青蒿琥酯对氟化钠所致骨细胞MLO-Y4凋亡和线粒体自噬的影响  

Effect of artesunate on NaF-induced apoptosis and mitophagy in osteocytes MLO-Y4

作　　者：王子晗 莘裕辰 楼凯 沈方敏 王钰婷 鲍欣怡 章璐彬 张云[1] WANG Zihan;SHEN Yuchen;LOU Kai;SHEN Fangmin;WANG Yuting;BAO Xinyi;ZHANG Lubin;ZHANG Yun(College of Medicine,Shaoxing University,Shaoxing 312000,China)

机构地区：[1]绍兴文理学院医学院,浙江绍兴312000

出　　处：《中国药理学与毒理学杂志》2024年第3期183-193,共11页Chinese Journal of Pharmacology and Toxicology

基　　金：浙江省自然科学基金(LY21H060001)。

摘　　要：目的 探讨青蒿琥酯(Art)对氟化钠(NaF)所致骨细胞MLO-Y4损伤的改善作用及其分子机制。方法 NaF(2 mmol·L^(-1))与MLO-Y4细胞共孵育48 h,构建MLO-Y4细胞损伤模型。细胞分为细胞对照组、NaF组及NaF+Art 0.25,0.50和1.00μmol·L^(-1)组(Art预孵育2 h后,加NaF继续孵育12或48 h)。MTT法检测细胞存活率。钙黄绿素乙酰甲酯(calcein-AM)染色观察细胞活性。化学比色法检测乳酸脱氢酶(LDH)水平,DCFH-DA染色检测活性氧(ROS)水平,化学比色法检测丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,Hoechst33342染色和Annexin-V/PI染色分析细胞凋亡,JC-1染色检测线粒体膜电位(MMP),Lyso-Tracker和Mito-Tracker染色观察自噬泡形成和线粒体形态,荧光素酶法检测腺嘌呤核苷三磷酸(ATP)含量,免疫荧光染色检测微管相关蛋白轻链3(LC-3)在线粒体中的表达,Western印迹法检测LC-3、p62、PTEN诱导假定激酶1(PINK1)和E3泛素-连接酶(Parkin)蛋白表达水平。结果 与细胞对照组比较,NaF组细胞存活率和细胞活性显著降低(P<0.01),LDH含量、ROS水平、MDA含量、细胞凋亡率和自噬泡形成明显增加(P<0.01),PINK1和Parkin蛋白表达增加(P<0.01),LC3在线粒体中的表达增加且LC-3Ⅰ向LC-3Ⅱ转换增加(P<0.01),SOD活性、MMP水平、ATP水平和P62蛋白表达显著降低(P<0.05,P<0.01)。与NaF组比较,NaF+Art 0.25,0.50和1.00μmol·L^(-1)组细胞活性和存活率显著增加(P<0.01),LDH含量、ROS水平、MDA含量、细胞凋亡率和自噬泡形成明显减少(P<0.05,P<0.01),PINK1和Parkin蛋白表达下降(P<0.01),LC3在线粒体中的表达下降且LC-3Ⅰ向LC-3Ⅱ转换减少(P<0.01),SOD活性、MMP、ATP水平和P62蛋白表达显著增加(P<0.05,P<0.01)。结论 Art对NaF所致MLO-Y4细胞氧化损伤具有改善作用,其机制可能与减少细胞凋亡和线粒体自噬水平有关。OBJECTIVE To investigate the protective effect of artesunate(Art)against apoptosis and mitophagy induced by NaF in osteocytes MLO-Y4,and to explore the molecular mechanism.METHODS MLO-Y4 cells were treated with NaF(2 mmol·L^(-1))for 48 h to establish an in vitro model of osteocytes injuries,and the cells were divided into the cell control group,NaF(2 mmol·L^(-1))group and NaF+Art 0.25,0.50 and 1.00μmol·L^(-1) groups.The cells were pretreated for 2 h and NaF was added for 48 h.The cell survival of MLO-Y4 cells was detected by MTT assay.The cell viability of MLO-Y4 cells was measured by Calcein-AM staining.The lactate dehydrogenase(LDH)content in the supernatant was examined by the LDH detection kit.The level of intracellular reactive oxygen species(ROS)was examined by DCFH-DA staining.The malondialdehyde(MDA)content and superoxide dismutase(SOD)activity were detected by chemical colorimetry.Apoptosis was measured by Hoechst33342 staining and Annexin-V/PI staining.The level of mitochondrial membrane potential(MMP)was measured by JC-1 staining.The formation of autophagic vacuoles and morphological mitochondrial changes were observed via Lyso-tracker staining and Mito-Tracker staining.The ATP content was detected with the luciferase method.The expression of microtubule-associated protein light chain 3(LC-3)in mitochon-dria was examined by immunofluorescence staining.Protein expressions of LC-3,P62,E3 ubiquitin-ligase(Parkin)and PTEN-induced putative kinase 1(PINK1)were detected by Western blotting.RESULTS Compared with the cell control group,the cell survival rate and cell viability were significantly reduced in the NaF group(P<0.01),LDH content in the supernatant,the level of intracellular ROS,the MDA content,apoptosis rate and autophagic vesicle formation were remarkably increased(P<0.01),protein levels of Parkin and PINK1,and the conversion of LC-3Ⅱfrom LC-3Ⅰwere markedly upregulated along with the elevation of LC-3 in damaged mitochondria(P<0.01),while P62 levels,SOD activity,MMP and ATP contents were red

关 键 词：青蒿琥酯 氟化物 骨细胞 氧化应激 凋亡 线粒体自噬 

分 类 号：R966[医药卫生—药理学] R285.5[医药卫生—药学]