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Role of the hippo signaling pathway in the extracellular matrix degradation of chondrocytes induced by fluoride exposure
Ecotoxicology and Environmental Safety ( IF 6.8 ) Pub Date : 2021-09-21 , DOI: 10.1016/j.ecoenv.2021.112796
Fang-Fang Yu 1 , Juan Zuo 1 , Xiaoli Fu 2 , Ming-Hui Gao 1 , Lei Sun 1 , Shui-Yuan Yu 1 , Zhiyuan Li 2 , Guo-Yu Zhou 1 , Yue Ba 1
Affiliation  

To identify the role of the Hippo signaling pathway in the extracellular matrix degradation of chondrocytes induced by fluoride exposure. Environmental response genes (ERGs) of bone injury induced by fluoride exposure were obtained from the Comparative Toxicogenomics Database, and annotated by STRING for KEGG pathway enrichment analysis. The CCK-8 kit was used to measure the proliferation of ATDC5 cells. The malondialdehyde (MDA), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-PX) levels in ATDC5 cells were measured using oxidative stress detection kit. Western blot analysis was used to measure the p-MST1/2, p-LATS1/2, and p-YAP/YAP1 expression levels in the Hippo pathway and the COL2A1, ACAN and MMP13 expression levels in the cartilage matrix. Localizations of YAP1 and COL2A1 proteins in chondrocytes were performed using cell immunofluorescence. Continuous data from the multiple groups were compared using one-way analysis of variance, and then the differences between groups were tested with Dunnett's t-test, with the test level α = 0.05. The 145 ERGs of bone injury induced by fluoride exposure were identified, and KEGG enrichment analysis revealed Hippo signaling pathways significantly related to bone injury. A CCK-8 assay revealed that the viability of the ATDC5 cells was significantly decreased with increased fluorine concentration. The MDA content in 20 mg/L sodium fluoride (NaF) exposure group was significantly higher than that in the control group, the T-SOD, T-AOC and GSH-PX activities in 15 and 20 mg/L NaF exposure groups were significantly lower than those in the control group (P < 0.05). Western blot results showed the protein levels of p-MST1/2, p-LATS1/2 and p-YAP1 in 15 and 20 mg/L NaF exposure groups were significantly lower than those in the control group, while the YAP1 protein level in 20 mg/L NaF group was significantly higher than that in the control group. The COL2A1 and ACAN proteins in 20 mg/L NaF group were significantly decreased, while the MMP13 protein level in 15 and 20 mg/L NaF groups were significantly increased (P < 0.05). It was observed that the expression of YAP1 protein expression level in the cytoplasm decreased with the increased fluoride exposure, whereas that the expression level of YAP1 protein in the nucleus increased. Fluoride inhibited the proliferation of ATDC5 cells, induced oxidative stress, inhibited the activity of the Hippo pathway, and eventually led to cartilage matrix degradation.



中文翻译:

河马信号通路在氟暴露诱导的软骨细胞胞外基质降解中的作用

确定 Hippo 信号通路在氟化物暴露诱导的软骨细胞细胞外基质降解中的作用。氟化物暴露引起的骨损伤的环境反应基因 (ERG) 从比较毒物基因组学数据库中获得,并通过 STRING 注释用于 KEGG 通路富集分析。CCK-8 试剂盒用于测量 ATDC5 细胞的增殖。使用氧化应激检测试剂盒测量 ATDC5 细胞中的丙二醛 (MDA)、总抗氧化能力 (T-AOC)、总超氧化物歧化酶 (T-SOD) 和谷胱甘肽过氧化物酶 (GSH-PX) 水平。Western印迹分析用于测量Hippo途径中p-MST1/2、p-LATS1/2和p-YAP/YAP1的表达水平以及软骨基质中COL2A1、ACAN和MMP13的表达水平。使用细胞免疫荧光对软骨细胞中的 YAP1 和 COL2A1 蛋白进行定位。使用单因素方差分析比较来自多个组的连续数据,然后使用Dunnett's检验组间的差异t检验,检验水平α  = 0.05。鉴定了 145 个氟化物暴露引起的骨损伤 ERG,KEGG 富集分析显示 Hippo 信号通路与骨损伤显着相关。CCK-8 测定表明,ATDC5 细胞的活力随着氟浓度的增加而显着降低。20 mg/L氟化钠(NaF)暴露组MDA含量显着高于对照组,15和20 mg/L NaF暴露组T-SOD、T-AOC和GSH-PX活性显着升高低于对照组(P < 0.05)。Western blot结果显示,15和20 mg/L NaF暴露组p-MST1/2、p-LATS1/2和p-YAP1蛋白水平显着低于对照组,而20 mg/L NaF暴露组中YAP1蛋白水平显着降低。 mg/L NaF组显着高于对照组。20 mg/L NaF组COL2A1和ACAN蛋白水平显着降低,而15和20 mg/L NaF组MMP13蛋白水平显着升高(P < 0.05)。观察到细胞质中 YAP1 蛋白表达水平的表达随着氟化物暴露的增加而降低,而细胞核中 YAP1 蛋白的表达水平增加。氟化物抑制ATDC5细胞的增殖,诱导氧化应激,抑制Hippo通路的活性,最终导致软骨基质降解。

更新日期:2021-09-21
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