Effect and Mechanism of Probucol on Oxygen-glucose Deprivation/Reoxygenation Injury in Renal Tubular Epithelial Cells

doi:10.12114/j.issn.1007-9572.2019.00.237

Abstract

Abstract: Background　The main pathophysiological basis of renal ischemia/reperfusion injury is lipid peroxidation injury of renal tubular epithelial cells，which directly affects the formation of urine.Probucol is a hypolipidemic drug with strong antioxidant effect. It can reduce the level of lipid peroxidation by increasing the activity of antioxidant enzymes in cells, and has better tissue or cell protective effect. However, there is no report about the effect of probucol on the damage of HK-2 human renal proximal tubule cells（RPTC）induced by oxygen-glucose deprivation/reoxygenation（OGD/R）.Objective　To explore effect and potential mechanism of probucol on OGD/R in HK-2 RPTC，giving new ideas for the new use of old drugs.Methods　From December 2017 to September 2018，HK-2 RPTC were conventionally cultured to adhere on the culture plate completely and then divided into normal control group，OGD/R group，OGD/R+25 μmol/L probucol group，OGD/R+50 μmol/L probucol group，and OGD/R+100 μmol/L probucol group，respectively.The normal control group received no intervention.OGD/R models were established in other four groups.After successfully modeling，OGD/R group were given no intervention，OGD/R+25 μmol/L probucol group，OGD/R+50 μmol/L probucol group，and OGD/R+100 μmol/L probucol group were treated with 25，50 and 100 μmol/L probucol，respectively.At 12，24 and 48 hours after treatment，the proliferation activities of cells in each group were calculated to determine the optimal time and concentration of probucol for subsequent experiments.HK-2 cells were cultured to adhere on the culture plate completely and then divided into normal control group（no treatment），OGD/R group（establishing OGD/R model），probucol group（treatment with the optimal concentration of probucol），and OGD/R + probucol group（first establishing OGD/R model，then intervened with the optimal concentration of probucol）．At the optimal treatment time of probucol，the morphology of cells in each group was observed，and the activities of lactate dehydrogenase（LDH）and glutathione peroxidase（GPX），levels of malondialdehyde（MDA），glutathione（GSH）and GPX4 were detected.Results　The proliferative activities of cells in OGD/R group，OGD/R+25 μmol/L probucol group，OGD/R+50 μmol/L probucol group and OGD/R+100 μmol/L probucol group were lower than those in normal control group at 12，24 and 48 hours after intervention（P<0.05）．The cell proliferation activities of OGD/R+50 μmol/L probucol group and OGD/R+100 μmol/L probucol group at 24 and 48 hours after intervention were higher than those of OGD/R group and OGD/R+25 μmol/L probucol group（P<0.05）．The cell proliferation activities at 24 and 48 hours after intervention increased significantly compared with those at 12 hours after intervention in both OGD/R+50 μmol/L probucol group and OGD/R+100 μmol/L probucol group（P<0.05）．Because the principle of drug use is that under similar action，lower concentration and shorter action time are generally chosen，the following experiment was carried out with 50 μmol/L probucol for 24 hours.In OGD/R group，the number of suspended cells increased，the cells shrank，the volume decreased，the normal morphology lost，and the refractive index decreased.The cells in probucol group had normal morphological structure and complete adherence.In OGD/R+probucol group，the number of suspended cells decreased，but the refractive index of cells was comparatively good.The activities of LDH in OGD/R group and OGD/R+probucol group were higher than those in normal control group（P<0.05）．The activities of LDH in probucol group and OGD/R+probucol group were lower than those in OGD/R group（P<0.05）．The activity of LDH in OGD/R+probucol group was higher than that in probucol group（P<0.05）．The level of MDA in the control group was lower than that of OGD/R group and probucol group（P<0.05）．The level of MDA in OGD/R group was higher than that of probucol group and OGD/R+probucol group（P<0.05）．The level of MDA in OGD/R+probucol group was higher than that in probucol group（P<0.05）．The activity of GPX in OGD/R group was lower than that in normal control group（P<0.05）．The activities of GPX in both probucol group and OGD/R+probucol group were higher than those in OGD/R group（P<0.05）．The activity of GPX in OGD/R+probucol group was lower than that in probucol group（P<0.05）．The level of GSH in OGD/R group was lower than that in normal control group（P<0.05）．The level of GSH in OGD/R group was lower than that of both probucol group and OGD/R+probucol group（P<0.05）．The level of GPX4 in OGD/R group was lower than that in normal control group（P<0.05）．The levels of GPX4 in probucol group and OGD/R+probucol group were higher than those in OGD/R group（P<0.05）．Conclusion　Probucol can protect HK-2 RPTC from OGD/R-induced injury，possibly by activating GPX，especially by increasing the level of GPX4，directly or indirectly inhibiting the excessive production of lipid peroxides.

