Effects Of Cistanche Glycosides On The Immune Function Of D-galactose-induced Aging Model Mice

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

〔Abstract〕 Objective:

To study the immunological mechanism of the anti-aging effect of Cistanche glycosides on D-galactose-induced aging mice. Methods: The 3 H-TdR incorporation method was used to determine the lymphocyte transformation ability, the neutral red test was used to determine the phagocytic function of mouse peritoneal macrophages, the immunofluorescence method was used to detect lymphocyte subsets, and the radioimmunoassay was used to determine the level of IL-2 in peripheral blood. , Trace NAG enzyme release method to measure NK cell activity. Results: The immune function of D-galactose-induced aging mice was significantly decreased, which was manifested in the ability of lymphocyte transformation, peripheral blood IL-2 content, CD4+ T cell, and CD8+ T cell content, a phagocytic function of peritoneal macrophages, and NK cell activity. Decrease (P <0.05); the cistanche of glycoside can improve the lymphocyte transformation ability, peripheral blood IL-2 content, a phagocytic function of peritoneal macrophages, NK cell activity, CD4 + T and CD8 + T cell content (P <0.05). 0. 05). Conclusion: the cistanche of glycoside can significantly enhance the immune function of D-galactose-induced aging mice, restore or approach the state of young mice, and have the effect of delaying aging.

[Keywords]: Cistanche total glycosides; D-galactose; aging model; immunity

Cistanche of glycoside: anti-aging

Cistanche is the most frequently used drug in anti-aging and longevity formulas. Cistanche total glycosides (GCS) is the general term for a class of phenylethanoid glycosides in cistanche and is the main biological activity extracted from the salt-producing cistanche in the northern Xinjiang Component [1], GCS has been proved to have an anti-lipid peroxidation effect [2], but there are few research reports on its anti-aging immune mechanism.

1. Materials and Methods

1. 1 Animal grouping and medicine

60 Kunming mice, male and female, 3 months old, weighing 18-21 g, provided by the Animal Room of the Institute of Biological Products, Ministry of Health. The mice in each group were housed in the same room in separate cages, and they were free to eat and drink. The animals were randomly divided into three groups: the youth control group, the aging model group, and the model administration group, with 20 animals in each group. GCS was extracted and identified by the Department of Phytochemistry, School of Pharmacy, Jiamusi University according to the literature [1].

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1.2 Method

The young group was injected with normal saline subcutaneously in the neck for 30 days every day and was given warm water for 15 days from the 15th day. The mice in the aging model group were injected with D-galactose (autoclaved) 100 mg/kg subcutaneously on the back of the neck every day. Bodyweight for 30 consecutive days. In the model administration group, D-galactose 100 mg/kg body weight was injected subcutaneously on the back of the neck every day, and GCS 125 mg·kg-1·d-1 was administered at the same time from the 15th day, and the administration was continuously administered for 15 days. All animals were sacrificed at the same time on the 30th day, and various observation indexes were determined.

1. 2. 1 Lymphocyte transformation experiment

The mouse spleen cell suspension was adjusted to a cell concentration of 2×106, and Con A was used as a stimulus to be cultured in vitro for 5 days, and its transformation ability was tested by the 3 H-Td R incorporation method. The results are expressed as the stimulus index SI (SI=ConA stimulation group/control group).

1. 2. 2 Mouse peritoneal macrophage phagocytic function (neutral red test [3])

Collect the mouse peritoneal fluid macrophage suspension aseptically. After centrifugation, float the precipitate in the cell culture medium. Use a hemocytometer to count and adjust the cell concentration of the solution to 2×106. Place it in a 37℃ incubator for 2 hours. , Discard the supernatant and non-adherent cells. Take the wall macrophages and add 0.1% neutral red physiological saline to 37℃ and continue to culture for 30 min, discard the neutral red and repeatedly wash the macrophages with 37℃ PBS, dilute with 50% acetic acid and 50% ethanol, set 722 Spectrophotometer colorimetric at 540 nm wavelength, measured absorbance A value.

