Intervention Research Of Phenylethyl Alcohol Glycosidesin Cistanche On Perimenopausal Syndrome Animal Model Ⅲ
Apr 16, 2024
Experiment 2
Effect of Cistanche phenylethanol glycoside on mouse perimenopausal model Materials and methods
Experimental materials
1.1 Experimental animals
SPF grade mice, Kunming species, female, 20-25g, provided by Wuhan Institute of Biological Products. Certificate number: 42000400000611; 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.
Result 1 Effect on autonomous activities in mouse perimenopausal model
The results of the autonomous activity test of mice in each experimental group are shown in Table 2 and Figure 2.
1.3 Experimental reagents
Sodium carboxymethyl cellulose, 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 (analytical grade), Yantai City Shuangshuang Chemical Co., Ltd., production batch number: 20130902; 0.9% sodium chloride injection, Chenxin Pharmaceutical Co., Ltd.; specification: 250ml, production batch number: 1301265303;
Chloral hydrate, Tianjin Guangfu Fine Chemical Research Institute, batch number 20120827; mouse E2 ELISA detection kit, R&D Company, batch number: 20131001A; mouse T ELISA detection kit, R&D Company, batch number: 20131001A; mouse LH ELISA detection Test kit, R&D Company, batch number: 20131001A;
Mouse FSH ELISA detection kit, R&D Company, batch number: 20131001A. 1.4 Experimental instruments
Electronic scale, Shanghai Minqiao Medical Instrument Co., Ltd., model: JY601; electronic analytical balance, Ohaus (Shanghai) Company, model: AR1140/C; high-speed desktop centrifuge, Shanghai Anting Scientific Instrument Factory, model: TGL-168; Electric thermostatic water bath, Shanghai Yiheng Scientific Instrument Co., Ltd., model: HWS12; Mouse autonomous activity tester, Chengdu Taimeng Technology Co., Ltd., model: ZZ-6; Mouse dark avoidance meter, Chengdu Taimeng Technology Co., Ltd., model: BA-200; high-speed refrigerated centrifuge, Zhongjia Branch of HKUST Innovation Co., Ltd., model: KDC-160HR; adjustable pipette, Shanghai Leibo Analytical Instrument Co., Ltd.;
Microplate reader, American BIO-RAD Company, model: 680;
Electric microscope, Japan OLYMPUS company, model: BX61
HOW LONG DOES IT TAKE FOR CISTANCHE TO WORK?
2 Experimental methods
2.1 Modeling and Drug Administration
Modeling method: Take 100 female Kunming mice weighing 23-25g, randomly select 12 mice as the blank group, and perform sham surgery, and the remaining mice are used to create menopausal models. After weighing, the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.03ml/10g) and fixed in the abdominal position. Then, the hair was cut from the back of the mouse under the last rib at the intersection of the midaxillary line and about 1 cm from the outside of the spine, and then disinfected and cut. The skin and back muscles are opened about 0.5-1cm, and a milky white shiny fat mass can be seen in the incision field, 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 tubes (including fat) under the ovaries with thin threads, and then remove the ovaries. After the operation, the uterine horns are put back into the abdominal cavity, the muscles and skin are sutured, and both ovaries are removed in the same way. After the operation, the animals were carefully raised and penicillin 200,000 u/kg (0.1 mL each) was intramuscularly injected to prevent infection, once a day for 3 consecutive days. Vaginal smear examination was performed on each mouse 5 days after the operation, once a day for 5 consecutive days to determine whether the ovaries were completely removed. The mice showing estrous reaction in the smear were discarded, and 72 completely castrated mice were randomly and evenly divided into 6 groups for experimental purposes, namely model group, Gengniang'an capsule group, soy isoflavone soft capsule group, and large group. , medium and small doses of Cistanche phenylethanol glycoside group. 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 the Cistanche phenylethanol glycoside group were 200mg/kg, 100mg/kg and 50mg/kg respectively (administration volume 0.1ml/10g). Preparation method: Weigh 2000mg, 1000mg, and 500mg of Cistanche phenylethanoid glycoside respectively, dissolve it with a small amount of 0.5% CMC, then adjust the volume to 100ml, mix well, and that's it. Gengniangan Capsules (675mg/kg, mixed with distilled water to 67.5 mg/ml, 0.1ml/10g, equivalent to 15 times the clinical dosage); preparation method: take 9 Gengniangan Capsules, first use a small amount of 0.5% CMC Dissolve, then adjust the volume to 40ml, and mix well to obtain the dose of Gengnianan Capsule Suspension (675mg/kg). Soybean isoflavone vitamin E soft capsule (250mg/kg, use distilled water to prepare 25mg/ml, 0.1ml/10g, equivalent to 15 times the clinical dosage); preparation method: take 2 soybean isoflavone capsules, first use a small amount of 0.5 Dissolve %CMC, then adjust the volume to 40ml, and mix well to obtain the dose of soy isoflavone capsule suspension (250mg/kg).
