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摘要：
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目的:探讨施他宁对急性胰腺炎腺泡细胞损伤的影响及其可能作用机制。方法:雨蛙素(Cerulein)诱导大鼠胰腺腺泡细胞AR42J建立急性胰腺炎模型。AR42J细胞随机分为NC组(空白对照)、Cerulein组(Cerulein处理AR42J细胞)、低剂量组(20 μg·ml-1施他宁处理AR42J细胞)、中剂量组(40 μg·ml-1施他宁处理AR42J细胞)、高剂量组(80 μg·ml-1施他宁处理AR42J细胞)、Cerulein+anti-miR-NC组(anti-miR-NC转染至AR42J细胞后,用Cerulein处理AR42J细胞)、Cerulein+anti-miR-126-5p组(anti-miR-126-5p转染至AR42J细胞后,用Cerulein处理AR42J细胞)、miR-NC+高剂量组(miR-NC转染至AR42J细胞后,用Cerulein和80 μg·ml-1施他宁处理AR42J细胞)、miR-126-5p+高剂量组(miR-126-5p mimics转染至AR42J细胞后,用Cerulein和80 μg·ml-1施他宁处理AR42J细胞);流式细胞术检测细胞凋亡率;试剂盒检测丙二醛(MDA)水平及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性;酶联免疫吸附试验(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)的水平;实时荧光定量聚合酶链反应(qRT-PCR)检测miR-126-5p的表达量;Western blot实验检测B淋巴细胞瘤-2相关蛋白(Bax)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-caspase-3)、B淋巴细胞瘤-2(Bcl-2)蛋白表达量。结果:施他宁处理后,Cerulein诱导的AR42J细胞凋亡率及Bax、Cleaved-caspase-3、磷酸化p65(p-p65)、磷酸化核因子κB抑制蛋白α(p-IκBα)蛋白水平降低(P<0.05),miR-126-5p的表达量及MDA、TNF-α、IL-6的水平降低(P<0.05),Bcl-2的表达量升高(P<0.05),SOD、CAT活性升高(P<0.05);与Cerulein+anti-miR-NC组比较,Cerulein+anti-miR-126-5p组细胞凋亡率及Bax、Cleaved-caspase-3蛋白水平降低(P<0.05),MDA、TNF-α、IL-6水平降低(P<0.05),SOD、CAT活性升高(P<0.05);与miR-NC+高剂量组比较,miR-126-5p+高剂量组细胞凋亡率和Bax、Cleaved-caspase-3蛋白水平升高(P<0.05),MDA、TNF-α、IL-6水平升高(P<0.05),Bcl-2蛋白水平及SOD、CAT活性降低(P<0.05)。结论:施他宁可通过下调miR-126-5p及抑制NF-κB信号通路活化而抑制Cerulein诱导的胰腺腺泡细胞凋亡、氧化应激及炎症反应从而减轻细胞损伤。 |
Objective:To explore the effect of stilamin on acinar cell injury in acute pancreatitis and its possible mechanism.Methods:Cerulein induces rat pancreatic acinar cells AR42J to establish an acute pancreatitis model.AR42J cells were randomly divided into NC group (blank control),Cerulein group (Cerulein-treated AR42J cells),low-dose group (20 μg·ml-1 stilamin-treated cells),medium-dose group (40 μg·ml-1 stilamin-treated cells),high-dose group (80 μg·ml-1 stilamin-treated cells),Cerulein+anti-miR-NC group (after anti-miR-NC was transfected into AR42J cells,AR42J cells were treated with Cerulein),Cerulein+anti-miR-126-5p group (after anti-miR-126-5p was transfected into AR42J cells,AR42J cells were treated with Cerulein),miR-NC+high-dose group (after miR-NC was transfected into AR42J cells,cells were treated with Cerulein and 80 μg·ml-1 stilamin),miR-126-5p+high-dose group (after miR-126-5p mimics were transfected into AR42J cells,cells were treated with Cerulein and 80 μg·ml-1 stilamin).Flow cytometry was used to detect the apoptosis rate.The levels of malondialedhyde (MDA),superoxide dismutase (SOD),and catalase (CAT) were tested according to the kit instructions.ELISA method was used to detect the levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6).qRT-PCR experiment was used to detect the expression of miR-126-5p.Western blot experiment was used to detect the protein expression of Bcl-2-associated X protein (Bax),cleaved-caspase-3 and B-cell lymphoma-2(Bcl-2).Results:After stilamin treatment,Cerulein-induced apoptosis rate and protein levels of Bax,cleaved-caspase-3,phosphorylated p65(p-p65) and phosphorylated nuclear factor κB inhibitory protein α(p-IκBα) in AR42J cells were decreased (P<0.05),the expression of miR-126-5p and the levels of MDA,TNF-α,and IL-6 were decreased (P<0.05),while the expression of Bcl-2 was increased (P<0.05),and the activities of SOD,CAT were increased (P<0.05).Compared with Cerulein+anti-miR-NC group,cell apoptosis rate,and the protein levels of Bax,cleaved-caspase-3 in Cerulein+anti-miR-126-5p group were decreased (P<0.05),and the levels of MDA,TNF-α,and IL-6 were decreased (P<0.05),while the activities of SOD,CAT were increased (P<0.05).Compared with the miR-NC+high-dose group,cell apoptosis rate,and the protein levels of Bax,cleaved-caspase-3 in the miR-126-5p+high-dose group were increased (P<0.05),and the levels of MDA,TNF-α and IL-6 were increased (P<0.05),while the expression of Bcl-2 and the activities of SOD,CAT were decreased (P<0.05).Conclusion:Stilamin can inhibit Cerulein-induced pancreatic acinar cell apoptosis,oxidative stress and inflammation by down-regulating miR-126-5p and inhibiting the activation of NF-κB signaling pathway,thereby reducing cell damage. |
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