白果内酯对IL-1β诱导的ATDC5软骨细胞自噬、增殖和凋亡的影响  

Effects of Bilobalide on Autophagy, Proliferation and Apoptosis of IL-1β-induced ATDC5 Chondrocytes

作　　者：马天文 于跃 吕良钰 贾丽娜 阮红日 王颢然 王新宇 张雨欣 张建涛[1] 高利[1] MA Tianwen;YU Yue;Lü Liangyu;JIA Lina;RUAN Hongri;WANG Haoran;WANG Xinyu;ZHANG Yuxin;ZHANG Jiantao;GAO Li(Heilongjiang Provincial Key Laboratory of Pathogenesis and Comparative Medicine of Animal Diseases,College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学动物医学学院黑龙江省动物疾病致病机制与比较医学重点实验室,哈尔滨150030

出　　处：《畜牧兽医学报》2023年第2期837-846,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技重大专项和重点研发项目省级资金资助项目(GX18B023);内蒙古自治区重点研发和成果转化计划项目(2022YFSH0052);黑龙江省应用技术研究与开发计划重大项目(GA18B203);新疆维吾尔自治区科技支疆项目计划(指令性)项目(2020E0236)。

摘　　要：旨在探讨白果内酯对白介素1β(IL-1β)诱导的ATDC5软骨细胞自噬、增殖和凋亡的影响。采用10 ng·mL^(-1)IL-1β诱导ATDC5软骨细胞构建体外炎症模型,随机分为对照组、IL-1β组、白果内酯组(低、中、高剂量)。利用EdU检测细胞新合成的DNA,并结合细胞核标记物(Hoechest)进行双重标记检测细胞增殖速度。使用膜Annexin-V/PI通过流式细胞仪分析ATDC5软骨细胞凋亡情况。通过mRFP-GFP-LC3双荧光系统的组合测量方法检测自噬流。蛋白免疫印迹法(Western blot)和实时荧光定量PCR(qRT-PCR)方法检测各组软骨细胞中LC3-Ⅱ、Beclin1、BAX、Caspase-3和Bcl2的蛋白和mRNA表达情况。结果显示,IL-1β诱导ATDC5软骨细胞后,细胞增殖减弱,下调自噬促进凋亡,而白果内酯干预能够过促进自噬和细胞增殖,抑制凋亡。白果内酯促进ATDC5软骨细胞的增殖(P<0.05),与IL-1β组相比,抑制了LC3-II、Beclin1、BAX和Caspase-3的蛋白和mRNA表达(P<0.05),促进Bcl2蛋白和mRNA表达(P<0.05),激活自噬并上调自噬流,凋亡率显著降低(P<0.05)。结果提示,白果内酯能够促进IL-1β诱导ATDC5软骨细胞的自噬并且抑制细胞凋亡,促进细胞增殖进而发挥保护软骨细胞作用。The purpose of this study was to investigate the effect of bilobalide on IL-1β-induced autophagy, proliferation and apoptosis of ATDC5 chondrocytes. 10 ng·mL^(-1)IL-1β was used to induce ATDC5 chondrocytes to construct an in vitro inflammatory model, and then the cells were randomly divided into control group, IL-1β group and bilobalide groups(low-, medium-and high-dosage). The newly synthesized DNA of cells was detected by EdU, and the cell proliferation rate was detected by double labeling combined with the nuclear marker(Hoechest). The apoptosis of ATDC5 chondrocytes was analyzed by flow cytometry using membrane Annexin-V/PI. Autophagic flux was detected by a combined measurement of the mRFP-GFP-LC3 dual fluorescence system. Western blot and qRT-PCR methods were used to detect the protein and mRNA expression levels of LC3-II, Beclin1, BAX, Caspase-3 and Bcl2 in chondrocytes of each group. The results showed that after IL-1β-induced ATDC5 chondrocytes, cell proliferation was attenuated, autophagy was down-regulated and apoptosis was improved. Bilobalide intervention can promote autophagy, proliferation and inhibit apoptosis. Bilobalide promoted the proliferation of IL-1β-induced ATDC5 chondrocytes(P<0.05). Compared with IL-1β group, bilobalide inhibited the protein and mRNA expression of LC3-II, Beclin1, BAX and Caspase-3(P<0.05), and promoted the protein and mRNA expression of Bcl2(P<0.05). Autophagy flux was activated, and the apoptosis rate was significantly reduced(P<0.05). The results suggest that bilobalide can promote autophagy of IL-1β-induced ATDC5 chondrocytes and inhibit cell apoptosis, promote cell proliferation and protect chondrocytes.

关 键 词：关节软骨 白果内酯 自噬 凋亡 细胞增殖 软骨细胞 

分 类 号：S853.74[农业科学—临床兽医学]