山奈酚通过激活PI3K/AKT/mTOR信号通路改善视网膜氧化应激损伤

Kaempferol ameliorates oxidative stress injury in the retina through activating the PI3K/AKT/mTOR signaling pathway

目的探讨山奈酚减轻视网膜氧化应激损伤的作用机制。方法H_(2)O_(2)处理ARPE-19细胞,构建体外视网膜氧化应激损伤模型,CCK-8评估细胞活力,流式细胞仪检测细胞凋亡。25只雄性SD大鼠随机分为5组:对照组、模型组及山奈酚低剂量(12.5 mg/kg)组、中剂量(25 mg/kg)组和高剂量(50 mg/kg)组;对照组动物正常饲养,模型组和治疗组动物给予急性视网膜缺血/再灌注损伤处理,构建视网膜氧化应激损伤模型。检测超氧化物歧化酶(SOD)和丙二醛(MDA)的细胞内活性,H-E染色观察视网膜结构完整性,酶联免疫吸附试验(ELISA)检测炎性细胞因子的表达,Western blot评价凋亡自噬的表达和磷脂酰肌醇3激酶(PI3K)/丝氨酸/苏氨酸激酶(AKT)/雷帕霉素机制靶蛋白(mTOR)信号通路的影响。结果体外细胞实验表明,与对照组相比,山奈酚能显著提高ARPE-19氧化应激损伤细胞的活力和减少细胞凋亡(P<0.05)。体内实验表明,与模型组相比,山奈酚能减轻视网膜氧化应激损伤;显著降低MDA表达和升高SOD表达(P<0.05);显著降低血管内皮生长因子(VEGF)和γ-干扰素(IFN-γ)的分泌,增加白细胞介素-10(IL-10)的分泌(P<0.05);同时能显著增强B细胞淋巴瘤/白血病-2(BCL-2)、微管相关蛋白1A/1B-轻链3(LC3)、PI3K、磷酸化mTOR(p-mTOR)和磷酸化AKT(p-AKT)蛋白表达,抑制Bcl-2同源结构域蛋白1抗体(Beclin1)表达(P<0.05)。结论山奈酚通过激活PI3K/AKT/mTOR信号通路减轻视网膜氧化应激损伤。

Objective To investigate the mechanism of kaempferol in alleviating retinal oxidative stress injury.Methods ARPE-19 cells were treated with H_(2)O_(2)to establish an in vitro model of retinal oxidative stress injury.The cell viability was assessed by CCK-8 assay.The apoptosis was detected by flow cytometry.Twenty-five male SD rats were randomly divided into five groups:a control group,a model group,a low-dose(12.5 mg/kg)kaempferol group,a medium-dose(25 mg/kg)kaempferol group and a high-dose(50 mg/kg)kaempferol group.Rats in the control group were kept normally,while those in the model and treatment groups were subject to acute retinal ischemia/reperfusion injury to construct a model of retinal oxidative stress injury.The intracellular activities of superoxide dismutase(SOD)and malondialdehyde(MDA)were detected,the structural integrity of the retina was observed by H-E staining.The expression of inflammatory cytokines was detected by enzyme-linked immunosorbent assay(ELISA).The expression of apoptotic autophagy and the effect of PI3K/AKT/mTOR signaling pathway were evaluated by Western blot.Results In vitro cellular experiments showed that kaempferol significantly increased the viability and decreased the apoptosis of ARPE-19 cells induced by oxidative stress,compared with the control group(P<0.05).In vivo experiments showed that compared with the model group,kaempferol attenuated retinal oxidative stress injury;significantly decreased MDA expression and elevated SOD expression(P<0.05);significantly decreased the secretion of vascular endothelial growth factor(VEGF)andγ-interferon(IFN-γ)and increased the secretion of interleukin-10(IL-10)(P<0.05);and significantly enhanced the protein expression of B cell lymphoma/leukaemia-2(BCL-2),microtubule-associated protein 1A/1B-light chain 3(LC3),phosphatidylinositol 3 kinase(PI3K),phosphorylated target of rapamycin mechanism(p-mTOR)and phosphorylated serine/threonine kinase(p-AKT)and inhibited the protein expression of the antibody to the Bcl-2 cognate structural domain protein 1(Beclin1)(P<0.05).Conclusions Kaempferol attenuates retinal oxidative stress injury by activating the PI3K/AKT/mTOR signaling pathway.