参苓白术散通过自噬调节肠隐窝上皮细胞损伤的作用机制

目的探究参苓白术散通过细胞自噬调节脂多糖(LPS)诱导的肠隐窝上皮细胞(IEC-6)损伤的作用与机制。方法 IEC-6细胞分组:空白组、LPS模型组、LPS+参苓白术散含药血清低、中、高(5%、10%、20%)剂量组、自噬抑制剂3-甲基腺嘌呤组(3-MA)、自噬诱导剂雷帕霉素靶蛋白(mTOR)阻断剂雷帕霉素组(Rapamycin)。采用MTT方法检测LPS的细胞毒性、检测参苓白术散含药血清浓度梯度活性;通过ELISA试剂盒测定IEC-6细胞上清液中白介素1β(IL-1β)、白介素8(IL-8)、白介素10(IL-10)和肿瘤坏死因子α(TNF-α)的浓度;通过RT-PCR法检测ATG5、ATG12、ATG13、ATG16的mRNA水平;使用RFP-GFP-LC3腺病毒转染IEC-6细胞,测定IEC-6细胞自噬体的数目。结果 LPS浓度为100μg·mL-1时,IEC-6细胞抑制率为29.3%,以该浓度进行后续实验;不同浓度参苓白术散含药血清对细胞活力均无明显作用;模型组与空白组比较,促炎因子IL-1β、IL-8和TNF-α浓度上升,抑炎因子IL-10浓度下降,ATG5、ATG13、ATG16L2 mRNA水平上升(均P <0.05),模型组中没有明显的自噬体斑点形成;含药血清组与模型组比较,促炎因子IL-1β、IL-8和TNF-α浓度下降,抑炎因子IL-10浓度上升,ATG5、ATG13、ATG16L2 mRNA水平下降(均P <0.05),ATG12和ATG16L1 mRNA水平无显著性差异(P> 0.05),含药血清组和自噬诱导剂Rapamycin组自噬斑点的形成明显增多;自噬抑制剂3-MA组与模型组比较,自噬体斑点没有差别。结论参苓白术散含药血清能抑制促炎症因子IL-1β、IL-8和TNF-α的释放,增加抑炎症因子IL-10的释放,减轻IEC-6细胞的炎症损伤,这一过程可能与调控自噬相关通路Beclin1有关。

Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae

Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentia-tion in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.

Inflammatory bowel disease:Genetic and epidemiologic considerations

Genome-wide association studies have firmly established that many genomic loci contribute to inflammatory bowel disease, especially in Crohn’s disease. These studies have newly-established the importance of the interleukin 23 and autophagy pathways in disease pathogenesis. Future challenges include: (1) the establishment of precisely causal alleles, (2) definition of altered functional outcomes of associated and causal alleles and (3) integration of genetic findings with environmental factors.

Hsp90 regulates processing of NF-κB2 p100 involving protection of NF-κB-inducing kinase (NIK) from autophagy-mediated degradation

NF-κB-inducing kinase （NIK） is required for NF-κB activation based on the processing of NF-κB p100. Here we report a novel mechanism of NIK regulation involving the chaperone 90 kDa heat shock protein （Hsp90） and autophagy. Functional inhibition of lisp90 by the anti-tumor agent geldanamycin （GA） efficiently disrupts its interaction with NIK, resulting in NIK degradation and subsequent blockage of p100 processing. Surprisingly, GA-induced NIK degradation is mediated by autophagy, but largely independent of the ubiquitin-proteasome system. Hsp90 seems to be specifically involved in the folding/stabilization of NIK protein, because GA inhibition does not affect NIK mRNA transcription and translation. Furthermore, Hsp90 is not required for NIK-mediated recruitment of the α subunit of IκB3 kinase to p 100, a key step in induction of p100 processing. These findings define an alternative mechanism for Hsp90 client degradation and identify a novel function ofautophagy in NF-κB regulation. These findings also suggest a new therapeutic strategy for diseases associated with p 100 processing.

Overview of macroautophagy regulation in mammalian cells

Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGyrelated genes has greatly increased our knowledge about the mechanism responsible for antophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.

Effects of Qingguang’an Granules on mitochondrial autophagy of retinal ganglion cells in rats with chronic ocular hypertension

