20-Hydroxyecdysone from Dacrycarpus imbricatus bark inhibits the proliferation of acute myeloid leukemia cells

Objective:To investigate the anti-proliferative effects of 20-hydroxyecdysone isolated from the bark of Dacrycarpus imbricatus(Blume) de Laub.Methods:Column chromatography was used for isolation of compounds from plant material.The structure of the isolated compound was identified by mass spectrometry and nuclear magnetic resonance techniques,including HSQC,HMBC,NOE-difference experiments.The isolated compound was tested for its anti-proliferative activity in acute myeloid leukemia(AML) and OCI-AML cells.Results:Compound 1 was isolated from the ethyl acetate fraction of Dacrycarpus imbricatus barks by column chromatography.Its chemical structure was identified as 20-hydroxyecdysone(20HE),a cholestane-type ecdysteroid,by a combination of mass spectrometry and nuclear magnetic resonance spectrometric analyses.Our goal was to test the anti-proliferative activity of 20 HE using the OCI-AML cell line.20 HE significantly decreased OCI cell number at a concentration of 1 mg/mL,whereas lower concentrations were ineffective.Moreover,this decrease was due to partial blockage of the G_1/S phase of the cell cycle,with a reduction of cells in the G_2M phase,not due to increased apoptosis.Conclusions:This indicates that 20 HE significantly decreases the number of cells in the G_1/S phase of the cell cycle in human AML cells.This is the first time that the anti-proliferative activity of 20 HE against a human tumor cell line has been reported.

20-Hydroxyecdysone Improves Neuronal Differentiation of Adult Hippocampal Neural Stem Cells in High Power Microwave Radiation-Exposed Rats

Objective The hippocampus is thought to be a vulnerable target of microwave exposure.The aim of the present study was to investigate whether 20-hydroxyecdysone(20E)acted as a fate regulator of adult rat hippocampal neural stem cells(NSCs).Furthermore,we investigated if 20E attenuated high power microwave(HMP)radiation-induced learning and memory deficits.Methods Sixty male Sprague-Dawley rats were randomly divided into three groups:normal controls,radiation treated,and radiation+20E treated.Rats in the radiation and radiation+20E treatment groups were exposed to HPM radiation from a microwave emission system.The learning and memory abilities of the rats were assessed using the Morris water maze test.Primary adult rat hippocampal NSCs were isolated in vitro and cultured to evaluate their proliferation and differentiation.In addition,hematoxylin&eosin staining,western blotting,and immunofluorescence were used to detect changes in the rat brain and the proliferation and differentiation of the adult rat hippocampal NSCs after HPM radiation exposure.Results The results showed that 20E induced neuronal differentiation of adult hippocampal NSCs from HPM radiation-exposed rats via the Wnt3a/β-catenin signaling pathway in vitro.Furthermore,20E facilitated neurogenesis in the subgranular zone of the rat brain following HPM radiation exposure.Administration of 20E attenuated learning and memory deficits in HPM radiation-exposed rats and frizzled-related protein(FRZB)reduced the 20E-induced nuclear translocation ofβ-catenin,while FRZB treatment also reversed 20E-induced neuronal differentiation of NSCs in vitro.Conclusion These results suggested that 20E was a fate regulator of adult rat hippocampal NSCs,where it played a role in attenuating HPM radiation-induced learning and memory deficits.

