钌络合物通过诱发高尔基体应激在Walker-256荷瘤大鼠中发挥抗肿瘤作用的机制

摘要 目的：探讨钌络合物通过诱发高尔基体应激在Walker-256荷瘤大鼠中发挥抗肿瘤作用的机制。方法：以30只雄性Wistar大鼠为研究对象，通过Walker-256细胞右骨盆肢体皮下注射建立荷瘤大鼠模型，然后根据实验目的将大鼠分为3组，对照组（正常大鼠，PBS干预），肿瘤模型组（荷瘤模型大鼠，PBS干预）和钌络合物组荷瘤模型大鼠，管饲法给予5 mg / kg钌络合物溶液（由含2% Tween的PBS溶解）]，各10只。通过测厚仪和电子秤分别计算大鼠肿瘤体积及重量；酶联免疫吸附试验试剂盒检测大鼠刚脏组织匀浆中氧化应激水平；蛋白印迹和荧光探针DCFH-DA试剂盒分析LC3 II/I表达和ROS活性；蛋白印迹分析高尔基应激相关蛋白GOLPH3、GRASP65的表达；实时定量PCR分析Bax、Bcl-2和Caspase-3的mRNA表达。结果：钌络合物组较肿瘤模型组肿瘤重量降低（P<0.05），肿瘤模型组较对照组体重增加降低（P<0.05），钌络合物组较肿瘤模型组体重增加（P<0.05）。肿瘤模型组较对照组SOD活性和LPO升高（P<0.05），CAT、GST和GSH活性降低（P<0.05），钌络合物组较肿瘤模型组LPO降低（P<0.05），CAT、GST和GSH活性升高（P<0.05）。肿瘤模型组较对照组LC3 II/I蛋白表达和ROS活性升高（P<0.05），钌络合物组较肿瘤模型组LC3 II/I蛋白表达和ROS活性降低（P<0.05）。肿瘤模型组较对照组GOLPH3、GRASP65的蛋白表达升高（P<0.05），钌络合物组较肿瘤模型组GOLPH3、GRASP65的蛋白表达降低（P<0.05）。肿瘤模型组较对照组Bax和Caspase-3的mRNA表达升高（P<0.05），Bcl-2 mRNA表达降低（P<0.05），钌络合物组较肿瘤模型组Bax和Caspase-3的mRNA表达降低，Bcl-2 mRNA表达升高（P<0.05）。结论：钌络合物通过调节高尔基应激反应，削弱氧化磷酸化从而促进Walker-256细胞死亡发挥抗肿瘤活性。

The Mechanism of Ruthenium Complex Exerting Anti-tumor Effect in Walker-256 Tumor-bearing Rats by Inducing Golgi Stress

ABSTRACT Objective: To investigate the mechanism of ruthenium complexes exerting anti-tumor effects in Walker-256 tumor-bearing rats by inducing Golgi stress. Methods: Thirty male Wistar rats were used as research objects. Walker-256 cells were injected subcutaneously into the right pelvic limb to establish a tumor-bearing rat model, then divided into 3 groups according to the experimental purpose, the control group (normal rats, PBS intervention), tumor model group (tumor-bearing model rats, PBS intervention) and ruthenium complex group tumor-bearing model rats, given 5 mg/kg ruthenium complex solution by gavage (dissolved in PBS containing 2% Tween)], 10 each. The volume and weight of the rat tumor were calculated by the thickness gauge and the electronic scale; ELISA kit was used to detect the level of oxidative stress in rat visceral tissue homogenate; Western blot and fluorescent probe DCFH-DA kit were used to analyze LC3 II/I expression and ROS activity; Western blotting was used to analyze the expression of Golgi stress-related proteins GOLPH3 and GRASP65; RT-PCR was used to analyze the mRNA expression of Bax, Bcl-2 and Caspase-3 in real time. Results: The weight of the tumor in the ruthenium complex group was lower than that in the tumor model group (P<0.05), the weight of the tumor model group was lower than that in the control group (P<0.05), and the weight of the ruthenium complex group was higher than the tumor model group (P<0.05 ). Compared with the control group, the tumor model group had higher SOD activity and LPO activity (P<0.05), CAT, GST and GSH activity decreased (P<0.05), the ruthenium complex group had lower LPO than the tumor model group (P<0.05), CAT, The activities of GST and GSH increased (P<0.05). The tumor model group had higher LC3 II/I protein expression and ROS activity than the control group (P<0.05), and the ruthenium complex group had lower LC3 II/I protein expression and ROS activity than the tumor model group (P<0.05). The protein expression of GOLPH3 and GRASP65 in the tumor model group was higher than that in the control group (P<0.05), and the protein expression of GOLPH3 and GRASP65 in the ruthenium complex group was lower than that in the tumor model group (P<0.05). The mRNA expression of Bax and Caspase-3 in the tumor model group was higher than that in the control group (P<0.05), and the expression of Bcl-2 mRNA was lower (P<0.05). The ruthenium complex group was compared with the mRNA expression of Bax and Caspase-3 in the tumor model group. The expression decreased, and the expression of Bcl-2 mRNA increased (P<0.05). Conclusion: Ruthenium complexes can regulate the Golgi stress response and weaken oxidative phosphorylation to promote Walker-256 cell death to exert anti-tumor activity.