Experiment 3 Effect Of Cistanche Deserticola Phenylethanol Glycoside On Rat Perimenopausal Model Ⅲ

Experiment 3 Effect of Cistanche deserticola phenylethanol glycoside on rat perimenopausal model

1 Experimental materials

1.1 Experimental animals

Wistar rats, SPF grade, female, provided by Shandong Lukang Pharmaceutical Co., Ltd., rat certificate number for this batch: 0017216; laboratory certificate number SYXK (Yu) 2010-001. 

1.2 Experimental drugs

Cistanche deserticola phenylethanol glycoside was prepared according to the method of Experiment 1, and the content reached 66.47%. Gengnian'an Capsule, ingredients: Rehmannia glutinosa, Rehmannia glutinosa, Alisma, Ophiopogon japonicus, Scrophulariaceae, peony bark, Poria cocos, mother-of-pearl, curculigo, Schisandra chinensis, magnetite, Polygonum multiflorum vine, Uncaria, floating wheat, polygonum multiflorum. Functions and Indications: Nourish yin and subdue yang, relieve troubles and calm the mind. Used for menopausal hot flashes and sweating, dizziness, tinnitus, irritability and insomnia. Specifications: 0.3g per capsule. Usage and dosage: Oral, 3 capsules at a time, 3 times a day. Manufacturer: Shanxi Tianxing Pharmaceutical Co., Ltd. Production batch number: 121104. Approval number: National Drug Approval No. Z14021848.

Soy isoflavones and vitamin E soft capsules, ingredient list: soy isoflavones powder, vitamin E, sunflower oil, gelatin, glycerin, water, tartrazine, titanium dioxide. Iconic ingredients and content: Each 100g contains 55.2 mg of soy isoflavones (calculated as genistein) and 608.5 mg of vitamin E. Health functions: Increase bone density. Instructions and dosage: Take 1 capsule 2 times a day with warm water. Specifications: 500mg×100 capsules. Manufacturer: Weihai Ziguang Biotechnology Development Co., Ltd. Production batch number: 13070302. Approval number: National Food Health G20080032.

HOW LONG DOES IT TAKE FOR CISTANCHE TO WORK?

1.3 Experimental reagents

Carboxymethylcellulose sodium, Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd., batch number: 20120418; penicillin sodium for injection, North China Pharmaceutical Co., Ltd., specification: 4 million units, production batch number: c1206807;

Formaldehyde solution (analytically pure), Yantai Shuangshuang Chemical Co., Ltd., production batch number: 20130902; 0.9% sodium chloride injection, Henan Shuanghe Huali Pharmaceutical Co., Ltd.; specification: 250ml, production batch number: 13082405B;

Chloral hydrate, Tianjin Guangfu Fine Chemical Research Institute, batch number 20120827; Rat E2 ELISA detection kit, R&D Company, batch number: 20140101A; Rat T ELISA detection kit, R&D Company, batch number: 20140101A; Rat LH ELISA detection Kit, R&D Company, batch number: 20140101A; Rat FSH ELISA detection kit, R&D Company, batch number: 20140101A;

2 Experimental methods

2.1 Modeling and Drug Administration

Modeling method: Take 100 female wistar rats with a body weight of 210~230g, randomly select 12 rats as a blank group and undergo sham surgery, and the remaining rats will be used to create menopausal models. After the rats were weighed, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3ml/100g) and the abdominal position was fixed. Then, the rats were trimmed from under the last rib on their back at the intersection of the mid-axillary line and about 2 cm from the lateral side of the spine, and then disinfected. The skin and back muscles are incised about 1cm, and a milky white shiny fat mass can be seen in the field of view of the incision, and the ovary is embedded in it. Use small tweezers to gently grasp the fat mass and pull it out of the incision, separate the fat mass, and you will see a group of thin, irregular, yellow-red ovaries. When cutting, first ligate the fallopian tube (including fat) under the ovary with a thin thread, completely remove the left ovary, and remove 80% of the right ovary. After the operation, the uterine horns were put back into the abdominal cavity, the muscles and skin were sutured, and both ovaries were removed in the same way. After the operation, the animals were carefully raised and intramuscularly injected with 200,000 u/kg penicillin (0.1ml each) to prevent infection, once a day for 3 consecutive days. 5 days after the operation, vaginal smear examination of rats was started one by one, once a day for 5 consecutive days. Rats with estrous reactions in the smear were discarded. 72 completely castrated rats were selected and randomly divided into 6 groups. For experimental purposes, they are the model group, Gengnianan group, soy isoflavone group, and large, medium, and small dose Cistanche deserticola phenylethanol glycoside group.

