Notoginsenoside R1 Has Thrombopoietic Effect in Irradiated Mice

Objective: Chinese herbs have been reported to be effective and safe as a treatment for thrombocytopenia. Sanqi, Radix Notoginseng, is the dried roots of Panax notoginseng (Burk.) F. H. Chen (Araliaceae). It has been prescribed in several Chinese formulas including "Yunnan Bai Yao", which is used for treatment of trauma and bleeding due to internal and external injury. Its main constituents are ginsenosides (a kind of saponins), as well as notoginsenosides (only rich in Notoginseng species). Although Sanqi is a well-known haemostatic drug, its effects and mechanisms on megakaryocyte/platelet production have not been well studied. The objective of this study was to compare the effect of a purified notoginsenoside R1 (NR1) and thrombopoietin (TPO) on thrombopoiesis in irradiated mice.

Methods: NR1 (2.5 mg) and TPO (0.25 ug) were dissolved in distilled water and given by intra-peritoneal injection daily for 14 days starting from the day after radiotherapy. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from NR1, TPO, and vehicle control groups on day 0, 7 and 14. On day 14, the mice were sacrificed and bone marrow cells were harvested for CFU-MK, CFU-GM, BFU-E and CFU-F (fibroblastoid) assays.

Results: Our results showed that NR1 enhanced the recovery of platelets, WBC, and RBC count. Moreover, NR1 also promoted the CFU-F (12 + 0.8 vs 20 + 0.4 colonies/2 x 10⁶ cells, p=0.003), CFU-MK (22 + 1.8 vs 26 + 3.6 colonies/2 x 10⁵ cells, p=0.025), CFU-GM (26 + 5.0 vs 38 + 4.2 colonies/2 x 10⁵ cells, p=0.002), and BFU-E (12 + 2.6 vs 18 + 1.8 colonies/2 x 10⁵ cells, p=0.003) formation. Similar results were obtained in TPO-treated group. In in-vitro study, we further analyzed the effect of NR1 (0-50mM) on mouse CFU-MK formation using a plasma clot colony assay. The results showed that NR1 (20 mM) enhanced TPO (50 ng/ml)-induced CFU-MK formation (18 + 2.2 vs 30 + 6.0 colonies/2 x 10⁵ cells, p=0.022, n=6). Furthermore, the effect of NR1 (5-50 mM) on the growth of bone marrow stromal cells was also investigated using CFU-F assay. NR1 (50mM) had a promoting effect on CFU-F growth (18 + 3.6 vs 24 + 1.8 colonies/2 x 10⁶ cells, p=0.04, n=6). Our studies showed that NR1 enhances thrombopoiesis in vivo and the growth of bone marrow stromal cells as well as megakaryocytes in vitro. Therefore, we speculate that the thrombopoietic activity of NR1 may be mediated via promoting the progenitor of platelet, megakaryocytes, and bone marrow stromal cells. Consequently, we tested the anti-apoptotic effect of NR1 using megakaryocytic cell line M-07e. Data demonstrated that nutrient deprivation significantly increased cell death (n = 6, control vs. normal), and NR1 significantly reduced apoptosis and total cell death (n = 6, NR1 vs. control). TPO treatment also significantly reduced M-07e cell death (n = 6, TPO vs. control). Data also demonstrated that nutrient deprivation increased caspase-3 expression (n = 6, normal vs. control), and NR1 (NR1 vs. control) or TPO (TPO vs. control) treatment significantly reduced caspase-3 expression. M-07e cells were stained with JC-1 reagent. Control cultures had increased proportion of cells containing JC-1 monomers, the percentage of R2 populations are significant higher in the normal, NR1 and TPO treated groups comparing to those of the control group (n = 6). R1, a transitional cell subset containing both monomers and aggregates, was not significantly affected by these reagents. R2: a cell subset showing mitochondria membrane damage.

Conclusions: Here we reported that the effect of NR1 is comparable with TPO on the production of platelets in irradiated mice. Therefore, the effect of NR1 on thrombopoiesis may be mediated via anti-apoptosis on megakaryocytic cells.