Effects of Salinity Fluctuation Frequency on the Growth, Molting Rate and Hemolymph 20-Hydroxyecdysone Concentration in Juvenile Chinese shrimp, Fenneropenaeus chinensis

This experiment was conducted to examine the effects of salinity fluctuation frequency on the growth, molting rate and hemolymph 20-hydroxyecdysone (20-HE) concentration for juvenile Fenneropenaeus chinensis with an initial wet body weight of 1.115 g±0.012g. The salinity of the control treatment (represented by D0) was 28 throughout the experiment, whereas treatments D2, D4,D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The range of salinity fluctuation was 4. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8 d, respectively; each original salinity was decreased abruptly to salinity 24, which first lasted for another 2 d and was then raised to its initial value 28. This constituted a salinity fluctuation cycle and afterwards the cycle repeated. It was found that: 1) The weight gain of shrimp in treatment D4 was 202.9% and significantly higher than that in treatment D0 (P<0.05). The molting rate in treatment D2 was the lowest, while that in treatment D6 was the highest and significantly higher than those in treatments D0 and D2 (P<0.05). 2) The hemolymph 20-HE concentration of shrimps in all treatments was at low levels and increased first gradually during the inter-molt period and then increased sharply. It reached a peak value during the pre-molt stage, and then decreased abruptly during the post-molt stage when the lowest level occurred. 3) During the post-molt stage, the hemolymph 20-HE concentration in treatment D2 was 7.47 pg μL-1 and significantly higher than that in treatment D0 (P<0.05). There were, however, no significant differences in hemolymph 20-HE concentration among all five treatments during the inter-molt stage (P>0.05). During the pre-molt stage, the hemolymph 20-HE concentration in treatment D6 was significantly higher than that in treatment D0 (P<0.05), whereas the hemolymph 20-HE concentration in treatment D2 was significantly lower than that in other treatments (P<0.05). Adequate salinity fluctuation promoted the molting rate through increasing hemolymph 20-HE concentration at the pre-molt stage.

Epidermal collagenase activity and its induction by 20-hydroxyecdysone in the fiddler crab Uca pugilator

The epidermal collagenase activity and its induction by 20-hydroxyecdysone in Uca pugilator were investigated. Zymographic electrophoresis showed four bands of collagenase activity,16,19,22 and 29 kDa in molecular weight,with the former two accounting for 60% and 36%,respectively,of the total collagenase activity. The collagenase activity varies during the molting cycle. Among the molt stages tested,Premolt Stage D0 exhibited the highest epidermal collagenase activity for both the 16 and 19 kDa isoenzymes and,as the molt stage proceeded,the enzymatic activity of these two isoenzymes decreased,with the lowest activity for both found in Premolt Stage D 3-4. Injection of 20-hydroxyecdysone significantly induced the activity of the 16 kDa collagenase in the epidermis of Uca pugilator,suggesting that the activity of this isoenzyme is under molting hormone control. Although 20-hydroxyecdysone injection did not result in a statistically significant increase in the activity of the 19 kDa isoenzyme,a tendency of the induction was nonetheless demonstrated. This is the first report on epidermal collagenase activity and its induction by the molting hormone in a

Genome-wide CRISPR screening reveals key genes and pathways associated with 20-hydroxyecdysone signal transduction in the silkworm(Bombyx mori)

Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E).Despite important progress in understanding various aspects of silkworm biology,the hormone signaling pathway in the silkworm remains poorly understood.Genome-wide screening using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-based libraries has recently emerged as a novel method for analyzing genome function,enabling further research into essential genes,drug targets,and virus-host interaction.Previously,we constructed a genome-wide CRISPR/Cas9-based library of the silkworm(Bombyx mori)and successfully revealed the genes involved in biotic or abiotic stress factor responses.In this study,we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action.Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus.Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity,interfere with intracellular nutrition and energy metabolism,and eventually cause cell apoptosis.The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes,which had increased tolerance to 20E.Our findings provide a panoramic overview of signaling in response to 20E in the silkworm,underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.

