Melatonin rescued interleukin 1β-impaired chondrogenesis of human mesenchymal stem cells

Abstract

Background: Osteoarthritis (OA) is a widespread arthritic disease and a primary cause of disability. Increasing evidence suggests that inflammation has a pivotal part in its pathogenesis. Interleukin-1β (IL-1β) is a primary mediator of local inflammatory processes in OA. Current therapies for OA mainly focus on the symptoms of the advanced stage of the disease. The possible utilization of bone marrow mesenchymal stem cells (BMSCs) to regenerate cartilage is an appealing method, but in the case of OA requires chondrogenesis to take place within an inflamed environment. Our previous study showed that melatonin (MLT) can promote chondrogenic differentiation of MSCs, but whether MLT can rescue IL-1β-impaired chondrogenesis in human BMSCs has not yet been established. MLT, which can have anti-inflammatory and prochondrogenic effects, has demonstrated potential in defeating IL-1β-induced inhibition of chondrogenesis and further study should be conducted.

Methods: Human bone marrow-derived MSCs were separated and cultured based on our system that was already documented. A high-density micromass culture system was used for the chondrogenic differentiation of human BMSCs, which was also described previously. Human BMSCs were induced for chondrogenesis for 7, 14, and 21 days with the treatment of IL-1β and MLT. The cultured cartilage pellets were then evaluated by morphology, extracellular matrix accumulation, and chondrogenic, metabolic, and apoptotic marker expression. Furthermore, cell apoptosis was assessed by TUNEL assay. The phosphorylation level P65 and IκBα of the NF-κB pathway activity was explored on day 21 of chondrogenic differentiation of BMSCs.

Results: The current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs in different aspects. Firstly, MLT can restore the chondrogenic pellet size, and rescue matrix synthesis and accumulation. Secondly, MLT can upregulate chondrogenic marker COL2A1 expression at both mRNA and protein levels, and also regulate the expression levels of other chondrogenic markers like ACAN, SOX9, and COL10A1 in the presence of IL-1β. Thirdly, MLT can maintain the metabolic balance of the chondrogenic process by suppressing expression of catabolic genes, such as MMP, MMP13, and ADAMTS4. Furthermore, MLT can subdue IL-1β-induced cell apoptosis of BMSCs throughout chondrogenesis. Meanwhile, MLT suppressed the phosphorylation level of P65 and IκBα, which were elevated by IL-1β treatment, indicating that MLT can attenuate the IL-1β-induced activation of NF-κB signaling.

Conclusion: The current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs by restoring the pellet size and matrix accumulation, and maintaining the metabolic balance, reducing cell apoptosis. Our study also showed that MLT can attenuate the IL-1β-induced activation of the NF-κB signaling pathway, which is the most important pathway downstream of IL-1β, and plays a crucial role in inflammation, apoptosis, and metabolism. Thus, MLT has prospects for treating OA due to its multifaceted functions, such as mitigating inflammation, maintaining metabolic balance, and mitigating apoptosis.

Keywords: Apoptosis; Chondrogenesis; Human mesenchymal stem cells; Interleukin-1β; Melatonin; Metabolic balance.

Fig. 1

Impact of IL-1β and MLT on cartilage pellet development throughout chondrogenesis of human mesenchymal stem cells (hMSCs). hMSCs prompted into chondrogenesis in chondrogenic medium with vehicle, 10 ng/ml IL-1β and vehicle, or 10 ng/ml IL-1β and 50 nM MLT via high-density micromass culture system. Cartilage pellets cultured for 7 (a), 14 (c), and 21 (e) days and observed by stereoscopic microscope in representative samples. Scale bars: 0.5 mm. Perimeters of pellets cultured for 7 (b), 14 (d) and 21 (f) days of chondrogenesis , 12 pellets used for measurement in each group. g Growth tendency of pellets from day 7 to day 21 in different groups. ^aP < 0.05 versus control group; ^bP < 0.05 versus IL-1β group. IL-1β interleukin-1β, MLT melatonin

Fig. 2

MLT rescued IL-1β-impaired matrix accumulation during chondrogenesis process. Alcian blue staining performed to visualize matrix accumulation. Pellets of different stages harvested. Paraformaldehyde 4% used for fixation. Paraffin sections (4 μm thick) prepared for staining. Cartilage pellets cultured for 7 (a), 14 (c), and 21(e) days observed by microscope in representative samples. Scale bars: 0.1 mm. Quantitative analysis of glycosaminoglycan (GAG) evaluated typical matrix accumulation during chondrogenesis. GAG content quantitatively examined and normalized by DNA content on days 7 (b), 14 (d), and 21 (f). Outcomes representative of three separate experiments. g Changing pattern of GAG/DNA from day 7 to day 21 in different groups. ^aP < 0.05 versus control group; ^bP < 0.05 versus IL-1β group. IL-1β interleukin-1β, MLT melatonin

Fig. 3

MLT reversed IL-1β-inhibited COL2A1 expression at both mRNA and protein levels. qPCR and IHC analysis assessed COL2A1 expressions at 7, 14 and 21 days. mRNA expression of COL2A1 measured by qPCR and normalized to GAPDH. Relative expression levels of gene on 7 (b), 14 (d), and 21 (f) days representative of three independent experiments. ^aP < 0.05 versus control group; ^bP < 0.05 versus IL-1β group. IHC staining of collagen type II performed after 7 (a), 14 (c), and 21 (e) days of differentiation. Scale bars: 0.1 mm. IL-1β interleukin-1β, MLT melatonin

Fig. 4

IL-1β and MLT treatment affected chondrogenic and apoptotic marker expression at mRNA level. Expression of ACAN (a), SOX9 (b), COL10A1 (c), MMP9 (d), MMP13 (e), and ADAMTS4 (f) measured by qPCR and normalized to GAPDH. Relative expression level of each gene on different stages of differentiation representative of three independent experiments. ^aP < 0.05 versus control group; ^bP < 0.05 versus IL-1β group. IL-1β interleukin-1β, MLT melatonin

Fig. 5

MLT protected human MSCs from IL-1β-induced apoptosis in process of chondrogenesis. TUNEL assay showed apoptotic MSCs (green fluorescence) with PI-labeled nuclei (blue fluorescence) on days 7 (a), 14 (c), and 21 (e). Positive rates statistically analyzed on right correspondingly (b, d, f), and data represent mean ± SD of three independent experiments. Scale bars = 50 μm. ^aP < 0.05 versus control group; ^bP < 0.05 versus IL-1β group. DAPI 4′,6-diamidino-2-phenylindole, IL-1β interleukin-1β, MLT melatonin, TUNEL terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling

Fig. 6

MLT downregulated IL-1β-enhanced phosphorylation of P65 and IκBα in NF-κB pathway. Immunoblotting (a) conducted to detect expression and total and phosphorylated P65 and IκBα to reflect activation of NF-κB signaling. Phospho-P65, P65, phospho-IκBα, and IκBα protein levels established (b). Ratio of relative protein expression of p-P65 to relative protein expression of P65 (p-P65/P65) (c), and ratio of relative protein expression of p-IκBα to relative protein expression of IκBα (p-IκBα/IκBα) examined (d). ^aP < 0.05 versus control group; ^bP < 0.05 versus IL-1β group. IL-1β interleukin-1β, MLT melatonin