藻蓝蛋白对小鼠精原细胞氧化损伤的保护作用及其机制

PROTECTIVE EFFECT OF C-PHYCOCYANIN AGAINST OXIDATIVE INJURY IN MOUSE SPERMATOGONIAL CELLS AND RELATED MECHANISM

目的探讨藻蓝蛋白(C-PC)对H_(2)O_(2)诱导的小鼠精原细胞(GC-1 spg)氧化损伤的保护作用及其机制。方法制备H_(2)O_(2)诱导的GC-1 spg氧化损伤模型。实验分为对照组、模型组及C-PC低、中、高3个剂量预处理组。C-PC各剂量组用不同浓度C-PC预处理24 h后,除对照组以外,其余各组细胞培养液中均加入H_(2)O_(2),37℃孵育24 h。采用CCK-8方法检测各组细胞的增殖活性;采用二氯荧光黄双乙酸盐(DCFH-DA)染色检测各组细胞产生活性氧(ROS)情况;采用流式细胞术检测各组细胞的坏死情况;采用Western blot方法测定各组细胞中受体相互作用蛋白-1(RIP-1)、RIP-3的表达情况。结果与对照组比较,模型组GC-1 spg增殖活性下降(F=76.86,q=23.01,P<0.05),细胞内ROS水平显著升高,坏死细胞增多(F=206.9,q=60.07,P<0.05);与模型组比较,C-PC低、中、高剂量组细胞增殖活性显著增加(q=4.45~10.83,P<0.05),细胞内ROS水平显著降低,坏死细胞减少(q=9.09~32.41,P<0.05)。Western blot分析显示,与模型组比较,C-PC预处理可下调细胞中RIP-1、RIP-3表达(F=541.2、297.5,q=27.97~62.98,P<0.05)。结论C-PC对H_(2)O_(2)诱导的GC-1 spg氧化损伤具有明显的保护作用,可能与C-PC提高GC-1 spg的抗氧化能力,抑制GC-1 spg中RIP-1、RIP-3蛋白表达,降低细胞坏死有关。

Objective To investigate the protective effect of C-phycocyanin(C-PC)against H_(2)O_(2)-induced oxidative injury in mouse GC-1 spg spermatogonial cells and related mechanism.Methods GC-1 spg cells were used to establish a model of H_(2)O_(2)-induced oxidative injury.The cells were divided into control group,model group,and low-,middle-,and high-dose C-PC pretreatment groups.The cells in the low-,middle-,and high-dose C-PC pretreatment groups were pretreated with different concentrations of C-PC for 24 h,and then H_(2)O_(2) was added to the cell culture medium of all groups except the control group and was incubated at 37℃for 24 h.CCK-8 assay was used to measure cell proliferation viability;DCFH-DA staining was used to measure the production of reactive oxygen species(ROS)in cells;flow cytometry was used to measure cell necrosis;Western blot was used to measure the protein expression of receptor-interacting protein-1(RIP-1)and receptor-interacting protein-3(RIP-3)in cells.Results\Compared with the control group,the model group had a significant reduction in the proliferative activity of GC-1 spg cells(F=76.86,q=23.01,P<0.05)and significant increases in the level of ROS and the number of necrotic cells(F=206.9,q=60.07,P<0.05).Compared with the model group,the low-,middle-,and high-dose C-PC pretreatment groups had a significant increase in the proliferative activity of cells(q=4.45-10.83,P<0.05)and significant reductions in the level of ROS and the number of necrotic cells(q=9.09-32.41,P<0.05).Western blot showed that compared with the model group,the C-PC pretreatment groups had significantly downregulated protein expression of RIP-1 and RIP-3(F=541.2,297.5,q=27.97-62.98,P<0.05).Conclusion C-PC exerts a marked protective effect against H_(2)O_(2)-induced oxidative injury in GC-1 spg cells,possibly by improving the antioxidant capacity of GC-1 spg cells,inhibiting the protein expression of RIP-1 and RIP-3 in cells,and reducing cell necrosis.