Pseudomonas aeruginosa homoserine lactone triggers apoptosis and Bak/Bax‐independent release of mitochondrial cytochrome C in fibroblasts

Pseudomonas aeruginosa use N‐(3‐oxododecanoyl)‐homoserine lactone (C12) as a quorum‐sensing molecule to regulate gene expression in the bacteria. It is expected that in patients with chronic infections with P. aeruginosa, especially as biofilms, local [C12] will be high and, since C12 is lipid soluble, diffuse from the airways into the epithelium and underlying fibroblasts, capillary endothelia and white blood cells. Previous work showed that C12 has multiple effects in human host cells, including activation of apoptosis. The present work tested the involvement of Bak and Bax in C12‐triggered apoptosis in mouse embryo fibroblasts (MEF) by comparing MEF isolated from embryos of wild‐type (WT) and Bax^?/?/Bak^?/? (DKO) mice. In WT MEF C12 rapidly triggered (minutes to 2 h): activation of caspases 3/7 and 8, depolarization of mitochondrial membrane potential (Δψ_mito), release of cytochrome C from mitochondria into the cytosol, blebbing of plasma membranes, shrinkage/condensation of cells and nuclei and, subsequently, cell killing. A DKO MEF line that was relatively unaffected by the Bak/Bax‐dependent proapoptotic stimulants staurosporine and etoposide responded to C12 similarly to WT MEF: activation of caspase 3/7, depolarization of Δψ_mito and release of cytochrome C and cell death. Re‐expression of Bax or Bak in DKO MEF did not alter the WT‐like responses to C12 in DKO MEF. These data showed that C12 triggers novel, rapid proapoptotic Bak/Bax‐independent responses that include events commonly associated with activation of both the intrinsic pathway (depolarization of Δψ_mito and release of cytochrome C from mitochondria into the cytosol) and the extrinsic pathway (activation of caspase 8). Unlike the proapoptotic agonists staurosporine and etoposide that release cytochrome C from mitochondria, C12's effects do not require participation of either Bak or Bax.     

The Effects of Consuming Frequent,Higher Protein Meals on Appetite and Satiety During Weight Loss in Overweight/Obese Men

The purpose of this study was to determine the effects of dietary protein and eating frequency on perceived appetite and satiety during weight loss. A total of 27 overweight/obese men (age 47 ± 3 years; BMI 31.5 ± 0.7 kg/m²) were randomized to groups that consumed an energy‐restriction diet (i.e., 750 kcal/day below daily energy need) as either higher protein (HP, 25% of energy as protein, n = 14) or normal protein (NP, 14% of energy as protein, n = 13) for 12 weeks. Beginning on week 7, the participants consumed their respective diets as either 3 eating occasions/day (3‐EO; every 5 h) or 6 eating occasions/day (6‐EO; every 2 h), in randomized order, for 3 consecutive days. Indexes of appetite and satiety were assessed every waking hour on the third day of each pattern. Daily hunger, desire to eat, and preoccupation with thoughts of food were not different between groups. The HP group experienced greater fullness throughout the day vs. NP (511 ± 56 vs. 243 ± 54 mm · 15 h; P < 0.005). When compared to NP, the HP group experienced lower late‐night desire to eat (13 ± 4 vs. 27 ± 4 mm, P < 0.01) and preoccupation with thoughts of food (8 ± 4 vs. 21 ± 4 mm; P < 0.01). Within groups, the 3 vs. 6‐EO patterns did not influence daily hunger, fullness, desire to eat, or preoccupation with thoughts of food. The 3‐EO pattern led to greater evening and late‐night fullness vs. 6‐EO but only within the HP group (P < 0.005). Collectively, these data support the consumption of HP intake, but not greater eating frequency, for improved appetite control and satiety in overweight/obese men during energy restriction‐induced weight loss.     

