原花青素减轻TCP磨损颗粒诱导的骨细胞氧化损伤  

Effects of procyanidins on oxidative damage of osteocytes caused by TCP wear particles

作　　者：金晶晶 楼佳琪 王脉 陈晓斌 杨子键 黄君瑶 张云[1] JIN Jing-jing;LOU Jia-qi;WANG Mai;CHEN Xiao-bin;YANG Zi-jian;HUANG Jun-yao;ZHANG Yun(College of Medicine, Shaoxing University, Shaoxing 312000, China)

机构地区：[1]绍兴文理学院医学院

出　　处：《中国病理生理杂志》2019年第9期1676-1682,共7页Chinese Journal of Pathophysiology

基　　金：浙江省自然科学基金资助项目(No.LY17H060007);国家级大学生创新创业训练计划项目(No.201910349035);绍兴市大学生科技创新项目(No.SXSDC201809)

摘　　要：目的:研究原花青素(PC)对磷酸三钙(TCP)磨损颗粒诱导的骨细胞氧化损伤的影响,并探讨其可能的作用机制。方法:将TCP磨损颗粒(0.1 g/L)与小鼠长骨MLO-Y4骨细胞共孵育构建骨细胞体外损伤模型,实验分为4组:正常对照(control)组、TCP组、PC(10 μmol/L)组和PC(50 μmol/L)组。应用Calcein-AM染色和MTT等方法检测骨细胞活力;ELISA检测细胞培养上清液中牙本质基质蛋白1(DMP-1)、骨硬化蛋白(SOST)及白细胞介素1β(IL-1β)水平;流式细胞术定量分析骨细胞凋亡情况;化学比色法检测骨细胞中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,以及细胞培养上清液中乳酸脱氢酶(LDH)释放量;Western blot法检测骨细胞中NOD样受体蛋白3(NLRP3)、含CARD的凋亡相关斑点样蛋白(ASC)、cleaved caspase-1和IL-1β蛋白水平的变化。结果:与control组比较,TCP组MLO-Y4细胞的损伤、凋亡率及MDA含量显著增加( P <0.05),SOD活性显著降低( P <0.05),NLRP3、ASC、cleaved caspase-1和IL-1β蛋白水平显著上调,上清液中IL-1β和LDH的水平明显增加( P <0.05);与TCP组比较,PC组MLO-Y4细胞的损伤明显减轻,凋亡率显著降低( P <0.05),NLRP3、ASC、cleaved caspase-1和IL-1β的蛋白水平显著下调( P <0.05),IL-1β和LDH水平明显降低( P <0.05)。结论: PC可明显抑制TCP磨损颗粒诱导的骨细胞氧化损伤,其机制可能与减轻NLRP3炎症小体的活化和细胞焦亡相关。AIM: To investigate the effects of procyanidins (PC) on oxidative damage of osteocytes caused by tricalcium phosphate (TCP) wear particles, and to explore the underling mechanism. METHODS: Mouse long bone osteocyte MLO-Y4 cells were treated with TCP wear particles (0.1 g/L) for 48 h to establish the model of osteocyte injuries. The MLO-Y4 cells were divided into 4 groups: control group, TCP group, PC (10 μmol/L) group and PC (50 μmol/L) group. Calcein-AM staining and MTT assay were used to observe the viability of MLO-Y4 cells. The levels of dentin matrix protein 1 (DMP-1), sclerostin (SOST) and interleukin-1β(IL-1β) in the culture media were examined by ELISA. The apoptosis of MLO-Y4 cells was analyzed by flow cytometry with Annexin V/PI double staining. The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity of MLO-Y4 cells, and lactate dehydrogenase (LDH) release in the culture media were measured by chemical colorimetry. The protein levels of NOD-like receptor protein 3 (NLRP3), apoptosis -associated speck-like protein containing a CARD (ASC), cleaved caspase-1 and IL-1β in the MLO-Y4 cells were determined by Western blot.RESULTS: Compared with control group, MLO-Y4 cell injuries, apoptosis rate and MDA level were obviously increased in TCP group, while SOD activity was significantly decreased ( P <0.05) The protein levels of NLRP3, ASC, cleaved caspase-1 and IL-1β were remarkably up-regulated ( P <0.05) in the MLO-Y4 cells, and the level of IL-1β and LDH release were increased in the culture media ( P <0.05). Compared with TCP group, the injuries of MLO-Y4 cells, apoptosis rate and MDA level were decreased obviously ( P <0.05) in PC groups, whereas SOD activity was increased ( P <0.05). The protein levels of NLRP3, ASC, cleaved caspase-1 and IL-1β were down-regulated remarkably in the MLO-Y4 cells ( P <0.05), and the level of IL-1β and LDH release were significantly decreased in the culture media ( P <0.05).CONCLUSION: PC obviously inhibit oxidative damage of osteocytes caused by T

关 键 词：磷酸三钙磨损颗粒 骨细胞 原花青素 氧化应激 NLRP3炎症小体 细胞焦亡 

分 类 号：R285.5[医药卫生—中药学] R363.2[医药卫生—中医学]