研究丹参新酮(miltirone)对人急性髓系白血病THP-1细胞的细胞毒作用及其机制。通过中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)筛选出丹参新酮活性成分...研究丹参新酮(miltirone)对人急性髓系白血病THP-1细胞的细胞毒作用及其机制。通过中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)筛选出丹参新酮活性成分及其对应靶点,利用UniProt数据库将靶蛋白转化成基因;利用GeneCards和DisGeNET数据库收集急性白血病疾病相关基因;运用Venny 2.1构建韦恩图并得到交集靶点;利用STRING数据库和Cytoscape 3.8.2软件构建蛋白相互作用(PPI)网络;选取对数生长期的THP-1细胞,用DMSO,2.5、5、10、15及20μmol·L^(-1)miltirone处理24 h;采用羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)标记法检测细胞增殖;Annexin V-PE/7AAD双染流式细胞术检测凋亡率,吖啶橙染色观察细胞形态;实时荧光定量PCR(qPCR)检测NCOA2、PARP1、Bax、Bcl-2、caspase-3 mRNA的表达;检测caspase抑制剂Z-VAD-FMK对miltirone诱导凋亡的影响。筛选出26个miltirone靶蛋白,急性白血病疾病相关基因1046个,6个潜在作用靶点与急性白血病相关。流式结果显示,10μmol·L^(-1)miltirone可显著抑制THP-1细胞增殖并促进凋亡;吖啶橙染色可见细胞萎缩、细胞核碎裂、染色体聚集等典型的凋亡形态学改变;NCOA2、PARP1 mRNA表达水平降低,Bax/Bcl-2比例增大,促凋亡蛋白caspase-3活性增强。Z-VAD-FMK能减弱miltirone诱导凋亡的作用。研究结果表明miltirone能抑制THP-1细胞的增殖并诱导其凋亡,其机制可能与下调NCOA2、PARP1基因的表达,Bax/Bcl-2比值增大,激活促凋亡因子caspase-3有关。展开更多
Objectives: The existing inflammatory models are concentrated in relatively complex medical fields, and most of them use a single type of cell, and the induction conditions are not uniform, so the current LPS-induced ...Objectives: The existing inflammatory models are concentrated in relatively complex medical fields, and most of them use a single type of cell, and the induction conditions are not uniform, so the current LPS-induced inflammation model is less conducive to the study of skin inflammation. The aim of this research is to enhance the existing LPS-induced inflammation model and establish a skin inflammation model that is suitable for the swift screening of anti-inflammatory agents in the cosmetics industry. Methods: LPS was used to induce inflammatory responses in KC and THP-1 cells. Enzyme-linked immunosorbent assay (ELISA) was employed to assess the levels of IL-1α, IL-8, and TNF-α in the two cell types, while the DCFH-DA probe was utilized to label the levels of reactive oxygen species (ROS) in both cell types. Results: In KC cells, 10 μg/mL of LPS induced a significant upregulation of IL-8 but did not result in elevated expression of IL-1α. However, at 100 μg/mL of LPS, both IL-8 and IL-1α were highly expressed in KC cells. LPS concentrations ranging from 0.01 to 100 μg/mL failed to stimulate TNF-α production in KC cells but induced a gradient increase in ROS levels. In THP-1 cells, LPS concentrations from 0.01 to 100 μg/mL did not induce IL-1α production but significantly elevated IL-8 and led to a gradient increase in TNF-α and ROS. After treatment with 100 μg/mL of LPS, the cosmetic ingredient Rucika KGM mitigated the elevated levels of IL-1α, IL-8, and ROS in LPS-induced KC cells and IL-8 and ROS in THP-1 cells. Conclusion: This study has successfully developed an application-oriented model suitable for investigating skin inflammation, enabling the rapid and comprehensive screening of cosmetic ingredients with anti-inflammatory activity. .展开更多
文摘研究丹参新酮(miltirone)对人急性髓系白血病THP-1细胞的细胞毒作用及其机制。通过中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)筛选出丹参新酮活性成分及其对应靶点,利用UniProt数据库将靶蛋白转化成基因;利用GeneCards和DisGeNET数据库收集急性白血病疾病相关基因;运用Venny 2.1构建韦恩图并得到交集靶点;利用STRING数据库和Cytoscape 3.8.2软件构建蛋白相互作用(PPI)网络;选取对数生长期的THP-1细胞,用DMSO,2.5、5、10、15及20μmol·L^(-1)miltirone处理24 h;采用羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)标记法检测细胞增殖;Annexin V-PE/7AAD双染流式细胞术检测凋亡率,吖啶橙染色观察细胞形态;实时荧光定量PCR(qPCR)检测NCOA2、PARP1、Bax、Bcl-2、caspase-3 mRNA的表达;检测caspase抑制剂Z-VAD-FMK对miltirone诱导凋亡的影响。筛选出26个miltirone靶蛋白,急性白血病疾病相关基因1046个,6个潜在作用靶点与急性白血病相关。流式结果显示,10μmol·L^(-1)miltirone可显著抑制THP-1细胞增殖并促进凋亡;吖啶橙染色可见细胞萎缩、细胞核碎裂、染色体聚集等典型的凋亡形态学改变;NCOA2、PARP1 mRNA表达水平降低,Bax/Bcl-2比例增大,促凋亡蛋白caspase-3活性增强。Z-VAD-FMK能减弱miltirone诱导凋亡的作用。研究结果表明miltirone能抑制THP-1细胞的增殖并诱导其凋亡,其机制可能与下调NCOA2、PARP1基因的表达,Bax/Bcl-2比值增大,激活促凋亡因子caspase-3有关。
文摘Objectives: The existing inflammatory models are concentrated in relatively complex medical fields, and most of them use a single type of cell, and the induction conditions are not uniform, so the current LPS-induced inflammation model is less conducive to the study of skin inflammation. The aim of this research is to enhance the existing LPS-induced inflammation model and establish a skin inflammation model that is suitable for the swift screening of anti-inflammatory agents in the cosmetics industry. Methods: LPS was used to induce inflammatory responses in KC and THP-1 cells. Enzyme-linked immunosorbent assay (ELISA) was employed to assess the levels of IL-1α, IL-8, and TNF-α in the two cell types, while the DCFH-DA probe was utilized to label the levels of reactive oxygen species (ROS) in both cell types. Results: In KC cells, 10 μg/mL of LPS induced a significant upregulation of IL-8 but did not result in elevated expression of IL-1α. However, at 100 μg/mL of LPS, both IL-8 and IL-1α were highly expressed in KC cells. LPS concentrations ranging from 0.01 to 100 μg/mL failed to stimulate TNF-α production in KC cells but induced a gradient increase in ROS levels. In THP-1 cells, LPS concentrations from 0.01 to 100 μg/mL did not induce IL-1α production but significantly elevated IL-8 and led to a gradient increase in TNF-α and ROS. After treatment with 100 μg/mL of LPS, the cosmetic ingredient Rucika KGM mitigated the elevated levels of IL-1α, IL-8, and ROS in LPS-induced KC cells and IL-8 and ROS in THP-1 cells. Conclusion: This study has successfully developed an application-oriented model suitable for investigating skin inflammation, enabling the rapid and comprehensive screening of cosmetic ingredients with anti-inflammatory activity. .
