微小RNA-204-5p靶向蛋白质酪氨酸磷酸酶1B基因对缺氧复氧诱导的大鼠心肌细胞氧化应激的影响

目的 探讨miR-204-5p对缺氧复氧诱导大鼠心肌细胞H9C2氧化应激的调控作用机制.方法 体外培养大鼠胚胎心肌细胞H9C2,构建心肌细胞缺氧/复氧模型.实验分为空白组、缺氧复氧组、缺氧复氧+miR-con组、缺氧复氧+miR-204-5p组、缺氧复氧+miR-204-5p+pcDNA组、缺氧复氧+miR-204-5...     

缺氧心肌细胞来源外泌体对Gli1~+细胞纤维化表型转换的作用机制

目的观察缺氧心肌细胞来源外泌体对Gli1~+细胞纤维化过程的影响,并探讨其可能的机制。方法分离Gli1~+细胞和SD乳鼠心肌细胞。心肌细胞分别进行常氧和缺氧培养,收集培养基并提取外泌体,用透射电镜、Nanosight、Western blot等方法对其鉴定。两种外泌体分别与Gli1~+细胞共培养。RT-qPCR法检测这两种外泌体并对比分析,明确其中起关键调控作用的miRNA。转染miRNA模拟物观察Gli1~+细胞中纤维化相关蛋白的表达水平。结果 Gli1~+细胞强阳性表达CD29、CD105和Gli1,不表达CD31、CD34和CD45。心肌细胞表达cTnT和α-Actinin,其外泌体直径约100 nm,并检测到Flotillin-1、Alix、HSP-70和CD63。缺氧处理外泌体中miR-223明显上调(P<0.01);Gli1~+细胞经缺氧外泌体和miR-223 mimic处理,纤维化相关的蛋白α-SMA(P<0.01,P<0.05)、DDR-2(P<0.01,P<0.01)和collagen I(P<0.05,P<0.01)的表达都明显升高。结论缺氧心肌细胞外泌体能促进Gli1~+细胞内纤维化相关蛋白的表达,使其向纤维化表型转换,这一作用可能与外泌体中高表达的miR-223有关。     

circ_0000285靶向miR-625对缺氧/复氧诱导的心肌细胞氧化应激损伤的影响

目的 探讨环状RNA 0000285(circ_0000285)对缺氧/复氧诱导的心肌细胞氧化应激损伤的影响,并分析其机制是否与调控miR-625表达有关.方法 体外培养大鼠心肌细胞H9C2构建缺氧/复氧(H/R)细胞损伤模型.将H9C2细胞随机分为对照(Con)组、H/R组、H/R+si-NC组、H/R+si-cir...     

米非司酮通过抑制Rab27a和Rab27b的表达对子宫内膜癌细胞增殖的抑制作用

目的 研究米非司酮在子宫内膜癌中的功能和潜在分子机制。方法 通过细胞增殖实验研究不同质量浓度（10、50、100 μg/mL）米非司酮以及随着时间变化米非司酮对子宫内膜癌细胞的影响;蛋白免疫印记实验及免疫荧光实验研究米非司酮（50 μg/mL）对子宫内膜癌细胞内外泌体分泌、外泌体调控关键蛋白的表达、外泌体关键调控蛋白对外泌体分泌的影响。结果 随着加入子宫内膜癌细胞内的米非司酮质量浓度升高和时间推移,子宫内膜癌细胞增殖速率显著减慢,同时细胞状态变差,数目显著减少;米非司酮显著降低外泌体蛋白胞外分泌,包括外泌体总蛋白以及标志蛋白CD63和Gp96等,使其滞留在胞内;米非司酮显著降低外泌体分泌调控蛋白Rab27a和Rab27b的表达;敲低外泌体分泌调控蛋白Rab27a和Rab27b的表达显著降低外泌体总蛋白以及标志蛋白CD63的胞外分泌。结论 米非司酮通过抑制外泌体分泌调控蛋白Rab27a和Rab27b的表达,从而抑制外泌体分泌,最终导致子宫内膜癌细胞增殖显著减慢。     

miR-152调控糖尿病心肌病心肌成纤维细胞增殖的作用

目的探究miR-152调控糖尿病心肌病心肌成纤维细胞(CFs)增殖的作用。方法应用链脲佐菌素(STZ)构建糖尿病大鼠模型,应用高糖(33.3 mmol·L-1)诱导CFs增殖模型。多聚甲醛固定心肌组织后,行HE和Masson染色;Western blot检测心肌组织和CFs中α-SMA、CollagenⅠ的蛋白表达;qPCR检测心肌组织和CFs中miR-152的表达;MTT检测miR-152对细胞增殖活性的影响。结果HE和Masson染色显示,糖尿病组大鼠心肌胶原含量明显增多,细胞排列紊乱;在高糖模型中,α-SMA和CollagenⅠ表达增加,而miR-152表达降低;乳鼠CFs转染miR-152模拟物后,α-SMA和CollagenⅠ表达降低,同时CFs增殖活性减弱。结论miR-152可能在糖尿病心肌病CFs增殖中起重要作用,提示其可能改善糖尿病心肌病。     

