The Effect Of Cistanche Tubulosa Polysaccharides On Immune Cells And Telomerase Activity in Aging Mice

Abstract: Objective: To study the effects of polysaccharide of Cistanche deserticola (PCD) on the content of malondialdehyde (MDA), telomerase activity, and immune function in the tissue of subacute aging mice. Methods. The subacute aging model of mice was induced by D-galactose. The content of MDA was detected by thiobarbituric acid colorimetry; Telomerase activity was detected by PCR-ELISA; The lymphocyte proliferation reaction was measured by the MTT method; The phagocytic function of mouse peritoneal macrophages was measured by neutral red test; The content of IL-2 in serum was determined by enzyme-linked immunosorbent assay (ELISA). Result. The content of MDA in the heart, liver, and brain of the model group increased significantly, while the telomerase activity, lymphocyte proliferation reaction, peritoneal macrophage phagocytosis, and the content of IL-2 in peripheral blood decreased significantly. After administration of PCD, the MDA content in the heart, liver, and brain of mice was significantly decreased, and the telomerase activity in heart and brain tissue, lymphocyte proliferation reaction, peritoneal macrophage phagocytosis, and the content of IL-2 in peripheral blood were significantly increased. Conclusio: PCD can antagonize free radical damage, enhance telomerase activity in heart and brain tissue and immune function of aging mice, and improve the aging induced by D-galactose.

Key words: Cistanche deserticola polysaccharide; Malondialdehyde; Telomerase; Immunization; be senile

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Chinese Library Classification No.: R284 Document Code: A Article No.: 1001-2494 (2011) 14-1081-05

Cistanche deserticola is a dried fleshy stem with phosphorus leaves of Cistanche deserticola, which has the function of tonifying the liver and kidney and benefiting the essence and qi [1]. Cistanche deserticola polysaccharide is a crude polysaccharide separated from Cistanche deserticola, which is removed from water-soluble small molecular impurities, deproteinized by the Sevag method, and repeatedly treated with water and alcohol to obtain PCD [2]. This experiment starts with the immune theory and telomere theory of aging and adopts the D-galactose aging mouse model to observe the effect of PCD on the content of malondialdehyde (MDA) and telomerase activity in the heart, liver, and brain, and preliminarily explores its mechanism.

1. Material and methods

1.1 Animals, drugs, reagents, and instruments

3-month-old ICR mice, clean grade, 26-32g, half male and half female [Yangzhou University Experimental Animal Center, Experimental Animal Quality Certificate No.: SCXK (Su) 2002-0009]. The room temperature is 20~25 ℃, the natural lighting, and the environment is suitable for feeding for one week before the experiment. PCD is provided by Nanjing Wildlife Research Institute and dissolved in distilled water; D-galactose (D-Gal, Shanghai Second Reagent Factory); Telomerase activity test kit (MB Company, USA); MDA detection kit, and mouse IL-2 ELISA kit (Nanjing Jiancheng Biological Engineering Research Institute). ELX800UV enzyme marker (BIO-TEK, USA); PT-MR3100D tissue homogenizer (Polytron, Switzerland); MODEL550 desktop freezing centrifuge (Eppendorf, Germany); BP110S electronic scale (Sartorius, Germany).

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1.2 Animal grouping, model preparation, and administration

The aging mouse model was prepared according to the method in the literature [3]. 10% D-Gal was prepared with water for injection, and 10 mL · kg-1 was subcutaneously injected into the neck and back of mice once a day for 6 weeks. The mice were randomly divided into five groups: the normal control group, the model control group, PCD25, 5,0, and 100mg · kg-1 groups (PL, PM, PH groups), with 12 mice in each group. The mice in the model group and the PCD administration group were established according to the law, and PCD25, 50, and 100 mg · kg-1 with a volume of 20 mL · kg-1 were given by gavage each day. The normal control group mice were injected with the same volume of water for injection and given the same volume of normal saline by gavage.

1.3 Determination of MDA content and telomerase activity

24 hours after the last administration, the eyeball was removed and blood was taken. The mice were killed, the heart, liver, and brain were quickly removed, and fresh tissue homogenate was prepared under low temperature, centrifugation, and the supernatant was taken. The content of MDA was determined by thiobarbituric acid colorimetry. The telomerase activity was detected by PCR-ELISA. The operation steps were carried out according to the instructions on the kit. The A value was measured at 450nm of the enzyme marker.

1.4 Determination of lymphocyte proliferation reaction [4]

Take out the mouse spleen aseptically in the ultra-clean workbench, prepare the mouse spleen cell suspension, and adjust the cell density to 1 × 109 · L-1. Concanavalin A (ConA) was cultured as a stimulator in vitro for 48 hours, and the cell proliferation reaction was measured by the MTT method. The value of A was measured at the wavelength of 570 nm by the enzyme linker, and the result was expressed as the mean value of three complex holes.

