Repairing effect of apigenin-7-glucoside on oxalate-mediated HK-2 cell damage via the lRE1/ASK1/P38 MAPK pathway
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摘要: 目的 探讨自然界中常见的类黄酮糖苷化合物芹菜素-7-葡萄糖苷(A7DG)对草酸诱导的人近端肾小管上皮细胞HK-2损伤是否具有修复作用及其具体作用途径。方法 培养HK-2细胞到80%生长密度后分成3组并进行不同处理：①NC组：换用基础培养液继续培养；②OX组：换用含浓度为2 mmol/L草酸的基础培养液进行培养；③A7DG药物干预组：换用含2 mmol/L草酸和不同浓度的A7DG的基础培养液进行培养。经不同条件处理24 h后，分别对3组HK-2细胞进行细胞活力检测(CCK-8)、乳酸脱氢酶(LDH)及抗氧化能力相关指标还原型谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、丙二醛(MDA)检测，使用荧光显微镜检测线粒体膜电位(MMP)的变化，使用蛋白质印迹法(Western blot)检测细胞内NOX4、NOX2、p22、GRP78/BIP、IRE1、p-IRE1、p-ASK1、p38、p-p38、CHOP以及炎症相关因子IL-2、IL-6的蛋白表达情况。结果 CCK-8实验结果表明OX组细胞存活率较NC组明显降低，而A7DG减轻了OX对HK-2细胞的损伤。LDH、GSH、SOD及MDA检测结果表明A7DG可降低草酸预处理后HK-2细胞NADPH氧化酶的释放，通过干预草酸介导的活性氧(ROS)减轻草酸对HK-2细胞产生的损伤，并使异常的MMP恢复至正常。此外，蛋白质印迹显示A7DG干预下氧化应激特征蛋白如GRP78，CHOP以及IRE1途径关键蛋白p-IRE1、p-ASK、p-MAPK的蛋白表达显著降低，氧化应激损伤途径中IL-2、IL-6等炎性因子的表达也降低。结论 本研究结果证实A7DG在草酸诱导的HK-2细胞损伤中具有积极修复作用，且通过IRE1/ASK1/P38MAPK信号通路发挥积极地抗氧化作用，A7DG可为草酸介导的肾损伤和尿路结石的治疗提供新的参考。Abstract: Objective To investigate whether apigenin-7-glucoside (A7DG) has a repairing effect on oxalic acid-induced damage in HK-2 cells and its specific mechanism.Methods HK-2 cells were cultured to 80% growth density and divided into 3 groups for different treatments: ①NC group: cultured in basal medium; ②OX group: cultured in basal medium containing 2 mmol/L oxalic acid concentration; ③A7DG drug intervention group: cultured in basal medium containing 2 mmol/L oxalic acid and different concentrations of A7DG. After 24 hour of treatment, three groups of HK-2 cells were extracted for cell viability test (CCK-8), lactate dehydrogenase (LDH) test and related antioxidant capacity-related indicators such as reduced glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA) test. Mitochondrial membrane potential (MMP), the protein expression of NOX4, NOX2, p22, GRP78/BIP, IRE1, p-IRE1, p-ASK1, p38, p-p38, CHOP and inflammation-related factors IL-2 and IL-6 were also tested.Results The results of CCK-8 assay showed that the cell growth rate of OX group was significantly lower than that of NC group, and A7DG attenuated the damage of OX on HK-2 cells. The results of LDH, GSH, SOD and MDA assays showed that A7DG reduced the release of NADPH oxidase from HK-2 cells after oxalic acid pretreatment, attenuated the damage of oxalic acid on HK-2 cells by interfering with oxalic acid-mediated reactive oxygen species (ROS) damage and restored the abnormal mitochondrial membrane potential to normal. In addition, the results of WB test showed that the protein expression of oxidative stress signature proteins such as GRP78, CHOP and key proteins of the IRE1 pathway, p-IRE1, p-ASK and p-MAPK, as well as the expression of inflammatory factors such as IL-2 and IL-6 in the oxidative stress damage pathway were significantly reduced by A7DG intervention.Conclusion The results of this study confirmed that A7DG has a positive repairing effect on oxalic acid-induced HK-2 cell injury and exerts a positive antioxidant effect through the IRE1/ASK1/P38MAPK signaling pathway, which means it may provide a new reference for the treatment of oxalate-mediated renal injury and nephrolithiasis.
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Key words:
- apigenin-7-glucoside /
- oxalate /
- apoptosis /
- kidney stones /
- oxidative stress /
- reactive oxygen species
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