1. 2. 3 Detection of lymphocyte subsets

Take 0.1 ml of 1×107/ml lymphocyte suspension, add 10 μl mouse anti-mouse CD3, CD4, and CD8 monoclonal antibodies respectively, mix well, incubate at 4°C for 30 min, 1% FC S in PBS (0. 2M, pH7.4) Wash 3 times, flow cytometry detection: laser flow cytometer (FACScan, USA BD company), argon laser, excitation wavelength 488 nm, FITC detection spectrum 480～630 nm, count 800/ s cells, 200,000 cells in total. The results were analyzed and recorded with a computer HP340 and professional software LYSI SⅡ.

1. 2. 4 Determination of IL-2 in peripheral blood by radioimmunoassay

Take 1 ml of blood from the mouse eyeballs in a test tube. After coagulation, the serum is separated. According to the kit operation, the CPM number is measured on the counter.

1. 2. 5 NK cell activity

Take mouse eye blood aseptically and add heparin for anticoagulation. Refer to the literature [4] for details. 1.3 Statistical processing The experimental data is expressed as x±s, processed by the S AS software, and analyzed by variance analysis, F test, and t-test.

benefit of cistanche: Improve Immunity

2. results

2.1 The effects of GCS on the transformation ability of lymphocytes and T cell subsets in each group of mice are shown in Table 1.

Table 1. The effect of GCS on the transformation ability of lymphocytes and T cell subsets in each group of mice (n= 16,x±s)

Compared with Ⅰ: 1) P <0.05, 2) P. 05; compared with Ⅱ: 2) P <0.05; the same as the table below

2.2 The effects of GCs on the phagocytic function, IL-2 content, and NK cell activity of peritoneal macrophages in each group of mice are shown in Table 2.

Table 2. The effect of GCS on the phagocytic function, NK cell activity and IL-2 content of peritoneal macrophages in each group of mice (n= 16,x±s)

3. Discussion

In this experiment, the phagocytic function of peritoneal macrophages and NK cell activity of the aging model mice were reduced (P <0.05). GCs can increase the phagocytic function and NK cell activity of peritoneal macrophages in aging model mice (P<0.05), which is close to the youth model group. From this, it can be inferred that GCs can improve the body's non-specific cellular immunity and immune regulation functions, and enhance Anti-tumor and anti-infection of the aging body is one of the ways to delay aging [4]. IL-2 is an indispensable cytokine for the proliferation and differentiation of T lymphocytes, and it can regulate the functions of lymphocytes, monocytes-macrophages, and NK cells. IL-2 can promote the proliferation and differentiation of T lymphocytes, regulate the immune response, induce Tc activity, and promote the production of antibodies and interferon-α. It plays an important role in the immune system. In this experiment, the IL-2 content of aging model mice decreased (P<0.05). After GCs were given, the IL-2 content increased (P<0.05), which was close to the youth model group, indicating that GCs produced IL by increasing T lymphocytes. -2, enhance the immune function of elderly mice and achieve the effect of delaying immune aging. The content of CD4+ T cells and CD8+ T cells decreased significantly with increasing age. The low T cell function was also manifested by the decline of specific antigen and mitogen Con A, and the decline of the ability of T cells to divide repeatedly. In this experimental model, the lymphocyte transformation ability, CD4+ T cell and CD8+ T cell content of the mouse was reduced (P <0.05), and GCs increased the mouse lymphocyte transformation ability, CD4 + T cell and CD8 + T cell content (P <0.05). 05), close to the youth model group. It shows that GCs can make the body resist aging and tend to be younger by promoting the proliferation of lymphocytes and improving the specific cellular immune function of mice.

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4. References

1. Du Niansheng, Wang Hong. Isolation and identification of phenoxyethanol glycosides from Cistanche [J]. Natural Products Research and Development, 1993; 5(4): 7-8.

2. Li Linlin, Wang Xiaowen, Wang Xuefei, et al. Anti-lipid peroxidation and anti-radiation effects of the total glycosides of Cistanche 〔J〕. Chinese Journal of Chinese Materia Medica, 1997; 22(6): 364-367.

3. Xu Shuyun, Bian Rulian. Experimental Methodology of Pharmacology [M]. 2nd Edition. Beijing: People's Health Publishing House, 1994: 1221.

4. Liu Xiongbo, Liu Caiyu, Yang Xiuyun, et al. Detection of NK cell activity by micro-NAG release method [J]. Journal of Clinical Laboratory Science, 1996; 14(2): 66-67.