TIPE2 deficiency accelerates neointima formation by downregulating smooth muscle cell differentiation

Phenotypic switching of vascular smooth muscle cells (VSMCs) is known to play a key role in the development of atherosclerosis. However, the mechanisms that mediate VSMC phenotypic switching are unclear. We report here that TIPE2, the tumor necrosis factor (TNF) α-induced protein 8-like 2 (TNFAIP8L2), plays an atheroprotective role by regulating phenotypic switching of VSMCs in response to oxidized low-density lipoprotein (ox-LDL) stimuli. TIPE2-deficient VSMCs treated with ox-LDL expressed lower levels of contractile proteins such as SMαA, SM-MHC and calponin, whereas the proliferation, migration and the synthetic capacity for growth factors and cytokines were increased remarkably. Furthermore, TIPE2 inhibited VSMCs proliferation by preventing G₁/S phase transition. Interestingly, these effects of TIPE2 on VSMCs were dependent on P38 and ERK1/2 kinase signals. As a result, neointima formation was accelerated in the carotid arteries of TIPE2-deficient mice. These results indicate that TIPE2 is a potential inhibitor of atherosclerosis.     

人特异性CHRFAM7A基因抑制小鼠肾缺血再灌注损伤炎症反应

本研究探讨人特异性CHRFAM7A基因抑制小鼠肾缺血再灌注损伤早期炎症作用及其机制。 12只野生型（wild type, WT）C57BL/6成年雄性小鼠随机分为2组：野生型假手术组（WT Sham）和野生型肾缺血再灌注损伤（WT RIRI）组。12只性别、年龄匹配的 CHRFAM7A转基因（transgenic mice, GT）小鼠也随机分组为：转基因型假手术组（GT Sham）和转基因型肾缺血再灌注损伤（GT RIRI）组,每组各6只。除WT 和GT假手术组仅行剖腹手术外,所有小鼠均夹闭双侧肾蒂40 min,再灌注24 h后,留取各组小鼠血清及肾组织标本。生化分析仪检测血清中的尿素氮（BUN）和肌酐（Scr）水平;ELISA法检测白介素-8（IL-8）、肿瘤坏死因子（TNF-α）和胱天蛋白酶7水平;免疫组织化学染色法检测高迁移率族蛋白1（HMGB1）表达水平;苏木-伊红（HE）染色和原位末端标记法（TUNEL）染色观察肾组织病理损伤;流式检测HK-2细胞凋亡率。结果显示,与WT Sham组对比,WT RIRI组血清中,BUN、Scr、IL-8和TNF-α含量增加（P<0.0001）;肾组织中,胱天蛋白酶7水平增高（P＜0.001）,HMGB1平均光密度值增加(P<0.0001),肾组织细胞凋亡指数（AI%）增高(P<0.0001)。与GT Sham组对比, GT RIRI组血清BUN、Scr、IL-8和TNF-α含量增加（P<0.05,P<0.0001, P<0.01, P<0.01）;肾组织中,胱天蛋白酶7水平明显增高（P＜0.01）,HMGB1平均光密度值增加(P<0.0001),肾组织细胞AI%增高(P=0.0005)。与WT RIRI组对比,GT RIRI组血清中的BUN、Scr水平降低（P<0.0001）,IL-8、TNF-α、HMGB1和胱天蛋白酶7的水平明显下降（P<0.01, P<0.0001, P<0.0001, P<0.01）,肾组织细胞凋亡指数（AI%）下降（P=0.0003）.与pLVX空载质粒+氯化钴组相比,pLVX-CHRFAM7A+氯化钴组的凋亡率(19.31%±1.45 vs 34.92%±4.21, P<0.001)明显下降。上述结果表明,人特异性CHRFAM7A基因在小鼠肾缺血再灌注损伤中,通过抑制早期的炎症反应,对肾组织发挥保护作用。     

Interference of IP-10 Expression Inhibits Vascular Smooth Muscle Cell Proliferation and Intimal Hyperplasia in Carotid Artery: A New Insight in the Prevention of Restenosis

