X-D Lu1, Y-R Liu, Z-Y Zhang. 1. Department of Orthopedics, First Hospital of Shanxi Medical University, Taiyuan, China. lyrsci888@163.com.
Abstract
OBJECTIVE: The intervertebral disc contains abundant extracellular matrix (ECM) imbued with proteoglycans, collagens, and water. With the development of intervertebral disc degeneration (IVDD), the ECM undergoes changes characterized by loss of water content, proteoglycans, and collagen content. The purpose of this study was to explore the vital role of Matrilin-3, an ECM protein involved in the progress of IVDD. MATERIALS AND METHODS: NP cells were isolated from the patients' disc samples and exposed to recombinant human (rh)-Matrilin-3 protein (MATN3), and IL-1β is used as a reducer of nucleus pulposus (NP) cells degeneration. Matrilin-3 and IL-1 receptor antagonist (IL-1Ra) were knocked down by siRNA transfection. Messenger RNA expressions of IL-1Ra, Collagen II, aggrecan, MMP-13, and ADAMTS-5 were determined using Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). Later, the protein levels of IL-Ra, Collagen II, and aggrecan were also detected by Western blot. The IL-1Ra, MMP-13, and ADAMTS-5 dose of the supernatants in the culture medium was determined by enzyme linked immunosorbent assay (ELISA). Finally, immunofluorescence was used to expose the expression of Collagen II, aggrecan, and Collagen X. RESULTS: It was found that the expression of IL-1Ra was markedly increased in the present of MATN3 or IL-1β, especially these two at once. Besides, MATN3 could upregulate Collagen II and aggrecan expressions, as well as inhibit the MMP-13 and Collagen X production of NP cells. However, the protective effects of Collagen II and aggrecan were abolished after Matrilin-3 silenced. Furthermore, IL-1β downregulated the Collagen II and aggrecan but promoted the MMP-13 and Collagen X levels of NP cells, which were antagonized by the action of MATN3. Surprisingly, silencing of IL-1Ra significantly abolished the MATN3-induced the protective effects of ECM in NP cells. CONCLUSIONS: This study provides a novel viewpoint of Matrilin-3 in the ECM stability of NP due to its ability to activate IL-1Ra. It is considered that MATN3 efficiently protects ECM degeneration of human NP cells related to maintain the content of Collagen II and aggrecan, as well as inflammatory inhibition.
OBJECTIVE: The intervertebral disc contains abundant extracellular matrix (ECM) imbued with proteoglycans, collagens, and water. With the development of intervertebral disc degeneration (IVDD), the ECM undergoes changes characterized by loss of water content, proteoglycans, and collagen content. The purpose of this study was to explore the vital role of Matrilin-3, an ECM protein involved in the progress of IVDD. MATERIALS AND METHODS: NP cells were isolated from the patients' disc samples and exposed to recombinant human (rh)-Matrilin-3 protein (MATN3), and IL-1β is used as a reducer of nucleus pulposus (NP) cells degeneration. Matrilin-3 and IL-1 receptor antagonist (IL-1Ra) were knocked down by siRNA transfection. Messenger RNA expressions of IL-1Ra, Collagen II, aggrecan, MMP-13, and ADAMTS-5 were determined using Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). Later, the protein levels of IL-Ra, Collagen II, and aggrecan were also detected by Western blot. The IL-1Ra, MMP-13, and ADAMTS-5 dose of the supernatants in the culture medium was determined by enzyme linked immunosorbent assay (ELISA). Finally, immunofluorescence was used to expose the expression of Collagen II, aggrecan, and Collagen X. RESULTS: It was found that the expression of IL-1Ra was markedly increased in the present of MATN3 or IL-1β, especially these two at once. Besides, MATN3 could upregulate Collagen II and aggrecan expressions, as well as inhibit the MMP-13 and Collagen X production of NP cells. However, the protective effects of Collagen II and aggrecan were abolished after Matrilin-3 silenced. Furthermore, IL-1β downregulated the Collagen II and aggrecan but promoted the MMP-13 and Collagen X levels of NP cells, which were antagonized by the action of MATN3. Surprisingly, silencing of IL-1Ra significantly abolished the MATN3-induced the protective effects of ECM in NP cells. CONCLUSIONS: This study provides a novel viewpoint of Matrilin-3 in the ECM stability of NP due to its ability to activate IL-1Ra. It is considered that MATN3 efficiently protects ECM degeneration of human NP cells related to maintain the content of Collagen II and aggrecan, as well as inflammatory inhibition.