金钱草总黄酮对草酸钙结晶肾损伤的作用机制

摘要

目的:探讨金钱草总黄酮对一水合草酸钙(COM)诱导肾小管上皮细胞凋亡所致肾损伤的影响及其机制.方法:应用不同浓度COM处理肾小管上皮细胞(HK-2)建立体外泌尿系结石细胞模型,将肾小管上皮细胞分为对照组、金钱草总黄酮处理组(TF组)、P38丝裂原活化蛋白激酶(p38/MAPK)抑制组(SB组)、COM处理组(COM组)、COM和金钱草总黄酮处理组(CTF组)、COM和p38/MAPK抑制组(CSB组)6组,WST-1法检测细胞存活率,Western blot检测人肾损伤分子(KIM-1)、p-p38、p38、自噬相关蛋白LC3-Ⅱ的表达,流式细胞术检测不同处理组的细胞凋亡数.结果:COM浓度为0.5、1、2、5mmol/L时,细胞存活率分别为(85.02±5.50)％、(70.27±3.78)％、(58.09±3.23)％、(42.56±4.13)％,较对照组(99.83±3.65)％降低(P＜0.05).在COM处理的肾小管上皮细胞中,随着时间的增加,KIM-1、p-p38及LC3-Ⅱ表达增加.COM可诱导HK-2细胞过度自噬和凋亡,CSB组细胞凋亡率较COM组降低[(7.91±0.70)％vs(29.90±1.07)％,P＜0.05].CSB组LC3-Ⅱ表达较COM组降低[(0.52±0.04)vs(1.17±0.04),P＜0.05].CTF组p-p38蛋白水平较COM组降低[(0.66±0.06) vs(1.01±0.06),P＜0.05].随着金钱草总黄酮浓度的增加,HK-2细胞存活率也逐渐增加.CTF组细胞凋亡率较COM组降低[(10.54±1.07)％vs(29.90±1.07)％,P＜0.05].CTF组LC3-Ⅱ表达较COM组降低[(0.82±0.10) vs(1.17±0.04),P＜0.05].结论:金钱草总黄酮可通过阻断p38/MAPK通路抑制肾小管上皮细胞的过度自噬和凋亡,减少肾小管上皮细胞的损伤,从而抑制泌尿系结石的形成.%Objective To discuss the effect of total flavonoids of Lysimachiae Herba (TFLH) on kidney injury induced by calcium oxalate monohydrate (COM).Methods Human kidney proximal tubular epithelial cell line (HK-2 cells) was treated with different concentration of COM to simulate urinary calculi cell model in vitro.Then,cells were divided into six groups,namely control group,TFLH treated group (TF group),p38/ mitogen-activated protein kinase (MAPK) inhibitor group (SB group),COM treated group (COM group),COM and TFLH treated group (CTF group) and COM and p38/MAPK inhibitor group (CSB group).WST-1 assay was used to measure cell viability.Kidney injury molecule-1 (KIM-1),p-p38,p38 and cell autophagy related proteins LC3-Ⅱ were tested by Western blotting.Flow cytometry was used to detect the number of apoptotic cells.Results With the increase of COM concentration (0.5,1,2 and 5 mmol/L),cell viability [(85.02 ± 5.50)％,(70.27 ± 3.78)％,(58.09 ± 3.23)％ and (42.56 ± 4.13)％] was significantly decreased compared with control group (99.83 ± 3.65)％ (P ＜ 0.05).In renal tubular epithelial cells treated with COM,the expression of KIM-1,p-p38 and LC3-II were increased with the increase of time.COM could induce excessive autophagy and apoptosis in HK-2 cells,and the apoptosis rate in CSB group was lower than that in COM group [(7.91 ± 0.70)％ vs (29.90 ± 1.07)％,P ＜ 0.05].LC3-II expression in CSB group was lower than that in COM group [(0.52 ± 0.04) vs (1.17 ± 0.04),P ＜ 0.05].The level of p-p38 decreased in CTF group compared with COM group [(0.66 ± 0.06) vs (1.01 ± 0.06),P ＜ 0.05].With the increase of TFLH concentration,the survival rate of HK-2 cells increased gradually.The rate of apoptosis in CTF group was lower than that in COM group [(10.54 ± 1.07)％ vs (29.90 ± 1.07)％,P ＜ 0.05].The expression of LC3-II in CTF group was lower than that in COM group [(0.82 ± 0.10) vs (1.17 ± 0.04),P ＜ 0.05].Conclusion TFLH could inhibit excessive autophagy and apoptosis of renal tubular epithelial cells treated with COM by blocking the p38/MAPK pathway,reduce the injury of renal tubular epithelial cells,thus inhibit the formation of urinary calculi.