After vascular angioplasty, vascular smooth muscle cell (VSMC) proliferation causes atherosclerosis and intimal hyperplasia leading to restenosis. Interferon-γ-inducible protein (IP)-10 plays a role in atherogenesis, but the mechanism remains unclear. We evaluated the role of IP-10 in intimal hyperplasia and restenosis. IP-10 expression was determined in arterial specimens from 20 arteriosclerotic obliteration patients and 6 healthy individuals. VSMCs were stimulated in vitro with IFN-γ and transfected with IP-10 siRNA. Silencing was verified with RT-PCR/Western blot; cell proliferation rate was detected by methyl-thiazol-tetrazolium. The carotid artery model of atherosclerosis injury was established with IP-10 siRNA. IP-10 expression was detected at 1 and 4 weeks using RT-PCR and immunohistochemistry. Artery morphology was assessed with hematoxylin-and-eosin staining, and intimal hyperplasia was evaluated by electron microscopy. IP-10 was overexpressed in arteriosclerotic obliteration group compared with control group (P < 0.05). IP-10 expression in transfected group was significantly lower than in untransfected group. The intima-to-media ratio of transfected group at 4 weeks was lower than that of untransfected group (P < 0.01). The transfected group exhibited more regular intimal structure and less hyperplasia under electron microscopy. We, therefore, concluded that IP-10 played an important role in intimal hyperplasia as siRNA-mediated IP-10 silencing inhibited aberrant VSMCs hyperplasia and reduced restenosis.     

A Plasmodium α/β‐hydrolase modulates the development of invasive stages

The bud emergence (BEM)46 proteins are evolutionarily conserved members of the α/β‐hydrolase superfamily, which includes enzymes with diverse functions and a wide range of substrates. Here, we identified a Plasmodium BEM46‐like protein (PBLP) and characterized it throughout the life cycle of the rodent malaria parasite Plasmodium yoelii. The Plasmodium BEM46‐like protein is shown to be closely associated with the parasite plasma membrane of asexual erythrocytic stage schizonts and exo‐erythrocytic schizonts; however, PBLP localizes to unique intracellular structures in sporozoites. Generation and analysis of P. yoelii knockout (Δpblp) parasite lines showed that PBLP has an important role in erythrocytic stage merozoite development with Δpblp parasites forming fewer merozoites during schizogony, which results in decreased parasitemia when compared with wild‐type (WT) parasites. Δpblp parasites showed no defects in gametogenesis or transmission to mosquitoes; however, because they formed fewer oocysts there was a reduction in the number of developed sporozoites in infected mosquitoes when compared with WT. Although Δpblp sporozoites showed no apparent defect in mosquito salivary gland infection, they showed decreased infectivity in hepatocytes in vitro. Similarly, mice infected with Δpblp sporozoites exhibited a delay in the onset of blood‐stage patency, which is likely caused by reduced sporozoite infectivity and a discernible delay in exo‐erythrocytic merozoite formation. These data are consistent with the model that PBLP has an important role in parasite invasive‐stage morphogenesis throughout the parasite life cycle.     

Polymorphism of chicken myocyte-specific enhancer-binding factor 2A gene and its association with chicken carcass traits

Myocyte-specific enhancer-binding factor 2A (MEF2A) gene is a member of the myocyte-specific enhancer-binding factor 2 (MEF2) protein family which involved in vertebrate skeletal muscle development and differentiation. The aim of the current study is to investigate the potential associations between MEF2A gene SNPs (single nucleotide polymorphisms) and the carcass traits in 471 chicken samples from four populations. Three new SNPs (T46023C, A72626G, and T89232G) were detected in the chicken MEF2A gene. The T46023C genotypes were associated with live body weight (BW), carcass weight (CW), eviscerated weight, semi-eviscerated weight (SEW), and leg muscle weight (LMW) (P < 0.05); the A72626G genotypes were associated with BW, CW, LMW (P < 0.01) and breast muscle weight (BMW), leg muscle percentage (LMP) (P < 0.05); whereas the T89232G genotypes were associated with carcass percentage (CP) and semi-eviscerated percentage (SEP) (P < 0.05). The haplotypes constructed on the three SNPs were associated with BW, CW, LMW (P < 0.01), SEW, BMW, CP (P < 0.05). Significantly and suggestive dominant effects of diplotype H1H2 were observed for BW, CW, SEW, BMW and CP, whereas diplotype H5H5 had a negative effect on BW, CW, SEW, BMW and LMW. Our results suggest that the MEF2A gene may be a potential marker affecting the muscle trait of chickens.     

