豹皮樟总黄酮通过增强小鼠睾丸Leydig细胞缝隙连接功能减轻奥沙利铂的细胞毒作用

Total flavonoids of Litsea Coreana decreases the cytotoxicity of oxaliplatin in TM3 Leydig cells via enhancing the function of gap junction

目的:探讨豹皮樟总黄酮(TFLC)对体外培养的小鼠睾丸Leydig细胞系(TM3)缝隙连接(GJ)功能的作用,以及TFLC是否减轻化疗药奥沙利铂(OHP)的细胞毒作用。方法:荧光示踪法测定体外培养的TM3细胞之间荧光传递,反映GJ的功能;Western印迹法检测TFLC对连接蛋白43(Cx43)表达的影响;细胞免疫荧光法观察TFLC对TM3细胞胞膜Cx43蛋白表达的影响;MTT法检测OHP的细胞毒作用,以及TFLC对OHP细胞毒的影响。结果:在TM3细胞中GJ的功能随着TFLC浓度的增加而增强;Western印迹和免疫荧光实验结果显示TFLC可显著增强TM3细胞中Cx43蛋白和膜Cx43蛋白的表达;MTT结果表明,高密度接种细胞(生长融合,有GJ形成),20μg/ml TFLC增强细胞GJ功能,且减轻OHP的细胞毒作用[细胞存活率(89.5±1.8)%高于单用ONP组(80.0±1.5)%,(P<0.05)];低密度接种细胞(生长未融合,无GJ形成),TFLC对OHP的细胞毒作用没有影响(P>0.05)。结论:TFLC增强睾丸正常细胞TM3中Cx43的表达,并增强细胞间的GJ功能;TFLC减轻OHP在TM3细胞的毒性作用,并可能与增强GJ功能有关。

Objective ： To investigate the effects of total flavonoids of Litsea Coreana （TFLC） on the gap junction （GJ） intercellular communication in TM3 testicular Leydig cells and whether TFLC can reduce the cytotoxicity of oxaliplatin （OHP） in vitro. Methods ： We detected the effect of TFLC on the dye spread of the in vitro cultured TM3 ceils by parachute assay, observed changes in the expression of connexin 43 （ Cx43 ） total protein in the TFLC-treated TM3 cells by Western blot, and determined the effects of TFLC on the expression of Cx43 on the membrane of the TM3 cells by immunofluorescence assay and on the cytotoxicity of OHP by MTT assay. Results： TFLC obviously enhanced the GJ function with the increasing of the TFLC concentration in the TM3 cells. Western blot and immunofluorescence assay confirmed that TFLC significantly enhanced the expression of Cx43 total protein and Cx43 expression on the membrane of the TM3 cells. MTT assay showed that at a high cell density （ confluent with GJ formation ） , 20 μg/ml TFLC enhanced the GJ function of the TM3 cells and reduced the cytotoxicity of OHP （ P 〈 0.05 ）, while at a low density （ preconfluent with no GJ formation）, TFLC exhibited no effect on the cytotoxicity of OHP （ P 〉 0. 05 ）. Conclusion ： TFLC increases the Cx43 expression and GJ function in normal TM3 Leydig cells, and the enhancement of GJ function reduces the cytotoxicity of OHP.Natl J Androl , 2014, 20 （5）： 400-404