Key words: Anoxia, Reperfusion injury, Probucol, Oxygen-glucose deprivation/reoxygenation, HK-2, Molecular mechanisms of pharmacological action

摘要： 背景　肾缺血-再灌注损伤的主要病理生理基础是肾小管上皮细胞发生脂质过氧化损伤，而肾小管上皮细胞损伤直接影响尿液的形成。普罗布考是具有很强抗氧化作用的降血脂药，其可以通过提高细胞内抗氧化酶活性降低脂质过氧化水平，具有较好的组织或细胞保护作用。但目前尚未见关于普罗布考对氧糖剥夺/复氧（OGD/R）引起的人肾小管上皮细胞株（HK-2）损伤影响的报道。目的　探讨普罗布考对HK-2 OGD/R损伤的影响及其机制，为老药新用开辟新思路。方法　2017年12月—2018年9月，取HK-2，常规培养至完全贴壁后将其分为正常对照组、OGD/R组、OGD/R+25 μmol/L普罗布考组、OGD/R+50 μmol/L普罗布考组、OGD/R+100 μmol/L普罗布考组。正常对照组不予处理，OGD/R组、OGD/R+25 μmol/L普罗布考组、OGD/R+50 μmol/L普罗布考组、OGD/R+100 μmol/L普罗布考组进行OGD/R模型的建立，之后OGD/R组不予处理，OGD/R+25 μmol/L普罗布考组、OGD/R+50 μmol/L普罗布考组、OGD/R+100 μmol/L普罗布考组分别加入25、50、100 μmol/L普罗布考；分别于12、24、48 h时，计算各组细胞增殖活性，确定普罗布考最佳作用时间与浓度，用于后续实验。取HK-2，常规培养至完全贴壁后将其分为正常对照组（不予处理）、OGD/R组（建立OGD/R模型）、普罗布考组（给予最佳作用浓度的普罗布考）、OGD/R+普罗布考组（建立OGD/R模型后给予最佳作用浓度的普罗布考）；于普罗布考最佳作用时间，观察各组细胞形态，检测各组乳酸脱氢酶（LDH）活性、丙二醛（MDA）水平、谷胱甘肽过氧化物酶（GPX）活性、谷胱甘肽（GSH）水平、GPX4水平。结果　OGD/R组、OGD/R+25 μmol/L普罗布考组、OGD/R+50 μmol/L普罗布考组、OGD/R+100 μmol/L普罗布考组12、24、48 h时细胞增殖活性低于正常对照组（P<0.05）；OGD/R+50 μmol/L普罗布考组、OGD/R+100 μmol/L普罗布考组24、48 h时细胞增殖活性高于OGD/R组、OGD/R+25 μmol/L普罗布考组（P<0.05）。OGD/R+50 μmol/L普罗布考组、OGD/R+100 μmol/L普罗布考组24、48 h时细胞增殖活性高于本组12 h时（P<0.05）。由于用药原则为在相似的作用下，一般选择较低浓度及较短作用时间，因此采用50 μmol/L普罗布考作用24 h进行后续实验。OGD/R组悬浮细胞增多，细胞皱缩体积减小，失去正常形态，折光性减弱；普罗布考组细胞形态结构正常，贴壁完整；OGD/R+普罗布考组悬浮细胞减少，细胞折光性较好。OGD/R组、OGD/R+普罗布考组LDH活性高于正常对照组（P<0.05）；普罗布考组、OGD/R+普罗布考组LDH活性低于OGD/R组（P<0.05）；OGD/R+普罗布考组LDH活性高于普罗布考组（P<0.05）。OGD/R组、OGD/R+普罗布考组MDA水平高于正常对照组（P<0.05）；普罗布考组、OGD/R+普罗布考组MDA水平低于OGD/R组（P<0.05）；OGD/R+普罗布考组MDA水平高于普罗布考组（P<0.05）；OGD/R组GPX活性低于正常对照组（P<0.05）；普罗布考组、OGD/R+普罗布考组GPX活性高于OGD/R组（P<0.05）；OGD/R+普罗布考组GPX活性低于普罗布考组（P<0.05）。OGD/R组GSH水平低于正常对照组（P<0.05）；普罗布考组、OGD/R+普罗布考组GSH水平高于OGD/R组（P<0.05）。OGD/R组GPX4水平低于正常对照组（P<0.05）；普罗布考组、OGD/R+普罗布考组GPX4水平高于OGD/R组（P<0.05）。结论　普罗布考对OGD/R引起的HK-2损伤具有保护作用，可能机制是通过激活GPX，尤其是提高GPX4水平，直接或间接抑制脂质过氧化物的过度产生。

关键词: 缺氧, 再灌注损伤, 普罗布考, 氧糖剥夺/复氧, 人肾小管上皮细胞株, 药理作用分子作用机制

REN Guangwei,GONG Miao,LI Mingming, et al. Effect and Mechanism of Probucol on Oxygen-glucose Deprivation/Reoxygenation Injury in Renal Tubular Epithelial Cells　[J]. Chinese General Practice, 2019, 22(18): 2211-2216. DOI: 10.12114/j.issn.1007-9572.2019.00.237.
任广伟,龚淼,李明明等. 普罗布考对肾小管上皮细胞氧糖剥夺/复氧损伤的影响及其机制研究[J]. 中国全科医学, 2019, 22(18): 2211-2216. DOI: 10.12114/j.issn.1007-9572.2019.00.237.

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