Decreased bone mineral density and reduced bone quality in H(+) /K(+) ATPase beta-subunit deficient mice

Proton pump inhibitors (PPIs) are widely used against gastroesophageal reflux disease. Recent epidemiological studies suggest that PPI users have an increased risk of fractures, but a causal relationship has been questioned. We have therefore investigated the skeletal phenotype in H⁺/K⁺ATPase beta‐subunit knockout (KO) female mice. Skeletal parameters were determined in 6‐ and 20‐month‐old KO mice and in wild‐type controls (WT). Whole body bone mineral density (BMD) and bone mineral content (BMC) were measured by dual energy X‐ray absorptiometry (DXA). Femurs were examined with µCT analyses and break force were examined by a three‐point bending test. Plasma levels of gastrin, RANKL, OPG, osteocalcin, leptin, and PTH were analyzed. KO mice had lower whole body BMC at 6 months (0.53 vs. 0.59 g, P = 0.035) and at 20 months (0.49 vs. 0.74 g, P < 0.01) compared to WT as well as lower BMD at 6 months (0.068 vs. 0.072 g/cm², P = 0.026) and 20 months (0.067 vs. 0.077 g/cm², P < 0.01). Mechanical strength was lower in KO mice at the age of 20 months (6.7 vs. 17.9 N, P < 0.01). Cortical thickness at 20 months and trabecular bone volume% at 6 months were significantly reduced in KO mice. Plasma OPG/RANKL ratio and PTH was increased in KO mice compared to controls. H⁺/K⁺ATPase beta subunit KO mice had decreased BMC and BMD, reduced cortical thickness and inferior mechanical bone strength. Whereas the mechanism is uncertain, these findings suggest a causal relationship between long‐term PPI use and an increased risk of fractures. J. Cell. Biochem. 113: 141–147, 2012. © 2011 Wiley Periodicals, Inc.     

MEF2C在湿性年龄相关性黄斑变性中的表达及其对脉络膜新生血管和巨噬细胞极化的影响

摘要 目的：揭示肌细胞增强因子2C（MEF2C）在湿性年龄相关性黄斑变性（AMD）中的表达及其对脉络膜新生血管（CNV）和巨噬细胞极化的影响。方法：通过qRT-PCR法检测30例湿性AMD患者（AMD组）和30例健康体检者（健康对照组）的血清MEF2C水平。将MEF2C过表达慢病毒（MEF2C-LV组）和阴性对照过表达慢病毒（NC-LV组）转染至恒河猴脉络膜血管内皮细胞系（RF/6A）。转染后,将RF/6A细胞分为常氧组（Normoxia组）、低氧组（Hypoxia组）、低氧+NC-LV组（Hypoxia+NC-LV组）、低氧+MEF2C-LV组（Hypoxia+MEF2C-LV组）。转染及缺氧处理后,分别测定各组细胞进行Matrigel小管。通过激光诱导CNV C57BL/6J小鼠模型,将建模成功的C57BL/6J小鼠随机分为模型组、NC-LV组和MEF2C-LV组,每组10只,未建模的小鼠作为对照组。然后对NC-LV组和MEF2C-LV组小鼠玻璃体腔注射NC-LV或MEF2C-LV,对照组和模型组小鼠不进行治疗。治疗7 d后进行眼底荧光血管造影（FFA）和眼球苏木精伊红（HE）染色。通过qRT-PCR和Western blot检测MEF2C、VEGFA、VEGFR2、IL-12p35、IL-12p40和IL-10的mRNA和蛋白表达。结果：与Healthy组相比,AMD组患者的血清MEF2C水平显著降低（1.00±0.23 vs 0.48±0.29,t=7.689,P<0.001）。与Normoxia组相比,Hypoxia组的闭合管腔数量增加（P<0.05）。与Hypoxia组相比,Hypoxia+MEF2C-LV组的闭合管腔数量减少（P<0.05）。与模型组相比,MEF2C-LV组视网膜和脉络膜病变程度减轻,结构基本恢复正常,脉络膜组织厚度降低,血管生成减少。与模型组相比,MEF2C-LV组的CNV相对荧光强度降低,脉络膜组织中MEF2C、VEGFA和VEGFR2的mRNA和蛋白表达水平均降低（P<0.05）。与模型组相比,MEF2C-LV组脉络膜组织中IL-12p35和IL-12p40的mRNA和蛋白表达水平均升高,IL-10均降低（P<0.05）。结论：MEF2C在湿性AMD患者血清中低表达,上调MEF2C可抑制脉络膜血管生成,并促进巨噬细胞从M2型向M1型的转换。     