Preparation method: Preparation method: 0.5% CMC Preparation method: Weigh 4g of carboxymethyl cellulose sodium and mix it with distilled water to make 800ml. The dosages of large, medium, and small doses of Cistanche deserticola phenylethanol glycoside group were 133.33mg/kg, 66.67mg/kg, and 33.33mg/kg respectively (administration volume 1ml/100g). Preparation method: Weigh 1333.3mg, 666.7mg, and 333.3mg of total raspberry flavonoids respectively, dissolve them with a small amount of 0.5% CMC, then adjust the volume to 100ml, mix well, and that's it. Gengniangan Capsules (450mg/kg, mixed with distilled water to 45 mg/ml, 1ml/100g, equivalent to 10 times the clinical dosage); Preparation method: Take 15 Gengniangan Capsules, first dissolve them with a small amount of 0.5% CMC , then adjust the volume to 100ml and mix well, which is the dose of Gengnianan Capsule Suspension (450mg/kg). Soybean Isoflavone Vitamin E Soft Capsules (166.67mg/kg, use distilled water to make 16.67mg/ml, 1ml/100g, equivalent to 10 times the clinical dosage); Preparation method: Take 4 Soybean Isoflavones Vitamin E Soft Capsules, first Dissolve with a small amount of 0.5% CMC, then adjust the volume to 120ml, and mix well to obtain the dose of soy isoflavone vitamin E soft capsule suspension (16.67mg/kg).

Administration method: Animals in each group were given corresponding drugs on the 10th day after surgery. The blank group and the model group were gavaged with the same volume of 0.5% CMC solution (the gavage volume was 1ml/100g), and the Gengnianan group was gavaged with Gengnianan capsule suspension (450mg/kg, equivalent to 10 times the clinical dose). ), the soybean isoflavone group was orally administered soybean isoflavone vitamin E soft capsule suspension (166.67 mg/kg, equivalent to 10 times the clinical dose), and the large, medium, and small dose Cistanche deserticola phenylethanol glycoside groups were orally administered according to the group. Large, medium and small doses of Cistanche deserticola phenylethanol glycoside (the dosages are 133.33mg/kg, 66.67mg/kg, and 33.33mg/kg respectively, the dosage volume is 1ml/100g), administered once a day by intragastric administration, continuously. medicine for 30 days.

2.2 Observation items and detection methods

The horizontal-vertical movement scores of rats in each group were measured 29 days after administration. 2 hours after the last gastric administration (fasting for 15 hours), remove the eyeballs to collect blood, separate serum, and plasma, and measure the contents of E2, T, LH, FSH, GnRH, and BGP in the serum, and the content of β-EP in the plasma; the rats were dissected. Remove the thymus, spleen, uterus and the remaining 20% of the ovarian tissue, weigh their wet weights and calculate the organ index of the thymus, spleen and uterus (organ index = organ wet weight mg/rat weight g), and then remove the brain For the hypothalamus and pituitary gland, tissue homogenates were prepared from the hypothalamus, pituitary gland and 1/2 of the uterus, and the content of estrogen receptors in the hypothalamus, pituitary gland and uterine tissue homogenates and the androgen receptor content in the hypothalamic tissue homogenates were determined. The thymus, spleen, uterus, and ovary were fixed in 10% formaldehyde solution, embedded in paraffin, sectioned, and stained with HE. The histomorphological changes in each group were observed under a light microscope.

2.2.1 Open field method test

The experimental device is a cubic open box with a height of 40cm and a length and width of 80cm. The surrounding walls and bottom are black. The bottom is composed of 25 blocks of equal area, divided by white lines. During the experiment, the rat was placed in the square in the center of the open box, and the horizontal activity score of the number of blocks the rat crossed on the bottom within 5 minutes (squares with all four paws entered can be counted), and the number of times the hind limbs stood upright (two front paws were in the air). (or clinging to a wall) vertical activity score. The feces must be completely removed after each experiment, and each rat is measured once 2 hours after administration. This experiment was conducted in a quiet room.

2.2.2 Kit determination method

Obtain serum specimens: Use test tubes that are free of pyrogens and endotoxins. Avoid any cell stimulation during the operation. After collecting the blood, centrifuge it at 3000 rpm for 10 minutes to quickly and carefully separate the serum and red blood cells. Obtain plasma specimens: anticoagulated with heparin anticoagulant tubes. Centrifuge at 3000 rpm for 30 minutes and take the supernatant. Obtain tissue homogenate specimens: Add an appropriate amount of physiological saline to the tissue and mash it. Centrifuge at 3000 rpm for 10 minutes and take the supernatant.

The determination method of rat (Rat) estradiol (E2) ELISA kit is the same as Experiment 2. The concentrations of standards (S0-S5) are 0, 4, 8, 16, 32, and 64 pmol/L.

