Panax ginseng modulates cytokines in bone marrow toxicity and myelopoiesis: ginsenoside Rg1 partially supports myelopoiesis

Abstract

In this study, we have demonstrated that Korean Panax ginseng (KG) significantly enhances myelopoiesis in vitro and reconstitutes bone marrow after 5-flurouracil-induced (5FU) myelosuppression in mice. KG promoted total white blood cell, lymphocyte, neutrophil and platelet counts and improved body weight, spleen weight, and thymus weight. The number of CFU-GM in bone marrow cells of mice and serum levels of IL-3 and GM-CSF were significantly improved after KG treatment. KG induced significant c-Kit, SCF and IL-1 mRNA expression in spleen. Moreover, treatment with KG led to marked improvements in 5FU-induced histopathological changes in bone marrow and spleen, and partial suppression of thymus damage. The levels of IL-3 and GM-CSF in cultured bone marrow cells after 24 h stimulation with KG were considerably increased. The mechanism underlying promotion of myelopoiesis by KG was assessed by monitoring gene expression at two time-points of 4 and 8 h. Treatment with Rg1 (0.5, 1 and 1.5 µmol) specifically enhanced c-Kit, IL-6 and TNF-α mRNA expression in cultured bone marrow cells. Our results collectively suggest that the anti-myelotoxicity activity and promotion of myelopoiesis by KG are mediated through cytokines. Moreover, the ginsenoside, Rg1, supports the role of KG in myelopoiesis to some extent.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Invivo experimental design (A), C57BL6 (n = 40) were treated with 5FU (200 mg/kg, i.p) on day zero and post-treated (24 h) with KG (25, 50 and 100 mg/kg) for six days, daily once.

Complete blood count (CBC) was taken at 0, 3, 5, 7 10 and 14 day. C57BL6 mice (n = 40) were treated with 5FU (200 mg/kg, i.p) on day zero and post-treated (24 h) with KG (25, 50 and 100 mg/kg) for six days, sacrificed on day 7 for cytokines, histopathology, gene expression and colony forming studies (B). Bone marrow cells isolated from C57BL6 were cultured and treated with KG (0.2, 2, 20 and 200 µg/ml) or Rg1 (0, 0.5, 1 and 1.5 µmol/L) at for cytokines (ELISA) and gene expression by semi quantitative and quantitative PCR (C).

Figure 2. Effects of KG on Haematologic parameters in 5FU treated mice.

Before and after 24 h of 5FU injection and KG treatment, about 60–100 µl of retro orbital sinus blood was collected using heparin coated capillary tube at different days (0, 3, 5 7 10 and 14) and haematological parameters such as (A) white blood cells (WBC), (B) neutrophils, (C) lymphocytes (absolute number) and (D) platelets were measured. Data were expressed as means ± SD (n = 8). *P<0.05 **P<0.01, normal Vs. 5FU and KG Vs. 5FU.

Figure 3. Effects of KG on Haematologic parameters and body weight changes in 5FU treated mice.

Before and after 24 h of 5FU injection and KG treatment, about 60–100 µl of retro orbital sinus blood was collected using heparin coated capillary tube at different days (0, 3, 5 7 10 and 14) parameters such as (A) hemoglobin, (B) red blood cells (RBC), (C) hematocrit and (D) body weight were measured. Data were expressed as means ± SD (n = 8). *P<0.05 **P<0.01, normal Vs. 5FU and KG Vs. 5FU.

Figure 4. Effects of KG on Organ weight and Histopathology.

After 24 h of 5FU injection and 6 days of KG treatment, group of mice were sacrificed under ether anesthesia on day 7 and organ weights such as spleen and thymus were measured using an automated electronic balance (A) Organs such as spleen, thymus and bone marrow were removed and processed for Haematoxylin and Eosin (B). The stained tissues were examined under a light microscope (200X magnifications). Data were expressed as means ± SD. *P<0.05 **P<0.01, a – normal Vs. 5FU, b – KG Vs. 5FU.

Figure 5. Effects of KG on 5FU-induced changes gene expression in spleen.

Total RNA was extracted from the spleen cells, mRNA of (A) IL-1, (B) SCF, (C) c-Kit and (D) IL-4 were analyzed by semi quantitative PCR using specific forward and reverse primers. Data (means ± SD). *p<0.05, Normal Vs. 5FU and KG Vs. 5FU.

Figure 6. Effects of KG on 5FU induced changes IL-3, GM-CSF and CFU-GM.

Serum separated from blood collected from abdominal aorta of mice was subjected to cytokine analysis using Elisa kit (A). Bone marrow cells isolated from mice were grown in MethoCult methylcellulose-based complete medium in a 5% CO2 incubator for 14 d (B). Isolated bone marrow cells cultured in 6 well plated treated with KG (0.2, 2, 20 200 µg/ml) for 24 h and conditioned media was subjected to cytokine (pg/ml) analysis using commercial kit method (C &D). Data were expressed as means ± SD. *P<0.05 **P<0.01 compared with normal, a – normal Vs. 5FU, b – KG Vs. 5FU.

Figure 7. Effects of KG on genes c-Kit, FasL, IL-1, IL-2 and IL-6 in bone marrow cells.

Bone marrow cells treated with KG (0.2, 2 20 and 200 µg/ml) for 4 and 8 h (n = 3). Total RNA was isolated from bone marrow cells using RNAase mini kit and analyzed by semi quantitative PCR using mouse specific primers. The relative intensities of the bands (F) were determined with the use of the ratios to β actin (normalized) and expressed as percentage (A–E).

Figure 8. Effects of KG on genes IL-12, MCP1, TGF-β, IFN-γ and TNF-α in bone marrow cells.

Bone marrow cells treated with KG (0.2, 2 20 and 200 µg/ml) for 4 and 8 h (n = 3). Total RNA was isolated from bone marrow cells using RNAase mini kit and analyzed by semi quantitative PCR using mouse specific primers. The intensities of the bands (F) were determined with the use of the ratios to β actin (normalized) and expressed as percentage (A–E).

Figure 9. Compositional analysis of KG and role of Ginsinoside Rg1 on IL-6, c-Kit and TNF-α in bone marrow cells.

Column was eluted with solvents A (18% Acetonitrite) and B (80% Acetonitrite) at flow rate of 2.5 ml/min. Solution 100% A and 0% B changing over 32 min, 80% A and 20% B to 80 min, 0% A and 100% B to 100 min, and 100% A and 0% B to 110 min were used, amount of ginsinosides present (n = 3) were expressed in mg/kg (A). (b–d) Bone marrow cultured cells were treated with Ginsinoside Rg1 (0.5, 1 and 1.5 µmol/L) for 4 and 8 h. mRNA isolated from bone marrow cells using Trireagent and products were analyzed by quantitative PCR using specific primers. Results were normalized by using the reference gene, actin, and are represented as percentage versus the reference gene. Data were expressed as means ± SD. *p<0.05, **p<0.01 compared with 4 h blank control (Untreated), #p<0.05, ##p<0.01 compared with 8 h blank.