Therapeutic Effect Of Phenylethanoid Glycosides On Cyclopfosphamide-induced Dyszoospermia in Mice And Its Mechannism
Mar 08, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
ZHAO Dong-hail， ZHANG Lei?， ZHANG Yan²， QI Ling'， ZOU Xiang-ming'
Abstract: Objective To explore the therapeutic effect of phenylethanoid glycosides on cyclophosphamide(CTX)-induced dyszoospermia in mice and to preliminary elucidate the mechanisms involved in the process. Methods Phenylethanoid glycoside was extracted by ethanol extraction, Forty male BALB/C mice were randomly divided into control group, model group, low dose of phenylethanoid glycosides group (50 mg·kg-I), and high dose of phenylethanoid glycosides group(100 mg·kg-). Except for the control group, the dyszoospermia mouse model was established by peritoneal injection of CTX at the daily dose of 80 mg· kg-i, once daily for successive 5 d. After modeling, phenylethanoid glycosides were intragastrically administered at corresponding doses to each phenylethanoid glycosides group. An equal volume of normal saline was given to the mice in the control group and model group by gastrogavage. All the medication was performed once daily for successive 30 d. The testis tissue was obtained 24 h after the last intragastric administration. The level of testosterone in the testis tissue homogenate was determined by enzyme-linked immunosorbent assay. The sperm counts, the motility rates, and the teratozoospermia rates in various groups were compared. The morphological changes of the testis tissue were observed using HE staining. Results Compared with the control group, the sperm count and the motility rate were decreased, the teratozoospermia rate was increased, and the testosterone level in the testis tissue homogenate was decreased in the model group(P<0.01). Compared with the model group, the sperm counts and the motility rates were increased, the teratozoospermia rates were decreased, and the testosterone levels in the testis tissue homogenate were increased in phenylethanoid glycosides groups(P<0.05 or P<0,01). The histological results showed atrophy and degeneration of seminiferous tubule, thicker seminiferous epithelium, and azoospermic lumina in the model group; the number of seminiferous epithelial layers was increased and the seminiferous cells orderly arranged, and many sperms were found in the tubules in phenylethanoid glycosides groups. Conclusion Phenylethanoid glycosides have obviously therapeutical effects on CTX-induced dyszoospermia in mice, and their mechanisms might be correlated with recovering the testosterone level.
Key words: phenylethanoid glycosides; cyclophosphamide; spermatogenic impairment; mice, BALB/C
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Due to the acceleration of social rhythm, bad living habits and environmental pollution, and other factors, abnormal sperm quality such as oligospermia and asthenospermia has become the main cause of male infertility [1]. At present, there are fewer drugs for treating oligospermia and asthenospermia, and the therapeutic effect is not obvious. Traditional Chinese medicine prescriptions or active ingredients have good effects in improving male reproductive function [2-6]. However, due to the wide variety of prescription drugs, the relationship between the pharmacological effects and the effective ingredients of the drug is not clear, which limits the promotion and application of spermatogenic drugs. The research and development of effective ingredients of traditional Chinese medicine and the study of its therapeutic effects in reproductive injury have good application value. The phenylethanoid glycosides in Cistanche cistanche are one of the most important active ingredients in Cistanche. They are usually natural glycosides containing ester and oxo-glycoside bonds with β-glucose as the nucleus. They are widely present in dicotyledonous plants 7]. Studies [8] have shown that Cistanche phenethanol glycosides can protect rat sperm from oxidative damage in vitro, but there are no reports about its spermatogenesis. This experiment studies the therapeutic effects of Cistanche phenylethanoid glycosides on the spermatogenesis of mice with cyclophosphamide-induced spermatogenesis and provides an experimental and theoretical basis for the application of Cistanche phenethyl alcohol glycosides in the treatment of spermatogenesis disorders.
1 Material and methods
1.1 Experimental animals, main reagents, and instruments
40 SPF adult BALB/C mice, 6-8 weeks old, weight 20-22 g, purchased from the Animal Experimental Center of Jilin University School of Basic Medicine, animal certificate number: SCXK ( Kyrgyzstan) 2008-0005. The mice were reared adaptively for 5 days before the experiment. Feeding conditions: 22℃～25℃, circadian rhythm (12 h/12 h), free drinking water and food. Cistanche is a product of Xinjiang Dakang Trading Company, cyclophosphamide for injection was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd. (batch number 11061921), testicular tissue homogenate testosterone enzyme-linked immunoassay kit was purchased from Tianjin Jiuding Medical Bioengineering Co., Ltd. (batch number 20120812), other reagents are all domestically produced analytical grades. PLUS384 microplate reader (MDC, USA), TE2000U inverted microscope (Nikon, Japan), J-26XP centrifuge (Nanodrop, USA).
1.2 Preparation of cistanche phenylethanoid glycosides
10 g of Cistanche tubulosa powder was refluxed with 70% ethanol, twice, 2 hours each time to obtain the phenylethanoid glycoside concentrate. The concentrated solution was subjected to D101 macroporous resin column chromatography and eluted with 70% ethanol to obtain the Cistanche phenylethanoid glycoside alcohol. The UV spectrophotometer identified the extract at a wavelength of 333 nm.
