UPLC-Q-TOF/MS

UPLC - Q - TOF / MS
  • 文章类型: Journal Article
    基于网络药理学和实验验证,本研究旨在筛选六味地黄汤(LW)对轻度认知功能障碍(MCI)的潜在作用靶点。
    基于网络药理学,本研究初步探讨了LW治疗MCI的作用靶点和分子机制。结果表明,LW对MCI的作用机制可能与cAMP通路有关。然后,建立衰老细胞和动物模型,进一步验证其分子机制。
    在LW中鉴定出总共23种活性成分。此外,通过网络药理学分析,我们在LW中发现了22种抗MCI活性成分,其中阿利沙醇B的作用最显著,并预测了LW可能通过cAMP信号通路改善MCI的潜在机制途径。进一步的体内和体外实验证实,LW可以缓解衰老小鼠的认知功能障碍,减少D-半乳糖诱导的衰老细胞,这可能是通过激活cAMP/PKA/CREB信号通路。
    这项研究发现,中药配方LW可能通过调节cAMP/PKA/CREB信号通路来改善MCI,为进一步开展LW抗MCI作用及其分子机制的临床研究提供参考。
    UNASSIGNED: Based on network pharmacology and experimental validation, this study aimed to screen the potential targets of Liuwei Dihuang decoction (LW) against mild cognitive impairment (MCI).
    UNASSIGNED: Based on network pharmacology, this study preliminarily explored the targets and molecular mechanisms of LW in the treatment of MCI. The results showed that the mechanism of action of LW against MCI may be related to the cAMP pathway. Then, an aging cell and animal model was established to further verify its molecular mechanism.
    UNASSIGNED: A total of 23 active ingredients were identified in LW. In addition, through network pharmacological analysis, we found 22 anti-MCI active ingredients in LW, of which alisol B had the most significant effect, and predicted the potential mechanism pathway by which LW may improve MCI through the cAMP signaling pathway. Further in vivo and in vitro experiments confirmed that LW can alleviate cognitive dysfunction in aging mice and reduce D-galactose-induced senescent cells, which may be through activation of the cAMP/PKA/CREB signaling pathway.
    UNASSIGNED: This study found that the traditional Chinese medicine formula LW may play a role in improving MCI by regulating the cAMP/PKA/CREB signaling pathway, which provides a reference for further clinical research on the anti-MCI effect of LW and its molecular mechanism.
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  • 文章类型: Journal Article
    研究表明,许多中药可以提高人体的免疫力。当迪口服液(DDO)主要由当归(Oliv。)迪尔斯(当归),地黄。(地黄),牛膝Bl.(牛溪),甘草。(甘草)。在这项研究中,采用快速超高效液相色谱-四极杆飞行时间质谱(UPLC-Q-TOF/MS)法鉴定DDO潜在有效化合物。然后通过淋巴细胞增殖测定DDO的免疫活性,巨噬细胞吞噬功能,NK细胞活性,迟发型超敏反应,溶血菌斑数,sIgA含量和免疫器官指数。结果表明,共鉴定出51个化合物。此外,DDO能显著促进淋巴细胞增殖,提高巨噬细胞吞噬能力,NK细胞活性,溶血菌斑数,sIgA含量及免疫器官指数与对照组比较,中剂量组疗效最佳(P<0.05)。这些结果表明DDO可以增强小鼠的免疫力。
    Studies have shown that a lot of traditional Chinese medicines could improve the immunity of the body. Dangdi oral liquid (DDO) was mainly composed of Angelica sinensis (Oliv.) Diels (Danggui), Rehmannia glutinosa Libosch. (Dihuang), Achyranthes bidentata Bl. (Niuxi), Glycyrrhiza uralensis Fisch. (Gancao). In this study, the rapid ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) method was used to identify the potentially effective compounds of DDO. Then the immune activity of DDO was measured by lymphocyte proliferation, macrophage phagocytic function, NK cell activity, delayed type hypersensitivity reaction, hemolytic plaque number, sIgA content and immune organ index. The results showed that a total of 51 compounds were identified. In addition, DDO could significantly promote the lymphocyte proliferation, improve macrophage phagocytic ability, NK cell activity, hemolytic plaque number, sIgA content and immune organ index compared with control group, and the medium dose possessed the best efficacy (P<0.05). These results indicated that DDO could enhance the immunity of mice.
