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by Xue Li, Jared Sipple, Qishen Pang, and Wei Du
Salidroside stimulates DNA repair enzyme Parp-1 activity in mouse HSC maintenance by Xue Li, Jared Sipple, Qishen Pang, and Wei Du Blood Volume 119(18): May 3, 2012 ©2012 by American Society of Hematology
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Salidroside prevents oxidative stress–induced HSC loss.
Salidroside prevents oxidative stress–induced HSC loss. (A) salidroside partially limits H2O2-induced LSK expansion. BM cells from WT C57BL/6 mice pretreated with or without salidroside (75μg/g body weight) followed by H2O2 (0.25 μmol/g body weight) injection were harvested for LSK (Lin-Sca1+c-kit+) cell frequency assessment using flow cytometry. (B) Quantification of LSK frequency in mice described in panel A. (C) SD prevents H2O2-induced LSK CD34-Flt3- cell loss. BM cells described in panel A were subjected to clow cytometry analysis for LSK CD34−Flt3− frequency. (D). Quantification of LSK CD34−Flt3− frequency in mice described in panel A. (E) SD prevents H2O2-induced LSK CD150+CD48− cell loss. BM cells described in panel A were subjected to Flow Cytometry analysis for LSK CD150+CD48− frequency. (F) Quantification of CD150+CD48− frequency in mice described in panel A. Results are means ± standard deviation (SD) of 3 independent experiments (n = 9 per group). Xue Li et al. Blood 2012;119: ©2012 by American Society of Hematology
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Salidroside enhances HSC repopulation.
Salidroside enhances HSC repopulation. (A) Salidroside enhances the repopulating ability in recipient mice. LSK (Lin-Sca1+c-kit+) cells from WT C57BL/6 mice (CD45.2+) pretreated with or without salidroside (75μg/g body weight) followed by H2O2 (0.25 μmol/g body weight) treatment were isolated by cell sorting using FlowAria II. One thousand sorted LSK cells plus 1 million c-kit–depleted competitors (CD45.1+) were injected to lethally irradiated recipients. Donor chimerism was examined at 4 months after transplantation using peripheral blood from recipients. Representative images (left) and quantifications (right) were shown. Results are means ± SD of 3 independent experiments (n = 15 per group). (B) Salidroside increases HSC-enriched LSK cells in recipient mice. One thousand sorted LSK cells from WT C57BL/6 mice (CD45.2+) pretreated with or without salidroside followed by H2O2 treatment were injected along with 1 million c-kit–depleted competitors (CD45.1+) to lethally irradiated recipients. BM cells from recipient mice were harvested and subjected to flow cytometry analysis for LSK frequency. (C) Salidroside enhances competitive reconstitution of stressed HSCs. Various numbers of donor (CD45.2+) LSK cells (50, 100, or 1000) from stressed WT C57BL/6 mice were mixed with 1000 competitor LSK (CD45.1+) cells and the mixtures were transplanted intravenously into lethally irradiated congenic (CD45.1+) recipients. Donor-derived hematopoietic reconstitution was analyzed by flow cytometry 8 and 16 weeks after transplantation using peripheral blood from recipients. (D) Salidroside increases HSC-enriched LSK in recipient mice. BM cells from recipient mice described in panel C were subjected to flow cytometry analysis for LSK frequency. (E-F) Salidroside enhances LT-HSC repopulation. BM cells from primary recipient mice described in panel B were used for secondary transplantation by injecting 5 × 106 BM cells to lethally irradiated recipients. Donor-derived chimerism (E) and LSK (F) were analyzed by flow cytometry 16 weeks after BMT. Results are means ± SD of 2 independent experiments (n = 10 per group). Xue Li et al. Blood 2012;119: ©2012 by American Society of Hematology
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Xue Li et al. Blood 2012;119: ©2012 by American Society of Hematology
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Xue Li et al. Blood 2012;119: ©2012 by American Society of Hematology
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Salidroside reduces DNA strand breaks but not ROS
Salidroside reduces DNA strand breaks but not ROS. (A) Salidroside does not reduce H2O2-induced ROS. WT C57BL/6 mice were pretreated with one dose of salidroside (75μg/g body weight), NAC (50μg/g body weight), or MnTBAP (10μg/g body weight), followed by H2O... Salidroside reduces DNA strand breaks but not ROS. (A) Salidroside does not reduce H2O2-induced ROS. WT C57BL/6 mice were pretreated with one dose of salidroside (75μg/g body weight), NAC (50μg/g body weight), or MnTBAP (10μg/g body weight), followed by H2O2 (0.25 μmol/g body weight). BM cells were then harvested and labeled with CM-H2DCFDA for flow cytometry analysis of ROS in the LSK-gated cells. Representative images (left) and quantifications (right) were shown. (B) Salidroside reduces DNA strand breaks. BM cells from mice described in panel A were isolated by magnetic beads depletion, and then subjected to analysis for DNA strand breaks by the comet assay. The mean tail moment of untreated vehicle sample is expressed as 100%. Larger tail moment represents higher levels of DNA damage. For each treatment, 50 cells were scored from random sampling. (C) Salidroside reduces 8-oxodG. Protein extractions were prepared using low density BM cells from mice described in panel A. Whole cell lysates were then subjected to SDS-PAGE and immunoblotted with Western Blot antibodies for 8-oxodG and β-actin. (D) Repair kinetics. Low-density BM cells were pretreated with salidroside (250μM), NAC (500μM), or MnTBAP (250μM) for 2 hours, followed by H2O2 for additional 2 hours, and then released for indicated time intervals. Whole cell lysates were prepared and subjected to SDS-PAGE and immunoblotted with antibodies for 8-oxodG and β-actin. Xue Li et al. Blood 2012;119: ©2012 by American Society of Hematology
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Salidroside stimulates PARP-1 activity.