Storage method: refrigerate, keep warm during use.
Administration method: Animals in each group were given corresponding drugs on the 10th day after surgery. The Gengnian'an capsule group was orally administered Gengniang'an capsule suspension 675 mg·kg-1, and the soy isoflavone soft capsule group was orally administered 250 mg·kg-1 soy isoflavone soft capsule suspension, and large, medium and small doses of Cistanche benzene were administered. The ethanol glycoside group was orally administered large, medium and small doses of Cistanche phenylethanoid glycoside 200mg·kg-1, 10mg·kg-1, 50mg·kg-1, 0.1ml/10g respectively. The blank group and the model group were gavaged with the same volume of 0.5% CMC solution once a day for 21 consecutive days.
Mouse FSH ELISA detection kit, R&D Company, batch number: 20131001A. 1.4 Experimental instruments
Electronic scale, Shanghai Minqiao Medical Instrument Co., Ltd., model: JY601; electronic analytical balance, Ohaus (Shanghai) Company, model: AR1140/C; high-speed desktop centrifuge, Shanghai Anting Scientific Instrument Factory, model: TGL-168 ; Electric thermostatic water bath, Shanghai Yiheng Scientific Instrument Co., Ltd., model: HWS12; Mouse autonomous activity tester, Chengdu Taimeng Technology Co., Ltd., model: ZZ-6; Mouse dark avoidance meter, Chengdu Taimeng Technology Co., Ltd. , model: BA-200; high-speed refrigerated centrifuge, Zhongjia Branch of HKUST Innovation Co., Ltd., model: KDC-160HR; adjustable pipette, Shanghai Leibo Analytical Instrument Co., Ltd.;
Microplate reader, American BIO-RAD Company, model: 680;
Electric microscope, Japan OLYMPUS company, model: BX61
2 Experimental methods
2.1 Modeling and drug administration
Modeling method: Take 100 female Kunming mice weighing 23-25g, randomly select 12 mice as the blank group, and perform sham surgery, and the remaining mice are used to create menopausal models. After weighing, the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.03ml/10g) and fixed in the abdominal position. Then, the hair was cut from the back of the mouse under the last rib at the intersection of the midaxillary line and about 1 cm from the outside of the spine, and then disinfected and cut. The skin and back muscles are opened about 0.5-1cm, and a milky white shiny fat mass can be seen in the incision field, 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 tubes (including fat) under the ovaries with thin threads, and then remove the ovaries. After the operation, the uterine horns are put back into the abdominal cavity, the muscles and skin are sutured, and both ovaries are removed in the same way. After the operation, the animals were carefully raised and penicillin 200,000 u/kg (0.1 mL each) was intramuscularly injected to prevent infection, once a day for 3 consecutive days. Vaginal smear examination was performed on each mouse 5 days after the operation, once a day for 5 consecutive days to determine whether the ovaries were completely removed. The mice showing estrous reaction in the smear were discarded, and 72 completely castrated mice were randomly and evenly divided into 6 groups for experimental purposes, namely model group, Gengniang'an capsule group, soy isoflavone soft capsule group, and large group. , medium and small doses of Cistanche phenylethanol glycoside group. 