Caspase依赖的氧化应激对脓毒症肾小管上皮细胞损伤的作用及机制
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1.南京医科大学附属儿童医院;2.南京中医药大学附属中西医结合医院

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The mechanism of Caspase-dependent oxidative stress onSepsis renal tubular epithelial cell injury
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1.Children’s Hospital of Nanjing Medical University,Pediatric Research Center,Nanjing;2.AffiliatedSHospitalSofSIntegratedSTraditionalSChineseSandSWesternSMedicine,SNanjing

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    摘要:

    摘要:目的:观察不同活性氧(Reactive oxygen species,ROS)及氧化应激水平对脓毒症肾小管上皮细胞增殖、凋亡、周期等生物学效应影响,探索氧化应激水平和线粒体-Caspase途径是否能作为脓毒症肾小管上皮损伤的治疗靶点。方法:用终浓度为10 μg/ml的脂多糖(Lipopolysaccharide,LPS)处理人肾小管上皮细胞HK-2细胞12 hr。将细胞分为H2O2组、Caspase抑制剂组、Caspase抑制剂 + H2O2组、阴性对照组和空白对照组。Western Blot检测Cleaved-Caspase 3和Cleaved-多聚ADP核糖多聚酶(Poly ADP-ribose polymerase,PARP)蛋白表达,MTT检测细胞增殖,流式细胞术检测细胞ROS水平、细胞凋亡和细胞周期水平。结果:H2O2组肾小管上皮细胞Cleaved-Caspase 3和Cleaved-PARP蛋白表达较阴性对照组和空白对照组显著升高(p<0.05)。脓毒症肾小管上皮细胞中ROS水平高于空白对照组细胞(p<0.05),细胞增殖率较空白对照组降低(p<0.05),凋亡率升高(p<0.05),G1期细胞所占比例高于空白对照组(p<0.05)。H2O2组细胞中ROS水平高于阴性对照组(p<0.05),细胞增殖率较阴性对照组降低(p<0.05),凋亡率升高(p<0.05),处于细胞周期G1期的细胞比例升高(p<0.05)。Caspase抑制剂+ H2O2组细胞中ROS水平较H2O2组降低(p<0.05),但仍高于阴性对照组(p<0.05)。Caspase抑制剂+ H2O2组细胞增殖率较H2O2组升高(p<0.05),凋亡率降低(p<0.05),处于细胞周期G1期的细胞比例减少(p<0.05)。结论:脓毒症肾小管上皮细胞中存在异常升高的氧化应激反应。ROS能通过线粒体-Caspase凋亡途径,抑制脓毒症肾小管上皮细胞增殖、促进细胞凋亡和引起细胞周期G1期阻滞。抑制Caspase对ROS引起的脓毒症肾小管上皮细胞损伤有一定保护作用。

    Abstract:

    Abstract: Objective: To observe the effects of different Reactive oxygen species (ROS) and oxidative stress levels on cell proliferation, apoptosis, cycle in the renal tubular epithelial model of sepsis, and to explore whether oxidative stress and mitochondrial-Caspase pathway can be used to be the therapeutic target of sepsis renal tubular epithelial injury. Methods: The human renal tubular epithelial cells HK-2 cells were treated with Lipopolysaccharide (LPS) at a final concentration of 10 μg/ml for 12 hr to establish a sepsis cell model. The cells were divided into H2O2 group, Caspase inhibitor group, Caspase inhibitor + H2O2 group, negative control group and normal control group. Western Blot detects the expression of Cleaved-Caspase-3 and Cleaved- Poly ADP-ribose polymerase (PARP) protein, MTT detects cell proliferation, and flow cytometry detects cell ROS levels, apoptosis and cell cycle. Results: The expressions of Cleaved-Caspase 3 and Cleaved-PARP in H2O2 group were significantly higher than those in the negative control group and the blank control group (p<0.05). The level of ROS in sepsis renal tubular epithelial cells was higher than that of normal control cells (p<0.05), the cell proliferation rate was lower (p<0.05), the apoptosis rate was higher, and the proportion of cells in G1 phase was higher than the normal control group (p<0.05). The level of ROS in the H2O2 group was higher than that in the negative control group (p<0.05), the cell proliferation rate was lower (p<0.05), the apoptosis rate was higher (p<0.05), and the proportion of cells in the G1 phase of the cell cycle was lower (p<0.05) than that of the negative control group. The level of ROS in the cells of the Caspase inhibitor + H2O2 group was lower than that of the H2O2 group (p<0.05), but still higher than the negative control group (p<0.05). The cell proliferation rate of the Caspase inhibitor + H2O2 group was higher (p<0.05), the apoptotic rate was lower (p<0.05), and the proportion of cells in the G1 phase of the cell cycle was lower (p<0.05) than that of the H2O2 group. Conclusion: The oxidative stress level of renal tubular epithelial cells in sepsis is abnormally increased. Excessive ROS can inhibit the proliferation of septic renal tubular epithelial cells, promote cell apoptosis and cause cell cycle G1 block through the mitochondrial-Caspase pathway. Inhibition of Caspase has a certain protective effect on renal tubular epithelial cell injury in sepsis caused by ROS.

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  • 收稿日期:2021-02-10
  • 最后修改日期:2021-11-12
  • 录用日期:2022-01-24
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