Nogo-B在血管损伤、组织修复和炎症反应中发挥重要作用.然而,Nogo-B在动脉粥样硬化中的作用仍不明确.本研究拟在巨噬细胞中探讨Nogo-B对巨噬细胞泡沫化的影响.在RAW264.7细胞中沉默Nogo-B后,采用氧化低密度脂蛋白(Ox-LDL)或DiI修饰的Ox-LDL诱导巨噬细胞泡沫化;通过激光共聚焦显微镜观察巨噬细胞中荧光脂质,并在透射电镜下观察各组细胞中自噬泡;采用Western印迹分析Plin2、p62和LC3-Ⅱ的蛋白质水平;采用实时荧光定量PCR检测p62 mRNA水平;采用氯喹处理以及mRFP-GFP-LC3双荧光体系分析自噬流功能;进一步过表达Nogo-B后,比较巨噬细胞中脂质负荷程度以及Plin2、p62和LC3-Ⅱ的蛋白质水平.结果 显示,DiI-Ox-LDL处理后,Nogo-B沉默组细胞中脂质负荷程度高于对照组(2.34±0.67 vs.0.69±0.14,P＜0.05);Ox-LDL处理后,Nogo-B沉默组细胞中自噬泡数量(8.67±0.58 vs.4.33±0.58,P＜0.01)、Plin2(4.65±0.50 vs.3.24±0.71,P＜0.05)、p62(10.13±1.79 vs.5.76±1.84,P＜0.05)和LC3-Ⅱ(4.38±0.20 vs.2.33±1.56,P＜0.01)的蛋白质水平均显著高于对照组,而p62 mRNA水平无差异(P＞0.05);进一步研究发现,Nogo-B沉默组的自噬流被抑制了;过表达Nogo-B后,虽然p62蛋白质水平无明显变化,但是细胞中脂质负荷程度显著低于对照组(1.68±1.06 vs.4.94±0.70,P＜0.05),Plin2和LC3-Ⅱ的蛋白质水平也明显降低.上述结果表明,Nogo-B通过促进自噬流抑制了Ox-LDL诱导的巨噬细胞泡沫化,Nogo-B可能具有抗动脉粥样硬化的作用.

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome that elevates the risk of hepatocellular carcinoma (HCC). Although alteration of lipid metabolism has been increasingly recognized as a hallmark of cancer cells, the deregulated metabolic modulation of HCC cells in the NAFLD progression remains obscure. Here, we discovers an endoplasmic reticulum-residential protein, Nogo-B, as a highly expressed metabolic modulator in both murine and human NAFLD-associated HCCs, which accelerates high-fat, high-carbohydrate diet-induced metabolic dysfunction and tumorigenicity. Mechanistically, CD36-mediated oxLDL uptake triggers CEBPβ expression to directly upregulate Nogo-B, which interacts with ATG5 to promote lipophagy leading to lysophosphatidic acid-enhanced YAP oncogenic activity. This CD36-Nogo-B-YAP pathway consequently reprograms oxLDL metabolism and induces carcinogenetic signaling for NAFLD-associated HCCs. Targeting the Nogo-B pathway may represent a therapeutic strategy for HCC arising from the metabolic syndrome.

目的 探讨巨噬源性泡沫细胞中p62蛋白对脂质代谢相关的自噬以及炎症因子表达的影响,为p62在抗动脉粥样硬化中的应用提供参考.方法 利用Ox-LDL刺激RAW264.7细胞的方法模拟巨噬源性泡沫细胞的形成,通过Western blot及实时荧光定量PCR检测巨噬源性泡沫细胞中p62蛋白和mRNA水平.通过Western blot比较p62 siRNA组和对照组中Ox-LDL诱导的LC3剪切、脂滴相关蛋白Plin2和过氧化物酶体相关蛋白PEX2的蛋白水平,通过实时荧光定量PCR比较TNFα和IL-6 mRNA表达水平.结果 Ox-LDL对RAW264.7细胞中p62蛋白以及mRNA水平均有上调作用.干扰p62的表达之后,LC3-Ⅱ蛋白水平降低,Plin2的蛋白水平并无明显变化,PEX2的蛋白水平升高,TNFα和IL-6 mRNA表达升高.进一步研究发现,干扰Nrf2后能明显抑制Ox-LDL对p62的上调作用.结论 巨噬源性泡沫细胞中Ox-LDL通过Nrf2介导p62蛋白的上调,p62可能具有抗动脉粥样硬化的作用.