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 phenylethanol glycoside group were 200mg/kg, 100mg/kg and 50mg/kg respectively (administration volume 0.1ml/10g). Preparation method: Weigh 2000mg, 1000mg, and 500mg of Cistanche phenylethanoid glycoside respectively, dissolve it with a small amount of 0.5% CMC, then adjust the volume to 100ml, mix well, and that's it. Gengniangan Capsules (675mg/kg, mixed with distilled water to 67.5 mg/ml, 0.1ml/10g, equivalent to 15 times the clinical dosage); preparation method: take 9 Gengniangan Capsules, first use a small amount of 0.5% CMC Dissolve, then adjust the volume to 40ml, and mix well to obtain the dose of Gengnianan Capsule Suspension (675mg/kg). Soybean isoflavone vitamin E soft capsule (250mg/kg, use distilled water to prepare 25mg/ml, 0.1ml/10g, equivalent to 15 times the clinical dosage); preparation method: take 2 soybean isoflavone capsules, first use a small amount of 0.5 Dissolve %CMC, then adjust the volume to 40ml, and mix well to obtain the dose of soy isoflavone capsule suspension (250mg/kg).
Storage method: refrigerate, keep warm during use.
Administration method: Animals in each group were given corresponding drugs on the 10th day after surgery. The Gengnian'an capsule group was orally administered Gengniang'an capsule suspension 675 mg·kg-1, and the soy isoflavone soft capsule group was orally administered 250 mg·kg-1 soy isoflavone soft capsule suspension, and large, medium and small doses of Cistanche benzene were administered. The ethanol glycoside group was orally administered large, medium and small doses of Cistanche phenylethanoid glycoside 200mg·kg-1, 10mg·kg-1, 50mg·kg-1, 0.1ml/10g respectively. The blank group and the model group were gavaged with the same volume of 0.5% CMC solution once a day for 21 consecutive days.
2.2 Observation items and detection methods
The number of spontaneous activities of mice in each group within 5 minutes was measured on the 18th day of administration. The latency of the mice entering the darkroom for the first time and the number of electric shocks received within 5 minutes of entering the darkroom were measured on the 19th to 20th day of administration. 2 hours after the last dose (12 hours without food or water), the mice were weighed, their eyeballs were removed, blood was collected, the serum was separated, and the contents of E2, T, LH, and FSH in the serum were measured; the mice were then sacrificed by cervical dislocation and dissected. , remove the thymus, spleen, and uterine tissue, weigh their wet weight and calculate the organ index (organ index = organ wet weight mg/mouse weight g), and then remove the pituitary gland; fix the thymus, spleen, uterus, and pituitary gland on In 10% formalin solution, embedded in paraffin, sectioned, HE stained, and the tissue morphological changes of each group were observed under a light microscope.
2.2.1 Test on the number of independent activities
The mice in each group were put into the voluntary activity apparatus, first adapted to the environment for 1 minute, and then the number of voluntary activities within 5 minutes was measured.
2.2.2 Dark avoidance test
Put the mice in each group into the test box with their tails facing the small opening into the darkroom, and conduct training. After 24 hours, the latency period of the mice entering the darkroom for the first time and the number of electric shocks received within 5 minutes after entering the darkroom were re-measured.
