Magnesium Inhibits Extracellular Matrix Calcification In Chondrocytes By Regulating Erk Cellular Signial And Autophagy Formation

OBJECTIVE:The most important cytopathological changes in osteoarthritis are chondrocyte hypertrophy,chondrocyte apoptosis,and chondrocyte replacement by osteoblasts.In the progression,extracellular matrix?ECM?is transformed from collagen to calcium phosphate,and ultimately leading to chondrocyte extracellular matrix being replaced by bone tissue to form the main histology features of arthritis.Magnesium is a constant element in human body.It is the second intracellular divalent cation to calcium ion.Magnesium plays an important role in maintaining normal physiological function of human body and promoting cell and biological enzyme activities.Magnesium participates in many enzymatic reactions,protein synthesis and energy metabolism;maintains the bone growth and excitability of muscle tissue as well as nerve;maintains the stability of gastrointestinal tract and hormone function.As the two most abundant bivalent cations in human body,the balance of extracellular fluid regulates calcium deposition and tissue calcification in extracellular microenvironment.At the same time,physiological bone formation,bone resorption,the balance of these two ions also affects pathological heterotopic ossification and chondrocyte calcification.At present,calcium content in calcified tendons and ligaments is higher than that in normal tissues,and increases with the severity of tendon degeneration and calcification,while magnesium content decreases with tendon aging and degeneration.Recent studies have shown that Mg²⁺is closely related to the calcification process of extracellular matrix,involving many common pathological conditions,such as osteoporosis and atherosclerosis.In addition,the effective inhibition of ectopic ossification of connective tissue by Mg²⁺has been confirmed in animal experiments;intra-articular injection of magnesium sulfate can also effectively inhibit articular cartilage to produce similar pathological changes in arthritis;the New England Journal also pointed out that the abnormal content of magnesium ion in blood can lead to calcium pyrophosphate deposition between chondrocytes,which can lead to cartilage calcinosis.According to the large sample study of the population,when the serum Mg²⁺concentration is increased or the diet is added with high concentration of magnesium,the imaging manifestation of knee osteoarthritis is decreased,and has statistical significance.It can be said that Mg²⁺plays a key role in regulating extracellular matrix calcification.ATDC5 cell line and DMM-induced osteoarthritis animal model are widely used at present.They are the most effective experimental in vitro and in vivo objects to study the pathogenesis of osteoarthritis.Compared with aging-induced spontaneous knee osteoarthritis model in mice,DMM-induced model can not only reproduce pathological changes of similar severity,but also the same location in a short time,because this model can lead to cartilage degeneration from the medial space of knee joint,and cause knee instability and pressure concentration in medial compartment.The pathological mechanism is related to human knee joint and the pathogenesis of arthritis is similar.Chondrocyte is the only cell in articular cartilage.Its main function is to synthesize abundant ECM and participate in the transformation of extracellular matrix.The transformation of ECM from collagen to bone tissue is the focus of this study.ATDC5 cell line was isolated from mouse tumors for the first time in 1990.Relevant studies have also found that ATDC5 cell line has strong chondrogenesis and cartilage differentiation ability compared with other established cell lines?C3H10T1/2 and RJC3.1?.In the process of cell culture,the cells showed typical chondrocyte characteristics,such as formation of cartilage nodules,secretion of type II collagen,secretion of aggregated proteoglycan and other ECM molecules.With the prolongation of culture time,hypertrophy of chondrocyte appeared,and the expression of collagen X protein increased.ECM calcification appeared 45 days after cultured in regular medium.ATDC5 chondrocyte has the characteristics of rapid proliferation and persistence in undifferentiated