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  • 文章类型: Journal Article
    中草药因其疗效显著、副作用小而受到广泛的重视。草药的定量分析对于确保其安全性和有效性至关重要。同时检测多种质量标记(Q标记)已成为草药质量控制的重要方法和趋势。近年来,非靶向筛查已成为发现和鉴定未知化合物的有效策略.这项研究开发了一种非针对性的筛查和定量分析策略,以发现,识别和量化真正代表五灵胶囊功效的多种成分。在这一战略中,通过分析质量裂解途径,从五灵胶囊中初步发现并鉴定了18种黄酮类化合物,化合物的精确分子量,并将数据与数据库进行比较。比较标准后确定了十种类黄酮。此外,在回归方程的评估之后,线性范围,检测限(LOD),定量限(LOQ),精度,重复性,并恢复了所提出的定量方法,对六种类黄酮进行了定量。该方法成功筛选,已识别,并量化了五灵胶囊中潜在的活性成分,为提高其他草药的质量控制标准提供见解。
    Herbal medicine has been widely valued because of its remarkable efficacy and minimal side effects. The quantitative analysis of herbal medicines is essential to ensure their safety and efficacy. The simultaneous detection of multiple quality markers (Q-markers) has emerged as an important approach and trend in herbal medicine quality control. In recent years, non-targeted screening has become an effective strategy for the discovery and identification of unknown compounds. This study developed a non-targeted screening and quantitative analysis strategy to discover, identify and quantify the multiple components that truly represent the efficacy of Wuling capsule. Within this strategy, 18 types of flavonoids were tentatively discovered and identified from Wuling capsule by analyzing mass cleavage pathways, the precise molecular weights of compounds, and comparing the data with a database. Ten types of flavonoids were determined after the comparison of the standards. Additionally, following the evaluation of the regression equation, linear range, limit of detection (LOD), limit of quantitation (LOQ), precision, repeatability, and recovery of the proposed quantitative method, six flavonoids were quantified. This method successfully screened, identified, and quantified the potential active components in Wuling capsule, providing insights for improving the quality control standards in other herbal medicines.
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  • 文章类型: Journal Article
    通过代谢组学和血清药物化学分析了“玄参贝母”的药效学物质及其对甲状腺肿治疗作用的分子机制。使用丙基硫氧嘧啶(PTU)建立大鼠甲状腺肿模型,用玄参贝母处理动物。“根据甲状腺组织病理学和血液生化指标评估药物对的疗效。通过UPLC-Q-TOF/MS分析大鼠的血清和尿液样本。采用主成分分析(PCA)和正交偏最小二乘判别分析(OPLS-DA)筛选尿液中潜在的生物标志物及相应的代谢途径。还鉴定了“玄参贝母”的血液成分,并分析了它们与尿液生物标志物的相关性,以筛选潜在的生物活性化合物。“玄参贝母”减轻了对甲状腺组织的损伤,并使甲状腺激素FT3,FT4和TSH的水平正常化。我们还确定了22种与甲状腺肿相关的尿液生物标志物,其中19个受玄参贝母调节。\"此外,尿液生物标志物参与色氨酸代谢,类固醇激素生物合成,和β-丙氨酸代谢,这些途径可能是药物对的目标。此外,通过血清药物化学检测了“玄参贝母”中的47种化合物,其中九个组成部分,即,丁香酸,丹皮,cedrol,和顺式阿魏酸,fetisinine,aucubicenin,亚麻酸,ussuriedine,和5-(甲硫基)戊腈,被确定为针对甲状腺肿的潜在有效物质。总结一下,我们表征了“玄参贝母”治疗甲状腺肿的化学成分和机理,为其临床应用提供了实验依据。
