The significant cytopathological changes of osteoarthritis are chondrocyte hypertrophy, proteoglycan loss, extracellular matrix (ECM) calcification, and terminally, the replacement of cartilage by bone. Meanwhile, magnesium ion (Mg 2+ ), as the second most abundant divalent cation in the human body, has been proved to inhibit the ECM calcification of hBMSCs (human bone marrow stromal cells), hVSMCs (Human vascular smooth muscle cells), and TDSCs (tendon-derived stem cells) in vitro studies. The ATDC5 cell line, which holds chondrocyte characteristics, was used in this study as an in vitro subject. We found that Mg 2+ can efficiently suppress the ECM calcification and downregulate both hypertrophy and matrix metalloproteinase-related genes. Meanwhile, Mg 2+ inhibits the formation of autophagy by inhibiting Erk phosphorylation signaling and lowers the expression of LC3, and eventually effectively reduces the formation of ECM calcification in vitro. In this study, we also used destabilization of the medial meniscus (DMM)-induced osteoarthritis (OA) animal model to further confirm the protective effect of Mg 2+ on articular cartilage. Compared with the control group (saline-injected), continuous intraarticular magnesium chloride (MgCl 2 ) injection can significantly alleviate the severity of cartilage calcification in OA animal model. Immunofluorescence staining also revealed that saline-injected DMM group had a higher positive rate of LC3 expression in cartilage chondrocytes, compared with MgCl 2 -injected DMM group. In general, Mg 2+ can significantly downregulate the hypertrophic gene Runx2, MMP13, and Col10α1, upregulate the chondrogenic genes Sox9 and Col1α1, inhibit the Erk phosphorylation signaling, reduce the expression of autophagy protein LC3, and effectively inhibit the ECM calcification of ATDC5. In vivo study also proved that intra-articular injection of Mg 2+ protected knee cartilage by inhibiting the autophagy formation. K E Y W O R D S autophagy, calcification inhibitive, chondrocyte hypertrophy, DMM-induced osteoarthritis, magnesium ion SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. How to cite this article: Yue J, Jin S, Gu S, Sun R, Liang Q. High concentration magnesium inhibits extracellular matrix calcification and protects articular cartilage via Erk/ autophagy pathway.

Background/Aims: The high mobility group box 1 (HMGB1) has been regarded as an important inflammatory mediator. Previous studies showed the involvement of HMGB1 protein in the dysfunction of endothelial barrier function during acute lung injury. However, the molecular mechanism remains unclear. Methods: In this study, we used recombinant human HMGB1 (rhHMGB1) and HMGB1 plasmid to treat human pulmonary microvascular endothelial cell (HPMECs). We examined endothelial permeability by measuring TEER value and HRP flux. Western blot and real-time PCR were used to examined change of endothelial-to-mesenchymal transition (EndoMT) markers and related pathways. Immunofluorescence was used to examine localization and expression of ZO-1 and VE-cadherin. SB203580.was used to block p38 pathway. Unfractionated heparin (UFH) and RAGE siRNA were also used to antagonize the effect of HMGB1. Results: We showed that HMGB1 induced EndoMT with downregulation of ZO-1 and VE-cadherin at both mRNA and protein levels in HPMECs. We also demonstrated that HMGB1 upregulated endothelial permeability by measuring TEER value and HRP flux. Moreover, HMGB1 activated p38/GSK3β/Snail signaling pathway and treatment with p38 inhibitor SB203580 abolished its biological effects. In addition, we found that UFH was able to reverse the effect of HMGB1 on EndoMT and endothelial permeability through inhibition of p38 signaling in a dose-dependent manner. We discovered that RAGE, a membrane receptor of HMGB1, transduced p38/Snail pathway to EndoMT. RAGE siRNA inhibited the effect of HMGB1 induced EndoMT in HPMECs. Conclusion: The present study demonstrated that HMGB1 induced EndoMT through RAGE receptor and p38/GSK3β/Snail pathway. While UFH antagonized HMGB1 and maintained the integrity of the endothelial barrier through p38 inhibition.

No abstract