Objective To investigate the effect and underlying mechanism of Qingguang’an Granules(青光安颗粒剂,QGAG)on mitochondrial autophagy(mitophagy)of retinal ganglion cells(RGCs)in rats with chronic ocular hypertension(COH).Methods Sixty Sprague Dawley(SD)rats,half males and half females,were randomly assigned to three groups:the control,model,and QGAG(2.5 g/kg)groups,with 20 rats in each group.Rats’model of COH was established by cauterizing episcleral veins in the model group and QGAG group.Three weeks after successful modeling,rats in the QGAG group were intra-gastrically administered with QGAG,while rats in the control group and the model group received an equal dose of normal saline.After three months of intragastric administration,intraocular pressure(IOP)of all rats was measured.The mitophagy was monitored by the immunofluorescence method,the mitochondrial membrane potential was measured using the JC-1 method,and the morphological changes of mitophagy in RGCs were observed by transmission electron microscopy.Meanwhile,rat RGCs were labeled using the fluorescent gold method,and RGCs density in each group was calculated.Moreover,RGCs apoptosis was observed by TdT-mediated dUTP Nick-End Labeling(TUNEL)assay.Finally,the expression levels of Parkin,optineurin,microtubule-associated protein 1 light chain 3-Ⅱ/microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅱ/LC3-Ⅰ),recombinant lysosomal associated membrane protein 1(LAMP1),and B-cell lymphoma-2(Bcl-2)in RGCs were determined by Western blot assay.The corresponding mRNAs were detected through quantitative real-time polymerase chain reaction(qRT-PCR).Results The QGAG reduced IOP in COH rats,and inhibited mitophagy and apoptosis of RGCs(P<0.05).Besides,the QGAG significantly increased the expression levels of Parkin and Bcl-2(P<0.05),and inhibited the expression levels of optineurin,LAMP1,and LC3-Ⅱ/LC3-Ⅰ(P<0.05)in RGCs of COH rats.Conclusion The QGAG can inhibit mitophagy in RGCs of COH rats and show a protective effect against optic nerve damage caused by glaucoma,which may be mediated through the mitophagy ubiquitination via the Parkin/PINK1-related pathway.

The convergent point of the endocytic and autophagicpathways in leydig cells

Endocytic tracers and marker enzyme of lysosomeswere used in the present study to analyze the processesof autophagocytosis and endocytosis, and the convergentpoint of these two pathways in Leydig cells. The endocyticand autophagic compartments call be easily identified inLeydig cells, which makes easier to define the stages of twopathways than was possible before. The evidences indicated that the late endosomes (dense MVBs) deliver theirendocytosed gold tracers together with lysosomal enzymesto the early autophagosomes and they are the convergentpoint of the two pathways. During this convergent process,the early autophagosomes transform into late autophagosomes and the late endosomes transform into mature lysosomes.

Preparation and in vitro Studies of Stealth PEGylated PLGA Nanoparticles as Carriers for Arsenic Trioxide

The aim of this study was to prepare arsenic trioxide （ATO）-loaded stealth PEGylated PLGA nanoparticles （PEG-PLGA-NPs） and to assess the merits of PEG-PLGA-NPs as drug carriers for ATO delivery. PEG-PLGA copolymer was synthesized with methoxypolyethyleneglycol （Mw=5000）, D, L-lactide, and glycolide by the ring-opening polymerization method. Amorphous ATO was transformed into cubic crystal form to increase its solu-bility in the organic solvent. ATO-loaded PEG-PLGA-NPs were prepared by the modified spontaneous emulsification solvent diffusion （SESD） method, and the main experimental factors influencing the characteristics of nanopar- ticles were investigated, to optimize the preparation. To confirm the escape of PEG-PLGA-NPs from phagocytosis by phagocytes, PEG-PLGA-NPs labeled rhodamine B uptake by murine peritoneal macrophages （MPM） were analyzed by flow cytometry. The results showed that the physicochemical characteristics of PEG-PLGA-NPs were affected by the type and concentration of the emulsifiers, polymer concentration, and drug concentration. ATO-loaded PEG-PLGA-NPs, with particle size of 120.8nm, zeta potential of-10.73mV, encapsulation efficiency of 73.6%, and drug loading of 1.36%, were prepared under optimal conditions. The images of transmission electron micros-copy （TEM） indicated that the optimized nanoparticles were near spherical and without aggregation or adhesion. The release experiments in vitro showed the ATO release from PEG-PLGA-NPs exhibited consequently sustained release for more than 26d, which was in accordance with Higuchi equation. The uptake of PEG-PLGA-NPs by MPM was found to decrease markedly compared to PLGA-NPs. The experimental results showed that PEG-PLGA-NPs were potential nano drug delivery carriers for ATO.

Ultracytochemical localization of H+-adenosine triphosphatase activity in autophagic vacuoles induced by vinblastine in rat liver

H+-adenosine triphosphatase (H+-ATPase) activity was demonstrated oytoohemioally in autophagio vaouoles (AVs) of rat hepatooytes using a modification of the method for the demonstration of neutral p-nitrophenyl phosphatase (p-NPPase) activity [1]. When an inhibitor of H+-ATPase, N-ethylmaleimide (NEM) or 4,4'-diisothiooyanostilbene-2,2'disalfonio aold, di-sodium salt (BIDS) was included in the incubation medium the enyzme activity was abolished indicating that p-NPPase demonstrated in this study represents H+-ATPase. Autophagy was induced by a single intraperitoneal injection of vinblastine sulfate (VBL). The number of AVs increased remarkably in hepatooytes from 40 min after VBL treatment. H+-ATPase activity was observed mainly on the membranes of lysosomes and AVs. However, early forms of AVs containing only incompletely digested material showed no H+-ATPase activity. Most AVs revealing a positive reaction seemed to be in advanced stages of development. Acid phosphatase aotioity was demonstrable in mature but not in early forms of AVs. The present investigation showed that membranes of advanced stage A Vs possess an H+-ATPase which may be derived from lysosomal membranes.