    The pharmacodynamic substances in \"Scrophulariae Radix-Fritillaria\" and the molecular mechanisms underlying its therapeutic effects against goiter were analyzed through metabolomics and serum pharmaco-chemistry. A rat model of goiter was established using propylthiouracil (PTU), and the animals were treated using \"Scrophulariae Radix-Fritillaria.\" The efficacy of the drug pair was evaluated in terms of thyroid gland histopathology and blood biochemical indices. Serum and urine samples of the rats were analyzed by UPLC-Q-TOF/MS. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to screen potential biomarkers in urine and the corresponding metabolic pathways. The blood components of \"Scrophulariae Radix-Fritillaria\" were also identified, and their correlation with urine biomarkers was analyzed in order to screen for potential bioactive compounds. \"Scrophulariae Radix-Fritillaria\" mitigated injury to thyroid tissues and normalized the levels of the thyroid hormones FT3, FT4, and TSH. We also identified 22 urine biomarkers related to goiter, of which 19 were regulated by \"Scrophulariae Radix-Fritillaria.\" Moreover, urine biomarkers are involved in tryptophan metabolism, steroid hormone biosynthesis, and beta-alanine metabolism, and these pathways may be targeted by the drug pair. In addition, 47 compounds of \"Scrophulariae Radix-Fritillaria\" were detected by serum pharmacochemistry, of which nine components, namely, syringic acid, paeonol, cedrol, and cis-ferulic acid, fetisinine, aucubigenin, linolenic acid, ussuriedine, and 5-(methylsulfanyl)pentanenitrile, were identified as potential effective substances against goiter. To summarize, we characterized the chemical components and mechanisms of \"Scrophulariae Radix-Fritillaria\" involved in the treatment of goiter, and our findings provide an experimental basis for its clinical application.
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  • 文章类型: Journal Article
    在这项研究中,开发了一种基于柠檬酸盐的QuEChERS方法,并用于分析新鲜红辣椒中的十二种新烟碱类农药,新鲜的绿色辣椒,干辣椒,超高效液相色谱-四极杆飞行时间质谱(UPLC-Q-TOF/MS)。在样品制备中,使用含1%甲酸的乙腈作为萃取溶剂。无水硫酸钠取代传统的无水硫酸镁用于除水,有效消除盐结块的问题。石墨化炭黑,十八烷基二氧化硅,和伯仲胺被用作清洁剂。该方法具有良好的灵敏度,新鲜辣椒的定量限低于0.03mg/kg,干辣椒的定量限低于0.15mg/kg。矩阵效应的值范围从-19.5%到8.4%,回收率为86.9%-105.2%。该分析方法为多种辣椒基质中新烟碱类农药残留的高通量检测提供了有效工具。
    In this study, a QuEChERS method based on citrate was developed and utilized for the analysis of twelve neonicotinoid pesticides in fresh red chilies, fresh green chilies, and dried chilies, coupled with ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS). In the sample preparation, acetonitrile containing 1% formic acid was used as the extraction solvent. Anhydrous sodium sulfate replaced the traditional anhydrous magnesium sulfate for water removal, effectively eliminating the issues of salt caking. Graphitized carbon black, octadecyl silica, and primary secondary amine were used as cleaning agents. The method showed good sensitivity, with the limits of quantification below 0.03 mg/kg for fresh chilies and below 0.15 mg/kg for dried chilies. Values of matrix effects ranged from -19.5% to 8.4%, and the recovery was 86.9% - 105.2%. The analytical method provided an effective tool for the high throughput detection of neonicotinoid pesticide residues in multiple chili matrices.
