| ObjectiveTo investigate the protective effects of holothurian spermatophore extract in model mice which genital system caused by cyclophosphamide with partial androgen deficiency in the aging male(PADAM).Methods:1. Get water-soluble matter from holothurian spermatophore by way of aqueous ultrasonic extraction; Get liposoluble substance from holothurian spermatophore by way of organic solvent extraction such as ethanol.2. Testing the microelement of power by ICP-MS. Testing the moisture, ash content, protein and fat according to standard of5009.3. Male Kunming mice were equally allotted to9groups:regular blank group, model control group, treated group with low, middle and high dosage holothurian spermatophore aqueous/alcohol extract, positive drug group. They were intraperitoneally injected with cyclophosphamide (CP,28mg/kg) once a day for5days to construct injured genital system and partial androgen deficiency model by CP except that the regular group were intraperitoneally injected with normal saline. Mice in treatment groups orally received holothurian spermatophore extraction (150,300,600mg/kg) and mice in positive drug group orally received methyltestosterone (1.5mg/kg) once a day for4weeks. Quantity,sportive rate and abnormal rate of sperm, visceral index, testosterone levels in serum, thickness of spermatogenic epithelium and number of spermatogenic cells were determined. Then total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) of testicle were detected; The apoptosis of testicular spermatogenic cells was examined by TUNEL method, and the distribution and expressions of Bcl-2, Bax and Caspase-3were detected by immunohistochemistry assay. And compared with those of model group. Results1. For the aqueous ultrasonic extraction, the rate is8.4%with the solvent-material ratio3and ultrasonic time25min and extracting time2h under30℃. For organic solvent extraction, the rate is23.9%with the solvent-material ratio10and extracting3h under30℃.2. The holothurian spermatophore lyophilized powder contains plenty of nutritional ingredient essential to human, such as protein, Fe, Ca, Mg, Zn, Se, the content is63.1%,5752,2696,87.6,10.5mg/kg.Meanwhile three kinds of harmful heavy metal content is far beyond the country’s limited healthy standard for the pollution-free aquatic products harmful heavy metal, the content is2.48,12,0.7mg/kg.3. Compared with blank group, the testosterone levels in serum, quantity, sportive rate of sperm, testis and thymus index in the model group decreased obviously, the abnormal rate of sperm increased obviously (P<0.01). The change of epididymis and spleen index had also a significant difference (P<0.05). Though the dose of holothurian spermatophore extracts had no obvious difference (P>0.05), for the model group, the above indicators of mice in treatment groups with damaged genital system were all significantly improved (P<0.01), even close to or higher than the normal level. The histopathology of testis showed that compared with blank group, the spermatogenic epithelium of mice in model groups became thinner and the difference had statistical significance (P<0.01). The number of spermatogenic cells decreased obviously (P<0.01). Compare to the model group, holothurian spermatophore extract can remit the toxic effect of the cyclophosphamide. The thickness of spermatogenic epithelium and number of spermatogenic cells were both improved obviously (P<0.01).4. Compared with blank group, the T-SOD and GSH-Px activity was significantly decreased, the MDA level was significantly increased (P<0.05,0.01). However, holothurian spermatophore extract can enhance the anti-oxidation ability of testis, reduce the MDA level, and they have significance in statistics compared with model group (P<0.05,0.01).5. The TUNEL showed that compared with blank group, the apoptotic index of spermatogenic cells in model group increased significantly (P<0.01); Holothurian spermatophore extract can decrease the apoptotic of spermatogenic cells, and they have significance in statistics compared with model group (P<0.05,0.01). The immunohistochemistry showed that compared with blank group, the expressions of Bax and Caspase-3of testis in model group increased and the expression of Bcl-2decreased, and the difference were statistically significant (P<..01). Then holothurian spermatophore extract had antagonism effect on the reproduction toxicity of CP, increased the expression of Bcl-2, decreased the expression of Bax and Caspase-3, and the differences compared with model group were significant (P<0.05,0.01).Conclusion:1. The holothurian spermatophore lyophilized powder contains plenty of microelement essential to human with a high nutritive value-high protein and low fat.2. Holothurian spermatophore extract can improve testosterone levels in serum content of the mice with damaged genital system caused by cyclophosphamide and have a effect on testosterone replacement, avoid the side effects caused by testosterone replacement of PAD AM patients. And the extract can improve the construction of seminiferous tubule, increase the number of spermatogenic cells and sperm and the sportive rate of sperm, decrease the abnormal rate of sperm, enhance the testis and epididymis index, and have a protective effect on the damaged genital system.3. Holothurian spermatophore extract can enhance the anti-oxidation ability of testis, remit the reproduction toxicity of CP, increase the expression of apoptosis gene, decrease the expression of anti-apoptosis gene, lower the activity of Caspase-3, then reduce the apoptotic of spermatogenic cells. |
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