Study of the Effects and Mechanisms of Ginsenoside Compound K on

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Bioactive Constituents, Metabolites, and Functions

Study of the Effects and Mechanisms of Ginsenoside Compound K on Myelosuppression Jiahong Han, Yu Wang, Enbo Cai, Lianxue Zhang, Yan Zhao, Nian Sun, Xiaoman Zheng, and Siqi Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06073 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019

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Journal of Agricultural and Food Chemistry

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Study of the Effects and Mechanisms of Ginsenoside Compound K on

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Myelosuppression

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Jiahong Han, Yu Wang, Enbo Cai*, Lianxue Zhang, Yan Zhao, Nian Sun,

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Xiaoman Zheng, Siqi Wang

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College of Chinese Medicinal Material, Jilin Agricultural University

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*Dr.

Enbo Cai, College of Chinese Medicinal Material, Jilin Agricultural University, Xincheng Street No. 2888, Changchun Jilin province, 130118, China *Corresponding

author: Enbo Cai, College of Chinese Medicinal Material, Jilin Agricultural University, 2888 Xincheng Street, Changchun Jilin province, 130118, China. E-mail:[email protected],tel.:13944816620. 1

ACS Paragon Plus Environment


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Study of the Effects and Mechanisms of Ginsenoside Compound K on

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Myelosuppression

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Abstract: Ginsenoside compound K (CK) is not a ginsenoside naturally exist in

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Panax ginseng Meyer. However, CK is a major metabolite of ginsenoside Rb1.,Rb2 or

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Rc in the intestine under the effects of bacteria. In this study, we first investigated the

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effects of CK on myelosuppression in mice induced by cyclophosphamide (CTX).

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The respective quantities of white blood cell (WBC), blood platelet (PLT) and bone

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marrow nucleated cells (BMNC) were determined to be 8.54±0.91 (109/L),

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850.90±44.11 (109/L) and 1.45±0.22 (109/L) in the CK-H group by detecting

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peripheral blood cells and BMNC. CK-H and CK-L both increased the thymus index

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by up to 0.62±0.06 (mg/g) and 0.52±0.09 (mg/g), respectively, and significantly

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increased the yields of CFU-GM and CFU-Meg. According to our study, CK could

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control apoptosis and promote cells enter the normal cell cycle by bcl-2/bax signaling

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pathway and MEK/ERK signaling pathway. Therefore, the BMNC could proliferate

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and differentiate normally after enter the normal cell cycle. So the peripheral blood

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cells could show a trend of returning to normal. The recovery of peripheral blood cells

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resulting in the level of cytokines tend to normal. This process may be the

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mechanisms of CK on myelosuppression. This study provides a reference for ginseng

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in the treatment of myelosuppression.

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Keywords: ginsenoside compound K (CK) ， myelosuppression ， chemotherapy, 2


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cytoxan(CTX), hematopoietic function

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INTRODUCTION

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Chemotherapy is the most widely used treatment for cancer since

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chemotherapeutic agents can act on the abnormally active cancer cells throughout the

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whole body 1. However, these agents also have destructive effects on normal rapidly

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proliferating cells such as blood cells, stomach cells, and especially hematopoietic

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stem cells (HSCs) while destroying cancer cells 2. CTX is the most widely used

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medicine with a high therapeutic index in chemotherapy. CTX can against a variety of

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cancers by damaging intracellular DNA molecules directly 3, affecting cell division

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and inducing apoptosis of HSC 4-5.

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Myelosuppression is a common toxic reaction of most chemotherapeutic agents.

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The clinical manifestation of myelosuppression is the decrease of blood cells in

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peripheral

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thrombocytopenia 7. The infection caused by leukopenia and the bleeding caused by

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thrombocytopenia both

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Therefore, determining how to effectively alleviate myelosuppression during

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chemotherapy is a problem that must be addressed.

blood

6.

Then

triggered

leukopenia,

neutropenia,

anemia

and

seriously affect the patient's treatment and prognosis 8.

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Ginseng is the root of Panax ginseng C. A. Mey. It is a traditional Chinese

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medicine and has been widely used all over the world nowadays. Ginsenosides are

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considered to be the main active compounds 9. Ginsenoside compound K (CK) does

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not exist in ginseng

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Rb1.,Rb2 or Rc in the intestine under the effects of bacteria 12. CK is considered a rare

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ginsenoside and has received much attention because of its bioactivities

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However, there have been no studies on the effects of CK on myelosuppression.

10-11.

Nonetheless, CK is a major metabolite of ginsenoside

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13-14.

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We speculate that CK has effect on myelosuppression mice. So in this study, we

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compared the count of blood cells, bone marrow nucleated cells (BMNC), and thymus

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and spleen index before and after treatment with CK.. We explored the mechanism of

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action by investigating the effects of CK on the proliferation and differentiation of

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hematopoietic progenitor cells, the influence on the bone marrow cell cycle and the

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level of hematopoiesis-related cytokines and the expression of proteins.

