Therapeutic Effect And Mechanism Of Cistanche Phenylethanol Glycosides On Cyclophosphamide-induced Spermatogenesis in Mice
Mar 11, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
ZHAODong-hai1, ZHANGLei2, ZHANGYan3, QILing1, ZOU Xiang-ming4
[Summary]
Purpose: To explore the therapeutic effect of Cistanche phenylethanoid glycosides on mice with cyclophosphamide-induced spermatogenesis and to preliminarily clarify its mechanism of action. Method: Ethanol extraction method to extract Cistanche phenylethanoid glycosides. Forty BALB/C mice were randomly divided into a control group, a model group, a low-dose Cistanche phenylethanoid glycoside group (50 mg·kg-1), and a high-dose group (100 mg·kg-1), with 10 mice in each group. Except for the control group, mice in the other groups were injected with cyclophosphamide (80 mg·kg-1) every day for 5 consecutive days to prepare mouse models of spermatogenesis disorders. After the last administration, mice in the low-dose and high-dose groups of Cistanche phenylethanoid glycosides were given the corresponding doses by gavage, and the mice in the control group and model group were given the same volume of normal saline by gavage, once a day for 30 days. Twenty-four hours after the last gavage, the mouse testis tissue was taken. An enzyme-linked immunosorbent assay was used to determine the level of testosterone in mouse testicular tissue homogenate. The sperm density, sperm motility, and sperm abnormality rate of each group were compared. The morphological changes of testicular tissue were observed by HE staining. Results: Compared with the control group, the sperm density and sperm motility rate of the model group decreased (P<0.01), the rate of sperm deformity increased (P<0.01), and the level of testosterone in the testicular tissue homogenate decreased (P<0.01). 0.01); Compared with the model group, the sperm density and sperm motility of mice in each dose group of Cistanche phenylethanoid glycosides were significantly increased (P<0.01), the rate of sperm deformity decreased (P<0.01), and the testicular tissue The level of testosterone in the homogenate increased (P<0.05 or P<0.01). Histological observations showed that the seminiferous tubules in the testis of the model group were atrophied and degenerated, the seminiferous epithelium was significantly thinner, the arrangement of spermatogenic cells was disordered, and the number of sperm in the cavity decreased; the number of the seminiferous epithelium in the testes of the Cistanche phenylethanoid glycoside group increased significantly, The layers are distinct, the spermatogenic cells are arranged neatly, tightly and regularly, and sperm can be seen in the lumen. Conclusion: Cistanche phenylethanoid glycosides have a significant therapeutic effect on the spermatogenesis disorder caused by cyclophosphamide in mice, and its mechanism may be related to the improvement of testosterone levels in testicular tissues.
[Keywords] Cistanche phenylethanoid glycosides; cyclophosphamide; spermatogenesis disorder; mice, BALB/C
Due to the acceleration of social rhythm, bad living habits and environmental pollution, and other factors, abnormal sperm quality such as oligospermia and asthenospermia have 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 the effective ingredients of Chinese medicine and the study of its therapeutic effects in reproductive injury have good application value. Cistanche phenylethanoid glycosides are one of the most important active ingredients in Cistanche deserticola. They are usually natural glycosides containing ester bonds and oxo-glycosidic bonds with β-glucose as the nucleus. They are widely found in dicotyledonous plants [7]. Studies [8] have shown that Cistanche phenylethanoid glycosides can protect rat sperm from oxidative damage in vitro, but there are no reports about its spermatogenesis. This experiment studies the therapeutic effect of Cistanche phenylethanoid glycosides on the spermatogenic function of mice with cyclophosphamide-induced spermatogenesis and provides an experimental and theoretical basis for the application of Cistanche phenylethanoid glycosides in the treatment of spermatogenesis disorders.
1. Materials and methods
1.1. Laboratory animals, main reagents, and instruments
40 SPF-class adult BALB/C mice, 6-8 weeks old, with a body mass of 20-22g, were purchased from the Animal Experimental Center of Jilin University School of Basic Medicine, and the animal certificate number: SCXK (Kyrgyzstan) 2008-005. The mice were bred adaptively for 5 days before the experiment. Feeding conditions: 22 ℃ ~ 25 ℃, circadian rhythm (12h/12h), 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 in the United States), TE2000U inverted microscope (Nikon, Japan), and J-26XP centrifuge (Nanodrop, United States).
