Effect of Melatonin on the Proliferation and Differentiation of Chondrocytes from Rat Vertebral Body Growth Plate In Vitro

Abstract

Purpose: Abnormal growth of vertebral body growth plate (VBGP) is considered as one of the etiologic factors in the adolescent idiopathic scoliosis (AIS). It was well-known that melatonin was correlated with the emergence and development of AIS. This study aimed to investigate the effect of melatonin on rat VBGP chondrocytes in vitro.Methods:Chondrocytes were isolated from rat VBGP, and treated with or without melatonin. Cell proliferation was measured by the Alamar Blue assay. Gene expression of collagen type II and aggrecan were evaluated by real-time PCR. Expression of the melatonin receptors (MT1, MT2), proliferating cell nuclear antigen (PCNA, a cell proliferation marker), Sox9 (a chondrocytic differentiation marker) and Smad4 (a common mediator in regulating the proliferation and differentiation of chondrocytes) were detected by Western blotting.Results: Expression of melatonin receptors (MT1, MT2) were detected in the rat VBGP chondrocytes. Melatonin, at 10 and 100 µg/mL concentration, significantly inhibited the proliferation of VBGP-chondrocytes and the gene expression of collagen type II and aggrecan, and down-regulated the protein expression of PCNA, Sox9 and Smad4. In addition, the inhibitory effect of melatonin was reversed by luzindole, a melatonin receptor antagonist.Conclusions: These results suggest that melatonin at high concentrations can inhibit the proliferation and differentiation of VBGP chondrocytes, which might give some new insight into the pathogenic mechanism of AIS.

Abnormal Response of the Proliferation and Differentiation of Growth Plate Chondrocytes to Melatonin in Adolescent Idiopathic Scoliosis

“…To the best of our knowledge, the current study constitutes the first report on the inhibitory effect of melatonin on the proliferation and differentiation of GPCs in human. In an in vitro study, melatonin at high concentrations (10, 100 µg/mL) showed an inhibitory effect on both the proliferation and differentiation of cultured rat vertebral body growth plate chondrocytes [ 26 ]. After incubation for 24 h in medium containing melatonin, the cell proliferation, gene expression of collagen type II and aggrecan, as well as protein expression of proliferating cell nuclear antigen (PCNA), Sox9 and Smad4 were significantly reduced.…”

“…After incubation for 24 h in medium containing melatonin, the cell proliferation, gene expression of collagen type II and aggrecan, as well as protein expression of proliferating cell nuclear antigen (PCNA), Sox9 and Smad4 were significantly reduced. Moreover, it was found that the effects of melatonin could be reversed by the melatonin receptor antagonist luzindole, indicating the involvement of membrane melatonin receptors in these functions [ 26 ]. In a broiler chicken model, Aota et al [ 28 ] noted that pinealectomized chickens had a significantly increased area of labeled hypertrophic zone per total hypertrophic zone compared with control chickens (32.8% ± 12.5% vs. 6.4% ± 5.0%, p < 0.005), as well as an increased number of hypertrophic and proliferative chondrocytes.…”

“…The functional outcome of abnormalities in the melatonin signaling pathway in the regulation of endochondral ossification in AIS, however, has not been reported. It has been documented that melatonin inhibited both proliferation and differentiation of rat vertebral body growth plate (VBGP) chondrocytes [ 26 ] with the involvement of MT1 and MT2 receptors. In addition, a recent study showed that melatonin enhanced the chondrogenic differentiation of human mesenchymal stem cells [ 27 ], mediated at least partially by the two membrane melatonin receptors.…”

Circadian production of melatonin in cartilage modifies rhythmic gene expression

“…Our results are largely consistent with these reports, confirming that melatonin enhances chondrocyte proliferation and the expression of cartilage-associated genes. However, a study using cultured chondrocytes from rat vertebral body growth plates showed that high concentrations of melatonin (4 × 10 −4 M) caused inhibition of cell proliferation and mRNA expression of Col2a1, Aggrecan, Sox9, and Smad4 (Zhong et al 2013).…”

Effect of Asymmetric Tension on Biomechanics and Metabolism of Vertebral Epiphyseal Plate in a Rodent Model of Scoliosis

“…The range of the load value and the time of its action are two important determinants of vertebral development. This view reflects our understanding of the complex biological and biomechanical multifactorial processes.…”