Li Li, Pan Min, Liu Ziao, Zhao Hongsu, Xu Fengqing, Tong Xiaohui, Wang Yajuan, Chen Bin, Yu Lihua, Wang Tongsheng
College of Integrated Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei, China.
Anhui Province Key Laboratory of Chinese Medical Formula, College of Pharmacy, Anhui University of Chinese Medicine, Hefei, China.
Comb Chem High Throughput Screen. 2024 Oct 15. doi: 10.2174/0113862073328011241004110538.
The TAp73 gene is an anti-cancer gene that also affects the junction between Sertoli and germ cells. Inhibition of this gene causes infertility in male mice. Our previous research proved that Wuzi-Yanzong-Wan (WZYZW) can protect spermatogenesis and maturation by preventing TAp73 inhibition.
This study aimed to investigate the effect of drug-containing serum of WZYZW on the defect of cell-cell junctions in the Sertoli-germ cells co-culture system in vitro.
LC-HRMS was used to analyze the content of active ingredients in WZYZWmedicated serum. Then, primary extraction and co-culture of germ cells and Sertoli cells were carried out. Co-cultured cells were added with PFT-α to induce the TAp73 inhibition model, with WZYZW-medicated serum at 2.5%, 5%, and 10% treated in parallel. Sloughing of germ cells from Sertoli cells was calculated. Transmission electron microscopy (TEM), Immunofluorescence, qRT-PCR, and western blot methods were employed.
The drug-containing serum of WZYZW contained schisandrin, hyperoside, geniposidic acid, ellagic acid, and quercetin. Using TEM assay, we observed restoration of the desmosomelike (Des), tight junctions (TJ), and basal ectoplasmic specialization (ES) structure following WZYZW treatment. WZYZW caused inhibition of peptidase and protease inhibitors (tissue inhibitor of metalloproteinase-1 (TIMP1), Serpina3n) by immunofluorescence analysis. Western blot and qRT-PCR analysis revealed that WZYZW was able to ameliorate the expressions of peptidase and protease inhibitors and cell adhesion factors, such as TAp73, TIMP1, Serpina3n, Desmocollin-3, N-cadherin, and Nectin-2.
WZYZW-medicated serum could prevent the defect of cell-cell junctions between Sertoli-germ cells co-culture system in vitro by up-regulating the expression of TAp73.
TAp73基因是一种抗癌基因，也影响支持细胞与生殖细胞之间的连接。该基因的抑制会导致雄性小鼠不育。我们之前的研究证明，五子衍宗丸（WZYZW）可通过防止TAp73抑制来保护精子发生和成熟。
本研究旨在探讨WZYZW含药血清对体外支持细胞-生殖细胞共培养系统中细胞间连接缺陷的影响。
采用液相色谱-高分辨质谱（LC-HRMS）分析WZYZW含药血清中活性成分的含量。然后，进行生殖细胞和支持细胞的原代提取及共培养。将共培养的细胞加入PFT-α以诱导TAp73抑制模型，同时平行处理2.5%、5%和10%的WZYZW含药血清。计算生殖细胞从支持细胞上的脱落情况。采用透射电子显微镜（TEM）技术、免疫荧光技术、qRT-PCR技术和蛋白质印迹法。
WZYZW含药血清中含有五味子醇甲、金丝桃苷、京尼平苷酸、鞣花酸和槲皮素。通过TEM检测，我们观察到WZYZW处理后，桥粒样（Des）、紧密连接（TJ）和基底外质特化（ES）结构得以恢复。通过免疫荧光分析发现，WZYZW可抑制肽酶和蛋白酶抑制剂（金属蛋白酶组织抑制剂-1（TIMP1）、丝氨酸蛋白酶抑制剂家族A成员3N（Serpina3n））。蛋白质印迹法和qRT-PCR分析显示，WZYZW能够改善肽酶和蛋白酶抑制剂以及细胞黏附因子的表达，如TAp73、TIMP1、Serpina3n、桥粒芯糖蛋白-3、N-钙黏蛋白和连接黏附分子-2。
WZYZW含药血清可通过上调TAp73的表达来预防体外支持细胞-生殖细胞共培养系统中细胞间连接的缺陷。