1.2 Preparation of Cistanche Phenylethanol Glucoside
10g of cistanche powder was extracted by refluxing with 70% ethanol, twice, 2h each time, to obtain the phenethyl alcohol glycoside concentrate. The concentrated solution was chromatographed on the D101 macroporous resin column and eluted with 70% ethanol to obtain Cistanche phenecoside alcohol. The UV spectrophotometer identified the extract at a wavelength of 333 nm.
1.3 Preparation of animal model and drug administration method
After 40 male BALB/C mice were adaptively fed for 5 days, the mice were randomly divided into 4 groups (10 mice in each group): control group, model group, Cistanche Cistanche phenylethanoid glycosides low-dose group, and Cistanche phenylethanoid glycosides high-dose 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 disorders. After the model was prepared, mice in the low and high-dose groups of Cistanche phenylethanoid were given 50 and 100 mg·kg-1·d-1 respectively. The mice in the control group and the model group were given equal volumes of normal saline. Once a day for 30 days. All mice were sacrificed 24 hours after the last drug administration and bilateral tests were taken. The right testis was fixed with formaldehyde. The epididymis and testis were taken from the left side. The testis was homogenized and centrifuged at 3,000 min for 10 min. The supernatant was taken and stored in an icebox 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 [9] in a constant temperature water bath to preheat it to 37°C, take a unilateral epididymis to remove connective tissue and fat, cut the tissue, and put it in a test tube at 37°C for 10 minutes 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 5μL of sperm suspension and the same amount of eosin-Y from the test tube Mix well, place it at room temperature for 10 minutes, add a cover glass, and observe with a light microscope. Dead sperm are stained pink, live sperm are not stained and the cytoplasm is transparent. Each specimen counts 200 sperm, calculates the sperm viability, the result is expressed as a percentage, sperm viability = a number of live sperm / the total number of sperm × 100%. Another 5 μL of sperm suspension was fixed in methanol for 5 minutes, 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 lens, and use a high-power lens to check the sperm morphology in order. Each mouse inspected 1,000 complete sperm. The sperm heads without hooks, banana-shaped, amorphous, fat heads and double heads 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 the membrane and placed in a vial dish, and the pre-cooled homogenization medium (0.01mol·L-1 sucrose, 0.01mol·L-1 Tris- HCl, 0.1mmol·L-1 EDTA-Na2 solution, pH 7.4), cut the testis tissue with ophthalmological scissors, pour it into a glass homogenization tube, and centrifuge at 4,000×g for 10 minutes at low temperature, and take the supernatant, Use enzyme-linked immunosorbent assay kit to measure testosterone level in testicular tissue homogenate and test 3 parallel samples for each sample. The experimental steps and methods were completed in accordance with the instructions of the kit.
1.6 Morphological observation of testis
Take paraffin sections of mouse testis, stained with HE, and observe the distribution of spermatogonia and spermatogenic cells in testis tissue under a light microscope.
1.7 Statistical analysis
Statistical analysis was performed using SPSS17.0 statistical software. Sperm density, sperm motility rate, sperm deformity rate, and testicular testosterone level of mice in each group were expressed as x±s, and the comparison between groups was performed by single-factor analysis of variance.
2. results
2.1. Sperm density, sperm motility, and sperm deformity rate of each group of mice
Compared with the control group, the sperm density and sperm viability 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 of Cistanche phenoxyethanol glycoside 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 had higher sperm density and sperm motility (P<0.05), and the rate of sperm deformity decreased (P<0.05). See Table 1.
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-1, 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 phenethyl alcohol glycoside group were (7.55±1.21) and (10.35±0.94) nmol·L-1. The level of testosterone in the low and high-dose groups of Cistanche cistanche ethanol glycosides was significantly higher than that of the model group (P<0.05 or P<0.01). The testosterone level in the testicular tissue homogenate of the Cistanche cistanche phenethyl glycoside high-dose 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 light microscope, the seminiferous tubules in the testes of the normal group were densely arranged, the level and number of germ cells were large, the seminiferous epithelium was arranged neatly, and the seminiferous tubules were all seen with sperm formation. In the model group, the seminiferous tubules in the testes of 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 low dose of Cistanche phenethyl alcohol glycoside.
