1.
[Mechanism of extract of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma on SIRT1 autophagy pathway of endothelial cell senescence induced by hydrogen peroxide].
Xiu, CK, Fu, YK, Wang, Q, Wang, X, Hu, YH, Wu, Y, Yang, J, Lei, Y
Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica. 2021;(23):6216-6223
Abstract
This study aims to explore the effect of extract of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Chuanxiong Rhizoma(hereinafter referred to as GNS) on the SIRT1-autophagy pathway of endothelial cell senescence induced by hydrogen peroxide(H_2O_2). To be specific, vascular endothelial cells were classified into the blank control group(control), model group(model), model + DMSO group(DMSO), resveratrol group(RESV), and GNS low-dose(GNS-L), medium-dose(GNS-M), and high-dose(GNS-H) groups. They were treated with H_2O_2 for senescence induction except the control. After intervention of cells in each group with corresponding drugs for 24 h, cell growth status was observed under an inverted microscope, and the formation of autophagosome under the transmission electron microscope. In addition, the changes of microtubule-associated protein 1 light chain 3β(LC3 B) were detected by immunofluorescence staining. The autophagy flux was tracked with the autophagy double-labeled adenovirus(mRFP-GFP-LC3) fusion protein. Dansylcadaverine(MDC) staining was employed to determine the autophagic vesicles, and Western blot the expression of sirtuin 1(SIRT1), ubiquitin-binding protein p62, and LC3Ⅱ. After H_2O_2 induction, cells demonstrated slow growth, decreased adhesion ability, raised number of SA-β-gal-stained blue ones, a certain number of autophagosomes with bilayer membrane and secondary lysosomes in the cytoplasm, and slight rise of autophagy flux level. Compared with the model group, GNS groups showed improved morphology, moderate adhesion ability, complete and smooth membrane, decreased SA-β-gal-stained blue cells, many autophagosomes, autophagic vesicles, and secondary lysosomes in the cytoplasm, increased autophagolysosomes, autophagy flux level, and fluorescence intensity of LC3 B and MDC, up-regulated expression of SIRT1 and LC3Ⅱ, and down-regulated expression of p62, suggesting the improvement of autophagy level. GNS can delay the senescence of vascular endothelial cells. After the intervention, the autophagy flux and related proteins SIRT1, LC3Ⅱand p62 changed significantly, and the autophagy level increased significantly. However, EX527 weakened the effect of Chinese medicine in delaying vascular senescence. GNS may delay the senescence of vascular endothelial cells through the SIRT1 autophagy pathway.
2.
[In vitro study on expression of tumor stem cell biomarker and transdifferentiation towards endothelial cells of retinoblastoma cells under hypoxia condition].
Zhang, LN, Zhao, GQ, Wang, Q, Niu, YJ, Xu, Q, Yang, WY
[Zhonghua yan ke za zhi] Chinese journal of ophthalmology. 2013;(8):736-43
Abstract
OBJECTIVE To investigate the capability of RB cells that represent characteristics of tumor stem cell and trans-differentiate towards endothelial cells under hypoxia microenvironment, as well as their mechanism. METHODS Experimental research. RB cell line Y79 was cultured in hypoxia environment with or without rapamycin treatment. Morphological changes, expression of neuron specific enolase (NES) and ATP binding cassette transporters G2 (ABCG2), and expression of HIF-1α protein and mRNA were detected. After been induced toward endothelial cells, expression of CD31 and vWF, up-taking of Dil-acLDL, vasculogenic mimicry (VM) formation in 3D culture and expression of HIF-1α,EphA2 and PI3K were detected. The ANOVA test was performed to compare the differences among groups, and SNK-q test was performed to further comparison. RESULTS Y79 cells in normal oxygen group were single or agminated suspending cells. In hypoxia group, part of Y79 cells became adherent. No adherent cells were observed in rapamycin fore-treated group. Positive rates of NES and ABCG2 had significant difference among these three groups (FNES = 698.45, FABCG2 = 864.48, all P < 0.01). Compared with normal group [(98.2 ± 2.5)%, (2.1 ± 2.1)%],NES positive rate [(35.1 ± 3.4)%] was significantly reduced and ABCG2 positive rate [(67.4 ± 3.6)%] was significantly enhanced in hypoxia group (q = 46.11, 50.89; both P < 0.01), no significant changes were observed in rapamycin fore-treated group(P > 0.05). Expressions of HIF-1α protein and mRNA showed significant difference among these three groups (Fprotein = 314.85, FmRNA = 132.01, all P < 0.01). Compared with normal oxygen group (0.165 ± 0.056,0.927 ± 0.715) , HIF-1α protein (1.094 ± 0.077) and mRNA (6.408 ± 0.686) were significantly increased in hypoxia group(q = 31.81, 18.40; both P < 0.01), HIF-1α mRNA (7.219 ± 0.591) was significantly increased but no increased protein expression (0.218 ± 0.061) was observed in rapamycin fore-treated group. After transdifferentiation induction,Y79 cell in hypoxia group expressed CD31 and vWF, acquired the abilities of acLDL up-taking and VM formation. Compared with normal group(0.327 ± 0.108, 0.194 ± 0.033, 0.402 ± 0.068), expression of HIF-1α,EphA2 and PI3K protein (1.440 ± 0.089,0.377 ± 0.056,0.762 ± 0.090) was significant increased in hypoxia group(q = 8.72-23.00, all P < 0.01). CONCLUSIONS Under hypoxic condition, part of RB cells can express tumor stem cell markers, transdifferentiate towards endothelial cells and acquire part of endothelial cell function and VM formation capability. HIF-1α through EphA2/PI3K pathway may play an important role in VM formation.