Effect of retinoic acid and delta-like 1 homologue (DLK1) on differentiation in neuroblastoma

The principal objective of this study was to evaluate the chemopreventive and therapeutic effects of a combination of all-trans-retinoic acid (RA) and knockdown of delta-like 1 homologue (Drosophila) (DLK1) on neuroblastoma, the most common malignant disease in children. As unfavorable neuroblastoma is poorly differentiated, neuroblastoma cell was induced differentiation by RA or DLK1 knockdown. Neuroblastoma cells showed elongated neurite growth, a hallmark of neuronal differentiation at various doses of RA, as well as by DLK1 knockdown. In order to determine whether or not a combination of RA and DLK1 knockdown exerts a greater chemotherapeutic effect on neuroblastoma, cells were incubated at 10 nM RA after being transfected with SiRNA-DLK1. Neuronal differentiation was increased more by a combination of RA and DLK1 knockdown than by single treatment. Additionally, in order to assess the signal pathway of neuroblastoma differentiation induced by RA and DLK1 knockdown, treatment with the specific MEK/ERK inhibitors, U0126 and PD 98059, was applied to differentiated neuroblastoma cells. Differentiation induced by RA and DLK1 knockdown increased ERK phosphorylation. The MEK/ERK inhibitor U0126 completely inhibited neuronal differentiation induced by both RA and DLK1 knockdown, whereas PD98059 partially blocked neuronal differentiation. After the withdrawal of inhibitors, cellular differentiation was fully recovered. This study is, to the best of our knowledge, the first to demonstrate that the specific inhibitors of the MEK/ERK pathway, U0126 and PD98059, exert differential effects on the ERK phosphorylation induced by RA or DLK1 knockdown. Based on the observations of this study, it can be concluded that a combination of RA and DLK1 knockdown increases neuronal differentiation for the control of the malignant growth of human neuroblastomas, and also that both MEK1 and MEK2 are required for the differentiation induced by RA and DLK1 knockdown.     

全反式维甲酸对大鼠肾脏MMPs/TIMPs影响

目的 观察全反式维甲酸(ATRA)对肾小球硬化(GS)大鼠肾脏基质金属蛋白酶及其组织抑制因子的影响,并探讨其作用机制.方法 80只Wistar大鼠随机分为假手术组、模型组、苯那普利组和ATRA组,每组20只.用单侧肾切除加尾静脉注射阿霉素建立GS模型,造模后12周末处死.肾脏病理切片行苏木素-伊红(HE)染色,计算肾小球硬化指数(GSI);免疫组化法检测肾组织Ⅵ型胶原(Col-Ⅳ)、纤维连接蛋白(FN)的蛋白表达;实时定量反转录-聚合酶链反应(Realtime RT-PCR)法检测肾组织基质金属蛋白酶-2、9(MMP-2、9)及其组织抑制因子-1(TIMP-1)mRNA的表达.结果 与模型组比较,ATRA组与苯那普利组大鼠GSI均显著降低(均P＜0.01),Col-Ⅳ和FN的蛋白表达均显著减少(均P＜0.05),TIMP-1 mRNA表达显著下调(P＜0.05),MMP-2和MMP-9的mRNA表达均显著上调(均P＜0.05),但2组间差异无统计学意义(P＞0.05).结论 ATRA可能通过下调GS大鼠肾组织TIMP-1的表达,上调MMP-2和MMP-9的表达,促使肾小球细胞外基质(ECM)的降解增加,减少ECM积聚等机制,起到减轻GS的作用.     

Zinc deficiency regulates hippocampal gene expression and impairs neuronal differentiation

Abstract

Objectives

Proliferating adult stem cells in the subgranular zone of the dentate gyrus have the capacity not only to divide, but also to differentiate into neurons and integrate into the hippocampal circuitry. The present study identifies several hippocampal genes putatively regulated by zinc and tests the hypothesis that zinc deficiency impairs neuronal stem cell differentiation.

Methods

Genes that regulate neurogenic processes were identified using microarray analysis of hippocampal mRNA isolated from adult rats fed zinc-adequate or zinc-deficient (ZD) diets. We directly tested our hypothesis with cultured human neuronal precursor cells (NT2), stimulated to differentiate into post-mitotic neurons by retinoic acid (RA), along with immunocytochemistry and western analysis.