The assay method of Rat (Rat) Testosterone (T) ELISA Kit is the same as the assay method of the Estradiol ELISA Kit. The concentrations of standards (S0-S5) are 0, 20, 40, 80, 160, and 320 pg/mL. The determination method of the rat (Rat) luteinizing hormone (LH) ELISA detection kit is the same as the determination method of the estradiol ELISA detection kit. The concentrations of standards (S0-S5) are 0, 3, 6, 12, 24, and 48 ml/ml. The assay method of rat (Rat) follicle-stimulating hormone (FSH) ELISA kit is the same as the assay method of estradiol ELISA kit. The concentrations of standards (S0-S5) are 0, 0.75, 1.5, 3, 6, and 12 IU/L. The determination method of the rat (Rat) gonadotropin-releasing hormone (GnRH) ELISA detection kit is the same as the determination method of the estradiol ELISA detection kit. The concentrations of standards (S0-S5) are 0, 5, 10, 20, 40, and 80 mlU/ml.

The determination method of rat (Rat) osteocalcin (BGP/OCN) ELISA detection kit is the same as the determination method of the estradiol ELISA detection kit. The concentrations of standards (S0-S5) are 0, 0.75, 1.5, 3, 6, and 12 ng/ml. The determination method of the rat (Rat) β-endorphin (β-EP) ELISA detection kit is the same as the determination method of the estradiol ELISA detection kit. The concentrations of standards (S0-S5) are 0, 3, 6, 12, 24, and 48 pg/mL. The determination method of the rat (Rat) estrogen receptor (ER) ELISA detection kit is the same as the determination method of the estradiol ELISA detection kit. The concentrations of standards (S0-S5) are 0, 4, 8, 16, 32, and 64 pg/mL. The determination method of the rat (Rat) androgen receptor (AR) ELISA detection kit is the same as the determination method of the estradiol ELISA detection kit. The concentrations of standards (S0-S5) are 0, 25, 50, 100, 200, and 400 pg/mL.

2.3 Statistical processing methods

For data analysis, the SPSS17.0 medical statistical package was used for the statistical processing of data. Measurement data were expressed as mean ± standard deviation (-x±s). One-way analysis of variance was used for comparison between groups. The LSD method was used to test for homogeneity of variances. , the Games-Howell test was used for uneven variances, and the Ridit test was used for grade data.

It can be seen from Table 11 and Figure 14 that compared with the blank group, the thymus index, spleen index, and uterine index of the rats in the model group were significantly decreased (P<0.01), indicating that the perimenopausal rat model was caused by incomplete removal of the ovaries. Atrophy of the thymus, spleen, and uterus occurs. Compared with the model group, each medication group can significantly improve the thymus and spleen index of perimenopausal model rats (P<0.01), and the Gengnianan, soy isoflavones, large and medium-dose Cistanche deserticola phenylethanol glycoside groups can significantly improve The uterine index of perimenopausal model rats was increased (P<0.01), and the low-dose Cistanche deserticola phenylethanol glycoside group significantly increased the uterine index of perimenopausal model rats (P<0.05).

3 Effects on blood biochemical index levels in perimenopausal model rats

The serum E2, T, LH, FSH, GnRH and BGP contents and plasma β-EP contents of rats in each group are shown in Tables 12~13 and Figures 15~17.

It can be seen from Tables 12~13 and Figures 15~18 that compared with the blank group, the serum E2 and T levels of rats in the model group were significantly decreased (P<0.01), and the LH and FSH levels were significantly increased (P<0.01 ), indicating that incomplete ovarian removal causes sex hormone disorders in the perimenopausal rat model, and the perimenopausal rat model was successfully replicated. Compared with the model group, each medication group could significantly increase serum E2 and T levels (P<0.01) and reduce FSH levels; the medium-dose Cistanche deserticola phenylethanol glycoside, soybean isoflavones, and menganianan groups could significantly reduce the elevated levels. The LH level in the high-dose Cistanche deserticola phenylethanoid glycoside group can be significantly reduced (P<0.05); the elevated LH level in the low-dose Cistanche deserticola phenylethanol glycoside group has a decreasing trend.