state,which effectively ensures the effective implementation of in vitro experiments in this study.This study will focus on the molecular biological mechanism and related cellular signaling pathways of Mg²⁺inhibits ATDC5 chondrocyte extracellular matrix calcification and alleviate the progression of knee osteoarthritis,so as to provide new theoretical basis and new therapeutic ideas for the effective prevention of knee osteoarthritis and slow down the progression of disease.METHODS:In this study,chondrocyte ATDC5 cell line and DMM-induced osteoarthritis animal model were used as the main research objects.The molecular biological mechanism and related cellular signaling pathways of Mg²⁺inhibiting ATDC5 chondrocyte extracellular matrix calcification and alleviating the progression of osteoarthritis were investigated.1.ATDC5 chondrocytes were cultured in regular medium,osteogenic induction medium,and gradient Mg²⁺osteogenic induction medium?1.2 mM/L,1.6 mM/L,2.0mM/L and 2.4 mM/L?,respectively.After 14 days of continuous culture,alizarin red staining was performed to evaluate the osteogenic effect.2.ATDC5 chondrocytes were cultured in regular medium,osteogenic induction medium or osteogenic induction medium containing 2.4 mM/L magnesium ion for 3,7 and 15 days.The osteogenic related genes ALP,Runx 2,chondrocyte hypertrophic marker gene Col10?1,MMP13,chondrocyte marker gene Sox9,Col1?1 and calcification suppressor gene MGP were detected by qPCR.3.ATDC5 chondrocytes were cultured in regular medium,osteogenic induction medium or osteogenic induction medium containing 2.4mM/L magnesium ion,and detected by Western blotting.Phosphorylated ERK1/2 protein and total ERK1/2protein were detected 24 hours later,LC3 and p62 protein were detected 72 hours later,Runx2,MMP13,Col1?1 and Col10?1 were detected 7 days later.4.ATDC5 chondrocytes were incubated in regular medium,osteogenic induction medium,and osteogenic induction medium containing 2.4mM/L magnesium ion and5 mm/L 3-MA for 3 or 6 hours.The positive expression of LC3 protein in ADTC5cells was detected by FACScan flow cytometry.5.DMM was performed on the right knee of 12-week-old male mice to induce knee osteoarthritis.Opening and exposing the knee joint cavity,without any manipulation of articular cartilage or meniscus,we performed sham operation on the left knee of the same mouse as a negative control.6.From 2 to 11 weeks after operation,MgCl₂?50 ug,dissolved in 10?l saline,experiment group?and 10?l saline in the control group were injected into the knee joint cavity every week under anesthesia?Avertin 240 mg/kg body weight?.Samples of two knee joints were collected 12 weeks after operation,decalcified,treated,embedded and sectioned for histological study.7.Continuous sagittal sections?3 um thick?of the knee joints of mice were stained with Alsine Blue/Hematoxylin and Eosin?AB/H&E?for morphological analysis.8.Histomorphological measurements were performed with OsteoMetrics?Inc.,Atlanta,GA,USA?.The blue area of articular cartilage in AB/H&E stained area was depicted on the projection image of each tissue slice,and the statistical analysis and comparison were made.9.According to the scoring system developed by Glasson et al.,the severity of the progress of knee osteoarthritis in each mouse was evaluated.10.Immunofluorescence?IF?staining was performed on tissue sections 3-micron thick.The fluorescence signal of LC3 positive expression was detected by Streptomyces antibiotic protein?488 conjugate,Thermo Fisher?.A liquid seal with DAPI was used,in which the nucleus could be dyed blue.Results:High concentration of Mg²⁺could effectively inhibit the extracellular matrix calcification of chondrocyte ATDC5,and intra-articular injection of MgCl₂could effectively inhibit the progress of knee osteoarthritis induced by DMM operation.1.When the content of magnesium ion in osteogenic induction medium was 1.6mM/L or higher,the degree of extracellular matrix calcification of ATDC5 was significantly inhibited,the quantity of alizarin red S was significantly decreased,and the inhibition of extracellular matrix by high concentration of Mg²⁺was dose-dependent.2.Total RNA extracted from ATDC5 for 3 days,7 days or 15 days showed that high concentration of Mg²⁺had no regulatory effect on ALP expression.Osteogenic induction