Steroid hormone 20-hydroxyecdysone disturbs fat body lipid metabolism and negatively regulates gluconeogenesis in Hyphantria cunea larvae

The steroid hormone 20-hydroxyecdysone(20E)has been described to regulate fat body lipid metabolism in insects,but its accurate regulatory mechanism,especially the crosstalk between 20E-induced lipid metabolism and gluconeogenesis remains largely unclear.Here,we specially investigated the effect of 20E on lipid metabolism and gluconeogenesis in the fat body of Hyphantria cunea larvae,a notorious pest in forestry.Lipidomics analysis showed that a total of 1907 lipid species were identified in the fat body of H.cunea larvae assigned to 6 groups and 48 lipid classes.The differentially abundant lipids analysis showed a significant difference between 20E-treated and control samples,indicating that 20E caused a remarkable alteration of lipidomics profiles in the fat body of H.cunea larvae.Further studies demonstrated that 20E accelerated fatty acidβ-oxidation,inhibited lipid synthesis,and promoted lipolysis.Meanwhile,the activities of pyruvate carboxylase,phosphoenolpyruvate carboxykinase,fructose-1,6-bisphosphatase,and glucose-6-phosphatase were dramatically suppressed by 20E in the fat body of H.cunea larvae.As well,the transcriptions of genes encoding these 4 rate-limiting gluconeogenic enzymes were significantly downregulated in the fat body of H.cunea larvae after treatment with 20E.Taken together,our results revealed that 20E disturbed fat body lipid homeostasis,accelerated fatty acidβ-oxidation and promoted lipolysis,but negatively regulated gluconeogenesis in H.cunea larvae.The findings might provide a new insight into hormonal regulation of glucose and lipid metabolism in insect fat body.

BmMD-2A responds to 20-hydroxyecdysone and regulates Bombyx mori silkworm innate immunity in larva-to-pupa metamorphosis

20E-hydroxyecdysone(20E)plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response.Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens.20E regulates a series of antimicrobial peptides(AMPs)through the immunodeficiency(IMD)pathway activation in Drosophila;nevertheless,whether other immune pathways are involved in 20E-regulated insect immunity is unknown.Our previous studies showed that BmMD-2A is a member of the MD-2-related lipid recognition(ML)family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway.In this study,we further demonstrate that BmMD-2A is also positively regulated by 20E,and the BmMD-2A neutralization experiment suggested that 20E activates some downstream immune effect factors,the AMP genes against Escherichia coli and Staphylococcus aureus,through the regulation of BmMD-2A in larval metamorphosis,implying that B.mori may use the Toll-ML signaling pathway to maintain innate immune balance in the larval-pupal metamorphosis stage,which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila.

Identification of LmUAP1 as a 20-hydroxyecdysone response gene in the chitin biosynthesis pathway from the migratory locust, Locusta migratoria

In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N-acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene Lm- CHS1, are expressed mainly in the integument and are responsible for cuticle formation. However, whether these genes are regulated by 20-hydroxyecdysone （20E） is still largely unclear. Here, we showed the developmental expression pattern ofLmUAP1, LmCHS1 and the corresponding 20E titer during the last instar nymph stage of locust. RNA interference （RNAi） directed toward a common region of the two isoforms of LmEcR （LmEcRcom） reduced the expression level of LmUAP1, while there was no difference in the expression of LmCHS1. Meantime, injection of 20E in vivo induced the expression of LmUAP1 but not LmCHS1. Further, we found injection-based RNAi of LmEcRcom resulted in 100% mortality. The locusts failed to molt with no apolysis, and maintained in the nymph stage until death. In conclusion, our preliminary results indicated that LmUAP1 in the chitin biosynthesis pathway is a 20E late-response gene and LmEcR plays an essential role in locust growth and development, which could be a good potential target for RNAi-based pest control.