Salidroside stimulates PARP-1 activity. (A) Salidroside fails to reduce H2O2-induced DNA strand breaks in Parp-1−/− cells. Lin− cells were isolated from Brca2−/−, DNA-PKcs3A/3A, Fancd2−/−, Parp-1−/−, or Xpc−/− mice. Cells were treated with or without salidroside, NAC, or MnTBAP in the presence of H2O2, and subjected to Comet Assay. The mean tail moment of H2O2-treated WT sample is expressed as 100%. For each sample, 50 cells were scored from random sampling. (B) Salidroside fails to reduce H2O2-induced 8-oxodG in Parp-1−/− cells. Protein lysates were prepared from low-density BM cells treated as described in panel A. Whole cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies against 8-oxodG and β-actin. (C) Salidroside stimulates Parp-1 activity. Low-density BM cells isolated from mice described in panel A were treated with or without H2O2 and salidroside, and BM cells were isolated and subjected to flow cytometry analysis for Parp-1 activity in the LSK population using antibody against PARP-1. (D) Salidroside activates Parp-1 in vivo. Low-density BM cells were isolated from WT mice, and treated with or without SD and H2O2 for 2 hours. Whole cell lysates were subjected to immunoprecipitation using PAR antibody. Precipitated samples were resolved by SDS-PAGE and blotted with antibody specific for PARP-1. Xue Li et al. Blood 2012;119: ©2012 by American Society of Hematology
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Inhibition of PARP-1 abrogates the effect of salidroside on HSC maintenance.
Inhibition of PARP-1 abrogates the effect of salidroside on HSC maintenance. (A) Deletion of Parp-1 abolishes salidroside-mediated increase in quiescent HSC frequency in stressed mice. Parp1−/− mice as well as their WT littermates were pretreated with or without salidroside (75 μg/g body weight) followed by H2O2 (0.25 μmol/g body weight). BM cells were subjected to flow cytometry analysis for quiescent HSC (G0 phase). Results are means ± SD of 3 independent experiments (n = 9 per group). (B) NU1025 treatment abolishes salidroside-mediated increase in quiescent HSC frequency in stressed mice. H2O2-treated (0.25 μmol/g body weight) WT mice were treated with or without salidroside (SD; 75μg/g body weight) and NU1025 (25 mg/kg body weight). BM cells were subjected to flow cytometry analysis for quiescent HSC. Results are means ± SD of 3 independent experiments (n = 9 per group). (C) The maintenance of HSC quiescence by salidroside requires Parp LSK cells from WT or Parp1−/− mice were injected to lethally irradiated WT recipients. Recipient mice were then subjected to salidroside and H2O2 treatments. Cycling donor-derived (CD45.2+) cells were assessed by Flow Cytometry analysis by pyronin Y staining. Representative images (top) and quantifications (bottom) were shown. Results are means ± SD of 3 independent experiments (n = 9 per group). (D) The enhancing effect of salidroside on the long-term repopulation abilities of stressed HSCs requires Parp1. Cells from primary recipients described in panel C were used for second transplantation by injecting 10 million whole BM cells to lethally irradiated WT recipients. Donor-derived cells were determined by flow cytometry analysis. Representative images (top) and quantifications (bottom) were shown. Results are means ± SD of 3 independent experiments (n = 9 per group). Xue Li et al. Blood 2012;119: ©2012 by American Society of Hematology
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