Experimental Study Of Anti-Sports Fatigue Biect Mechanisms Of Cistanche Deserticola

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

YANG Hong-Xin，YANG Yong²，YAN Xiao-hong'

Abstract: Objective To study the effect of cistanche deserticola on lactate dehydrogenase (LDH)isoenzyme, glycogen, and nitric oxide synthase 3 (NOS3) of the liver in the mice burden swimming and explore relevant molecular mechanisms of anti-sports fatigue. Method The mice were divided into the normal control group, the sport control group, and the cistanche deserticola experimental group. Each mouse of the normal control group and the sport control group was given saline 0.2 mL per day. Each mouse of the cistanche deserticola experimental group was given water decoction of cistanche deserticola 0.2 mL(3 g/kg per day. The administrations were for 15 days. The burden swimming for 90 minutes was carried out on the mice of the sport control group and the cistanche deserticola experimental group at 1 hour after the last administration. Livers of the mice were removed after 10 hours of swimming. One part of the liver was fixed in the neutral formalin liquid to prepare the paraffin sections and the others were used for measurement of LDH activity. The structure of the liver was observed by staining of HE and the liver glycogen was measured by staining of glycogen. NOS3 was examined by S-P immunohistochemical method. Rei The liver structure of the sport control group was injured seriously, the isoenzyme of LDH4 and LDH5 were higher, the liver glycogen was poor, NOS3 was decreased compared with the normal control group(P<0.05). The liver structure of the cistanche deserticola experimental group was good, LDH5 isoenzyme was low, the glycogen was rich and expression of NOS3 was upregulated compared with the sport control group (P<0.05). Conclusion: Cistanche deserticola could decrease LDH5, protect the liver of the mice's burden swimming, and accelerate glycogen accumulation by increasing the expression of NOS3 to protect the liver and improve physical capacity recovery.

Keywords; cistanche deserticola; liver glycogen; nitric oxide synthase 3; mice

Cistanche, also known as Dayun, is a two-year parasitic herb of the Lydanaceae family. It has dry fleshy stems with scales. It is mainly produced in sandy soils and semi-sandy grasslands in Inner Mongolia, Gansu, Xinjiang, and Qinghai. Cistanche in Inner Mongolia has the best quality among the production areas. Cistanche is sweet, salty, and warm in nature. It enters the kidneys and large intestine meridian. Its main function is to invigorate the kidney and strengthen the yang, nourish the essence and blood, and nourish the intestines. It is often used to treat impotence, infertility, weakness in the waist and knees, weakness of the muscles and bones, dry intestines, and constipation. . The purpose of this study is to observe the effects of Cistanche on the liver lactate dehydrogenase (LDH), liver glycogen and nitric oxide synthase 3 (NOS3) in weight-bearing swimming mice through a load-bearing swimming test in mice, and to explore its protective mechanism on the liver, Provide a theoretical basis for the in-depth research, development, and utilization of Cistanche in sports health food.

1 Experimental material

1.1 Animals 30 male Kunming rats, clean grade, weight (20±0.5) g, provided by the Animal Center of Inner Mongolia University.

1.2 Medicine Cistanche (Cistanche deserticola Y.C. Ma) is produced in Ejina Banner, Inner Mongolia, and has been identified. According to the original medicinal material: water=100g: 400mL, soak for 30min, heat, and boil, then decoct on warm fire for 30min, pour out the medicinal solution, and re-decorate at the ratio of 100g:200mL. Mix the two decoctions together, filter them with two layers of gauze, and then concentrate them to the equivalent of 1g of the original medicinal materials per milliliter of the original decoction, and store it in a refrigerator at 4°C. 1.3 Reagents and instruments NOS3 polyclonal antibody was purchased from Wuhan Boster Bioengineering Co., Ltd. SP kit was purchased from Fujian Maixin Reagent Company. The lactate dehydrogenase isoenzyme kit was purchased from Inner Mongolia Tongri Reagent Co., Ltd (wholly owned by Nissan). Nanjing JD-80 color image analyzer. American UVP gel image analysis system.