Aim To study the role of dendritic cells (DC) with lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) high expression in atherosclerosis in vitro and in vivo.Methods Western blot was used to examine the protein expression levels of LOX-1 in ox-LDL stimulated primary cultured bone marrow derived dendritic cells (BMDC)from C57 mice,flow cytometry was used to measure the ratio of LOX-1 high expression subsets and LOX-1 low expression subsets in BMDC,MACS was used to sort the two cell subsets with different LOX-1 expression and to observe the effects of ox-LDL on the ratio of two cell subsets and release of inflammatory factors.Results There were two different subsets of DC with high and low expression of LOX-1,the ratio of LOX-1high DC was increased after ox-LDL treatment.After the LOX-1 high DC was stimulated by ox-LDL,the TNF-α and IL-1 3 mRNA was up-regulated (P＜0.01),and the percentage of positive cells increased higher than that of negative cells.In hyperlipidemia model of C57 mice,total cholesterol levels were significantly increased after fed with high fat diet for 4,6 and 8 weeks.This was paralleled with an increase in DC number and in the ratio of LOX-1high DC and LOX-1low DC in the aorta from mice.Different concentrations of Dil-ox-LDL was used to stimulate DC2.4 cells,there was a concentration-dependent increase in the phagocytosis of Dil-ox-LDL by DC 2.4 cells.Western blot showed that the LOX-1 expression in DC2.4 cells could be induced by ox-LDL at 20 mg/L and 40 mg/L.Conclusions Two DC subsets of LOX-1high DC and LOX-1low DC were found in the present study,and the levels of expression of inflammatory cytokines in LOX-1high DC subsets were significantly higher than LOX-1low DC subsets.In hyperlipidemia model of mice in vivo,the ratio of LOX-1high DC and LOX-1low DC was significantly increased in the aorta.In DC2.4 cells,ox-LDL could up-regulate LOX-1 expression.

Immunotherapy is one of the key strategies for cancer treatment. The cGAS-cGAMP-STING-IRF3 pathway of cytosolic DNA sensing plays a pivotal role in antiviral defense. We report that the STING activator cGAMP possesses significant antitumor activity in mice by triggering the STING-dependent pathway directly. cGAMP enhances innate immune responses by inducing production of cytokines such as interferon-β, interferon-γ, and stimulating dendritic cells activation, which induces the cross-priming of CD8(+) T cells. The antitumor mechanism of cGAMP was verified by STING and IRF3, which were up-regulated upon cGAMP treatment. STING-deficiency dramatically reduced the antitumor effect of cGAMP. Furthermore, cGAMP improved the antitumor activity of 5-FU, and clearly reduced the toxicity of 5-FU. These results demonstrated that cGAMP is a novel antitumor agent and has potential applications in cancer immunotherapy.

Objective To study the protective effects and mechanism of Asiatic Acid (AA ) on traumatic brain injury . Methods The closed craniocerebral injury model made by the weight-drop method and fluid percussion brain injury model were used in the study .Brain water content ,blood brain barrier permeability and brain tissue pathology morphology were measured to study the neuroprotective effect of AA on traumatic brain injury .Moreover ,the inflammatory cytokines such as TNF-α,IL-β,IL-6 were examined by PCR and ELISA to study the mechanism of AA .Results Brain water content and permeability of blood brain barrier were significantly decreased ,the traumatic brain injury was nameliorated by AA .Moreover ,the expressions of inflammatory cytokines TNF-α,IL-β,IL-6 were inhibited by AA .Conclusion AA has the protective effects on traumatic brain injury and the mechanism is related to the anti-inflammatory effect .

The inflammation and apoptosis were vital important in the progress and recovery in myocardial infarction. Apoptosis was regulated by inflammation though TNF-α,CHOP,IL-10 andα7nAChR signal pathways and influenced the se-verity of the inflammation via feedback regulation.Inflammation and apoptosis affected myocardial infarction size and cardiac function recovery together.Inhibition of inflammation,reducing apoptosis had been proved to be the important parts in preven-tion of ventricular remodeling and regulating cardiac dysfunction after myocardial infarction,which had broad prospects.

