美洲大蠊小肽预处理对H_(2)O_(2)诱导人卵巢颗粒细胞氧化应激及凋亡的抑制作用

Inhibition of SPPA pretreatment on H_(2)O_(2)-induced oxidative stress and apoptosis in human ovarian granulosa cells

目的探讨不同浓度美洲大蠊小肽预处理对H_(2)O_(2)诱导人卵巢颗粒细胞(KGN细胞)氧化应激及凋亡的影响。方法取对数生长期的KGN细胞,分为空白组、H_(2)O_(2)组和美洲大蠊小肽50、100、150组。各组均常规培养24 h,空白组不予特殊处理,H_(2)O_(2)组加入250μmol/L H_(2)O_(2)作用6 h;美洲大蠊小肽50、100、150组分别加入50、100、150μg/mL美洲大蠊小肽进行预处理,然后加入250μmol/L H_(2)O_(2)作用6 h。采用CCK-8法检测空白组、H_(2)O_(2)组和美洲大蠊小肽50、100、150组分别预处理6、12、24 h的细胞存活率,并比较各组(美洲大蠊小肽50、100、150组均预处理12 h)细胞活性氧(ROS)、一氧化氮(NO)、丙二醛(MDA)表达。结果H_(2)O_(2)组细胞存活率低于空白组(P<0.05)。预处理6 h的美洲大蠊小肽50、100、150组细胞存活率与H_(2)O_(2)组比较差异均无统计学意义(P均>0.05),预处理12、24 h的美洲大蠊小肽50、100、150组细胞存活率均高于H_(2)O_(2)组(P均<0.05)。与预处理6 h比较,美洲大蠊小肽50、100、150组预处理12、24 h的细胞存活率均升高(P均<0.05);预处理12、24 h的美洲大蠊小肽50、100、150组细胞存活率比较差异均无统计学意义(P均>0.05)。与空白组比较,H_(2)O_(2)组细胞MDA、NO、ROS表达均升高(P均<0.05);与H_(2)O_(2)组比较,美洲大蠊小肽50、100、150组细胞MDA、NO、ROS表达均降低(P均<0.05),美洲大蠊小肽50、100、150组间比较差异均无统计学意义(P均>0.05)。结论美洲大蠊小肽预处理能够减轻H_(2)O_(2)诱导的KGN细胞氧化应激及凋亡,以50μg/mL作用12 h为最佳预处理条件。

Objective To investigate the effects of pretreatment with different concentrations of small peptides from Periplaneta americana(SPPA)on H_(2)O_(2)-induced oxidative stress and apoptosis in human ovarian granulosa cells(KGN cells).Methods KGN cells in the logarithmic growth phase were divided into the blank group,H_(2)O_(2) group and 50,100,and 150 SPPA groups.Cells in the five groups were cultured for 24 h.Cells in the blank group were not treated,and 250μmol/L H_(2)O_(2) was added to the H_(2)O_(2) group for 6 h.Cells in the 50,100,and 150 SPPA groups were pretreated with 50,100,and 150μg/mL SPPA,respectively,and then were treated with 250μmol/L H_(2)O_(2) for 6 h.CCK-8 was used to detect the cell survival rates in the blank group,H_(2)O_(2) group and 50,100,and 150 SPPA groups pretreated for 6,12 and 24 h,respectively,and we compared the expression levels of reactive oxygen species(ROS),nitric oxide(NO)and malondialdehyde(MDA)in the cells of all groups(50,100,and 150 SPPA groups were all pretreated for 12 h).Results The cell survival rate of the H_(2)O_(2) group was lower than that of the blank group(P<0.05).The differences in cell survival rates between the 50,100 and 150 SPPA groups pretreated for 6 h and the H_(2)O_(2) group were not statistically significant(all P>0.05),and the cell survival rates of the 50,100 and 150 SPPA groups pretreated for 12 and 24 h were higher than pre‐treatment were higher than those at 6 h of pretreatment(all P<0.05);no significant differences were found in the survival rates between the 50,100 and 150 SPPA groups at 12 and 24 h of pretreatment(all P>0.05).Compared with the blank group,the expression levels of MDA,NO and ROS in cells of the H_(2)O_(2) group increased(all P<0.05);compared with the H_(2)O_(2) group,the expression levels of MDA,NO and ROS in cells of the 50,100 and 150 SPPA groups decreased(all P<0.05),and no significant differences were found between the 50,100 and 150 SPPA groups(all P>0.05).Conclusion SPPA pretreatment was able to reduce H_(2)O_(2)-induced oxidative stress and apoptosis in KGN cells,and 50μg/mL for 12 h was the optimal condition.