Anti-fatigue cistanche supplement

2. Experimental methods

2.1 Grouping and administration Kunming mice were randomly divided into normal control group, exercise control group, and Cistanche experimental group, each with 10 mice. The normal control group and the exercise control group were given normal saline once a day, 0.2mL/time; the Cistanche experimental group was given a cistanche decoction (take 3mL of the original water decoction and add water to 10 mL), once a day, 0 ．2mL/time, a daily dosage of 3g/kg, continuous administration for 15 days. One hour after the last administration, the mice in the exercise control group and the Cistanche experiment group were loaded with a 1g lead weight on their tails, and they were placed in a pool with a height of 50cm, a length of 50cm, and a width of 35cm to swim. The temperature was kept at (30±0.5)℃ and kept constant. Observe the mouse, if it is found that the mouse stops swimming, use a wooden stick to stimulate its movement for 90 minutes. After exercise, eat a normal diet, rest for 10 hours and kill the mice together with the normal control group mice by de-neck method, take out the liver, fix a part with 10% neutral formaldehyde, dehydrate, transparent, soak in wax, and embed to make 4 thick continuous Slice, use HF staining method to observe the liver tissue structure of each group: the other part was washed with normal saline, frozen at 20℃, thawed at room temperature during measurement, dried with filter paper and weighed, and then liver homogenate was prepared with normal saline, 3500r Centrifuge for 15 min at /min, and take the supernatant to measure the activity of lactate dehydrogenase isoenzyme by electrophoresis.

2.2 Determination of lactate dehydrogenase activity Using polyacrylamide gel electrophoresis, the isoenzymes of LDH are separated according to their electrophoretic mobility differences. The separation gel concentration of the sample is 7%, pH 8.9, and the concentration of concentrated gel is 4%, pH 6.8.10. The sample is mixed with 1 drop of glycerin and bromophenol blue indicator. The electrode buffer is Tris-Glycine (pH 8.7), start to stabilize the pressure at 1o0～150V, add it to 2o0～250V after 30min, and perform electrophoresis for 4h. After electrophoresis, it was stained with the Lactate Dehydrogenase Isoenzyme Kit, stained at 37°C for 30 minutes, showing a blue lactate dehydrogenase isoenzyme band, then rinsed with water, fixed with glacial acetic acid, and used UVP gel image analysis system Perform data analysis.

2,3 Glycogen staining 4um thick serial sections are conventionally deparaffinized to water, put in 1% periodic acid solution at 37 ℃ for 15 minutes, rinsed in running water, put in Schiff solution for 60 minutes, rinsed in running water, dehydrated by gradient ethanol, xylene is transparent, neutral Mount the slides with gum and observe the staining results under a microscope.

2.4 The immunohistochemical staining section was routinely deparaffinized to water, and the antigen was repaired with citric acid buffer microwave, and the endogenous peroxidase was removed by liquid A for 10 minutes, and then blocked with liquid B (diluted goat serum) for 10 minutes. Add the primary antibody overnight at 4°C in a humid box, wash with pH 7.4 PBS solution for 5 min×3 times on the second day, add biotin-labeled solution C (secondary antibody working solution) for 10 min, wash with PBS solution for 5 min×3 times, and add Horseradish peroxidase-labeled D solution was incubated for 10min, washed with PBS solution for 5minX3 times, and finally developed in DAB solution for 5min, washed with tap water, HE counterstained, dehydrated with gradient alcohol, transparent with xylene, and mounted with neutral gum. After mounting the film, observe under a light microscope. In each batch of experiments, phosphate-buffered saline (PBS) was used as a negative control instead of the primary antibody.

2.5 Analysis of the expression of nitric oxide synthase 3 Preliminarily observed by the visual semi-quantitative method under a light microscope, the positive product of NOS3 is dark brown or brownish yellow, and the positive product is located in the cytoplasm of hepatocytes or between hepatocytes. In the cytoplasm of endothelial cells, the blue background is negative. Then, the color image analysis system is used to measure the gray value of the positive reactant, and the parameters of the 4 fields of view are randomly measured for each film under a 40-fold objective lens, and the average value is taken. This parameter is used to reflect the change of NOS3 dye intensity. 2.6 Statistical methods All data are represented by. SPSS11.0 statistical software package is used for t-test, and P<0.05 is considered a significant difference.

Anti-fatigue

3 Results

3.1 Observation of tissue structure under light microscope In the normal control group, the structure of liver lobules is clear, the cell cords are arranged neatly, the liver sinusoids are normal, the liver cells have no obvious lesions, and the nuclear structure is clear. In the exercise control group, the normal tissue structure disappeared, the lobules were unclear, the cell cords were disordered, most of the liver sinusoids disappeared, and the hepatocytes were extensive vacuolar degeneration, manifested by increased cell volume, loose cytoplasm, light staining, and even clear and transparent. Some liver cells showed a typical balloon-like change, with a small amount of focal or punctate necrosis. The liver cells of the Cistanche experimental group were mildly turbid, but they were arranged regularly, there were no obvious vacuoles and looseness in the cells, the cell cords were arranged neatly, and the nucleus structure was clear.