Metabolic Syndrome and Changes in Body Fat From a Low‐fat Diet and/or Exercise Randomized Controlled Trial

It is difficult to identify the successful component(s) related to changes in metabolic syndrome (MetS) from lifestyle interventions: the weight loss, the behavior change, or the combination. The purpose of this study is to determine the effects of a weight‐stable randomized controlled trial of low‐fat diet and exercise, alone and in combination, on MetS. Men (n = 179) and postmenopausal women (n = 149) with elevated low‐density lipoprotein cholesterol (LDL‐C) and low high‐density lipoprotein cholesterol (HDL‐C) were randomized into a 1‐year, weight‐stable trial with four treatment groups: control (C), diet (D), exercise (E), or diet plus exercise (D+E). MetS was defined using a continuous score. Changes in MetS score (ΔMetS) were compared between groups using analysis of covariance, stratified by gender and using two models, with and without baseline and change in percent body fat (ΔBF) as a covariate. In men, ΔMetS was higher for D vs. C (P = 0.04), D+E vs. C (P = 0.0002), and D+E vs. E (P = 0.02). For women, ΔMetS was greater for D vs. C (P = 0.045), E vs. C (P = 0.02), and D+E vs. C (P = 0.004). After adjusting for ΔBF, all differences between groups were attenuated and no longer significant. ΔMetS were associated with ΔBF for both men (P < 0.0001) and women (P = 0.004). After adjustment for ΔBF, low‐fat diet alone and in combination with exercise had no effect on MetS. The key component for MetS from low‐fat diet and/or increased physical activity appears to be body fat loss.     

脂多糖对离体培养大鼠血管平滑肌细胞增殖的影响

本研究观察到１０－７～１０－５ｋｇ／Ｌ脂多糖（ｌｉｐｏｐｏｌｙｓａｃｈａｒｉｄｅ,ＬＰＳ）可显著促进血管平滑肌细胞（ＶＳＭＣ）的增殖及ＤＮＡ的合成（Ｐ＜００５）。５×１０－４～１０－３ｋｇ／ＬＬＰＳ却抑制ＶＳＭＣ的增殖及ＤＮＡ的合成,降低其活力（Ｐ＜００１）,并呈时间依赖效应。一氧化氮合酶抑制剂ＮＮｉｔｒｏＬＡｒｇｉｎｉｎｅ（ＬＮＮＡ）可拮抗ＬＰＳ的抑制作用。大剂量ＬＰＳ作用组ＶＳＭＣ上清液中一氧化氮（ＮＯ）代谢产物ＮＯ－３和ＮＯ－２的含量与对照组相比显著增加（Ｐ＜００１）,４８ｈ组比２４ｈ组增加９１％,７２ｈ组比４８ｈ组增加４５％;同时,诱导性一氧化氮合酶（ｉｎｄｕｃｔｉｖｅｎｉｔｒｉｃｏｘｉｄｅｓｙｎｔｈａｓｅ,ｉＮＯＳ）免疫组化染色呈阳性。结果表明,低浓度ＬＰＳ促进ＶＳＭＣ增殖和ＤＮＡ合成,而高浓度ＬＰＳ却明显抑制ＶＳＭＣ增殖和ＤＮＡ合成,降低其活力。这种抑制作用可能与ＬＰＳ诱导ＶＳＭＣ产生的ＮＯ有关。     

