Abstract：
OBJECTIVE: This study aimed to investigate the potential mechanisms by which lysyl oxidase like 3 (LOXL3) affects the autophagy in chondrocytes in osteoarthritis (OA), specifically through the activation of mammalian target of rapamycin complex 1 (mTORC1).
METHODS: To establish an OA model, rats underwent anterior cruciate ligament transection (ACLT). Chondrocytes were isolated from cartilage tissues and cultured. Western blotting was performed to assess the expression of LOXL3, Rheb, phosphorylation of p70S6K (p-p70S6K, a downstream marker of mTORC1), and autophagy markers. The autophagy of chondrocytes was observed using an immunofluorescence assay.
RESULTS: The expression levels of both LOXL3 and Rheb proteins were upregulated in chondrocytes isolated from the OA model cartilage, in comparison to those from the normal cartilage. The silencing of LOXL3 resulted in a decrease in the protein levels of Rheb and p-p70S6K, as well as an increase in the expression of autophagy-related proteins. Additionally, the effect of LOXL3 could be reversed through the silencing of Rheb. The results of the immunofluorescence assay confirmed the impact of LOXL3 and Rheb on chondrocyte autophagy.
CONCLUSIONS: LOXL3 inhibits chondrocyte autophagy by activating the Rheb and mTORC1 signaling pathways.
METHODS: To establish an OA model, rats underwent anterior cruciate ligament transection (ACLT). Chondrocytes were isolated from cartilage tissues and cultured. Western blotting was performed to assess the expression of LOXL3, Rheb, phosphorylation of p70S6K (p-p70S6K, a downstream marker of mTORC1), and autophagy markers. The autophagy of chondrocytes was observed using an immunofluorescence assay.
RESULTS: The expression levels of both LOXL3 and Rheb proteins were upregulated in chondrocytes isolated from the OA model cartilage, in comparison to those from the normal cartilage. The silencing of LOXL3 resulted in a decrease in the protein levels of Rheb and p-p70S6K, as well as an increase in the expression of autophagy-related proteins. Additionally, the effect of LOXL3 could be reversed through the silencing of Rheb. The results of the immunofluorescence assay confirmed the impact of LOXL3 and Rheb on chondrocyte autophagy.
CONCLUSIONS: LOXL3 inhibits chondrocyte autophagy by activating the Rheb and mTORC1 signaling pathways.
摘要：
目的：本研究旨在探讨赖氨酰氧化酶样3（LOXL3）影响骨关节炎（OA）软骨细胞自噬的潜在机制。特异性通过激活哺乳动物雷帕霉素靶复合物1(mTORC1)。
方法：为了建立OA模型，大鼠接受前交叉韧带横切术（ACLT）。从软骨组织中分离软骨细胞并培养。进行蛋白质印迹以评估LOXL3、Rheb、磷酸化p70S6K(p-p70S6K，mTORC1的下游标记)，和自噬标记。使用免疫荧光测定法观察软骨细胞的自噬。
结果：LOXL3和Rheb蛋白的表达水平在从OA模型软骨分离的软骨细胞中上调，与正常软骨相比。LOXL3的沉默导致Rheb和p-p70S6K的蛋白质水平降低,以及自噬相关蛋白的表达增加。此外,LOXL3的作用可以通过沉默Rheb逆转。免疫荧光分析的结果证实了LOXL3和Rheb对软骨细胞自噬的影响。
结论：LOXL3通过激活Rheb和mTORC1信号通路抑制软骨细胞自噬。
方法：为了建立OA模型，大鼠接受前交叉韧带横切术（ACLT）。从软骨组织中分离软骨细胞并培养。进行蛋白质印迹以评估LOXL3、Rheb、磷酸化p70S6K(p-p70S6K，mTORC1的下游标记)，和自噬标记。使用免疫荧光测定法观察软骨细胞的自噬。
结果：LOXL3和Rheb蛋白的表达水平在从OA模型软骨分离的软骨细胞中上调，与正常软骨相比。LOXL3的沉默导致Rheb和p-p70S6K的蛋白质水平降低,以及自噬相关蛋白的表达增加。此外,LOXL3的作用可以通过沉默Rheb逆转。免疫荧光分析的结果证实了LOXL3和Rheb对软骨细胞自噬的影响。
结论：LOXL3通过激活Rheb和mTORC1信号通路抑制软骨细胞自噬。