转录因子EB在衰老心肌细胞自噬中的作用机制研究

目的 探讨转录因子EB(TFEB)在衰老心肌细胞自噬中的作用机制。方法 动物实验：将20只老年Wistar大鼠采取随机数字表法分为假手术组（Sham组）和缺血再灌注组（I/R组）。细胞实验：（1）体外培养大鼠心肌细胞，采用8 g/L D-半乳糖孵育8 d后分为常氧（Normoxia）组和缺氧/复氧（H/R）组。（2）在衰老心肌细胞中分别转染过表达和干扰TFEB腺病毒分为过表达对照（Ad-GFP）组、过表达对照+缺氧/复氧（Ad-GFP+H/R）组、过表达TFEB(AdTFEB)组、过表达TFEB+缺氧/复氧（Ad-TFEB+H/R）组、干扰对照（sh-NC）组、干扰对照+缺氧/复氧（sh-NC+H/R）组、干扰TFEB(sh-TFEB)组、干扰TFEB+缺氧/复氧（sh-TFEB+H/R）组。（3）分别用DNMT1、DNMT3a、DNMT3b的特异性抑制剂DC-05、TFD、NA处理缺氧/复氧后的衰老心肌细胞分为H/R组、DC-05组、TFD组、NA组。（4）在衰老心肌细胞中转染干扰DNMT3b腺病毒分为干扰对照（sh-NC）组、干扰对照缺氧/复氧（sh-NC+H/R）组、干扰DNMT...     

Linc-NeD125通过调控微小RNA-19b对缺氧/复氧诱导的心肌细胞增殖和凋亡的影响

目的 探究上调长链非编码RNA(lncRNA)Linc-NeD125对缺氧/复氧心肌细胞增殖和凋亡的影响及相关分子机制。方法 采用缺氧/复氧诱导HCM细胞建立损伤模型,通过实时定量聚合酶链反应（RT-q PCR）技术检测缺氧/复氧诱导的HCM细胞Linc-NeD125表达水平。将阴性对照慢病毒和Linc-NeD125慢病毒分别感染缺氧/复氧诱导的HCM细胞,HCM细胞分为NC组和Linc-NeD125组。细胞计数（CCK-8）法和流式细胞术分别检测NC组和Linc-NeD125组HCM细胞的增殖水平和凋亡率。双荧光素酶报告基因实验检测Linc-NeD125与微小RNA(miR)-19b的靶向关系。RT-qPCR检测NC组和Linc-NeD125组HCM细胞中miR-19b表达水平。蛋白免疫印迹法检测NC组和Linc-NeD125组HCM细胞中细胞周期蛋白（Cdk2、Cyclin E）、抗凋亡蛋白（Bcl-XL、Mcl-1）以及促凋亡蛋白Bax的表达。结果 缺氧/复氧诱导HCM细胞中LincNeD125表达水平低于正常HCM细胞（P<0.01）。Linc-NeD125组HCM细胞中...     

miR-30a-5p调控HDAC9对心肌细胞氧化应激损伤保护作用的机制研究

目的 探讨miR-30a-5p通过调控组蛋白去乙酰化酶9(HDAC9)对缺氧复氧诱导的心肌细胞凋亡、氧化应激的影响及作用机制.方法 本研究通过体外培养心肌细胞H9 c2,建立缺氧复氧损伤细胞模型,细胞分组为:Control组、H/R组、H/R+miR-NC组、H/R+miR-30a-5p组、H/R+si-NC组、H/R...     