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  • 文章类型: Journal Article
    作为著名的中医方剂,圣仙汤(SXD)已应用于心血管疾病治疗已有一个世纪,尤其是冠心病,但潜在有效的化合物和潜在机制仍不清楚.采用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF/MS)和网络药理学分析,本研究鉴定并揭示了SXD的潜在有效化合物及其抗CHD的药理机制。通过UPLC-Q-TOF/MS分析筛选出57个具有良好药代动力学特征和生物学活性的有效化合物,数据库和文献挖掘,与96个CHD相关靶标相互作用,以支持潜在的协同治疗作用。对PPI网络和微阵列数据的系统分析进一步揭示了六个核心目标,包括TNF,IL-1β,IL-6、TP53、VEGFA和PTGS2主要参与流体剪切应力和动脉粥样硬化,脂质和动脉粥样硬化,PI3K-Akt信号通路等。此外,有效化合物的拟议贡献指数表明这些化合物,包括异阿魏酸,槲皮素,calycosin,阿魏酸,山奈酚,calycosin7-O-糖苷,福蒙素,黄芪甲苷和柴胡皂苷D,作为SXD的核心化合物。分子对接结果证实了那些核心化合物-靶标对表现出强结合能。此外,我们验证了SXD可显着减轻CHD大鼠的心肌组织损伤,并通过减少TNF的产生来逆转H/R诱导的H9c2细胞活力降低,IL-6和IL-1β,根据RT-qPCR和Westernblot结果,通过下调TP53,caspase3和细胞色素CmRNA表达水平以及caspase3,caspase9和细胞色素C蛋白表达水平来减少心肌细胞凋亡。我们的发现解释了SXD在CHD治疗中的潜在的药理机制。为今后SXD的基础和临床研究奠定了基础。
    As a well-known classical Chinese medicine prescription, Shengxian Decoction (SXD) has been applied for a century to treat cardiovascular diseases, especially coronary heart disease (CHD), but the potentially effective compounds and underlying mechanisms remain unclear. With ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF/MS) and network pharmacology analysis, the potential effective compounds of SXD and their pharmacological mechanisms against CHD were identified and revealed. 57 effective compounds with favorable pharmacokinetic characteristics and biological activities were screened through UPLC-Q-TOF/MS analysis, database and literature mining, interacting with 96 CHD-related targets to support potential synergistic therapeutic actions. Systematic analysis of the PPI network and microarray data further revealed six core targets, including TNF, IL-1β, IL-6, TP53, VEGFA and PTGS2, which were mainly involved in fluid shear stress and atherosclerosis, lipid and atherosclerosis, PI3K-Akt signaling pathway et al. Moreover, the proposed contribution indexes of effective compounds indicated these compounds, including isoferulic acid, quercetin, calycosin, ferulic acid, kaempferol, calycosin 7-O-glycoside, formononetin, astragaloside IV and saikosaponin D, as the core compounds of SXD. The molecular docking results confirmed that those core compound-target pairs exhibited strong binding energy. Furthermore, we validated that SXD significantly alleviated myocardial tissue injury in CHD rats and reversed H/R-induced decreases in H9c2 cell viability by attenuating the production of TNF, IL-6 and IL-1β, and reducing cardiomyocyte apoptosis via down-regulating the TP53, caspase3 and cytochrome C mRNA expression levels as well as caspase3, caspase9 and cytochrome C protein expression levels according to RT-qPCR and Western blot results. Our findings explained the pharmacological mechanisms underlying the effectiveness of SXD in the treatment of CHD, and laid a foundation for future basic and clinical research of SXD.
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  • 文章类型: Journal Article
    天然靛蓝的制造需要长时间的叶片浸泡和石灰搅拌;所得的靛蓝纯度小于3.00%,靛蓝的产率(以茎和叶的重量测量)小于0.50%,使其不适合用于工业程序,如印染。为了产生高纯度的靛蓝,研究了不需要石灰的酶促方法。进行单因素试验以优化酶法制备条件。研究结果表明,60℃,pH5.5,200毫升叶提取物含0.45毫克/毫升印度,和4:1比例的酸性纤维素(活性:9000U/mL,液体)到in是酶法制备的理想参数。靛蓝的收率为40.32%,靛蓝和靛玉红的含量分别为37.37%和2.30%,分别。正离子模式的MALDI-TOF-MS和正离子和负离子模式的UPLC-Q-TOF-MS用于通过酶法制备分析BaphicacanthusCusia(Nees)Bremek的靛蓝提取物。已经发现13种生物碱,5有机酸,3萜类化合物,3类固醇,2个黄酮,和7种其他化合物存在于靛蓝提取物中。靛蓝的存在,靛玉红,异鼠李素,色胺酮,indidoleB,UPLC-Q-TOF-MS测定的吲哚C经MALDI-TOF-MS分析验证。靛蓝提取物的酶法制备保持与常规靛蓝相同的化学组成。热分析和SEM形态用于确认靛蓝提取物中没有石灰。在酶促过程中,Baphicacanthuscusia(Nees)Bremek被更有效地使用,提高靛蓝的产量和纯度。
