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目的:探讨五子衍宗丸含药血清对小鼠睾丸TM4支持细胞分泌功能及自噬的影响。方法:构建慢病毒空载体和细胞色素C氧化酶7a2(Cox7a2)慢病毒载体,并转染TM4细胞;制备正常小鼠空白血清,溶剂对照血清,五子衍宗丸低、高浓度含药血清,分别与转染Cox7a2慢病毒细胞共培养24h后,采用流式细胞仪检测细胞中ROS水平,Western blot法检测细胞中自噬基因LC3和Beclin 1的表达,ELISA法检测细胞中TF和INHB的含量。结果:与TM4正常细胞组比较,转染Cox7a2慢病毒细胞组中ROS水平明显升高(P<0.01),LC3、Beclin 1的表达均降低(P<0.01,P=0.32),TF、INHB的含量均下降(P<0.01,P=0.19);给予五子衍宗丸低、高浓度含药血清共培养后,转染Cox7a2慢病毒细胞中升高的ROS水平均降低(P<0.01,P=0.52),降低的LC3、Beclin 1表达均增高(P<0.05,P<0.01),TF、INHB的含量均明显升高(P<0.01)。结论:Cox7a2过表达可产生过量的ROS,TM4支持细胞自噬水平下降,导致细胞分泌功能障碍;五子衍宗丸含药血清可通过调节ROS和自噬水平,进而改善TM4支持细胞分泌功能。
Objective: To investigate the effect of Wuzi Yanzong Pill containing serum on the secretory function and autophagy of mouse testis TM4-supporting cells. Methods: Lentiviral empty vector and cytochrome C oxidase 7a2 (Cox7a2) lentiviral vector were constructed and transfected into TM4 cells. The normal mice blank serum, solvent control serum, Wuzi Yanzong Pill low and high concentration drug-containing serum were prepared respectively After co-cultured with Cox7a2 lentivirus for 24 hours, ROS levels were detected by flow cytometry. The expression of autophagy genes LC3 and Beclin 1 were detected by Western blot, and the contents of TF and INHB in the cells were detected by ELISA. Results: The levels of ROS in transfected Cox7a2 lentivirus group were significantly higher than those in TM4 normal group (P <0.01), while the expressions of LC3 and Beclin 1 were decreased (P <0.01, P = 0.32) (P <0.01, P = 0.19). After co-cultured with low and high concentrations of Wuzi Yanzong pill co-cultured, the elevated ROS levels in Cox7a2 lentivirus transfected cells decreased (P <0.01, P = 0.52 ), Decreased LC3 and Beclin 1 expression (P <0.05, P <0.01), while the levels of TF and INHB were significantly increased (P <0.01). Conclusion: Overexpression of Cox7a2 can produce excessive ROS, and the levels of autophagy in TM4-supporting cells decrease, resulting in dysfunction of cell secretion. WZK serum can improve the secretion of TM4-supporting cells by regulating ROS and autophagy.