by Western blot. RESULTS: Compared with the db/m mice, the blood glucose, glycosylated hemoglobin, serum creatinine, triglyceride and total cholesterol were increased in the db/db mice (P<0.01). Renal tubular epithelial cell vacuolar degeneration, renal tubular expansion and interstitial inflammatory cell infiltration in db/db mouse renal tissues with HE staining were observed. The images of Masson staining showed collagenous fiber-like substance deposition in the glomerular capillaries and renal interstitium, and disarrangement of tubular structure in the renal tissues of db/db mice. The protein expression levels of PTEN and LC3-Ⅱ were decreased (P<0.01 or P<0.05), while the protein levels of Notch1, P62, p-mTOR (Ser2448), p-Akt (Thr308), Col-I and Col-III were increased in the db/db mice as compared with the db/m mice (P<0.01). However, no significant change of total mTOR and Akt proteins between the 2 groups was found. CONCLUSION: Notch1 protein expression was increased, PTEN expression was significantly reduced, Akt/mTOR pathway was activated, autophagy was inhibited, and fibrosis was aggravated in the renal tissues of the diabetic mice.     

绵羊卵泡发育过程中关键信号通路研究进展

卵泡从原始卵泡发育为成熟卵泡，直至排卵、黄体发育等过程都受到精密的调控，产生大量的优势卵泡是绵羊产多羔及实现快速扩繁的关键因素。研究发现，相关信号通路和转录因子通过影响绵羊卵泡中卵母细胞、颗粒细胞的生长，进而调控卵泡的发育成熟，对这些信号通路进行深入了解，有助于探索卵泡发育的调控机制，早日实现绵羊高效繁育。Notch是卵泡发育过程中发挥重要作用的高度保守信号通路，PI3K/AKT/mTOR信号通路各成员都是广泛存在于细胞内的信号转导分子，在卵泡发育早期发挥了主要作用，还有间隙连接（gap junction，GJ）和跨带突触（transzonal projections，TZPs）等物理连接方式，在细胞间的交流通讯起到重要作用。作者详细介绍了Notch信号通路、PI3K/AKT/mTOR信号通路、间隙连接及跨带突触的结构功能在绵羊卵泡发育中的调控作用，为进一步探明绵羊卵泡发育的调控机制提供参考。     

必需氨基酸通过哺乳动物雷帕霉素靶蛋白信号通路调控乳蛋白合成的研究进展

最近的研究表明,必需氨基酸不仅可以作为合成乳蛋白的底物,还可以作为信号分子调控乳蛋白的合成.不同的必需氨基酸通过哺乳动物雷帕霉素靶蛋白(mTOR)信号通路调控乳蛋白合成的作用也不相同,既包括正调控作用,也包括负调控作用.研究不同必需氨基酸调控乳蛋白合成的分子机制将为提高乳蛋白合成率提供理论基础.本文综述了mTOR信号通路及必需氨基酸通过mTOR信号通路调控乳蛋白的合成的研究进展,为揭示必需氨基酸作为蛋白质合成底物及细胞信号分子在乳蛋白合成过程中的作用机制提供研究方向.     

mTOR对信号通路调控的研究进展

哺乳动物雷帕霉素靶蛋白(mTOR)信号通路是最近新出现的细胞内重要信号途径,该途径在进化上高度保守,主要通过PI3K/Akt/mTOR信号通路磷酸化激活来调控细胞分裂、促进转录、信号翻译等,从而控制蛋白合成来调节细胞生长。mTOR作为一种重要的调节基因通过调节细胞周期、蛋白质合成、细胞能量代谢等多种途径发挥重要的生理功能,在细胞增殖、生长、分化过程中起着中心调控点的作用。     

α-酮戊二酸对LPS慢性应激仔猪小肠黏膜形态与功能的影响

为了探讨α-酮戊二酸(AKG)能否缓解LPS慢性应激导致的仔猪小肠黏膜损伤及其机理,本试验研究了AKG对LPS慢性应激仔猪的小肠黏膜形态、血浆D-木糖的含量、血浆和小肠黏膜二胺氧化酶(DAO)活性及小肠黏膜mTOR及磷酸化的mTOR表达量的影响.18头(24±1)日龄健康断奶仔猪随机分成3个处理组(空白对照组、应激对照组、AKG组),每个处理6个重复.各组基础日粮一致,空白对照组和应激对照组饲喂基础日粮+1%淀粉,AKG组饲喂基础日粮+1%AKG.试验期为16 d.应激对照组和AKG组仔猪分别于第10、12、14和16天腹膜注射80μg·kg~(-1)BW的LPS,空白对照组注射相应剂量的灭菌生理盐水.第16天注射LPS 2 h后,按0.1g·kg~(-1)BW的剂量给仔猪灌服D-木糖溶液,注射LPS 3 h后,前腔静脉采血.第17天屠宰取小肠组织样,刮取肠黏膜及制作组织切片.结果表明:(1)与空白对照组相比,应激对照组十二指肠、空肠和回肠黏膜绒毛高度/隐窝深度、空肠和回肠磷酸化mTOR/mTOR(P-mTOR/mTOR)显著降低(P<0.05),血浆DAO活性显著升高(P<0.05).(2)与应激对照组相比,AKG组十二指肠、空肠和回肠黏膜绒毛高度/隐窝深度、空肠黏膜DAO活性、血浆D-木糖及十二指肠、空肠和回肠黏膜P-mTOR/mTOR显著升高(P<0.05).结果显示,日粮中添加1%AKG可在一定程度上改善仔猪的小肠组织学形态和吸收功能,缓解LPS慢性应激导致的仔猪小肠黏膜损伤,这与mTOR信号通路有关.