| 徐秀玲,金央,卢睿杰,等.加味五子衍宗方凝胶制剂贴敷治疗少弱精子症的实验研究[J].浙江中医药大学学报,2023,47(3):234-240. |
| 加味五子衍宗方凝胶制剂贴敷治疗少弱精子症的实验研究 |
| Experimental Study on the Acupoint Application Therapy with Jiawei Wuzi Yanzong Prescription Gel Preparation in the Treatment of Oligoasthenospermia |
| DOI：10.16466/j.issn1005-5509.2023.03.002 |
| 中文关键词: 少弱精子症 加味五子衍宗方 贴敷疗法 生精细胞 支持细胞 线粒体呼吸链复合物 再生 |
| 英文关键词: oligoasthenospermia Jiawei Wuzi Yanzong Prescription acupoint application therapy spermatogenic cell sertoli cell mitochondrial respiratory chain complex regeneration |
| 基金项目:浙江省中医药科技计划项目(2022ZA115) |
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| 中文摘要: |
| [目的] 研究加味五子衍宗方(Jiawei Wuzi Yanzong Prescription，JWYP)凝胶制剂对少弱精子症小鼠睾丸生精细胞和支持细胞损害的修复作用，探讨JWYP凝胶制剂经皮渗透贴敷的疗效及其作用机制。[方法] 将JWYP超细粉碎微粒与凝胶均匀混合，配制成凝胶贴敷制剂。24只雄性BALB/c小鼠随机分为经皮渗透组、灌胃染毒组、贴敷治疗组和对照组，经皮渗透组以荧光素钠(fluorescein sodium，FS)作为荧光染色剂，均匀混合标记JWYP凝胶制剂并贴敷于小鼠腹壁，扩散4～8 h后荧光显微镜观察其活体荧光成像；灌胃染毒组采用邻苯二甲酸(2-乙基己基)酯[di-(2-ethylhexyl)phthalate，DEHP]灌胃染毒法建立小鼠少弱精子症模型，将DEHP溶于食用大豆油并以20 mg/(kg·d)的剂量连续灌胃30 d染毒；贴敷治疗组将配制好的JWYP凝胶制剂贴敷于小鼠腹壁，每周5 d贴敷给药，连续4周，贴敷给药期间同步予以DEHP连续灌胃染毒；对照组小鼠同期以标准饲料添加食用大豆油同步喂养。检测各组小鼠睾丸的湿重和体积，采用光镜和透射电镜观察各组小鼠睾丸细胞的组织学形态结构，并同步检测小鼠睾丸细胞线粒体呼吸链复合物(mitochondrial respiratory chain complex，MRC)活性。[结果] 经皮渗透组小鼠腹壁皮下及腹膜腔内均显示有较强的荧光分布。与对照组比较，灌胃染毒组小鼠睾丸湿重和体积差异无统计学意义(P＞0.05)，贴敷治疗组小鼠睾丸湿重差异无统计学意义(P＞0.05)，睾丸体积明显增大(P＜0.05)。光镜和透射电镜观察显示，与对照组比较，灌胃染毒组小鼠睾丸生精细胞层形态结构基本正常，但细胞排列松散，管腔内无成熟精子生成，生精细胞空泡样变性退化，支持细胞的胞质内可见凋亡小体样结构；贴敷治疗组小鼠睾丸生精细胞层形态结构正常，支持细胞细胞排列密集，管腔内可见成熟精子，胞质内富含线粒体、溶酶体等细胞器。灌胃染毒组小鼠睾丸细胞MRC-Ⅰ、Ⅲ、Ⅳ的活性检测值明显低于对照组(P＜0.05)，贴敷治疗组小鼠睾丸细胞MRC-Ⅲ、Ⅳ的活性检测值明显高于灌胃染毒组(P＜0.05)。 [结论] JWYP凝胶制剂可通过贴敷给药的方式经皮渗透至小鼠皮下组织和腹腔，对DEHP连续灌胃染毒所致少弱精子症小鼠的睾丸损害具有治疗效果，其作用机制可能与增加细胞内线粒体和溶酶体等细胞器的数量，提高MRC活性，进而促进小鼠睾丸生精细胞和支持细胞的增殖再生有关。 |
| 英文摘要: |
| [Objective] To investigate the recovery effects of acupoint application therapy(AAT) with prepared Jiawei Wuzi Yanzong Prescription(JWYP) gel preparation on mice spermatogonia and sertoli cells and explore therapeutic results and its mechanism of percutaneous osmotic sticking therapy with JWYP gel preparation. [Methods] A JWYP gel preparation was prepared by superfine particles of JWYP Pills with gellan gum. Twenty-four male BALB/c mice were randomly divided into four groups: percutaneous infiltration(PI) group， di-(2-ethylhexyl) phthalate(DEHP) exposure by intragastric administration(DEHPe) group， AAT group and control group. In PI group， the JWYP gel preparation mixed evenly with fluorescein sodium(FS) as a fluorescent stain was applied to the abdominal wall of mice. The fluorescence imaging and distribution of FS in both skin and underlying peritoneum were observed under a fluorescent microscopy 4～8 hours after administration. DEHP dissolved in soya oil was given at the dose of 20 mg/(kg·d) by intragastric administration for 30 days consecutively to make an oligoasthenospermia animal model in DEHPe group. A prepared JWYP gel preparation was applied to the abdominal wall of mice 5 days a week for 4 weeks for male adult BALB/c mice in AAT group and during the period of AAT， DEHP dissolved in soya oil at the dose of 20 mg/(kg·d) was fed simultaneously. The mice in control group were given a standard diet with same dose of soya oil in the corresponding period. The wet weight and volume of mice testis were measured in all groups. The morphological structure and ultrastructure of mice testicular cells were investigated and observed under the both light microscopy and transmission electron microscopy. The activities of mitochondrial respiratory chain complex(MRC) in mice testicular cells were also detected. [Results] Brilliant distribution of FS in subcutaneous tissue of abdominal wall and underlying peritoneum rendered by fluorescence staining was imaged in PI group. Compared with control group， there was no significant difference in the wet weight and volume of testis in DEHPe group(P＞0.05). In AAT group， there was no significant difference in the wet weight compared with control group(P＞0.05)， but the testicular volume was significantly increased(P＜0.05). By contrast with control group， spermatogenic cells in mice testis were normal in shape and structure but arranged loosely under light microscopy， there was no mature spermatogenesis in seminiferous lumen， vacuolar degeneration of spermatogenic cells and apoptosis-like structure in the cytoplasm of sertoli cell were also detected under electron microscopy in DEHPe group. In AAT group， spermatogenic cells arranged in normal shape and structure， sertoli cells were closely packed and mature sperms were detected in seminiferous lumen， the increased count of organelle such as mitochodria and lysosome in the cytoplasm of sertoli cell were also observed under electron microscopy. The activities of MRC-Ⅰ， Ⅲ， Ⅳ of mice testicular cells in DEHPe group were significantly lower than that in control group(P＜0.05)， while the activities of MRC-Ⅲ， Ⅳ of mice testicular cells in AAT group were significantly higher than that in DEHPe group(P＜0.05). [Conclusion] The AAT of a prepared JWYP gel preparation， with the activity of transdermal osmotic drug delivery， may have treatment effects against testicular damage in mice caused by experimental exposure of DEHP via oral administration. The mechanism might be related to increasing the amount of mitochodria and lysosome in cytoplasm of sertoli cells， and enhancing the activities of MRC for promoting cell regeneration and spermatogenesis. |
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