2.2.3 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. Mouse estradiol (E2) ELISA detection kit assay method: Take out the required strips from the aluminum foil bag that has been equilibrated at room temperature for 20 minutes, and seal the remaining strips in a self-sealing bag, and return them to 4°C. Set up standard wells and sample wells, and add 50 μL of standards of different concentrations into each standard well. First, add 10 μL of the sample to be tested into the sample well, and then add 40 μL of sample diluent (that is, the sample is diluted 5 times); do not add it to the blank well. Except for the blank well, add 100 μL of horseradish peroxidase-labeled detection antibody to each well of the standard well and sample well, and seal the reaction well with a sealing film. Incubate in a 37°C water bath or incubator for 60 minutes. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, and let stand for 1 minute. Shake off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 5 times. First add 50 μL each of substrates A and B to each well, and incubate at 37°C in the dark for 15 minutes. Take out the enzyme plate, add 50 μL of stop solution to each well, and within 15 minutes, measure the absorbance value (OD value) of each well at a wavelength of 450 nm. According to the concentration of the standard product and the corresponding OD value, draw the standard product regression curve, and calculate the concentration value of each sample according to the curve equation. The final concentration is the actual measured concentration multiplied by the dilution factor. Among them, the concentrations of standard products (S0-S5) are 0, 4, 8, 16, 32, and 64 pmol/L. The concentrations of the mouse (Mouse) testosterone (T) ELISA detection kit standard (S0-55) are 0, 50, 100, 200, 400, and 800pg/mL;
The concentrations of the mouse (Mouse) luteinizing hormone (LH) ELISA detection kit standard (S0-55) are 0, 0.5, 1, 2, 4, 8mIU/mL;
The concentrations of the mouse (Mouse) follicle-stimulating hormone (FSH) ELISA detection kit standard (S0-55) are 0, 5, 10, 20, 40, and 80mIU/mL.
2.3 Statistical processing methods
For data analysis, 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.
Figure 3 Effect of Cistanche phenylethanol glycoside on the latent period of dark avoidance experiment in mouse perimenopausal model
Figure 4 The effect of Cistanche phenylethanol glycoside on the number of electric shocks in the dark avoidance experiment of the perimenopausal mouse model. It can be seen from Table 3 and Figures 3~4 that compared with the blank group, the incubation period and the number of electric shocks of the mice in the model group were significantly reduced ( P<0.01), reflecting the decline in memory in the mouse perimenopausal model. Compared with the model group, Gengnianan, soybean isoflavones, and large and medium doses of Cistanche phenylethanoid glycosides could significantly increase the latency of the dark avoidance test in mice, reduce the number of electric shocks (P<0.01), and improve the memory of mice.
3 Effects on organ index in mouse perimenopausal model
The thymus, spleen, and uterine indices of mice in each experimental group are shown in Table 4 and Figure 5.
As can be seen from Table 4 and Figure 5, compared with the blank group, the uterine index of the mice in the model group was significantly lower (P<0.01), indicating that removal of the ovaries caused uterine atrophy in the perimenopausal model mice, and removal of the ovaries resulted in insufficient estrogen secretion. Causes immune organ atrophy in perimenopausal mice. Compared with the model group, soy isoflavones, Gengnianan, large and medium doses of Cistanche phenylethanol glycoside can significantly improve the thymus, spleen and uterine index (P<0.01), and low dose Cistanche phenylethanol glycoside can significantly improve the perimenopausal model. Mouse uterine index (P<0.05). 4 Effects on serum sex hormone levels in mouse perimenopausal model
Contents of E2, T, FSH, and LH in the serum of mice in each group. See Tables 5~6 and Figures 6~9. It can be seen from Tables 5~6 and Figures 6~9 that compared with the blank group, the levels of E2 and T in the serum of mice in the model group were significantly decreased (P<0.01), and the levels of LH and FSH were significantly increased (P<0.01). , indicating that removal of ovaries leads to disorders of sex hormone levels in the serum of perimenopausal model mice and the perimenopausal mouse model was successfully replicated. Compared with the model group, each administration group can significantly reduce the elevated LH level in serum (P<0.01); large and medium doses of Cistanche phenylethanol glycoside, Gengniangan, and soybean isoflavones can significantly increase the levels of perimenopausal mice. E2 and T levels in the serum of the early-stage model (P<0.01) and reduced elevated FSH and LH levels in the serum. Low-dose Cistanche phenylethanol glycoside can significantly increase serum E2 levels and reduce serum FSH levels (P<0.05); low-dose Cistanche phenylethanoid glycoside has a tendency to increase serum T levels.