Steroid hormone 20-hydroxyecdysone promotes CTL1-mediated cellular immunity in Helicoverpa armigera

Mermithid nematodes,such as Ovomermis sinensis,are used as biological control agents against many insect pests,including cotton bollworm(Helicoverpa armigera).However,given the host's robust immune system,the infection rate of O.sinensis is low,thus restricting its widespread use.To understand the host defense mechanisms against mermithid nematodes,we identified and characterized a protein involved in the recognition of O.sinensis,the potential O.sinensis-binding protein C-type lectin 1(HaCTLla and/or HaCTLIb),which was eluted from the surface of O.sinensis after incubation with H.armigera plasma.HaCTLIb is homologous to the previously reported HaCTLla protein.HaCTLI was predominantly expressed in hemocytes and was induced by the steroid hormone 20-hydroxyecdysone through ecdysone receptor(HaEcR)or ultraspiracle(HaUSP),or both.Binding assays confirmed the interactions of the HaCTLI proteins with O.sinensis but not with Romanomermis wuchangensis,a parasitic nematode of mosquito.Moreover,the HaCTL1 proteins were secreted into the hemocoel and promoted hemocyte-mediated encapsulation and phagocytosis.A knockdown of HaEcR and/or HaUSP resulted in compromised encapsulation and phagocytosis.Thus,HaCTLI appears to modulate cellular immunity in the defense against parasitic nematodes,and the 20-hydroxyecdysone-HaEcR-HaUSP complex is involved in regulating the process.

Rapid Determination of Endogenous 20-Hydroxyecdysone in Plants on MALDI-TOF/TOF Mass Spectrometry via Chemical Labeling Based on Boronate Affinity

20-Hydroxyecdysone(20E)derived from plants has a wide range of physiological and pharmacological ef ects on animals and humans,and rapid and sensitive methods for screening of the endogenous 20E in plants are thus required.Herein,a matrixassisted laser desorption/ionization time-of-f ight tandem mass spectrometry(MALDI-TOF/TOF MS)method is described for rapid and sensitive determination of endogenous 20E in plants.It is based on the use of the(3-(acridin-9-ylamino)phenyl)boronic acid(AYPBA)as the mass tag to assist the MS and tandem MS(MS^(n))analysis of 20E on MALDI-TOF/TOF MS.Good linearity was obtained with a determination coef cient(R^(2))larger than 0.99 in the range of 0.025–2.5μΜ.The limit of detection(LOD)was 2.4 fmol.Acceptable precision and accuracy were gained by intra-and inter-day analysis with relative standard deviations less than 19.5%and relative recoveries ranging from 85.7 to 105.2%.In addition,the AYPBA labeled 20E produced abundant characteristic fragment ions under the high energy collision-induced dissociation,which facilitated the identif cation of 20E by MS^(2)analysis on MALDI-TOF/TOF MS.Using the method,we enabled the identif cation and quantif cation of endogenous 20E in four herbs including Cyanotis arachoidea,Achyranthes bidentata,Spinacia oleracea and Chenopodium quinoa willd.,demonstrating the feasibility of the proposed method for screening of the endogenous 20E in plants.

血清HMGB1、CCL20、HSP27水平与慢性牙周炎患者牙周病变程度的相关性分析

目的探讨血清高迁移率族蛋白1(HMGB1)、CC趋化因子配体20(CCL20)、热休克蛋白27(HSP27)水平与慢性牙周炎(CP)患者牙周病变程度的相关性。方法选取2021年9月至2023年8月在新乡医学院第一附属医院诊治的60例CP患者纳入观察组,根据1∶1原则,另选取同期牙周健康者60例纳入对照组。比较两组及不同牙周病变程度CP患者血清HMGB1、CCL20、HSP27水平,分析各指标水平与CP牙周病变程度的相关性及联合诊断价值,并分析不同血清水平患者发生CP的危险度。结果观察组血清HMGB1、CCL20、HSP27水平高于对照组(P<0.05);不同牙周病变程度CP患者血清HMGB1、CCL20、HSP27水平比较:轻度<中度<重度,且各指标水平与牙周病变程度均呈正相关(P<0.05);入院时HMGB1、CCL20、HSP27水平联合诊断CP的曲线下面积(AUC)为0.905,最佳诊断敏感度为91.67%,特异度为88.33%,约登指数0.800,且各指标高水平患者发生CP的危险度是低水平的1.105倍、1.034倍、1.105倍(P<0.05)。结论HMGB1、CCL20、HSP27在CP患者血清中呈异常高表达,各指标水平与牙周病变程度均呈正相关,且联合检测对CP具有较高诊断价值,可作为临床诊断CP、评估牙周病变程度的有效指标。