大血藤总酚酸调控微小 RNA-155对缺氧复氧心肌细胞的细胞活性凋亡及氧化应激的影响

目的 探讨大血藤总酚酸对缺氧复氧心肌细胞的细胞活性凋亡及氧化应激的影响及分子机制.方法 2019年12月至2020年8月,心肌细胞H9C2分为对照组(正常培养)、缺氧复氧组(缺氧4 h,复氧12 h)、大血藤总酚酸低、中、高浓度组(造模前分别用12.5、25.0、50.0 mg/L大血藤总酚酸培养2 h,然后进行缺氧复氧处理)、大血藤总酚酸-H+NC组(将miR-155模拟物阴性对照转染至H9C2中再用50.0 mg/L大血藤总酚酸培养,最后进行缺氧复氧处理)、大血藤总酚酸-H+miR-155 mimic组(将miR-155模拟物转染至H9C2中再用50.0 mg/L大血藤总酚酸培养,最后进行缺氧复氧处理).实时荧光定量PCR(RT-qPCR)检测微小RNA-155(miR-155)表达水平;MTT法检测细胞存活率;流式细胞术检测细胞凋亡;蛋白质印迹法(Western blotting)检测蛋白表达;试剂盒检测细胞培养液中肌酸激酶、乳酸脱氢酶(LDH)、丙二醛含量.结果 缺氧复氧处理的心肌细胞中miR-155表达水平升高,细胞吸光度降低,心肌细胞凋亡率升高,活化胱天蛋白酶-3(cleaved-caspase-3)表达水平升高,胱天蛋白酶-3前体(pro-caspase-3)表达水平降低,心肌细胞中肌酸激酶、LDH、丙二醛含量升高(P<0.05).大血藤总酚酸中、高浓度处理后缺氧复氧诱导的心肌细胞中miR-155表达水平降低,细胞吸光度升高,心肌细胞凋亡率降低,cleaved-caspase-3表达水平降低,pro-caspase-3表达水平升高,心肌细胞中肌酸激酶、LDH、丙二醛含量降低,且呈浓度依赖性(P<0.05);而大血藤总酚酸低浓度组各数据无显著变化.过表达miR-155可逆转大血藤总酚酸对缺氧复氧诱导的心肌细胞活性、凋亡[(22.73±0.58)％比(13.55±0.33)％,P<0.05]和肌酸激酶[(79.19±2.78)U/L比(41.22±2.31)U/L,P<0.05]、LDH[(44.20±2.80)U/L比(24.77±2.46)U/L,P<0.05]、丙二醛[(24.05±1.28)μmol/L比(12.73±0.59)μmol/L,P<0.05]含量的影响.结论 大血藤总酚酸可能通过下调miR-155抑制缺氧复氧心肌细胞的凋亡及氧化应激.     

IL-33通过miR-19a-3p/MAPK6通路影响心肌缺氧/复氧损伤的机制研究

目的 探讨白细胞介素-33(IL-33)通过miR-19a-3p/MAPK6通路对心肌缺氧/复氧(H/R)损伤的影响及可能的分子机制。方法 建立H/R诱导的新生小鼠心肌细胞体外模型,使用IL-33进行预处理,向心肌细胞中转入miR-19a-3p mimic,以上调miR-19a-3p表达,转入miR-19a-3p inhibitor,以抑制miR-19a-3p表达,转入pc DNA-MAPK6以上调MAPK6表达。通过Caspase-3活性、细胞凋亡ELISA法和原位细胞凋亡染色检测心肌细胞凋亡。Target Scan数据库预测miR-19a-3p的潜在靶点,双荧光素酶检测报告显示miR-19a-3p与MAPK6之间关系;RT-qPCR法检测心肌细胞miR-19a-3p和MAPK6 mRNA表达,Western blot法检测MAPK6蛋白表达。结果 IL-33预处理可以抑制H/R诱导的miR-19a-3p表达下调和细胞凋亡。双荧光素酶测定报告显示,miR-19a-3p靶向调控MAPK6。IL-33抑制了H/R诱导的MAPK6表达上调,过表达MAPK6减弱了IL-33对H/R的抗凋亡作...     

抑制miR-33表达对急性呼吸窘迫综合征大鼠肺纤维化的影响及机制研究

目的 探讨抑制miR-33表达对急性呼吸窘迫综合征(ARDS)大鼠肺纤维化的影响及其机制。方法 将60只SD大鼠按随机数字表法分成假手术组(Sham组)、ARDS模型组(Model组)、antagomir阴性对照组(antagomir-NC组)和miR-33 antagomir组(antagomir组),每组15只。除Sham组外,其他各组大鼠均通过颈部气管滴注脂多糖(LPS)建立ARDS模型。造模成功后给予miR-33 antagomir或antagomir-NC尾静脉注射。测定动脉血氧分压[p(O₂)]及氧合指数(OI);HE和Masson染色观察肺组织病理学变化;碱性水解法检测肺组织中羟脯氨酸(Hyp)含量;酶联免疫吸附试验(ELISA)检测肺泡灌洗液中炎性因子白细胞介素(IL)-1β、IL-6和肿瘤坏死因子(TNF)-α水平;实时荧光定量PCR检测肺组织中miR-33表达水平及转化生长因子β1(TGF-β1)、胶原蛋白(Collagen)Ⅰ和CollagenⅢmRNA表达水平;Western blot检测肺组织中TGF-β1、Smad2、p-Smad2、S...     