    The manufacturing of indigo naturalis requires prolonged leaf soaking and lime stirring; the resulting indigo purity is less than 3.00% and the yield of indigo (measured in stems and leaves weight) is less than 0.50%, making it unsuitable for use in industrial procedures like printing and dyeing. An enzymatic method of creating indigo without the requirement for lime was investigated in order to generate high purity indigo. Single factor tests were performed to optimize the enzymatic preparation conditions. The findings showed that 60 °C, pH 5.5, 200 mL of leaves extract containing 0.45 mg/mL indican, and a 4:1 ratio of the acidic cellulose (activity: 9000 U/mL, liquid) to indican were the ideal parameters for enzymatic preparation. The yield of indigo was 40.32%, and the contents of indigo and indirubin were 37.37% and 2.30%, respectively. MALDI-TOF-MS in positive ion mode and UPLC-Q-TOF-MS in both positive and negative ion modes were used to analyze indigo extracts from Baphicacanthus cusia(Nees) Bremek by enzymatic preparation. It has been discovered that 13 alkaloids, 5 organic acids, 3 terpenoids, 3 steroids, 2 flavones, and 7 other compounds are present in indigo extracts. The presence of the indigo, indirubin, isorhamnetin, tryptanthrin, indigodole B, and indigodole C determined by UPLC-Q-TOF-MS was verified by MALDI-TOF-MS analysis. The enzymatic preparation of indigo extracts kept the same chemical makeup as conventional indigo naturalis. Thermal analysis and SEM morphology were used to confirm that there was no lime in the indigo extract. During the enzymatic process, Baphicacanthus cusia (Nees) Bremek was employed more effectively, increasing the yield and purity of indigo.
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  • 文章类型: Journal Article
    血必净注射液(XBJ)在临床上广泛用于治疗肾病综合征(NS),但其生物活性成分和治疗机制尚不清楚。在这项研究中,采用超高效液相色谱-四极杆-飞行时间串联质谱(UPLC-Q-TOF/MS)测定XBJ的生物活性成分。在阿霉素诱导的BALB/c小鼠中评估XBJ对NS的治疗作用(ADR,10mg/kg)经由单尾静脉。使用脂多糖(LPS,4μg/mL)。结果表明,鉴定出XBJ的33个组分。此外,在血液中检测到12种生物活性成分,包括原儿茶酸,丹酚酸C,苯甲酰氧基烟草素,丹参素,丹酚酸A,丹酚酸B,儿茶素,咖啡酸,胆酰芍药苷,氧合烟苷,羟基红花黄色素A,迷迭香酸.生物活性成分的相对含量(%)依次为59.32、16.01、9.97、9.73、8.72、8.31、7.92、6.54、1.54、1.30、0.68和0.59。XBJ治疗后,肾功能,改善NS模型小鼠的高脂血症和肾脏病理损害。此外,足细胞中功能蛋白nephrin和desmin的水平被逆转,XBJ降低促炎因子水平。有趣的是,原儿茶酸和丹酚酸C对LPS诱导的MPC-5中的足细胞功能也具有良好的保护作用,并降低了炎症水平。该研究首次阐明XBJ的生物活性成分及其通过保护足细胞功能治疗NS的潜在治疗机制。
    Xuebijing injection (XBJ) is widely used to treat nephrotic syndrome (NS) in clinic, but its bioactive components and therapeutic mechanism are still unclear. In this study, the bioactive components of XBJ were determined by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS). The therapeutic effect of XBJ on NS was evaluated in BALB/c mice induced by adriamycin (ADR, 10 mg/kg) via a single tail vein. The protective effect of XBJ and its bioactive components on podocytes was demonstrated using mouse podocytes (MPC-5) induced by lipopolysaccharide (LPS, 4 μg/mL). The results show that 33 components of XBJ were identified. Furthermore, 12 bioactive components were detected in blood, including protocatechuic acid, salvianolic acid C, benzoyloxypaeoniflorin, danshensu, salvianolic acid A, salvianolic acid B, catechin, caffeic acid, galloylpaeoniflorin, oxypaeoniflorin, hydroxysafflor yellow A, rosmarinic acid. The relative content (%) of the bioactive components were 59.32, 16.01, 9.97, 9.73, 8.72, 8.31, 7.92, 6.54, 1.54, 1.30, 0.68 and 0.59 in this order. After XBJ treatment, the renal function, hyperlipidemia and renal pathological damage were improved in NS model mice. Moreover, the levels of nephrin and desmin which are functional proteins in podocytes were reversed, and the levels of pro-inflammatory factors were reduced by XBJ. Interestingly, protocatechuic acid and salvianolic acid C also showed good protective effects on podocyte function and reduced the level of inflammation in LPS-induced MPC-5. The study is the first time to elucidate the bioactive components of XBJ and its potential therapeutic mechanism for treating NS by protecting podocyte function.