类风湿关节炎患者血清SAA、CCL20、MMP-3表达水平及临床意义

目的探讨类风湿关节炎(RA)患者血清淀粉样蛋白A(SAA)、趋化因子配体20(CCL20)及基质金属蛋白酶3(MMP-3)水平变化及临床意义。方法回顾性选取2020年9月至2023年1月大同市第五人民医院收治的RA患者108例作为研究对象,依据28个关节的疾病活动度评分(DAS28)评分,将患者分为缓解期组(n=35,DSA28评分<2.6)与活动期组(n=73,DSA28评分≥2.6),并将活动期患者进一步分为轻度活动期(n=20,DSA28评分2.6~3.2)、中度活动期(n=29,DSA28评分>3.2~5.1)与重度活动期(n=24,DSA28为>5.1);另选取同期健康体检者80名作为对照组。所有受试者均检测血清SAA、CCL20、MMP-3水平。比较不同组别血清SAA、CCL20、MMP-3水平;使用Pearson相关分析血清SAA、CCL20、MMP-3水平与红细胞沉降率(ESR)、C反应蛋白(CRP)的相关性;使用受试者工作特征(ROC)曲线评价各指标对RA疾病活动度的诊断价值。结果RA组血清SAA、CCL20、MMP-3水平分别为(42.25±13.46)mg/L、(45.69±12.78)pg/mL、(85.41±25.14)ng/mL,均显著高于对照组[(7.14±2.12)mg/L、(18.12±5.47)pg/mL、(27.36±8.21)ng/mL],差异均有统计学意义(P<0.05)。RA活动期组患者血清SAA、CCL20、MMP-3水平分别为(51.78±10.71)mg/L、(53.14±10.45)pg/mL、(106.10±20.39)ng/mL,显著高于缓解期组[(22.37±7.14)mg/L、(30.15±8.24)pg/mL、(42.25±13.64)ng/mL],差异均有统计学意义(P<0.05)。重度活动度组血清SAA、CCL20、MMP-3水平均高于中度活动度组和轻度活动度组,差异均有统计学意义(P<0.05),随着疾病活动度提高,血清SAA、CCL20、MMP-3水平逐渐增高。Pearson相关分析显示,活动期RA患者血清SAA、CCL20、MMP-3水平与CRP、ESR均呈正相关(P<0.05)。ROC曲线分析得出,血清SAA、CCL20、MMP-3区分RA轻度活动度与中度活动度的曲线下面积(AUC)分别为0.852、0.756、0.725,区分中度活动度与重度活动度的AUC分别为0.852、0.756、0.725,均显示出一定的诊断能力。结论RA患者血清SAA、CCL20、MMP-3水平均呈高表达,这3项指标可作为评估疾病活动度的生物标志物,可为临床预测病情进展提供参考。

20钢/316L复合管固液复合制坯工艺研究

双金属复合管在石油运输行业中应用前景广阔,结合性能是考察复合管是否合格的重要指标之一。为了能在轧制成形过程中得到高品质的复合管材,在复合管制坯过程中要求实现界面处的冶金复合。以20钢和316L为原材料,基于结合温度及元素扩散情况,采用固液复合方式,并且结合界面的温度模型及通过Pro CAST软件模拟,获得了外模最佳预热温度为1050—1100℃。通过现场试制界面处实现了冶金复合,在最佳温度范围既保证了固态20钢和液态316L之间能达到冶金复合,又能使元素有充分的扩散时间而增强了界面结合强度,同时又可以防止因元素过渡扩散而导致316L腐蚀性能下降。该方法为后续复合管连轧提供了优良的管坯,可实现高品质复合管的现场生产。