AIMS:In our previous study, eEF1A1 was identified to be a new target for protecting brain ischemia injury, but the mechanism remains largely unknown. In this study, we screened the downstream cellular protein molecules interacted with eEF1A1 and found mechanism of eEF1A1 in brain ischemia protection.METHODS AND RESULTS:Through co-immunoprecipitation and mass spectrometry for searching the interaction of proteins with eEF1A1 in bEnd3 cells, HSC70 was identified to be a binding protein of eEF1A1, which was further validated by Western blot and immunofluorescence. eEF1A1 or HSC70 knockdown, respectively, increased OGD-induced apoptosis of brain vascular endothelial cells, which was detected by Annexin V-FITC/PI staining. HSC70 or eEF1A1 knockdown enhances phosphorylated JNK, phosphorylation of c-JUN (Ser63, Ser73), cleaved caspase-9, and cleaved caspase-3 expression, which could be rescued by JNK inhibitor.CONCLUSION:In summary, our data suggest that the presence of chaperone forms of interaction between eEF1A1 and HSC70 in brain vascular endothelial cells, eEF1A1 and HSC70 can play a protective role in the process of ischemic stroke by inhibiting the JNK signaling pathway activation.

Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury.

Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide produced by Entamoeba histolytica, has anti-inflammatory function and protective effect on ischemic stroke. In this study, we evaluated the effect of MLIF on myocardial ischemia. Mice were subjected to ischemia/reperfusion by occlusion of the left anterior descending artery (LAD). After sacrifice, the serum concentrations of cardiac troponin I (cTnI), creatine kinase (CK), lactate dehydrogenase (LDH) as well as the heart infarct size were measured. HE and TUNEL staining were used to observe the pathological damage and the apoptotic cells. For in vitro study, the oxygen-glucose deprivation(OGD) model was established in H9c2 cells. MTT assay and flow cytometry assay were performed to evaluate cell viability and apoptosis. The expression of JNK and caspase 3 was assessed by western blot analysis. Pull-down assay was used to detect the specific binding protein of MLIF in myocardial cells. MLIF significantly reduced the infarct size, and the cTnI, CK and LDH levels, amelioratived pathological damage and reduced the apopotosis compared with the myocardial I/R model group. MLIF improved cell survival and inhibited apoptosis and necrosis by inhibiting the p-JNK and cleaved caspase3 expression. Furthermore, the binding protein of MLIF in myocardial cells was vimentin. Inhibition of vimentin expression by withaferin A or vimentin siRNA repressed the protective effects of MLIF in OGD-provoked H9c2 cells. Taken together, our results demonstrate that the cardioprotective effects of MLIF on myocardial ischemia injury are related to reductions in the inflammatory response and apoptosis by targeting vimentin.

Monocyte locomotion inhibitory factor(MLIF)was an anti‐inflammatory pentapeptide produced by Entamoeba histolytica .In vivo and in vitro study showed that MLIF displayed anti‐inflammatory and immune protection effects and MLIF had protective effects on rheumatoid arthritis ,nerve injury ,myocardial ischemia ,cerebral ischemia and Alzheimer′s disease . Studies had shown that MLIF regulated inflammatory response and immune protection through NF‐κB and MAPK signal path‐ways .The sources and biological activities of MLIF were reviewed in this paper .

Objective Lox-1 ( lectin-like oxidized low-density lipoprotein receptor-1 ) was one of the main receptor of ox-LDL, which took an important role in vascular endothelial dysfunction , the formation of foam cells , and the stability of atherosclerotic plaques .To investigate the role of Lox-1 in cardiovascular diseases .Methods The literatures of Lox-1 in cardiovascular diseases retro-spectively were reviewed .Results As a new ox-LDL scavenger receptor , Lox-1 played an important role in cardiovascular diseases . Conclusion Lox-1 might provide new ideas for cardiovascular diseases .

