Most of hematopoietic growth factor regulates proliferation and differentiation of blood cells through JAKs-STATs signal transduction pathway. Total saponins of Panax ginseng (TSPG) can promote in vitro differentiation of CD34+ hematopoietic progenitor cells into erythroid cells, with similar effectiveness of hematopoietic growth factor. Erythropoietin receptor (EpoR) expression on the cell membrane of progenitor cells is critical during the erythroid differentiation process.OBJECTIVE:
To investigate the molecular mechanism of TSPG to induce erythroid cells through erythropoiesis and its receptor-mediated JAK2/STAT5 signal transduction.DESIGN, TIME AND SETTING:
An in vitro cytological observation. The study was performed at the Department of Histology and Embryology, Institute of Basic Medicine, Chongqing Medical University from May 2006 to October 2008.MATERIALS:
Umbilical cord blood of normal full-term pregnancy was provided by the First Hospital of Chongqing Medical University. TSPG, purity > 95%, provided by Chongqing Institute of Traditional Chinese Medicine, was diluted in RPMI-1640 for work concentration of 1 g/L and degermed by positive pressure filtration.METHODS:
①Colony-forming ability: 5 × 106/L mononuclear cells (MNCs) from human umbilical cord blood were incubated in RPMI-1640 culture solution containing horse serum, with various dilutions of TSPG (0 as blank control, 10, 25, 50, 75, 100 mg/L). The MNCs were cultured on 96-well culture plate, with 0.2 mL in each well. Early erythroid cells were counted on day 14, and late erythroid cells on day 6. ②100 μ L MNCs (5×108/L) was measured by MTT. ③0, 10, 25, 50, and 100 mg/L TSPG were separately cultured with 1 × 109/L MNCs for 24 hours for Western blot determination. ④1 × 109/L MNCs were harvested and cultured separately in RPMI-1640 culture solution containing 10% horse serum as control group and in TSPG (25 mg/L)- conditioned culture system as experimental group. 5 U/mL Epo was added for 0, 2, 5 and 30 minutes. Immunoprecipitation of JAK2/STAT5 was used for the effect of TSPG on Epo/EpoR-induced tyrosine phosphorylation of JAK2/STAT5.MAIN OUTCOME MEASURES:
Effect of TSPG on proliferation of erythroid progenitor cells from human umbilical cord blood; Effect of Epo on the proliferation of hematopoietic cells; Effect of TSPG on EpoR expression of the umbilical blood cells; tyrosine phosphorylations of JAK2 and STAT5.RERULTS:
TSPG (10-75 mg/L) promoted the colony formation of BEU-E, CFU-E, and the preferential differentiation into erythroid lineage cells was most induced from 25 mg/L of TSPG. Using the colorimetric MTT assay, MNCs exhibited proliferative responses to Epo (2-50 U/mL) reaching maximum at 5 U/mL Epo. The addition of TSPG did not increase the expression of EpoR after MNCs were incubated in the presence of with or without TSPG for 24 hours. The pretreatment with TSPG for 24 hours enhanced Epo-induced tyrosine phosphorylation of JAK2 and STAT5 (STAT5a and STAT5b).CONCLUSION:
The Epo/EpoR-mediated signals are enhanced and JAK2 / STAT5 signals play an essential role in the effect of TSPG on erythropoiesis, whereas the expression level of EpoR remains unchanged.