原油乳状液对20钢的腐蚀

以某油田原油和采出水为原料配制了不同含水率的油包水或水包油乳状液,利用失重法和电化学测试技术研究了20钢的腐蚀行为,分析了不同含水率和不同类型乳状液的腐蚀性。结果表明:乳状液的腐蚀性低于采出水的腐蚀性,且水包油乳状液腐蚀性大于油包水乳状液;由于油包水乳状液中原油在20钢表面吸附形成的油膜的完整性较好,20钢与水分子接触受到一定限制,腐蚀比较轻;随着含水率增大,油膜完整性逐渐受到破坏,乳状液腐蚀性明显变强;在水包油乳状液中,20钢与采出水直接接触,20钢表面只存在分散状的油膜,其对金属基体的保护作用较弱,随着含水率的增加,水包油乳状液对20钢的腐蚀接近于采出水;在油水界面处,20钢腐蚀严重,其腐蚀行为与在水包油乳状液中的类似。

小麦新品种济儒麦20的选育

济儒麦20是济宁市农业科学研究院于2012年以丰收60为母本、08鉴75为父本,利用常规育种技术育成的小麦新品种。该品种产量水平高,抗病性好,高抗叶锈病和条锈病,高抗干热风,落黄好,越冬抗寒性1级。2021-2022年度参加山东省高肥水地组生产试验平均产量为9745.5kg/hm2,比对照济麦22增产5.3%。2022年通过山东省农作物品种审定委员会审定,审定编号:鲁审麦20220005。

激光熔凝处理对P20钢组织及性能的影响

目的提高P20模具钢的表面硬度和耐磨性能。方法采用激光熔凝技术对P20模具钢表面进行强化处理。通过硬度梯度检测和摩擦磨损测试,分别评价熔凝层的硬度分布特征和耐磨性能,并通过光学显微镜和扫描电子显微镜对熔凝层及磨痕形貌进行分析。结果使用激光输出功率为500W、光斑直径为2.5mm、聚焦透镜距离为40 mm、扫描速度为6 mm/s、搭接率为45%、氮气保护的激光熔凝工艺所得熔凝层的组织细小,无脱碳、畸变、裂纹等缺陷,熔凝处理质量高。熔凝过程中单道激光熔凝层呈半椭圆形分布,最大深度为610~620μm。熔凝处理后表面硬度提升显著,熔凝层的硬度分布与熔凝层的区域位置有关,具有较高硬度且硬度保持基本稳定的熔凝层深度约为400μm;单道激光熔凝层最高硬度可达460~480HV,重叠影响区即双熔凝区的最高硬度在540~560HV之间,即熔凝层硬度普遍较基体硬度提高了60%以上。此外,P20模具钢经过激光熔凝处理后耐磨性能提升明显,其平均摩擦因数约为0.85,熔凝处理的磨损失重较未处理的试样减少了约61%,其磨损机制主要表现为磨粒磨损和少量的黏着磨损或剥落脱离。结论激光熔凝处理能够显著提高P20模具钢的表面硬度和耐磨性能,试验采用的激光熔凝工艺可在P20钢表面获得硬度较高且稳定可靠的熔凝层深度在400μm左右,能将P20模具钢的表面硬度及耐磨性提高60%以上。

粉末冶金法制造20CrMnTi钢汽车齿轮件代替传统工艺的研究

通过研究20CrMnTi齿轮材料使用粉末冶金方法制造代替传统锻造的方法,弥补了用传统方法制造齿轮的不足,验证了添加纳米粉体并采用合理的粉末冶金完全可以代替传统锻造方法制造汽车齿轮材料,为同行提供了借鉴。

东道国投资壁垒、制度质量与中国对外直接投资——来自G20国家的经验证据

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