Suppression and synthetic‐lethal genetic relationships of ΔgpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin‐binding protein interactions in Streptococcus pneumoniae D39

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side‐wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division‐protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co‐IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP‐PBP2x.     

研究报告: 基于AS1411适配体的DNA-RNA杂交载体的抗肿瘤研究

目的 研究连接适配体的DNA-RNA分子作为杂交载体靶向肿瘤细胞并导入功能性RNA分子进入细胞的有效性，以及对肿瘤细胞的影响。方法 设计合成短的互补DNA、RNA分子，组装成DNA-RNA杂合链；连接AS1411适配体为靶向分子，再分别连接p21 saRNA和TIGIT siRNA作为药物分子，记为P21 saRNA和TIGIT siRNA，构成杂交载体，通用结构式为AS1411-DNA/RNA-sxRNA；检测AS1411-DNA/RNA-sxRNA能否靶向结合并进入肿瘤细胞及其对瘤细胞生存、迁移、侵袭和凋亡的影响。结果 将设计的杂交载体各部分等摩尔加入杂交缓冲体系并于特定温度条件下孵育，TBM聚丙烯酰胺凝胶电泳检测到AS1411-DNA/RNA-sxRNA成功组装；AS1411-DNA/RNA-sxRNA杂交载体在10%血清条件下也显示出良好的抗降解稳定性；荧光显微镜和激光共聚焦显微镜下观察，SKOV3细胞表面及胞内存在绿色大量荧光信号，杂交载体成功进入肿瘤细胞。杂交载体孵育后：在mRNA水平上，p21基因表达（2.14±0.25）是对照组（1.02±0.10）2倍以上，P<0.05；TIGIT基因表达（0.63±0.09）低于对照组（1.09±0.15），P<0.05；在蛋白质水平上，p21基因表达（1.57±0.16）是对照组（1.10±0.09）1.5倍以上，P<0.05；TIGIT基因表达（0.61±0.12）低于对照组（1.01±0.07），P<0.05。CCK-8实验显示，P21 saRNA（3.10±0.13）和TIGIT siRNA（2.91±0.13）杂交载体孵育组与空白对照组（3.67±0.15）相比，卵巢癌细胞增殖能力显著下降（P<0.05）；划痕实验结果显示，P21 saRNA孵育组愈合率（42.53±2.90）%、TIGIT siRNA孵育组愈合率（36.23±3.43）%，明显低于空白对照组（76.47±3.64）%，P<0.05；Transwell检测迁移能力发现：P21 saRNA孵育组（128.25±5.36）、TIGIT siRNA孵育组（119.50±8.79）低于对照组（186.5±8.56）；侵袭能力：P21 saRNA孵育组（145.5±9.45）、TIGIT siRNA孵育组（112.25±5.63）也显著低于对照组（202.50±10.12），P<0.05；细胞凋亡率：P21 saRNA孵育组（11.74%±2.47%）、TIGIT siRNA孵育组（17.12%±2.04%）明显高于对照组（5.66%±1.44%），P<0.05。结论 所制备的AS1411-DNA/RNA-sxRNA杂交载体能够有效靶向肿瘤细胞，携带功能性小RNA靶向导入肿瘤细胞并调控目的基因表达，使肿瘤细胞的增殖、侵袭和迁移能力受到抑制；该结果为利用DNA-RNA偶联AS1411适配体作为靶向工具的杂交载体，靶向杀伤表面表达NCL蛋白的肿瘤细胞提供了实验基础。     

The apolipoprotein A‐I mimetic peptide,D‐4F,restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.     