1.3 Preparation of animal model and methods of administration
40 male BALB/C mice were adaptively fed for 5 days, and the mice were randomly divided into 4 groups (10 mice in each group): control group, model group, Cistanche cistanche low phenylethanoid glycosides Dose, and high-dose Cistanche phenethyl alcohol glycoside group. Except for the control group, the other three groups of mice were given an intraperitoneal injection of cyclophosphamide 80 mg·kg-1 for each mouse for 5 consecutive days to prepare animal models of spermatogenesis disorder. After the model was prepared, mice in the low and high-dose groups of Cistanche phenylethanoid were given respectively 50 and 100 mg·kg-'·d-'. , 1 time a day for 30 consecutive days. All mice were sacrificed 24 hours after the last administration, and bilateral tests were taken. The right testis was fixed in formaldehyde. The epididymis and testis were taken from the left side. The testis was homogenized and centrifuged at 3,000 r·min for 10 min. The supernatant was taken and stored in a refrigerator at 20°C for testing. The left epididymis was tested for sperm density, sperm motility, and deformity rate.
1.4 Determination of sperm density, sperm motility, and deformity rate
Place a test tube containing 10 mL of sperm nutrient solution in a constant temperature water bath to preheat to 37°C, remove the connective tissue and fat from one side of the epididymis, cut the tissue, and put it in the test tube at 37°C for 10 min. After the sperm is fully free, take 1 drop on the blood cell counting plate and calculate the sperm density according to the cell counting method. The result is expressed as the number of sperm per milliliter; in addition, take 5 μL of sperm suspension from the test tube and mix it with the same amount of Eosin-Y Evenly, place it at room temperature for 10 minutes, add a cover glass, and observe with a light microscope. The dead sperm are stained pink, and the live sperm are not stained but the cytoplasm is transparent. Count 200 sperm per specimen, calculate sperm viability, and the result is expressed as a percentage, sperm viability = a number of live sperm/a total number of sperm × 100%. Another 5 μL of sperm suspension was fixed in methanol for 5 min, dried and stained with 2% eosin aqueous solution for 1 hour, rinsed, and blow-dried. Look for parts with less sperm overlap under a low-power microscope, and use a high-power microscope to check the sperm morphology in sequence. Each mouse inspected 1000 complete sperm. The sperm heads were unhooked, banana-shaped, amorphous, fat-headed, double-headed, etc. were judged as deformities. The sperm deformity rate was calculated, and the results were expressed as a percentage. Sperm deformity rate = a number of deformed sperm/total number of sperm count × 100%.
1.5 Determination of reproductive hormones
The testis was peeled off and placed in a vial dish, and the pre-cooled homogenization medium (0.01 mol·L-sucrose, 0.01 mol·L-1Tris-HCl, 0.1 mmol· L-1EDTA-Naz solutions, pH 7.4), cut the testicular tissue with ophthalmological scissors, pour it into a glass homogenization tube and homogenize, centrifuge at 4 000×g for 10 min at low temperature, take the supernatant, and use enzyme-linked immunosorbent reagent The testosterone level in the testicular tissue homogenate was determined by the box, and 3 parallel samples were tested for each sample. The experimental steps and methods were completed in accordance with the kit instructions.
1.6 Morphological observation of testicular tissue
Take paraffin sections of mouse testis, HE stains, and observe the distribution of spermatogonia and spermatogenic cells in testis tissue under the light microscope.
1.7 Statistical analysis
Statistical analysis was performed using SPSS 17.0 statistical software. Sperm density, sperm motility rate, sperm deformity rate, and testicular testosterone level of mice in each group were expressed as z±s, and the comparison between groups was performed by one-way analysis of variance.
2. results
2.1 Sperm density, sperm viability, and sperm deformity rate of each group of mice
Compared with the control group, the sperm density and sperm motility rate of the model group decreased (P<0.01), and the sperm deformity rate increased (P<0.01). Compared with the model group, the sperm density and sperm motility rate of mice in each dose group of Cistanche phenethyl alcohol increased (P<0.05 or P<0.01), and the rate of sperm deformity decreased (P<0.05 and P<0.01). Compared with the low-dose Cistanche phenethyl alcohol glycoside group, the high-dose Cistanche phenethyl alcohol glycoside group increased sperm density and sperm motility (P<0.05), and the rate of sperm deformity decreased (P<0.05).
2.2 Testosterone levels in testis tissues of mice in each group
The testosterone levels in the testicular tissue homogenates of the control group and the model group were (10.26±0.57) and (5.33±0.84) nmol·L-', respectively. The testosterone level of the model group was significantly lower than that of the control group (P<0.01). The testosterone levels in the testicular tissue homogenate of the low-dose Cistanche phenylethanoid glycoside group were (7.55±1.21) and (10.35±0.94) nmol·L-1. The testosterone levels of the low and high-dose Cistanche phenylethanoid glycoside groups were significantly higher than those of the model group (P<0.05 or P<0.01). The level of testosterone in the testicular tissue homogenate of the high-dose Cistanche phenethyl alcohol glycoside group was significantly higher than that of the low-dose group (P<0.05).
2.3 Morphology of mouse testis in each group
Observed by a light microscope; the spermatogenic tubules in the testis of the normal group are densely arranged, the germ cell level and number are many, the seminiferous epithelium is arranged neatly, and the seminiferous tubules are all formed by sperm. In the model group, the seminiferous tubules in the testes of the mice were atrophied and degenerated, the seminiferous epithelium was significantly thinned, the number of epithelial layers was reduced, the arrangement was disordered, and the number of sperm in the cavity was reduced. Compared with the model group, the number of seminiferous tubules and seminiferous epithelial layers in the low-dose Cistanche phenylethanoid glycoside group increased, the epithelial arrangement was more regular, and the seminiferous tubule damage was significantly improved; the high-dose Cistanche phenylethanoid glycoside group improved the structure of the seminiferous tubules more significantly, The number and number of seminiferous epithelium have increased significantly, they are arranged neatly, there are mature sperm in the seminiferous tubules, and the structure has basically returned to normal. See Figure 1 (Inset 6).