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MATERIALS AND METHODS

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Chemicals. CK was obtained from the Jilin University (Changchun, China). It

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was 99.5% pure, as confirmed by HPLC. CTX was purchased from Jiangsu Shengdi

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Pharmaceutical Co., Ltd (Jiangsu, China). Mouse cytokines were obtained from

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novoprotein Scientific Inc. (Shanghai, China). ELISA kits was supplied by R&D

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systems (Minneapolis, MN, USA). Antibodies were obtained from Cell Signaling

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Technology (Danvers, MA, USA). The propidium iodide was supplied by Tianjin

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Sungene Biotech Co., Ltd (Tianjin, China). PVDF membrane (Millipore ， USA),

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Medical X-ray film (Eastman Kodak Company ，

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(AB85157, Abcam, UK), anti-Bcl-2 antibody (AB59348, Abcam, UK), anti-caspase-3

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antibody (AB13847, Abcam, UK), anti-MEK antibody (AB33918, Abcam, UK),

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anti-p-ERK antibody (AB194770, Abcam, UK).

USA), anti-Bax antibody

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Experimental Animals. BALB/c mice, male, 18 g ~ 22 g. Provided by the

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Laboratory Animal Quality Testing Center of Jilin Province (No: SCXK-2016-0003).

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After 7 days adaptation prior to the experiment. The temperature was controlled at

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22±2°C and humidity was at 50%±10%. Adjusted to 12 h light and 12 h dark. All

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efforts were to reduce the pain of the mice. Whole process obeid the rules of National 5



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Institute of Health Guide for the Care and Use of Laboratory Animals. The study was

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approved by the Animal Ethics Committee of the China Academy.

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The mice were divided into five groups: Normal, Model, Positive, CK-L and

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CK-H. Intraperitoneally (i.p.) injected the mice in 2-5 groups with CTX (100 mg·kg–1,

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0.1 mL·10 g–1) and in group 1 with equal volume of saline (0.9% NaCl aq) for 3 days.

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After CTX treatment for 24 hours, i.p. injected the Positive group with rhG-CSF

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(11.25 μg·kg–1), the CK-L group with CK (5 mg·kg–1), the CK-H group with CK (10

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mg·kg–1) and the Normal and the Model groups with equivalent volume of saline

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(0.9% NaCl aq) for 7 days.

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Determination of Peripheral Blood Cells. On Day 11, collected blood samples

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from the eye socket vein. after the eyeballs were removed, and the samples were

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placed into test tubes with ethylenediaminetetraacetic acid (EDTA). Counted white

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blood cells (WBC), red blood cells (RBC), hemoglobin (HGB) and platelets (PLT) by

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automated hematology analyzer (Sysmex KX-21N, Japan).

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Determination of Thymus and Spleen Index. Measured the weights of the

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mice after the last .i.p. injection. Sacrificed the mice and remove the thymus and

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spleen. Weighed and calculated the thymus and spleen index.

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The Preparation of Bone Marrow Nucleated Cell Suspension. Removed the

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femurs under aseptic conditions then used sterile phosphate-buffered saline (PBS)

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flushing bone marrow cells to form a single cell suspension. The bone marrow

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nucleated cell suspension from 10 mice was randomly divided into two parts; one was

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used for the count of BMNCs, hematopoietic progenitor cell culture and cell cycle,

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while the other was used for western blotting. 6


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Bone Marrow Nucleated Cells. Centrifuged the suspension for 10 min at 1200

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r/min then added RBC lysis buffer 2 ml. After standed for 3 min, centrifuged the

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mixture at 1200 r/min for 10 min. Rinsed the bone marrow karyocytes twice with

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sterile PBS 1 mL at 1200 r/min for 10 min. Counted by inverted microscope.

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HPCs Culture. Adjusted BMNC to a concentration of 105/mL. Inoculated bone

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marrow cells into the IMDM medium for colony formation units-granulocyte

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monocyte (CFU-GM), colony formation units-megakaryocytic(CFU-Meg), colony

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formation units-erythroid(CFU-E) and burst-forming unit-erythroid(BFU-E). Cultured

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BMNC at 37°C under 5% CO2 atmosphere. CFU-E were counted under inverted

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phase contrast microscope after 3 days of culture. BFU-E, CFU-GM and CFU-Meg

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were counted after 7 days of culture.

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The Levels of Hematopoiesis-related Cytokines in Plasma. Prepared plasma samples and measured the levels of EPO, TPO and GM-CSF by ELISA.

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Cell Cycle. Centrifuged single cell suspension after rinsed it twice with cold

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PBS. Added to 70% cold ethanol 2 mL and fix the cells at 4°C overnight. Then

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incubated cells with propidium iodide in the dark for 30 min. Measured cells by flow

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cytometry and calculated proliferation index (PI) by the following formula.

S  G2 / M  100% G 0 / G1  S  G 2 / M

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PI 

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Expression of Related Protein. Determined the ation of protein content by

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western blotting. Extracted the protein in BMNC and determined the protein

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concentration by the Bradford method. Separated protein samples by SDS-PAGE

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electrophoresis and transferred to PVDF membrane. Blocked in 5% skim milk 7



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solution for 1 hand added primary antibody. Incubated the samples at 4°C overnight.

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Incubated the samples for 1 h after added a secondary antibody. Chemiluminescence

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was used to develop the color. Analyzed the protein bands quantitatively after scanned

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the film. Obtained the expression level of protein.

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Statistical Analysis. Expressed data as the mean ± SD. The data were

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statistically analyzed by one way ANOVA, post-hoc Student-Newman-Keuls test.

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Considered a value of P

Study of the Effects and Mechanisms of Ginsenoside Compound K on

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