Epimedium on the regulation of immune function in mice
Authors: XIONG Ping-yuan Guo Kai-wen Zhao-Hui Tang
【Key Words】 epimedium;,, T lymphocytes;,, IL
Abstract: Objective: To investigate the Epimedium on
immune function in vitro. Methods: 3HTdR labeling method to measure NK cytotoxic activity of Epimedium on the impact and Epimedium on ConA induced activity of T-lymphocyte
transformation; by ELISA, of mouse spleen cells Epimedium IL2 in impact; the use of flow cytometry to detect T lymphocytes in Epimedium of CD3, CD4, CD8, CD4 / CD8-positive cells in the
percentage of impact. Results: In high-dose group can enhance
the cytotoxic activity of NK cells (P <0.01); middle-and high-
dose group can enhance the ConA induced T lymphocyte
transforming activity (P <0.01); each dose group were IL2 can enhance the production of spleen cells (P <0.01); each dose group significantly reduced the percentage of CD8 positive cells, CD4 / CD8-positive cells significantly increased the
percentage (P <0.01). Conclusion: Epimedium on immune function significantly in vitro.
Keywords: Epimedium; T lymphocytes; IL2; NK cells,
Epimedium (Epimedium Kereanum Nakai) has a kidney yang, strengthens bones and tendons, rheumatism effect. A long
history of medicinal Epimedium, Berberidaceae Epimedium from a variety of plants. Li's in the "Compendium of Materia Medica" and call it a "Yijing gas, Caine bones, complement waist and knee, strong effort," the effect. Modern Pharmacological
Experimental studies have shown that Epimedium can increase cardiovascular and cerebrovascular blood flow, promote hematopoietic function, immune function and bone metabolism, has anti-aging, anti-tumor effects and so on. This study in
mice NK cell activity tests, lymphocyte transformation test, IL2 activity tests, flow cytometry experiment to learn more about Epimedium on immune function in vitro.
Ba1b / c strain mice, weighing 18 ~ 22g, male and female in half, from the Experimental Animal Center of Peking
University Health Science. Epimedium purchase of the Central Pharmacy. ConA for the Sigma company's products, 3HTdR by the Chinese Academy of Sciences Institute of Atomic Energy Research Institute of Isotopes, mouse IL2 ELISA Kit for the
crystal-US Biological Engineering Co., Ltd. products. NK-
sensitive cell lines Yac1 cells from the Department of Immunology, Peking University Health Science Center to provide. Flow cytometry: FACS Can type, is the United States BactonDickson company's products.
2 Methods and Results
100% liquid preparation: Take dry crude drug 20g, add water 100 ~ 200ml, home refrigerator 24h, and then flooding crude drug Wuhuo boiling, and to slow fire boiling, maintaining 30min, coarse filtration with gauze, leaving the
filtrate, and then residual residue 100 ~ 200ml boiling water for 30min, and then filter residue. Two filtrate combined, concentrated to 20ml, containing per ml dose of 1g, adjusted pH to 7.4, and then to 1500rpm centrifuge 15min, the
supernatant backup . Mice were randomly divided into five
groups of 10 rats. The normal control group: each day with 0.25ml normal saline continuously fed 10d; making model control group: for 60CO irradiation modeling, a one-time 60CO
γ-rays 400 rad total body irradiation, dose rate of 213.93 Lun / min, time of 103 s [1 ]; drug test group: under the same conditions, a one-time 60CO γ-ray irradiation group were
given drug I epimedium 1g / (kg.d) body weight, administered orally 10d; Drugs II group were given Epimedium 10g / (kg.d) body weight, administered orally 10d; medicine III group were given Epimedium 50g / (kg.d) body weight, administered orally 10d .
21 NK cell activity tests taken Yac1 cells and effects of cell suspension of 50μl (effect-target ratio of 100:1),
adding 96-well plate, then add 0.1ml culture medium, while
effector cells and target cells based control, home 37 ? 5%
CO2 incubator 6h. Infiltration 0.5μCi/20mld the 3HTdR,
culture harvested after 6h. Collector with the long cells are collected in a cellulose membrane of cells, on repeated
washing with distilled water, and then dropping 50.0g / L trichloroacetic acid 2ml, the sample is fixed, 2ml ethanol eluted color flow, after placing 80 ? drying oven in 30min.
The dried membrane into the fluid containing 5ml scintillation measurement cup, placed in liquid scintillation instrument measured Cpm values. Each sample re-located three-hole
[2,4,6]. The results in Table 1.
Table 1 Epimedium on NK cell activity (omitted)
Table 1 The results showed that after treatment with
different concentrations of Epimedium effector cells co-
cultured with target cells after 6h, drugs and drug group ?
? group and the control group modeling, NK cell cytotoxic activity was significantly increased (P <0.01 ).
