中国大鲵肌肉蛋白肽对BPA诱导小鼠精子发生障碍的治疗作用及其机制研究

摘要 目的：研究中国大鲵肌肉蛋白肽（The protein and peptide extracts from muscles of Chinese giant salamanders，SP）对双酚A（bisphenol A，BPA）诱导的小鼠生精障碍的保护作用，并初步探讨其作用机制。方法：40只C57BL/6 雄性小鼠分为四组，分别为Control组、BPA组、BPA+CS（Compound Substance）组、BPA+SP组。BPA组使用50 mg/kg/d的BPA腹腔注射，BPA溶解于玉米油；Control组只腹腔注射玉米油。BPA+CS组灌胃其他有利于生精壮阳的复合物用作对照，复合物溶解于生理盐水中；BPA+SP组灌胃上述复合物联合大鲵肌肉蛋白肽，以上灌胃剂量均4 g/kg/d。连续造模28天，期间每周测定小鼠体重，造模结束后测定睾丸体积、睾体比，取附睾精子使用CASA计算机辅助分析系统测定精子数目、精子活力，ELISA法测定血清睾酮含量，HE染色、TUNEL染色分析睾丸组织病理学，最后采用免疫荧光染色及Western blotting分析转移相关基因2（MTA2）的表达情况、油红O染色观察支持细胞体内、体外吞噬作用。结果：与对照组比较，BPA组睾丸体积（P<0.05）、睾丸重量（P<0.05）、睾体比（P<0.05）、精子数目（P<0.05）、精子活力（P<0.05）、睾酮含量（P<0.01）均显著降低，睾丸组织形态受损（P<0.05）、生精细胞凋亡增多（P<0.01）、MTA2表达量降低（P<0.01）、支持细胞吞噬功能减弱（P<0.01）；BPA+CS组较BPA组无明显变化，BPA+SP组以上变化显著改善（P<0.05）。结论：大鲵肌肉蛋白肽对BPA诱导的小鼠生精功能障碍有明显的保护作用，作用机制可能与干预MTA2表达进而增强支持细胞的吞噬作用相关。

The Role and Mechanism of the Protein and Peptide Extracts from Muscles of Chinese Giant Salamanders Protects Mouse Testis from Deleterious Effects of Bisphenol A

ABSTRACT Objective: To explore the protective effect and its mechanism of the protein and peptide extracts from the muscle of Chinese giant salamanders (SP) on the protection of bisphenol A (BPA)-induced sperm disorder in mice. Methods: Forty male C57BL/6 mice were divided into four groups, Control group, BPA group, BPA+CS group and BPA+SP group. BPA group was injected with 50 mg/kg/d of BPA, which dissolved in corn oil. Control group was injected with corn oil only. The BPA+CS group was given compound substances, which have been proved to be beneficial for spermatogenesis, and were used as controls. The BPA+SP group was given the above mentioned compounds plus SP dissolved in physiological saline. Mice received the compounds plus SP by gavage. The doses were 4 g/kg/d for both groups. The model was established continuously for 28 days, during which the body weight of mice was measured weekly. Testicular volume and testosterone-body ratio were measured after establishment of animal model. Epididymal sperms were collected to measure sperm number and sperm motility by using the computer aided analysis system (CASA). The amount of testosterone in the serum was determined by ELISA. HE staining and TUNEL staining were used to analyze testicular histopathology. Finally, immunofluorescence staining and Western blotting were used to analyze the expression of metastasis associated protein 2 (MTA2), and Oil Red O staining was used to observe the phagocytosis of Sertoli cells in vitro and in vivo. Results: Compared with the control group, the testicular volume (P<0.05), testicular weight (P<0.05), testosterone-body ratio (P<0.05), sperm number(P<0.05), sperm motility (P<0.05), and the amount of testosterone in the serum of mice (P<0.01) in BPA group were significantly decreased. Testicular morphology was damaged and the number of apoptotic spermatogenic cells in BPA group was increased compared with control group(P<0.01). At the same time, the expression of MTA2 and the phagocytosis of Sertoli cells in BPA group were decreased compared with control group (P<0.01). There was no difference between BPA + CS group and BPA group. Surprisingly, the above changes in BPA group were significant difference by using SP(P<0.05). Conclusion: SP has obvious protective effect on BPA-induced sperm disorder in mice, and the mechanism of action may be related to enhanced phagocytosis of Sertoli cells by improving the expression of MTA2.