Pharmacoproteomic study of the effects of chondroitin and glucosamine sulfate on human articular chondrocytes

Abstract

IntroductionChondroitin sulfate (CS) and glucosamine sulfate (GS) are symptomatic slow-acting drugs for osteoarthritis (OA) widely used in clinic. Despite their widespread use, knowledge of the specific molecular mechanisms of their action is limited. The aim of this work is to explore the utility of a pharmacoproteomic approach for the identification of specific molecules involved in the pharmacological effect of GS and CS.MethodsChondrocytes obtained from three healthy donors were treated with GS 10 mM and/or CS 200 μg/mL, and then stimulated with interleukin-1β (IL-1β) 10 ng/mL. Whole cell proteins were isolated 24 hours later and resolved by two-dimensional electrophoresis. The gels were stained with SYPRORuby. Modulated proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry. Real-time PCR and Western blot analyses were performed to validate our results.ResultsA total of 31 different proteins were altered by GS or/and CS treatment when compared to control. Regarding their predicted biological function, 35% of the proteins modulated by GS are involved in signal transduction pathways, 15% in redox and stress response, and 25% in protein synthesis and folding processes. Interestingly, CS affects mainly energy production (31%) and metabolic pathways (13%), decreasing the expression levels of ten proteins. The chaperone GRP78 was found to be remarkably increased by GS alone and in combination with CS, a fact that unveils a putative mechanism for the reported anti-inflammatory effect of GS in OA. On the other hand, the antioxidant enzyme superoxide dismutase 2 (SOD2) was significantly decreased by both drugs and synergistically by their combination, thus suggesting a drug-induced decrease of the oxidative stress caused by IL-1β in chondrocytes.ConclusionsCS and GS differentially modulate the proteomic profile of human chondrocytes. This pharmacoproteomic approach unravels the complex intracellular mechanisms that are modulated by these drugs on IL1β-stimulated human articular chondrocytes.

Pharmacoproteomic Study of Three Different Chondroitin Sulfate Compounds on Intracellular and Extracellular Human Chondrocyte Proteomes

“…This protein was found in our previous pharmacoproteomic work as decreased by CS, alone or in combination with GS, in normal chondrocytes stimulated with IL-1␤ (13). Inversely, we found it increased in OA chondrocytes treated with CS1 in the present study (Table II): the DIGE analysis allowed the identification of three SOD2 protein isoforms (corresponding to spots 1214, 1215, and 1217), all of them exhibiting a CS1-dependent increase higher than 2-fold (Table I).…”

“…Chondrocytes were used at weeks 2-3 in primary culture (P1), after making them quiescent by incubation in a medium containing 0.5% FBS for 24 h. Finally, the cells were cultured in FBS-free medium containing chondroitin sulfate (200 g/ml), according to standard protocols (13). Three types of CS were tested: CS1 (porcine, purity 90.4%), CS2 (bovine, purity 96.2%), and CS3 (bovine, purity 99.9%).…”

“…In this step, an average of 1200 spots were matched between the gels, and the average relative abundances were calculated. We considered changes within a 95% confidence interval (p Յ 0.05) and standardized average spot volume ratios exceeding 1.2 with a statistical power of Ͼ0.8, based on previous experience by our group (13,17,18). This analysis resulted in the detection of 46 spots significantly and reproducibly altered: 27 modulated by CS1, 4 by CS2, and 15 by CS3.…”

Proteome Analysis During Chondrocyte Differentiation in a New Chondrogenesis Model Using Human Umbilical Cord Stroma Mesenchymal Stem Cells

“…Over the past year, our group has reported proteomic studies of chondrocytes in various pathologies and under differing culture conditions (11). This is the first report of a study of chondrogenic differentiation of MSCs from a novel source, the UC stroma.…”

Molecular effects of chondroguard in osteoarthritis and herniated discs

“…Для исследования всего комплекса действия ХС на клетки и ткани организма были проведены фармакопро-теомные исследования [7,8,[19][20][21][22][23] [19]. В фармакопротеомном исследовании у здоровых добро-вольцев, принимавших ХС (1200 мг/сут) и ГС (1500 мг/сут), установлено снижение активности цитокиновых сигнальных путей [8].…”