medium significantly inhibited the expression of MGP,Col1?1 and Sox9,while high concentration of Mg²⁺reversed the inhibition of osteogenic induction medium.Osteogenic induction medium could significantly increase the expression of Runx2,Col10?1 and MMP13,while high concentration of Mg²⁺could reverse the increase of Runx2,Col10?1 and MMP13.3.After 24 hours of culture,phosphorylated ERK was significantly activated in osteogenic induction medium.High concentration of Mg²⁺could inhibit the expression of p-ERK,while the total ERK remained stable.4.After 72 hours of culture,LC3 II was activated in osteogenic induction medium,and high concentration of Mg²⁺and 3-MA could reverse the activation.P62 was inhibited in osteogenic induction medium,while high concentration of Mg²⁺and3-MA increased the expression of p62.5.7 days after culture,osteogenic induction medium could activate the levels of Runx2,Col10?1 and MMP13 in ATDC5,while high concentration of Mg²⁺slowed down the increase of protein level.7 days after culture,osteogenic induction medium inhibited the chondrocyte marker Col1?1 in ATDC5,and high concentration of Mg²⁺reversed the inhibition effect and showed chondrocyte protection.6.Each group of samples was analyzed for 1×10⁴ ATDC5 cells.Osteogenic induction medium can significantly activate the expression of LC3,while high concentration of Mg²⁺and 3-MA can inhibit the expression of LC3.7.Continuous intra-articular injection of Mg²⁺after DMM can effectively alleviate the progress of osteoarthritis.Alsine blue/Hematoxylin and Eosin staining showed that 12 weeks after DMM operation,significant osteoarthritis-like phenotypes appeared in the knee joint of mice,including subchondral sclerosis,cartilage degeneration and chondrocyte hypertrophy.Mg²⁺can effectively alleviate the progress of osteoarthritis;reduce the occurrence of subchondral sclerosis,cartilage degeneration and chondrocyte hypertrophy.8.The articular cartilage area of tibial plateau was quantified by histomorphological measurement.The positive staining area of Alsine blue was tracked by OsteoMeasure system.The tibial cartilage area of DMM group injected with Mg²⁺was significantly higher than that of DMM group injected with saline.Two other observers evaluated the OARSI score of knee joint of mice by double-blinded method.The results showed that DMM operation caused significant osteoarthritis.High concentration of Mg²⁺can significantly protect articular cartilage.9.The expression of LC3 in animal models was detected by immunofluorescence.The positive chondrocytes of LC3 showed green fluorescence.Compared with sham-operated group,the LC3 positive expression of chondrocytes in DMM-induced animal model was higher,and MgCl₂ injection could also reduce the percentage of LC3 positive chondrocytes.Conclusion:High concentration of Mg²⁺can effectively inhibit the extracellular matrix calcification of chondrocyte ATDC5;inhibit osteoarthritis of knee joint induced by DMM operation,and produce cartilage protection effect.1.High concentration of Mg²⁺inhibits the extracellular matrix calcification of ATDC5 by inhibiting the expression of osteoblast-related transcription factor Runx2,chondrocyte hypertrophy gene Col10?1 and MMP13.High concentration of Mg²⁺protects the cartilage characteristics of ATDC5 chondrocytes by up-regulating the expression of chondrocyte marker genes Sox9,Col1?1 and calcification inhibitor gene MGP.2.High concentration of Mg²⁺can effectively inhibit the formation of autophagic protein LC3-II in ATDC5 chondrocyte by inhibiting the phosphorylation of ERK1/2protein in cell signaling pathway,increase the expression of autophagic binding ligand p62,and effectively inhibit the formation of autophagy activated by osteogenesis,causing the inhibition of ECM calcification in chondrocytes.3.Intra-articular injection of MgCl₂ can effectively alleviate knee osteoarthritis induced by DMM in mice,reduce subchondral sclerosis,cartilage degeneration and chondrocyte hypertrophy to a certain extent,effectively reduce knee OARSI score,partially retain articular cartilage area,and alleviate the progress of arthritis in DMM model.Immunofluorescence staining showed that intra-articular injection of MgCl₂could also effectively reduce the number of LC3 positive cells in articular cartilage.