3.2 Effects on the activity of liver lactate dehydrogenase isoenzymes in mice under load exercise (see Table 1)

Table 1 The effect of Cistanche on the liver lactate dehydrogenase isoenzyme of loaded Xiaofeng (x±s)

Note: Compared with the normal control group, P<0, 05; compared with the exercise control group, laP<0.05 (the same below)

3.3 Light microscope observation of liver glycogen staining. Although the glycogen content of the normal control group is not high, there are evenly distributed glycogen in each liver cell without vacuoles; the exercise control group has less glycogen, scattered in the liver Cells, vacuoles appeared in some areas; the Cistanche experimental group was rich in glycogen, but gathered a lot on one side of the cell. 3.4 Observation of the expression of nitric oxide synthase 3 with the light microscope and quantitative image analysis. The cytoplasm of normal control hepatocytes showed strong positive expression in diffuse distribution, especially in binuclear cells, and positive expression in endothelial cells. . The expression in the cytoplasm of hepatocytes and endothelial cells in the exercise control group was weakened. The cistanche experiment group showed positive expression in the cytoplasm of hepatocytes and strong positive expression in endothelial cells. The analysis results of the color image analyzer are shown in Table 2.

Table 2 Comparison of the expression difference of №s3 in hepatocytes and endothelial cells (x±s)

4 Discuss

That the human body needs energy for exercise, and the body also needs energy in the process of metabolism. Cell metabolism directly uses energy from the decomposition of adenosine triphosphate (ATP). The energy required for the synthesis of ATP is ultimately mainly provided by the catabolism of sugar. Studies have shown that after strenuous exercise, the body will produce a large amount of H’, free radicals, lactic acid, and other substances that affect the normal metabolism of cells. At the same time, the lack of glucose in the cells leads to exercise fatigue and cell damage. The results of this study show that liver tissue structure damage is very serious after a large number of exercises, and it also affects the synthesis of cells, such as the reduction of liver glycogen synthesis. After a lot of exercises, mice taking Cistanche can observe the reduction of liver damage, mainly due to the reduction of LDH5, and the liver glycogen content is significantly higher than that of the exercise control group, suggesting that Cistanche may achieve liver protection and maintain normal physiology by reducing LDH5 function. From the results of glycogen staining.

The glycogen content of the experimental group of Cistanche is higher than that of the normal control group. Does Cistanche increase liver glycogen storage before exercise or accelerate glycogen synthesis under stress after exercise. Because there is no non-exercise control of Cistanche in the experiment. It is impossible to draw a conclusion, and it needs to be confirmed in the next step, but the protective effect of Cistanche on the liver and the promotion of glycogen synthesis after a lot of exercises are obvious. N0 is a small molecule substance that has been highly valued by the sports community in recent years. NOS is the main rate-limiting factor for the production of N0. It has been confirmed that NOS has three types of isoenzymes, and NOSI is called neuronal nitric oxide synthase.

The enzyme, which exists in neuronal cells, skeletal muscle cells, etc., is mainly involved in neurodevelopment, neurosecretion, learning, and memory processes: NOS2 is an inducible nitric oxide synthase, mainly found in smooth muscle cells, macrophages, and lymphocytes Once this enzyme is induced, it can continue to synthesize NO until the substrate is exhausted or cell death. It is currently considered to be involved in cell damage; 'NOS3 is an endothelial nitric oxide synthase, which is mainly distributed in endothelial cells and hepatocytes, producing NO Mainly involved in physiological functions. The results of this study show that after a lot of exercises, the expression of NOS3 in endothelial cells and hepatocytes is weakened, and the resulting N0 decreases, so that blood vessels cannot be effectively expanded, blood flow is reduced, and the raw materials for the synthesis of glycogen cannot be transported to liver cells, so glycogen synthesis is reduced. ; Cistanche can up-regulate the expression of NOS3 in endothelial cells and liver cells, expand blood vessels reactively, increase blood flow, accelerate the ability to transport glycogen synthesis raw materials, accelerate glycogen synthesis, and maintain the body at a normal physiological metabolic level. Therefore, reducing LDH5 to protect the liver and up-regulating the expression of NOS3 to promote lactic acid gluconeogenesis may be an important mechanism for Cistanche to protect the liver and promote physical recovery. This research provides the scientific experimental basis for the further development and utilization of Cistanche in the field of sports medicine and also lays the foundation for the development and purification of Cistanche's effect substances.