Lymphocyte transformation test, 22 mice were killed off
cervical spine, aseptic spleen, Shredded, grinding, filtration, containing 10% fetal calf serum IMDM medium Preparation of spleen cell suspension, the cell concentration of 1 × 106A/ ml. Take 100μl in 96-well plate by adding 10.0
μg/mlConA, each hole final volume of 200μl. Cultured at
37 ?, after 48h by adding 3HTdR 2μlCi / hole, to continue to
foster 12h post-harvest. With long cell collector cells were collected on the cellulose membrane, on repeated washing with distilled water again after dropping 50.0g / L trichloroacetic acid 2ml, the sample is fixed, 2ml ethanol eluted color flow, after placing 80 ? drying oven in 30min. The dried membrane into the fluid containing 5ml scintillation measurement cup, placed in liquid scintillation instrument measured Cpm values. Each sample re-located three-hole [3,6]. The results in Table
2. Table 2 The results showed that different concentrations of Epimedium treated mouse lymphocytes after 48h co-culture with
ConA, compared with the control group, model making, drugs and drug group ? ? group can significantly enhance the activity of ConA-induced lymphocyte transformation ( P <0.01). Reposted elsewhere in the paper for free download http://
23 IL2 activity tests taken 1 × 106A/ ml concentration
of mouse spleen cell suspension 100μl in 96-well plate by
adding 10.0μg/ml ConA, each hole final volume of 200μl. Take
the supernatant frozen at -20 ?. Reference mouse IL2 ELISA
kit, measured OD450 value [2,5]. The results in Table 3.
Table 2 Epimedium on lymphocyte transformation activity slightly
Table 3 The results showed that after treatment with different concentrations of Epimedium ConA stimulated mouse lymphocytes in the ability to produce IL2, compared with the control group modeling, drug I group, drug group and drug group ? ? can markedly enhanced ConA-induced the ability to
produce IL2 cells (P <0.01).
24 flow cytometry tests obtained 1 × 107A/ ml
concentration of mouse spleen cell suspension 100μl in small
centrifuge tube were added to FTTC labeled CD3, PE labeled CD4, CD8 antibodies 10μl, oscillation mixing, at room
temperature dark place under 30min, washed two times with PBS, combined with 500μl PBS mixing, 10min later by flow cytometry using CD3, CD4, CD8, CD4 / CD8 positive expression rate of . The results in Table 4.
Table 4 Epimedium on T-lymphocyte subsets in slightly
Table 4 The results showed that after treatment with different concentrations of Epimedium can promote 60CO γ-ray
irradiated mice CD4/CD8 T-lymphocyte recovery, increase the ratio of CD4/CD8 T lymphocytes. Drug treatment groups compared with the control group modeling, P <0.01.
NK cells are non-specific immune cells, they do not need prior antigen sensitization, can directly kill certain tumor cells and virus-infected target cells in the body and early anti-tumor anti-viral and intracellular bacterial parasite infection may play an important role in the immune process . In the tumor or virus-specific IgG antibody in conditions, NK cells can be mediated through the surface of IgGFCR, identification of anti-IgG antibody with specific binding of tumor cells or virus-infected target cells. In addition, NK
cell activation, but also through the secretion of IFNγ, IL2,
TNF and other cytokines play a role in immune regulation. Thus, NK cells have anti-tumor, anti-infection, immune
regulation and other functions. Table 1 The results showed that after treatment with different concentrations of Epimedium effector cells co-cultured with target cells after
6h, drugs and drug group ? ? group and the control group
modeling, NK cell cytotoxic activity were significantly increased. Surface expression of T-lymphocyte mitogen can
combine a variety of membrane molecules, the binding
specificity of mitogen-point decision from the sugar Jeter. Combined with the appropriate mitogen, may directly induce resting T lymphocyte activation, proliferation and differentiation. Table 2 The results showed that different concentrations of Epimedium treated mouse lymphocytes after 48h co-culture with ConA, compared with the control group, model making, drugs and drug group ? ? group can
significantly enhance the activity of ConA-induced lymphocyte
transformation. IL2 mainly by activated T lymphocytes may act
on a variety of immune effector cells, including T lymphocytes, B lymphocytes, macrophages, NK cells and so on, and can induce a new type of killer cells, such as lymphokine-
activated killer cells (LAK). It has been demonstrated, T lymphocytes, B lymphocytes, macrophages, NK cell-surface
expression of both IL2R, IL2 to IL2R combination with the surface of these cells play a role in immune regulation. Table 3 The results showed that after treatment with different concentrations of Epimedium ConA stimulated mouse lymphocytes in the ability to produce IL2, compared with the control group modeling, drug I group, drug group and drug group ? ? can
markedly enhanced ConA-induced lymphocyte transforming
activity. Specific cell-mediated immune effector cells are
mainly Th1-type CD 4 cells and CD 8CTL cells. The former through the activation of MΦ and delayed-type
hypersensitivity induced inflammation in the host against intracellular pathogen infection plays an important role; the latter through the secretion of cytotoxic and induce apoptosis in target cells with a pathogen. Table 4 The results showed that after treatment with different concentrations of Epimedium can promote 60CO γ-ray irradiated mice CD4/CD8 T-
lymphocyte recovery, increase the ratio of CD4/CD8 T-
lymphocyte, drug treatment groups compared with the control group modeling. The results of this study by testing mice from NK cell killing activity, lymphocyte proliferation activity
and IL2 secretion activity and the ratio of CD4/CD8 T
lymphocytes and other proof of epimedium on immune function to enhance the role and regulation of the immune response , for development and utilization of this natural medicine, Epimedium has practical significance.
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