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  • 文章类型: Journal Article
    杜鹃花树皮已被用作食物中的成分和传统的草药药物多年。不幸的是,漆酚的存在,诱发过敏,限制了它的应用。这项研究使用了涡旋混合基质固相分散微萃取技术,从蛇皮树皮中提取了漆酚。HPLC用于评估提取的漆酚的量(15:0、15:1、15:2和15:3)。通过原位共沉淀方法制备了改性的磁性吸附剂,并使用多种技术进行了表征。优化的提取条件如下:以磁性沸石SoconyMobil-Five为吸附剂,a1:2的样品/吸附剂比例,涡流混合时间2.5min,以4mL的0.1%(V/V)三氟乙酸-甲醇为洗脱溶剂,超声时间8min。该方法线性好,重复性高。此外,漆酚的检出限范围为0.20至0.50μg/mL。在优化条件下,通过超高效液相色谱和四极杆飞行时间质谱鉴定了50个化合物。这些化合物包括8种酚酸,9单体漆酚,11漆酚二聚体,其他10个组件和11种类黄酮。建议的方法,与现有提取工艺相比,具有阶段少、提取效率高的优点,是获得和评估原料或提取物中漆酚的潜在有用方法。
    Toxicodendron vernicifluum bark has been used for many years as a component in foods and as a traditional herbal medication. Unfortunately, the presence of urushiols, which induce allergies, limits its application. This study used a vortex-blending matrix solid-phase dispersion microextraction technique to extract urushiols from Toxicodendron vernicifluum bark. HPLC was used to evaluate the amounts of the extracted urushiols (15:0, 15:1, 15:2, and 15:3). The modified magnetic adsorbent was prepared through an in situ coprecipitation method and characterized using a variety of techniques. The optimized extraction conditions are as follows: using magnetic Zeolite Socony Mobil-Five as an adsorbent, a 1:2 sample/adsorbent ratio, 2.5 min of vortex-blending time, 4 mL of 0.1% (V/V) trifluoroacetic acid-methanol as the elution solvent and 8 min of ultrasound time. There was good linearity and high repeatability in the method. Furthermore, the limits of detection for the urushiols ranged from 0.20 to 0.50 μg/mL. Under the optimized conditions, 50 compounds were identified by ultra high performance liquid chromatography and quadrupole time-of-flight mass spectrometry. These compounds included 8 phenolic acids, 9 monomeric urushiols, 11 urushiol dimers, 10 other components, and 11 flavonoids. The suggested approach, which has the advantages of few stages and high extraction efficiency over existing extraction procedures, is a potentially useful method for obtaining and evaluating urushiols in raw materials or extracts.
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  • 文章类型: Journal Article
    背景:糖尿病溃疡(DU)的慢性伤口组织中的活性氧(ROS)激增加剧了炎症反应。炎症过程中的氧化应激状态会加剧炎症,引起组织损伤,导致伤口愈合时间延长。生积化瘀方(SJHYF)是治疗糖尿病溃疡慢性伤口的著名中药方剂。越来越多的临床证据表明,SJHYF表现出优异的治疗效果,并具有良好的安全性。然而,SJHYF在DU病理条件下改善氧化损伤的潜在机制尚不清楚.