热休克蛋白70（heat shock protein 70,Hsp70）是生物体产生的一种可增强细胞对损伤的耐受程度、维持细胞正常代谢功能并提高细胞生存率的高度保守的应激蛋白。本文回顾了近几年有关Hsp70与缺血性脑卒中相关的报道,发现Hsp70可能通过提高细胞抗氧化应激能力、抑制炎症反应以及抑制细胞凋亡,在缺血性脑卒中发挥保护作用。本文对Hsp70在缺血性脑卒中进程中的保护作用及其机制进行了概述。

Three new polyhydroxysterols, named muriflasteroids A–C (1–3) were isolated from the South China Sea gorgonian Muriceopsis flavida, together with sixteen known analogs, cholest-3β,5α,6β-triol,3β-acetate (4), 5α-methoxycholest-3β,6β-diol (5), (22E)-cholest-22-en-3β,5α,6β-triol (6), cholest-3β,5α,6β-triol (7), (22E)-24-norcholest-22-en-3β,5α,6β-triol (8), (22E,24S)-ergost-22-en-3β,5α,6β-triol (9), ergost-24(28)-en-3β,5α,6β-triol (10), (22E)-cholest-7,22-dien-3β,5α,6β-triol (11), cholest-7-en-3β,5α,6β-triol (12), (22E)-24-norcholest-7,22-dien-3β,5α,6β-triol (13), ergost-7,24(28)-dien-3β,5α,6β-triol (14), (22E,24R)-ergost-7,22-dien-3β,5α,6β-triol (15), (22E)-cholest-22-en-1β,3β,5α,6β-tetrol (16), (22E)-24-norcholest-22-en-1β,3β,5α,6β-tetrol (17), cholest-1β,3β,5α,6β-tetrol (18), and (24ξ)-ergost-1β,3β,5α,6β-tetrol (19). The structures of the new compounds were elucidated by detailed spectroscopic analysis in combination with comparison of reported data. All the compounds are reported for the first time from the animal. In the bioassay in vitro, these compounds exhibited different levels of growth inhibition activity against A549 and MG63 cell lines. In particular, compound 18 displayed a considerable activity, being similar as that of positive control adriamycin. An annexin V analysis indicated that compounds 7 and 18 can significantly induce apoptosis in A549 cell, and compound 7 is more potent in the induction of apoptosis. Preliminary structure–activity analysis suggests that the acetylation on 3-OH and appearance of Δ7 may decrease the activity while substitution of 1-OH and the nature of side chain may also play an important role in the activity. Methylation of 5-OH contributed a little to the activity.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and established for the quantitative determination of monocyte locomotion inhibitory factor, a pentapeptide (Met-Gln-Cys-Asn-Ser) produced by Entamoeba histolytica in axenic culture, in dog blood. The main challenge was the chemical and enzymatic instability of the peptide which was successfully overcome. After a simple protein precipitation, MLIF was separated from AS-5 (Met-Gln-Gly-Asn-Ser), acted as an internal standard, on a Gemini C18 column (5 μm, 50 mm × 4.6 mm i.d.) using a gradient elution of acetonitrile (0.2% formic acid) and water (0.2% formic acid) and detected by electrospray ionization tandem mass spectrometry. Excellent linearity was achieved (r>0.9943) over the linear range 5-1000 ng/ml using 0.2 ml blood sample. The validation results demonstrated that this method was specific, accurate and precise. It was successfully applied in measuring MLIF following intravenous infusion its administration at 0.2, 0.4, and 0.8 mg/kg in beagle dogs to support the pre-clinical pharmacokinetic study.

The tumour stroma is an active participant during cancer progression. Stromal cells promote tumour progression and metastasis through multiple mechanisms including enhancing tumour invasiveness and angiogenesis, and suppressing immune surveillance. We report here that miR-126/miR-126*, a microRNA pair derived from a single precursor, independently suppress the sequential recruitment of mesenchymal stem cells and inflammatory monocytes into the tumour stroma to inhibit lung metastasis by breast tumour cells in a mouse xenograft model. miR-126/miR-126* directly inhibit stromal cell-derived factor-1 alpha (SDF-1α) expression, and indirectly suppress the expression of chemokine (C–C motif) ligand 2 (Ccl2) by cancer cells in an SDF-1α-dependent manner. miR-126/miR-126* expression is downregulated in cancer cells by promoter methylation of their host gene Egfl7. These findings determine how this microRNA pair alters the composition of the primary tumour microenvironment to favour breast cancer metastasis, and demonstrate a correlation between miR-126/126* downregulation and poor metastasis-free survival of breast cancer patients. Wang and colleagues show that miR-126 and miR-126* suppress metastasis by inhibiting the production of the Sdf-1α cytokine in mouse mammary tumours, resulting in decreased recruitment of mesenchymal stem cells and inflammatory monocytes to the tumour stroma.