In the group, the number of seminiferous epithelial layers of seminiferous tubules increased, the arrangement of the epithelium was more regular, and the damage of seminiferous tubules was obviously improved; the structure of seminiferous tubules in the high-dose Cistanche phenylethanoid glycoside group improved more significantly, and the number and number of seminiferous epithelial layers increased significantly. Arranged neatly, there are mature sperm in the seminiferous tubules, and the structure is basically back to normal. See Figure 1 (Inset 6).
3 Discussion
Cyclophosphamide is a commonly used anti-tumor drug and immunosuppressant, which mainly interferes with a variety of intracellular biological functions through the alkylation of DNA. Reproductive toxicity is one of the main side effects of cyclophosphamide [3], which can easily lead to sperm DNA damage and abnormal sperm development. Clinical studies [10-11] indicate that the reproductive toxicity of cyclophosphamide can cause azoospermia or oligospermia. , Leading to infertility or the occurrence of genetic defects or malformations in offspring.
Cistanche tubulosa is a perennial higher parasitic plant in the Leiden family. It is a well-known Chinese medicine that nourishes the kidney and yang, nourishes the blood, and nourishes the intestines. It is a commonly used prescription medicine for the traditional treatment of male reproductive dysfunction [12]. Phenylethanoid glycosides are one of the most important active components of Cistanche [13] and have a wide range of pharmacological effects, such as anti-oxidation, anti-aging, and anti-tumor [14].
In this study, cyclophosphamide was used to prepare a mouse model of spermatogenesis disorder, and the therapeutic effect of Cistanche phenylethanoid glycosides on spermatogenesis disorder caused by cyclophosphamide was studied. The results showed that compared with the normal group, the sperm density of the model group decreased, the survival rate decreased, and the deformity rate increased. Compared with the model group, all the dose groups of Cistanche phenoxyethanol glycosides can effectively increase sperm density, enhance sperm motility and reduce sperm deformity rate. The above results indicate that the cistanche phenoxyethanol glycosides can promote spermatogenesis and protect the damaged testis. The results of testis morphology showed that after treatment with Cistanche phenylethanoid glycoside, the number of the seminiferous epithelium in the testis of the model mice was significantly increased, the layers were distinct, the spermatogenic cells were arranged neatly, tightly, and regularly, and sperm can be seen in the lumen, suggesting that Cistanche phenylethanoid can change Shanhe restores the structure and morphology of seminiferous epithelium after reproductive injury.
Testosterone regulates the differentiation of male germ cells and is a necessary condition for the normal development of the testis and the normal occurrence of sperm. Some studies [15] reported that the selective knockout of androgen receptors in Sertoli cells of the testis caused sperm development disorders and male sterility. In this study, cyclophosphamide was used to produce spermatogenic.
After the obstacle injury model, the testosterone level in the testis tissue of the mice was significantly lower than that in the normal group. After the action of Cistanche phenylethanoid glycosides, the testosterone levels in each dose group increased, and the testosterone level in the high-dose group was significantly higher than that in the low-dose group. It is suggested that the cistanche phenoxyethanol glycosides may promote spermatogenesis by increasing the level of testosterone in the testicular tissue to treat spermatogenic dysfunction, but the mechanism of its effect on testosterone needs further research and discussion.
Testosterone regulates the differentiation of male germ cells and is a necessary condition for the normal development of the testis and the normal occurrence of sperm. Some studies [15] reported that the selective knockout of androgen receptors in Sertoli cells of the testis caused sperm development disorders and male sterility. In this study, cyclophosphamide was used to produce spermatogenic.
After the obstacle injury model, the testosterone level in the testis tissue of the mice was significantly lower than that in the normal group. After the action of Cistanche phenylethanoid glycosides, the testosterone levels in each dose group increased, and the testosterone level in the high-dose group was significantly higher than that in the low-dose group. It is suggested that the cistanche phenoxyethanol glycosides may promote spermatogenesis by increasing the level of testosterone in the testicular tissue to treat spermatogenic dysfunction, but the mechanism of its effect on testosterone needs further research and discussion.
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