Department of Internal Medicine, Erasmus MC, Erasmus University Medical Center, Rotterdam, the Netherlands.
Department of Internal Medicine, Erasmus MC, Erasmus University Medical Center, Rotterdam, the Netherlands.
Bone. 2022 Nov;164:116526. doi: 10.1016/j.bone.2022.116526. Epub 2022 Aug 19.
A major precursor of advanced glycation end-products (AGEs) - methylglyoxal (MG) - is a reactive carbonyl metabolite that originates from glycolytic pathways. MG formation and accumulation has been implicated in the pathogenesis of diabetes and age-related chronic musculoskeletal disorders. Human bone marrow-derived stromal cells (BMSCs) are multipotent cells that have the potential to differentiate into cells of mesenchymal origin including osteoblasts, but the role of MG on their differentiation is unclear. We therefore evaluated the effect of MG on proliferation and differentiation of BMSC-derived osteoblasts. Cells were treated with different concentrations of MG (600, 800 and 1000 μM). Cell viability was assessed using a Cell Counting Kit-8 assay. Alkaline phosphatase (ALP) activity and calcium deposition assays were performed to evaluate osteoblast differentiation and mineralization. Gene expression was measured using qRT-PCR, whereas AGE specific receptor (RAGE) and collagen 1 were examined by immunocytochemistry and Western blotting. RAGE knockdown was performed by transducing RAGE specific short hairpin RNAs (shRNAs) using lentivirus. During osteogenic differentiation, MG treatment resulted in reduction of cell viability (27.7 %), ALP activity (45.5 %) and mineralization (82.3 %) compared to untreated cells. MG significantly decreased expression of genes involved in osteogenic differentiation - RUNX2 (2.8 fold), ALPL (3.2 fold), MG detoxification through glyoxalase - GLO1 (3 fold) and collagen metabolism - COL1A1 (4.9 fold), COL1A2 (6.8 fold), LOX (5.4 fold) and PLOD1 (1.7 fold). MG significantly reduced expression of collagen 1 (53.3 %) and RAGE (43.1 %) at protein levels. Co-treatment with a MG scavenger - aminoguanidine - prevented all negative effects of MG. RAGE-specific knockdown during MG treatment did not reverse the effects on cell viability, osteogenic differentiation or collagen metabolism. In conclusion, MG treatment can negatively influence the collagen metabolism and differentiation of BMSCs-derived osteoblasts through a RAGE independent mechanism.
高级糖基化终产物（AGEs）的主要前体-甲基乙二醛（MG）-是一种来源于糖酵解途径的反应性羰基代谢物。MG 的形成和积累与糖尿病和与年龄相关的慢性肌肉骨骼疾病的发病机制有关。人骨髓基质细胞（BMSCs）是多能细胞，具有分化为间充质来源细胞的潜力，包括成骨细胞，但 MG 对其分化的作用尚不清楚。因此，我们评估了 MG 对 BMSC 来源成骨细胞增殖和分化的影响。细胞用不同浓度的 MG（600、800 和 1000 μM）处理。用细胞计数试剂盒-8 法评估细胞活力。碱性磷酸酶（ALP）活性和钙沉积试验用于评估成骨细胞分化和矿化。用 qRT-PCR 测量基因表达，通过免疫细胞化学和 Western blot 检测 AGE 特异性受体（RAGE）和胶原蛋白 1。通过慢病毒转导 RAGE 特异性短发夹 RNA（shRNA）进行 RAGE 敲低。在成骨分化过程中，与未处理的细胞相比，MG 处理导致细胞活力（27.7%）、ALP 活性（45.5%）和矿化（82.3%）降低。MG 显著降低了参与成骨分化的基因的表达- runt 相关转录因子 2（RUNX2，2.8 倍）、碱性磷酸酶（ALPL，3.2 倍）、通过甘油醛酶途径的 MG 解毒-GLO1（3 倍）和胶原蛋白代谢-COL1A1（4.9 倍）、COL1A2（6.8 倍）、赖氨酰氧化酶（LOX，5.4 倍）和脯氨酰 4-羟化酶 1（PLOD1，1.7 倍）。MG 显著降低了蛋白质水平的胶原蛋白 1（53.3%）和 RAGE（43.1%）的表达。与 MG 清除剂-氨基胍联合治疗可预防 MG 的所有负面影响。MG 治疗期间 RAGE 特异性敲低并未逆转对细胞活力、成骨分化或胶原蛋白代谢的影响。总之，MG 处理可通过 RAGE 非依赖性机制对 BMSC 来源成骨细胞的胶原蛋白代谢和分化产生负面影响。