    目的:研究SJHYF对过氧化氢(H2O2)诱导的人HaCaT角质形成细胞损伤的细胞保护特性,并探讨其使用RNA-seq治疗DU的潜在靶标和分子途径。
    方法:HaCaT细胞与H2O2孵育24小时,构建氧化应激细胞模型。使用MTT和EdU测定法测量细胞活力和增殖,分别。使用划痕试验评估细胞迁移,使用DCFH-DA探针测量ROS的荧光强度。通过UPLC-Q-TOF/MS分析SJHYF的化学成分,同时使用RNA-Seq分析SJHYF对H2O2诱导的HaCaT细胞的治疗作用。使用逆转录-定量聚合酶链反应(RT-qPCR)验证潜在的靶基因。同时,采用WesternBlot法探讨SJHYF对H2O2诱导的HaCaT细胞保护作用的通路表型表达。
    结果:以0.25mg/mL的浓度应用SJHY促进细胞增殖,细胞迁移,并减少了ROS的产生。此外,SJHYF检测到共有93种活性化合物,包括关键成分,如没食子酰-β-D-葡萄糖,丹参素,原花青素B2,儿茶素,和碱宁。RNA-seq分析确定了几个核心靶标,即KRT17,TGM1,JUNB,PRDX5、TXNIP、PRDX1、HSP90AA1、HSP90AB1、HSPA8和TNF-α。Westernblot显示存在JNK/c-Jun/MMPs通路及其相关转录因子。
    结论:SJHYF通过阻断JNK/c-Jun/MMPs通路对H2O2诱导的HaCaT细胞氧化损伤具有明显的保护作用。
    BACKGROUND: The reactive oxygen species (ROS) surge in the chronic wound tissue of diabetic ulcers (DUs) aggravates the inflammatory response. The oxidative stress state during inflammation will exacerbate inflammation and cause tissue damage, resulting in prolonged wound healing. Shengjihuayu Formula (SJHYF) is a renowned Chinese medicine prescription for treating chronic wounds in diabetic ulcers. Growing clinical evidence has demonstrated that SJHYF exhibits superior therapeutic efficacy and has a favorable safety profile. However, the underlying mechanisms by which SJHYF ameliorates oxidative damage under pathological conditions of DUs remain unclear.
    OBJECTIVE: To investigate the cytoprotective properties of SJHYF on hydrogen peroxide (H2O2)-induced cell damage in human HaCaT keratinocytes and to explore its potential targets and molecular pathways in treating DUs using RNA-seq.
    METHODS: HaCaT cells were incubated with H2O2 for 24 h to construct an oxidative stress cell model. Cell viability and proliferation were measured using the MTT and EdU assays, respectively. Cell migration was assessed using the scratch assay, and the fluorescence intensity of ROS was measured using the DCFH-DA probe. The chemical components of SJHYF were analyzed by UPLC-Q-TOF/MS, while the therapeutic effects of SJHYF on H2O2-induced HaCaT cells were analyzed using RNA-Seq. The potential target genes were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). At the same time, the pathway phenotype expression of SJHYF on the protection of H2O2-induced HaCaT cells was explored using Western Blot.
    RESULTS: The application of SJHY at a concentration of 0.25 mg/mL promoted cell proliferation, cell migration, and reduced ROS production. In addition, SJHYF was detected to have a total of 93 active compounds, including key components such as Galloyl-beta-D-glucose, Danshensu, Procyanidin B2, Catechin, and Alkannin. The RNA-seq analysis identified several core targets namely KRT17, TGM1, JUNB, PRDX5, TXNIP, PRDX1, HSP90AA1, HSP90AB1, HSPA8, and TNF-α. Western blot revealed the presence of the JNK/c-Jun/MMPs pathway and its related transcription factors.
    CONCLUSIONS: SJHYF displays significant protective effects on H2O2-induced oxidative cell damage in HaCaT cells via blocking the JNK/c-Jun/MMPs pathway.
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