4 Effects on serum sex hormone levels in mouse perimenopausal model
Contents of E2, T, FSH, and LH in serum of mice in each group. See Tables 5~6 and Figures 6~9.
It can be seen from Tables 5~6 and Figures 6~9 that compared with the blank group, the levels of E2 and T in the serum of mice in the model group were significantly decreased (P<0.01), and the levels of LH and FSH were significantly increased (P< 0.01), indicating that removal of ovaries leads to disorder of sex hormone levels in the serum of perimenopausal model mice, and the perimenopausal mouse model was successfully replicated. Compared with the model group, each administration group can significantly reduce the elevated LH level in serum (P<0.01); large and medium doses of Cistanche deserticola phenylethanol glycoside, Gengniannian, and soybean isoflavones can significantly reduce the level of LH in mice. E2 and T levels in serum of perimenopausal model (P<0.01) and reduced elevated FSH and LH levels in serum. Low-dose Cistanche deserticola phenylethanol glycoside can significantly increase serum E2 levels and reduce serum FSH levels (P<0.05); low-dose Cistanche deserticola phenylethanoid glycoside has a tendency to increase serum T levels. 5. Effects on organ tissue morphology in perimenopausal model mice
The pathological and histological observation results of the uterus, thymus, spleen, and pituitary gland of mice in each experimental group are as follows. The pathological photos are shown in Appendix 1 for the pathological photos of perimenopausal mice.
5.1 Effect on uterine tissue morphology of perimenopausal model mice
According to the different degrees of changes in the endometrium, glands, and myometrium of mice in each group of experiments, semi-quantitative standards were used to divide the pathological tissue morphology into four levels. The uteri of mice in each group of experiments were measured. The observation results are shown in Table 7. Table 7 Effect of Cistanche deserticola phenylethanol glycoside on uterine pathological changes in perimenopausal model mice Unit: only
"-" 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 the above table that compared with the blank group, the uterus of mice in the model group showed significant pathological tissue lesions (P<0.01). Compared with the model group, soy isoflavone vitamin E soft capsules, Gengnianan capsules, and high-dose Cistanche deserticola phenylethanol glycoside can significantly improve the uterine pathological tissue lesions of mice (P<0.01), and medium-dose Cistanche deserticola phenylethanol glycoside can Significantly improved uterine pathological tissue lesions in mice (P<0.05). 5.2 Effect on thymus and spleen tissue morphology in mouse perimenopausal model
Use a micrometer to measure the thickness of the thickest and narrowest parts of the thymus cortex of each mouse in each experimental group to obtain the average; use a micrometer to place the baseline on the splenic nodule, and measure the two thicknesses with the central artery as the center. The thickness of the splenic nodules on both sides was calculated and averaged; the results are shown in Table 8 and Figure 10.
Table 8 Effects of Cistanche deserticola phenylethanol glycoside on pathological changes of thymus and spleen in perimenopausal model mice (-x±s)
As can be seen from Table 8 and Figure 10, 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 mice. Compared with the model group, high-dose Cistanche deserticola phenylethanol glycoside can significantly thicken the thymic cortex (P<0.01), and medium-dose Cistanche deserticola phenylethanoid glycoside can significantly thicken the thymic cortex (P<0.05). Each medication group could significantly increase the volume of splenic nodules. 5.3 Effect on pituitary tissue morphology in mouse perimenopausal model
The high magnification field of view covers an area of 10000μm
2. Measure the number of basophil cells and anterior pituitary cells in the microrectangular frame. The results are shown in Table 9 and Figures 11~12.
It can be seen from Table 9 and Figures 11-12 that compared with the blank group, the number of basophil cells and anterior pituitary cells in the model group were significantly reduced (P<0.01), indicating that after the perimenopausal model was created in mice, secretion of The cellular source of gonadotropins is reduced. Compared with the model group, each medication group could significantly increase the number of pituitary basophil cells (P<0.01). Except for the low-dose Cistanche deserticola phenylethanol glycoside group, all other medication groups could significantly increase the number of anterior pituitary cells (P<0.01). P<0.01).