Results

Microarray analysis revealed the regulation of genes involved in cellular proliferation. This analysis also identified a number of genes known to be involved in neuronal differentiation, including the nuclear RA receptor, retinoid X receptor (RXR), doublecortin, and a transforming growth factor-beta (TGF-β) binding protein (P < 0.05). Zinc deficiency significantly reduced RA-induced expression of the neuronal marker proteins doublecortin and β-tubulin type III (TuJ1) to 40% of control levels (P < 0.01). This impairment of differentiation may be partially mediated by alterations in TGF-β signaling. The TGF-β type II receptor, responsible for binding TGF-β during neuronal differentiation, was increased 14-fold in NT2 cells treated with RA (P < 0.001). However, this increase was decreased by 60% in ZD RA-treated cells (P < 0.001).

Discussion

This research identifies target genes that are involved in governing neurogenesis under ZD conditions and suggests an important role for TGF-β and the trace metal zinc in regulating neuronal differentiation.     

红细胞铁蛋白含量对新生儿生长发育的影响

目的探讨红细胞铁蛋白(EF)对新生儿生长发育的影响。方法采用放射免疫法测定115对正常产妇及其新生儿EF。结果母血EF含量与新生儿脐血EF含量呈显著正相关(r=025,P<001);母血EF高值组新生儿,其Hb、体重、身长、头围、胎龄的均值优于母血EF低值组新生儿,差异显著(u=409、233、245、289、264;分别P<001、P<005、P<005、P<001、P<001)。结论母亲EF含量在新生儿生长发育中起着重要作用。     

Effect of pre- and postnatal essential fatty acid deficiency on brain development and myelination.

Pregnant mice were fed an essential fatty acid (EFA) deficient diet from day 1 of gestation. Several biochemical parameters of postnatal brain growth and myelination were measured on their progeny and compared with controls fed a normal diet containing 4% corn oil or a commercial breeder diet. Measurements of brain DNA, RNA and protein content of the EFA deficient mice suggested a retardation of brain growth and development of about 1 week compared to controls, with the most striking differences noted at ages below 15 days. DNA content of both control and experimental mice became comparable at 20 to 22 days but brain protein and RNA content remained lower in deficient mice at all ages studied. The levels of several myelin specific constituents were also measured in experimental and control brains. The brain galactolipid content was severely depressed at levels 21% of controls at 40 days of age. Proteolipid protein was also significantly reduced (23% of controls at 40 days of age). In contrast, the activity of the myelin marker enzyme, 2',3'-cyclic nucleotide-3'-phosphodiesterase, appeared to be totally unaffected by EFA deficiency. The results indicate that pre-and postnatal EFA deficiency results in a retardation of brain development and a profound reduction of some but not all myelin specific components.     

维生素A酸对体外培养小鼠囊胚发育的影响

目的:观察维生素A酸(RA)对小鼠囊胚细胞的凋亡效应,探讨RA对体外培养小鼠囊胚发育的影响。方法:获取妊娠3.5天小鼠囊胚,分别培养在含0、1μmol/L和10μmol/L的RA的M199培养基中24 h,用带有荧光(FITC)标记的原位末端标记检测法(TUNEL)检测RA对小鼠囊胚细胞凋亡的影响。结果:RA可诱导囊胚细胞凋亡。结论:维生素A酸对小鼠囊胚的发育具有细胞毒性作用。     

Increase of 63 kDa protein kinase in the nuclear matrix of HL-60 cells during differentiation by retinoic acid

The changes of protein kinases (PKs) in the nuclear matrix of HL-60 cells during the differentiation by retinoic acid (RA) were examined by in situ assay after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the nuclear matrix of HL-60, at least seven species of PKs (83, 63, 58, 46, 42, 38, and 31 kDa) were always detected. Among these PKs, 63 kDa PK was increased dramatically during differentiation by RA. Two-dimensional electrophoresis revealed that two species of 63 kDa PKs (pI 5.0 and 5.4) were presented in the nuclear matrix and increased similarly during the differentiation. This increase of 63 kDa PKs may be related to the lobulation of the nucleus and the nuclear matrix accompanying the differentiation by RA.     