Table 14 Effect of Cistanche deserticola phenylethanol glycoside on serum GnRH and plasma β-EP levels in rat perimenopausal model (-x±s)

It can be seen from Table 14 and Figures 19-20 that compared with the blank group, the serum GnRH level of rats in the model group was significantly increased and the plasma β-EP level was significantly decreased (P<0.01), indicating that incomplete removal of the ovaries caused The perimenopausal rat model has sex hormone disorders, and related hormones secreted by the hypothalamus are also disordered due to negative feedback regulation. Compared with the model group, the GnRH levels in each medication group were significantly reduced (P<0.01), and the β-EP levels in the medium-dose Cistanche deserticola phenylethanol glycoside group, soybean isoflavones group, and menanianan group were significantly increased (P<0.01). 0.01), the plasma β-EP level in the low-dose Cistanche deserticola phenylethanol glycoside group could be significantly increased (P<0.05).

4 Effects on ER and AR contents in tissues of perimenopausal model rats

The contents of ER and AR in relevant tissues of rats in each group are shown in Tables 16~17 and Figures 22~23.

It can be seen from Table 17 and Figure 23 that compared with the blank group, the AR level in the hypothalamus of the model group was significantly lower (P<0.01), indicating that the androgen receptors distributed in the hypothalamus of the perimenopausal rat model caused by incomplete ovary removal body, thereby reducing the biological effects of androgens. Compared with the model group, the AR levels in the hypothalamus of the large- and medium-dose Cistanche deserticola phenylethanoid glycoside group, Gengnianan group, and soybean isoflavone group were significantly increased (P<0.01). The hypothalamic AR level of the low-dose Cistanche deserticola phenylethanol glycoside group was rising trend.

5 Effects on organ tissue morphology in perimenopausal model rats

The pathological and histological observation results of the uterus, thymus, and spleen of rats in each experimental group are as follows. The pathological photos are shown in Appendix 1 for the pathological photos of perimenopausal model rats.

5.1 Effect on uterine tissue morphology in perimenopausal model rats

According to the varying degrees of changes in the endometrium, glands, and myometrium of the rats in each experimental group, semi-quantitative standards were used to divide the pathological tissue morphology into four levels, and the uteri of the rats in each experimental group were measured. The observation results are shown in Table 18.

"-" The endometrial epithelial cells, glands, myometrium, and serosa are all normal; "+" the endometrial epithelial cells and glands are atrophied, and the myometrium and serosa are normal; "++" the endometrial epithelium Cells and glands are partially atrophied, the muscle layer is slightly atrophied, and the serosa is normal; "+++" endometrial epithelial cells and glands are significantly atrophied, and the serosa is normal.

After Ridit test, it can be seen from Table 18 that compared with the blank group, the uterus of rats in the model group showed significant pathological tissue lesions (P<0.01). Compared with the model group, each medication group could significantly improve the uterine pathological tissue lesions of mice (P<0.01).

5.2 Effect on ovarian tissue morphology in perimenopausal model rats

According to the varying degrees of changes in follicles, corpus luteum, granulosa cells, and blood vessels in the ovaries of rats in each group of experiments, semi-quantitative standards were used to divide the pathological tissue morphology into four levels. The ovaries of rats in each group of experiments were measured and observed. The results are shown in Table 19.

Table 19 Effect of Cistanche deserticola phenylethanol glycoside on ovarian pathological changes in perimenopausal model rats Unit: only

"-" can show growing follicles, mature follicles and corpus luteum at all levels. The corpus luteum is well developed, with many layers of granulosa cells, rich follicular fluid and rich blood vessels; "+" can show growing follicles, mature follicles and corpus luteum, but the number of mature follicles and corpus luteum is small. , the follicles are smaller, and the granulosa cells are less layered; "++" can show mature follicles and the corpus luteum, with more corpus luteum, less granulosa cells, and fewer blood vessels; "+++" has no follicles, more corpus luteum, and the ovary Atrophic, avascular.

After Ridit test, it can be seen from Table 19 that compared with the blank group, the ovaries of the model group rats showed significant pathological tissue lesions (P<0.01). Compared with the model group, each medication group could significantly improve the ovarian pathological tissue lesions of rats (P<0.01).

5.3 Effects on thymus and spleen tissue morphology in perimenopausal model rats

Using a micrometer, measure the thickness of the thickest and narrowest parts of the thymic cortex of each rat in each experimental group to obtain the average;Use the micrometer's baseline to fall on the splenic nodules, measure the thickness of the splenic nodules on both sides with the central artery as the center, and calculate the average. The measured results are shown in Table 20 and Figure 24.

It can be seen from Table 20 and Figure 24 that compared with the blank group, the thickness of the thymus cortex and the volume of splenic nodules in the model group were significantly reduced (P<0.01), indicating that the thymus and spleen volumes atrophied after the perimenopausal model was created in rats. Compared with the model group, each medication group could significantly increase the thickness of the thymus cortex (P<0.01). Except for the low-dose Cistanche deserticola phenylethanoid glycoside group, all medication groups could significantly increase the volume of splenic nodules (P<0.01).