厄贝沙坦对链脲佐菌素和过氧化氢诱导的β细胞氧化损伤保护作用

Protective effects of Irbesartan against streptozotocin-and hydrogen peroxide-induced oxidative damage inβcells in vitro

目的 探讨厄贝沙坦对链脲佐菌素(STZ)和过氧化氢(H_(2)O_(2))诱导的β细胞损伤影响。方法 (1)将NIT-1细胞分为对照组及1、2、5 mmol/L STZ和300、500μmol/L H_(2)O_(2)组,处理30 min后,采用Hoechst 33342法检测各组细胞凋亡形态变化,流式细胞术检测细胞凋亡率,RT-qPCR检测Caspase3 mRNA水平。(2)将NIT-1细胞分为STZ及0.001、0.01、0.1 mmol/L厄贝沙坦组,作用24、48和72 h。(3)将NIT-1细胞分为H_(2)O_(2)及0.001、0.01、0.1 mmol/L厄贝沙坦组,作用24、48和72 h。采用流式细胞术检测细胞凋亡率和活性氧(ROS)含量,RT-qPCR检测血管紧张素II型1受体(AT1R)mRNA表达。结果 Hoechst 33342染色显示,5 mmol/L STZ组相较于1、2 mmol/L STZ组,500μmol/L H_(2)O_(2)组相较于300μmol/L H_(2)O_(2)组的细胞荧光染色较强,细胞碎片较多。5 mmol/L STZ组凋亡率为70.42%高于1、2 mmol/L STZ组的凋亡率(8.75%、47.2%)(均P<0.05)。500μmol/L H_(2)O_(2)组凋亡率为70.45%高于300μmol/L H_(2)O_(2)组的58.13%(P<0.05)。2 mmol/L STZ和300μmol/L H_(2)O_(2)诱导的NIT-1细胞中Caspase3 mRNA表达分别上调2.63、6.25倍(均P<0.05)。在STZ和H_(2)O_(2)模型组中,0.001、0.01、0.1 mmol/L厄贝沙坦组的细胞凋亡率、ROS水平和AT1R mRNA相对表达量均低于STZ组或H_(2)O_(2)组,差异均有统计学意义(均P<0.05)。同一浓度厄贝沙坦组中,72 h的细胞凋亡率和ROS水平均低于48 h和24 h,差异均有统计学意义(均P<0.05)。结论 2 mmol/L STZ和300μmol/L H_(2)O_(2)是诱导NIT-1细胞氧化急性损伤的适宜条件。厄贝沙坦可能通过抑制ROS生成和降低凋亡率来保护β细胞免受STZ和H_(2)O_(2)诱导的氧化损伤,其分子机制可能与AT1R mRNA水平的降低有关。厄贝沙坦有潜力成为预防和治疗2型糖尿病的新型药物。

Objective To establish a simple acute oxidative damage model and investigate the effect of Irbesartan on streptozotocin(STZ)and hydrogen peroxide(H_(2)O_(2))inducedβ-cell damage.Methods①NIT-1 cells were divided into control group,1 mmol/L STZ,2 mmol/L STZ,5 mmol/L STZ,300μmol/L H_(2)O_(2),500μmol/L H_(2)O_(2) groups.After treatment for 30 min,the morphological changes of apoptosis in each group were detected by Hoechst 33342 method,and the apoptosis rate was detected by flow cytometry.mRNA levels of Caspase3 were detected by RT-qPCR.②NIT-1 cells were divided into STZ,0.001 mmol/L Irbesartan,0.01 mmol/L Irbesartan,0.1 mmol/L Irbesartan group and cultured for 24,48 and 72 h.③NIT-1 cells were divided into H_(2)O_(2),0.001 mmol/L Irbesartan,0.01 mmol/L Irbesartan,0.1 mmol/L Irbesartan group,and cultured for 24,48 and 72 h.The apoptosis rate and the content of reactive oxygen species(ROS)were detected by flow cytometry.mRNA expression of angiotensin type Ⅱ 1 receptor(AT1R)was detected by RT-qPCR.Results Hoechst 33342 staining showed that the cell fluorescence staining of 5 mmol/LSTZ group was stronger than that of 1 and 2 mmol/L STZ groups,and that of 500μmol/L H_(2)O_(2) group was stronger than that of 300μmol/L H_(2)O_(2) group.The apoptosis rate of 5 mmol/L STZ group was 70.42%,which was higher than that of 1 and 2 mmol/L STZ groups 8.75%and 47.2%(P<0.05).The apoptosis rate of 500μmol/LH_(2)O_(2) group was 70.45%,which was higher than that of 300μmol/LH_(2)O_(2) group,which was 58.13%(P<0.05).The mRNA expression of Caspase3 in NIT-1 cells induced by 2 mmol/L STZ and 300μmol/L H_(2)O_(2) was up-regulated by 2.63 and 6.25 times,respectively(both P<0.05).In STZ and H_(2)O_(2) model groups,the apoptosis rate,ROS level and AT1R mRNA relative expression level in Irbesartan group were lower than those in STZ or H_(2)O_(2) group 0.001,0.01 and 0.1 mmol/L(P<0.05).In Irbesartan group with the same concentration,the apoptosis rate and ROS level at 72 h were lower than those at 48 h and 24 h,with statistical significance(P<0.05 for both).Conclusion 2 mmol/L STZ and 300μmol/L H_(2)O_(2) are suitable conditions for inducing acute oxidative injury of NIT-1 cells.Irbesartan may protectβcells from STZ and H_(2)O_(2)-induced oxidative damage by inhibiting ROS production and reducing apoptosis rate,and its molecular mechanism may be related to the decreased AT1R mRNA level.Irbesartan has the potential to be a novel drug for the prevention and treatment of type 2 diabetes.