视黄酸对肺过敏性炎症巨噬细胞功能影响

目的探讨视黄酸(RA)对肺过敏性炎症中巨噬细胞(Ma)功能的影响。方法采用鸡卵清蛋白(OVA)建立SD大鼠致敏模型,并持续吸入30 d激发大鼠哮喘;在石蜡油组、RA组、RA+皮质激素组雾化吸入治疗后,光学及电子显微镜观察各组大鼠肺、胸腺、脾脏过敏性炎症特点,以及Ma结构功能变化特征。结果激发后30~40 d,石蜡油组和RA+皮质激素组肺泡上皮细胞、内皮细胞与肺泡Ma均以空泡变性为主;石蜡油组致敏细胞较多,与肺泡Ma呈高度正相关(r=0.933);脾Ma退化伴随脾滤泡增生;RA+皮质激素组肺泡损伤和炎症较重,后期肺泡Ma与致敏细胞呈正相关(r=0.7);RA组肺泡受损较轻,超微结构恢复较好,肺泡Ma吞噬功能活跃,与致敏细胞呈负相关(r=-0.862),退化脾Ma较少,胸腺、脾增生渐弱。结论 RA可提高Ma吞噬功能,减少吸入抗原,保护肺泡结构;退化Ma可能导致内环境紊乱与免疫失衡。     

维甲酸对缺氧缺血性脑损伤新生鼠内源性神经干细胞巢蛋白表达的影响

【目的】研究维甲酸对缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)新生鼠内源性神经干细胞(NSCs)巢蛋白(Nestin)表达的影响。【方法】将56只7日龄HIBD新生SD乳鼠随机分为生理盐水组(对照组,n=28)及维甲酸治疗组(治疗组,n=28)。根据处死时相点每组分成3、7、14、21d4个小组,每小组7只。HIBD模型建立通过结扎7日龄新生SD乳鼠血管左颈总动脉后置于8%氧浓度的低氧环境中2.5h。HIBD的维甲酸治疗组在模型后开始每天腹腔注射维甲酸500μg/kg1次,对照组腹腔注射生理盐水。对各时相的神经功能缺损进行评分;采用免疫组织化学染色以及光镜技术分别对SD大鼠脑组织中的NSCs进行检测,Nestin阳性细胞标志神经干细胞,其数量增加表明神经干细胞的增殖。【结果】维甲酸治疗与对照组在各时相上的神经功能缺损评分比较差异均有显著性(P<0.01);第3、7、14d和21d在HIBD后的伤侧皮质及海马周围检出巢蛋白阳性细胞,分别在3、7d达高峰,随病程的延长而减少:维甲酸治疗组在巢蛋白阳性表达增高与对照组同时相上比较差异显著(P<0.01)。【结论】维甲酸具有促进HIBD新生鼠内源性神经干细胞巢蛋白阳性表达保持在较高水平的作用。     

Effect of all-trans retinol and retinoic acid nutriture on the immune system of chicks

The effects of all-trans retinol and retinoic acid (RA) on the humoral and cell-mediated immune response and on lymphoid organ weights of broiler chicks were examined. Chicks were fed diets supplemented with retinol or RA at 0, 0.2 and 2.0 micrograms/g diet. The diets were fed continuously from day of hatch or after depletion of hepatic vitamin A reserves. Rapid vitamin A deficiency was induced in chicks by initially feeding the diet containing 2.0 micrograms RA/g diet and subsequently feeding diets containing 0 or 0.2 microgram RA/g diet. Serum antibody hemagglutination titers, in response to intravenous injection of human serum albumin (HSA), were not affected by level or chemical form of vitamin A supplemented in diets. In vitamin A-deficient chicks, there was an inverse relationship observed between the number of peripheral blood lymphocytes obtained per milliliter of blood and their ability to proliferate in response to mitogenic stimulation. Growth of the bursa of Fabricius and the thymus was impaired in chicks fed a vitamin A-free diet. A partial deficiency of vitamin A adversely affected relative bursa weight but not that of thymus. In general, RA was inferior to retinol in maintenance of lymphoid tissue.     

全反式视黄酸对小鼠胚胎发育毒性的体外实验研究

目的为研究全反式视黄酸（atRA）的发育毒性及其可能的作用机制，本研究采用植入后体外全胚胎培养方法研究atRA对小鼠胚胎生长发育的影响。方法孕8．5日龄小鼠胚胎移植于atRA终浓度为0、0．01、0．1、0．2、0．4、1．0、10．0μmol／L的全胚胎培养基中，体外连续培养48h，观察atRA对胚胎发育和器官形态分化的影响。结果atRA可影响小鼠胚胎在体外的生长发育，并呈现剂量-效应关系；0．1μmol／L组，atRA明显影响卵黄囊直径、颅臀长、前脑、中脑、听觉系统、视觉系统、心脏、体位和前肢芽等指标，与对照组比较，差异有显著性（P〈0．05）；≥0．20μmol／L组，卵黄囊体积减小、表面皱缩、血管减少及分化程度降低，神经系统、心脏、腮弓和颅面等多种器官出现畸形，所有器官形态分化指标均显著低于对照组（P〈0．05）。结论atRA有致畸性和胚胎毒性，在器官形成期可以引起神经系统、心脏、腮弓和颅面等多种器官的发育畸形。     