ent-Kaurenoids from the roots of Cacalia pilgeriana

Two new ent-kaurenoids,19-acetoxyl-ent-3β,17-dihydroxykaur-15-ene (1), 19-acetoxyl-ent-3β-hydroxykaur-15-en-17-al (2), together with seven known ent-kaurenoids: ent-kaur-16-en-19-al (3), ent-kaur-16-en-19-oic acid (4), ent-kauran-16β,17-diol (5), ent-15β, 16β-epoxy-17-hydroxykauran-19-oic acid (6),19-acetyl-ent-3β-hydroxyl-kaur-16-ene (7), ent-3β,19-dihydroxykaur-16-ene (8), ent-17-hydroxykaur-15-ene (9), were isolated from Cacalia pilgeriana. Their structures were elucidated by spectroscopic methods including 2D NMR spectral analysis.     

Two new diterpenoid dimers,fritillebinide D and E,from bulbs of Fritillaria ebeiensis

Two new ent-kaurane diterpenoid dimers, fritillebinide D (1) and fritillebinide E (2), were isolated from bulbs of Fritillaria ebeiensis G.D. Yu et G.Q. Ji. Their structures have been determined to be ent-3β-acetoxy-kauran-16β, 17-acetal ent-3β-acetoxy-16β-kauran-17(R)-aldehyde (1) and ent-3β-acetoxy-kauran-16β,17-acetal ent-3β-acetoxy-16β-kauran-17(S)-aldehyde (2) by means of spectral analysis.     

MiR-22-3p in exosomes increases the risk of heart failure after down-regulation of FURIN

Heart failure (HF) is often the inevitable manifestation of myocardial ischemia. Hypoxia can induce cardiomyocytes to express many microRNAs (miRNAs), which are highly expressed in exosomes. In addition, miR-22-3p is a marker in heart failure. Therefore, miR-22-3p was taken as the research object to explore its role and mechanism in HF. HF differentially expressed miRNAs were screened by bioinformatic analysis. The HF rats model was constructed and identified by detecting serum brain natriuretic peptide (BNP) and ultrasound analysis [left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS)]. The extracted exosomes were identified by transmission electron microscopy, and Western blot was used to detect the expressions of Tsg101 and CD63. Quantitative real-time polymerase chain reaction detected miR-22-3p expression in serum, exosomes, and serum without exosomes, while the cardiomyocytes cytotoxicity was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and PKH26 staining. After overexpressing/silencing miR-22-3p in cells, cell viability, apoptosis, and apoptosis-associated markers were detected. Bioinformatic analysis screened the target gene of miR-22-3p, which was verified by dual-luciferase assay. Regulation of miR-22-3p on FURIN was measured by rescue tests. In vivo experiments were verified the above results. MiR-22-3p was identified as the research object. BNP was increased in the model group, while LVEF and LVFS were decreased. MiR-22-3p was overexpressed in HF-treated serum and exosomes. Normal exosomes did not affect cardiomyocyte function, while high concentrations of HF-treated exosomes were cytotoxic. By regulating apoptosis-related genes, overexpressed miR-22-3p inhibited cell activity and promoted cell apoptosis. Silenced miR-22-3p with opposite effects counteracted effects of HF-treated exosomes. FURIN, target gene of miR-22-3p, was negatively regulated by miR-22-3p, while overexpressed FURIN promoted cell activity and inhibited apoptosis. In vivo research was consistent with the results of cell experiments. By regulating FURIN, miR-22-3p in exosomes increases the risk of HF damage.     

Antibacterial constituents from Stemona sessilifolia

Bioassay-guided fractionation led to the isolation of eight compounds from Stemona sessilifolia. Of the eight isolates, three new bibenzyls, stilbostemins M-O (1-3), and a new tocopherol, 6-methoxy-3,4-dehydro-δ-tocopherol (4) were revealed together with four known compounds 3,5-dihydroxy-2'-methoxy bibenzyl (5), 3,5-dihydroxy bibenzyl (6), β-tocopherol (7), and γ-tocopherol (8). Compounds 5, 6, and 8 exhibited strong antibacterial activities against Staphylococcus aureus and S. epidermidis.     