1.5 Determination of phagocytic function of peritoneal macrophages by the neutral red method

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24 hours after the last administration, the mice were decapitated and killed. The peritoneal fluid of the mice was collected aseptically and centrifuged at 1000r · min-1. The cells were washed with Hank's solution three times, and the cell suspension was prepared with 1640 culture solution. Take 3mL and put it in a culture dish. Culture at 37 ℃ and 5% CO2 for 2h. Macrophages adhere to the surface of the container, blow and suck with 4 ℃ Hanks solution, collect adherent cells, count on the blood cell count plate, and adjust the cell density to 2 × 109 cells · L-1. Cultured in a 37 ℃ incubator for 2 hours, the supernatant and non-adherent cells were discarded. Take adherent macrophages, add 0.1% neutral red physiological saline, continue to the culture at 37 ℃ for 30min, discard neutral red, repeatedly wash macrophages with PBS at 37 ℃, dilute with 50% acetic acid and 50% ethanol by volume, and measure the value of A.

1.6 Determination of serum IL-2 level

Take blood from the eyeball, centrifuge (3000r · min-1, 10min) to separate the serum, and determine the content of IL-2 in the serum by enzyme-linked immunosorbent assay (ELISA). The specific operation is according to the instructions of the kit. The level of IL-2 in the serum of mice in each group is detected at 450nm with the enzyme marker.

1.7 Statistical analysis

The data were processed with SPSS 11.0, and the measurement data were expressed with mean ± standard deviation (x ± s), using one-way ANOVA and inter-group t-test.

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2. Results

2.1 The effect of PCD on the content of MDA in the heart, brain, and liver of aging mice

Was significantly higher than that of the normal control group (P<0.01). After intragastric administration of PCD25, 50 and 100 mg · kg-1, the content of MDA in the heart, brain, and liver of mice decreased in a dose-dependent manner compared with the model group (P<0.01, P<0.05), and the content of MDA in the high-dose group was close to the normal level (Table 1).

2.2 Effect of PCD on telomerase activity in heart, brain, and liver tissues of aging mice

Compared with the normal control group, the telomerase activity of the heart, liver, and brain tissue in the model group was significantly reduced. After intragastric administration of PCD, the activity of telomerase in heart and brain tissue was increased in a dose-dependent manner, and the level of the high-dose group was close to that of the normal control group. There was no significant effect on telomerase activity in the liver tissue of aging mice (P>0.05) (Table 2). 2.3 The effects of PCD on lymphocyte proliferation reaction, peritoneal macrophage phagocytosis, and serum IL-2 content in aged mice were significantly lower than those in the normal control group (P<0.01). After intragastric administration of PCD, the lymphocyte proliferation reaction, peritoneal macrophage phagocytosis, and serum IL-2 content of aging mice increased in dose dependence, which was significantly different from the model group (P<0.01, P<0.05) (Table 3).

3 . Discussion

The exact mechanism of aging is still unclear. Over the years, scholars at home and abroad have put forward dozens of aging theories, such as the free radical theory. Molecular oxygen is necessary for the survival of organisms, and nutrients in organisms release energy through oxidation. The oxidative state of cells plays an important role in aging, tumor occurrence, and development. However, free radicals will be formed in some oxidation processes, and aging is closely related to abnormal free radical reactions. When free radicals accumulate too much in the body, it will cause the peroxidation of unsaturated fatty acids in the body, especially in the biological membrane, resulting in changes in the structure of the cell membrane, damage to the membrane function, and production of MDA and lipofuscin (LPF) [5].

MDA is an extremely active cross-linking agent. It can covalently cross-link with proteins, enzymes, and free amino acids on nucleic acids to form Schiff bases. Because of its abnormal bonds, it cannot be digested by hydrolase after being swallowed by lysosomes, and it can gather in cells to form lipofuscin, which can poison cells, block the transmission of substances and messages in cells, and lead to nuclear acceleration of cell aging and death.

Therefore, MDA can be used as a reliable indicator of organ and cell aging. The telomere theory believes that the length of the chromosome (telomere) at the end of the chromosome is closely related to aging and life span. The length of the telomere is about 2 to 15 kb. Because of the problem of terminal replication, the telomere DNA will lose 50 to 200 bp every time the DNA is copied. With the increase in the number of cell divisions, the telomere DNA will gradually shorten. When it is shortened to a certain limit, it will not maintain the stability of staining, and the cell will lose the ability to divide and proliferate, and die of aging. This shortening is a sign of aging [6]. The cell enters the proliferative failure stage, and the cell continues to survive but cannot divide and proliferate [7]. Therefore, telomeres are also known as the "biological clock" of cells, and cell aging determines the aging of the whole body. Bryma's research found that the length of the telomere is related to the activity of telomerase. The higher the activity of telomerase, the longer the telomere, the better the stability and integrity of chromosomes, the more cell division times, and the longer the life span.

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The immune system, as a complete and autonomous system, plays an important role in the maintenance and adjustment of life activities, and the decline of immune function is the most prominent feature of aging. Immune dysfunction can accelerate the aging of the body [8]. The results of this experiment showed that PCD could significantly reduce the content of MDA in heart, brain, and liver tissues, increase the telomerase activity in heart and brain tissues of aging model mice, as well as the lymphocyte proliferation reaction, peritoneal macrophage phagocytosis, and the content of IL-2 in peripheral blood of aging mice. It can be concluded that PCD has a good role in antagonizing free radical damage, can promote lymphocyte proliferation, improve the nonspecific cellular immunity and immune regulation function of the body, and play a role in delaying the aging of the body.

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