Carnosic acid inhibits proliferation and augments differentiation of human leukemic cells induced by 1,25-dihydroxyvitamin D3 and retinoic acid

Carnosic acid, the polyphenolic diterpene derived from rosemary, is a strong dietary antioxidant that exhibits antimutagenic properties in bacteria and anticarcinogenic activity in various cell and animal models. In the present study, we show that carnosic acid (2.5-10 microM) inhibits proliferation of HL-60 and U937 human myeloid leukemia cells (half-maximal inhibitory concentration = 6-7 microM) without induction of apoptotic or necrotic cell death. Growth arrest occurred concomitantly with a transient cell cycle block in the G1 phase, which was accompanied by an increase in the immunodetectable levels of the universal cyclin-dependent kinase inhibitors p21WAFI and p27Kipl. Carnosic acid caused only a marginal induction of differentiation, as monitored by the capacity to generate superoxide radicals and the expression of cell surface antigens (CD11b and CD14) and receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine. However, at low concentrations, this polyphenol substantially augmented (100- to 1,000-fold) the differentiating effects of 1,25-dihydroxyvitamin D3 and all-trans retinoic acid. Furthermore, such combinations of carnosic acid and any of these differentiation inducers synergistically inhibited proliferation and cell cycle progression. These results indicate that carnosic acid is capable of antiproliferative action in leukemic cells and can cooperate with other natural anticancer compounds in growth-inhibitory and differentiating effects.     

Vitamin A prevents high fat diet-induced ACF development and modifies the pattern of expression of peroxisome proliferator and retinoic acid receptor m-RNA

Some dietary compounds, among them fats, are modulators of colon cancer risk. This study reports the modulating effects of n-6, with or without vitamin A, on promotion of colon preneoplasic lesions induced by 1,2-dimethylhydrazine (DMH) and on the expression of nuclear receptors (PPARgamma, RXRalpha, and RARbeta). One group of male Fisher rats was fed a basic diet (5% safflower oil) and two groups were fed a high-fat diet (HFD, 25% safflower oil). Of these, one was supplemented with 200 IU vitamin A for 5 mo. The safflower oil contained polyunsaturated fatty acids, mainly linoleic acid (73%). The data showed an increasing effect of safflower oil-enriched diet on aberrant crypt foci occurrence and multiplicity. This effect was impaired by vitamin A supplementation. In addition, an HFD-related up-regulation of PPARgamma and a concomitant down-regulation of RARbeta mRNA expression were observed with or without chemical initiation and were prevented by vitamin A. Moreover, when treated with DMH, HFD rats exhibited a dramatically decreased expression of RXRalpha mRNA (-49%). It was hypothesized that HFD, leading to hyperexpression of PPARgamma, would produce an alteration of retinoic acid signaling and, in this way, create a background modulating colon cancer risk.     

维甲酸致神经管畸形中牡蛎拮抗神经细胞凋亡的实验研究

目的 探讨维甲酸(retinoic acid,RA)致神经管畸形(neural tube defects,NTDs)中牡蛎拮抗神经细胞凋亡的分子机制。方法 建立NTDs模型并给予牡蛎拮抗,观察神经细胞的凋亡情况及凋亡相关蛋白Bcl-2、Bax与Caspase-3表达的变化。结果 1)RA组神经管上皮细胞凋亡指数明显高于对照组,牡蛎组凋亡指数明显低于RA组(P＜0.05)。2)RA组神经上皮细胞Bcl-2的表达明显低于对照组,牡蛎组Bcl-2的表达明显高于RA组(P＜0.05)。3)RA组Bax与Caspase-3的表达均明显高于对照组,牡蛎组Bax与Caspase-3的表达明显低于RA组(P＜0.05)。 结论 牡蛎可以拮抗RA引起的神经上皮细胞过度凋亡,其机理可能是通过上调Bcl-2的表达和下调Bax与Caspase-3的表达实现的。