Two new and four known polyphenolics obtained as new cell-cycle inhibitors from Rubus aleaefolius Poir

Two new polyphenolics, rubuphenol (1) and sanguiin H-2 ethyl ester (2), were isolated together with ellagic acid (3), ethyl gallate (4), 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (5) and 1,2,3,6-tetra-O-galloyl-β-D-glucopyranose (6) as new cell-cycle inhibitors from Rubus aleaefolius by bioassay-guided separation procedure and the structures of 1 and 2 were elucidated by spectroscopic method. Compounds 1 - 6 inhibited the cell cycle progression of tsFT210 cells at the G₀/G₁ phase with the MIC values of 14.6 μM (1), 22.1 μM (2), 10.3 μM (3), 7.8 μM (4), 7.9 μM (5) and 6.6 μM (6).     

外泌体传递miR-139调控卵巢癌顺铂敏感性的机制研究

目的探讨外泌体介导的miR-139传递对卵巢癌顺铂(cDDP)敏感性的影响。方法分别提取卵巢癌cDDP敏感细胞及耐药细胞分泌的外泌体,透射电镜观察外泌体形态,Western blotting检测外泌体标志蛋白的丰度,qPCR定量检测miR-139水平,MTT检测cDDP敏感细胞、miR-139敲低敏感细胞源性外泌体对耐药细胞cDDP敏感性的影响。结果卵巢癌细胞培养基上清液中存在大量椭圆形或圆形、粒径50~100 nm且高表达外泌体标志因子Alix、CD63、CD9的囊状结构。相较于卵巢癌cDDP耐药细胞源性外泌体,cDDP敏感细胞源性外泌体中miR-139的丰度更高,且将cDDP敏感细胞源性外泌体与耐药细胞株共同孵育可增强后者的cDDP敏感性,而下调外泌体中miR-139的表达可削弱其对耐药细胞的cDDP敏感性诱导作用。结论卵巢癌cDDP敏感细胞可通过外泌体传递miR-139,增强耐药细胞对cDDP的敏感性。     

Microbial hydroxylation of cinobufagin by Mucor spinosus

Mucor spinosus has been employed for the biotransformation of cinobufagin (1) to afford three metabolites. On the basis of their physico-chemical data, the structures of the transformation products have been characterized as 1β-hydroxy-cinobufagin (2), 12β-hydroxy-cinobufagin (3) and 1β,12β-dihydroxy-cinobufagin (4), of which metabolites 2 and 4 are new compounds. In vitro cytotoxic activities of the biotransformation products and the substrate-cinobufagin have been assayed against four tumor cell lines of Bel 7420, BGC 823, HeLa and HL 60; they all showed cytotoxic activities.     

Stilbene C-glucosides from Cissus repens

Four new stilbene C-glucosides, namely trans-3-O-methyl-resveratrol-2-C-β-glucoside (1), cis-3-O-methyl-resveratrol-2-C-β-glucoside (2), trans-3-O-methyl-resveratrol-2-(2-p-coumaric)-C-β-glucoside (cissuside A) (3), and trans-3-O-methyl-resveratrol-2-(3-p-coumaric)-C-β-glucoside (cissuside B) (4), were isolated from the aerial parts of Cissus repens, along with known trans-resveratrol (5), trans-resveratrol-2-C-β-glucoside (6) and cis-resveratrol-2-C-β-glucoside (7). Their structures were established by spectroscopic methods. Stilbene C-glucosides were found in the genus Cissus for the first time.     

Further flavonol and iridoid glycosides from Ajuga remota aerial parts

Five new iridoid glycosides characterised as 6-keto-8-acetylharpagide (1), 6,7-dehydro-8-acetylharpagide (2), 7,8-dehydroharpagide (3), 8-acetylharpagide-6-O-β-glucoside (4), harpagide-6-O-β-glucoside (5) together with three flavonol glycosides, myricetin 3-O-rutinoside-4'-O-rutinoside (6), myricetin 3-O-rutinoside-3'-O-rutinoside (7) and isorhamnetin 3-O-rutinoside-7-O-rutinoside-4'-O-β-glucoside (8) have been isolated from the aerial parts of Ajuga remota. Also isolated were two known compounds ajugarin IV and ajugarin V. Their structures were established using spectroscopic methods including UV, IR, FAB-MS, HR-MS, 1D and 2D NMR techniques.