SQSTM1/p62 and PPARGC1A/PGC-1alpha at the interface of autophagy and vascular senescence

ABSTRACT

Defective macroautophagy/autophagy and mitochondrial dysfunction are known to stimulate senescence. The mitochondrial regulator PPARGC1A (peroxisome proliferator activated receptor gamma, coactivator 1 alpha) regulates mitochondrial biogenesis, reducing senescence of vascular smooth muscle cells (VSMCs); however, it is unknown whether autophagy mediates PPARGC1A-protective effects on senescence. Using ppargc1a^?/- VSMCs, we identified the autophagy receptor SQSTM1/p62 (sequestosome 1) as a major regulator of autophagy and senescence of VSMCs. Abnormal autophagosomes were observed in VSMCs in aortas of ppargc1a^?/- mice. ppargc1a^?/- VSMCs in culture presented reductions in LC3-II levels; in autophagosome number; and in the expression of SQSTM1 (protein and mRNA), LAMP2 (lysosomal-associated membrane protein 2), CTSD (cathepsin D), and TFRC (transferrin receptor). Reduced SQSTM1 protein expression was also observed in aortas of ppargc1a^?/- mice and was upregulated by PPARGC1A overexpression, suggesting that SQSTM1 is a direct target of PPARGC1A. Inhibition of autophagy by 3-MA (3 methyladenine), spautin-1 or Atg5 (autophagy related 5) siRNA stimulated senescence. Rapamycin rescued the effect of Atg5 siRNA in Ppargc1a^+/+ , but not in ppargc1a^?/- VSMCs, suggesting that other targets of MTOR (mechanistic target of rapamycin kinase), in addition to autophagy, also contribute to senescence. Sqstm1 siRNA increased senescence basally and in response to AGT II (angiotensin II) and zinc overload, two known inducers of senescence. Furthermore, Sqstm1 gene deficiency mimicked the phenotype of Ppargc1a depletion by presenting reduced autophagy and increased senescence in vitro and in vivo. Thus, PPARGC1A upregulates autophagy reducing senescence by a SQSTM1-dependent mechanism. We propose SQSTM1 as a novel target in therapeutic interventions reducing senescence.     

下调血管生成素样蛋白7表达对血管紧张素Ⅱ介导的血管平滑肌细胞炎症反应的影响

目的探讨RNA干扰血管生成素样蛋白7 （Angptl7）基因对血管紧张素Ⅱ（AngⅡ）诱导的血管平滑肌细胞（VSMC）炎症因子的影响及其作用机制。 方法体外培养人VSMC,分为常规F12K培养基培养（对照）和1 μg/mL AngII培养24 h。VSMC用AngⅡ（1 μg/mL）处理24 h后,采用siRNA-Angptl7和阴性对照siRNA-NC在Lipofectamine 2000介导下转染VSMC。RT-qPCR检测mRNA表达水平;Griess反应测定一氧化氮（NO）含量;蛋白免疫印记法检测相关蛋白的改变;酶联免疫吸附法检测VSMC中炎症因子肿瘤坏死因子α（TNF-α）、白细胞介素-1β （IL-1β）和IL-6水平。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验,两组间比较采用独立样本t检验。 结果与对照比较,1 μg/mL AngⅡ处理可促进VSMC中Angptl7 mRNA （0.97±0.06比3.05±0.21）和蛋白表达（1.01±0.12比1.61±0.14）,亦可促进VSMC中IL-1β[（45.21±8.10）比（126.17±11.77） pg/mL]、IL-6[（50.50±7.51）比（108.50±9.51）pg/mL]和TNF-α的表达[（60.77±9.58）比（185.67±17.35）pg/mL],差异有统计学意义（P均< 0.01）。与对照和转染siRNA-NC相比,转染siRNA-Angptl7下调Angptl7蛋白表达（0.99±0.12,0.98±0.12比0.44±0.14,P < 0.01）。与AngⅡ干预组相比,siRNA-Angptl7降低AngⅡ介导的VSMC炎症反应相关蛋白TNF-α、IL-6和IL-1β的表达,核因子κB （NF-κB）/诱导型一氧化氮合酶（iNOS）/环氧化酶2 （COX-2）信号通路相关蛋白NF-κB、iNOS和COX-2表达及NO含量亦降低,差异有统计学意义（P均< 0.01）。与siRNA-NC相比,siRNA-Angptl7组AngⅡ诱导的VSMC炎症反应相关蛋白TNF-α （0.99±0.13比0.51±0.12）、IL-6 （1.00±0.12比0.38±0.05）和IL-1β的表达（0.99±0.14比0.48±0.11）,NF-κB （1.00±0.10比0.42±0.08）、iNOS （1.02±0.12比0.42±0.10）和COX-2表达（1.00±0.11比0.52±0.12）均降低,NO含量[（54.78±2.76）比（18.08±3.61）μmol/L]亦降低,差异有统计学意义（P均< 0.01）。 结论AngⅡ可通过Angptl7促进VSMC炎症反应,下调Angptl7蛋白表达可以抑制VSMC的炎症反应,其作用机制可能与抑制NF-κB/iNOS-COX-2信号通路有关。     

Δ133p53 decreases the chemosensitivity of carcinoma cell line H1299

The research evaluated the effect of Δ133p53 on the chemosensitivity of lung adenocarcinoma cell line H1299. By this study, the drug‐resistant molecular marker and a new target for cancer therapy could be provided. Δ133p53 or negative control plasmid were transferred into H1299 cells by lentivirus vector. The expression of Δ133p53 in transfected cells was examined using immunofluorescence. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) method and colony formation test were applied to detect drug sensitivity after cisplatin or 5‐fluorouracil (5‐FU) treatment. After cisplatin (CDDP)/FU treatment, MTT assay demonstrated that the inhibition rate of H1299/Δ133p53 cell was reduced compared with that of the H1299 and H1299/NEG cells at the same concentration of drug. The 50% inhibitory concentrations (IC ₅₀) of CDDP and 5‐FU rose by 36.1 and 30.2%, respectively (P < 0.05). The colony formation assay suggested that the cell proliferation ability of H1299/Δ133p53 cell was prominently increased when compared with that of control group H1299 and H1299 /NEG cells (P < 0.05). The present study demonstrated that the transfection of the Δ133p53 gene in H1299 cells led to the reduction of chemosensitivity.     

Asiaticoside might attenuate bleomycin‐induced pulmonary fibrosis by activating cAMP and Rap1 signalling pathway assisted by A2AR

Asiaticoside (AS) has been reported to have protective effect on pulmonary fibrosis (PF). In this study, we aimed to explore the potential mechanism of the therapeutic role of AS and its relationship with A2AR in PF. Adenosine 2A receptor gene knockout (A2AR^?/?) mice and wild‐type (WT) mice were used to establish bleomycin (BLM)‐induced PF models and were then treated with AS (50 mg/kg/d). Pulmonary inflammation and fibrosis were observed in the PF model with much higher severity in A2AR^?/?mice than that in WT mice and AS significantly alleviated lung inflammation and fibrosis; however, it was less effective in A2AR^?/? mice than in WT mice via histopathological analysis. Using RNA sequencing analysis, we found up‐regulated differentially expressed genes (DEGs) in BLM group were enriched in immune and inflammation‐associated pathways compared with control group. There were 242 common DEGs between down‐regulated in BLM vs control group and up‐regulated in BLM + AS vs BLM group, which were enriched in cAMP and Rap1 signalling pathways. Furthermore, the expression of five key factors of these two pathways including adenylate cyclase (ADCY1, ADCY5, ADCY8, cAMP and Rap1) were confirmed up‐regulated by AS with the presence of A2AR. Therefore, AS might attenuate BLM‐induced PF by activating cAMP and Rap1 signalling pathways which is assisted by A2AR, making it a promising therapeutic optional for PF.