Alpha‐pinene (α‐pinene) is an organic compound, found in the oils of many species of coniferous trees, especially pine. α‐Pinene reportedly has antioxidant and anti‐inflammatory activities; however, its effects on osteoblasts are unknown. This study investigated the effects of α‐pinene on osteoblast differentiation and tumour necrosis factor‐alpha (TNFα)‐induced inhibition of osteogenesis. Culture in control or osteogenic medium containing α‐pinene increased osteogenic marker expression. Alkaline phosphatase staining and alizarin red S staining confirmed that α‐pinene enhanced osteoblast differentiation. Also, α‐pinene attenuated TNFα‐induced inhibition of Smad1/5/9 phosphorylation and extracellular matrix mineralization. Taken together, our findings suggest that α‐pinene enhances osteoblast differentiation and mineralization in MC3T3‐E1 pre‐osteoblasts.
Objective
Vascular calcification requires the differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. This phenomenon can be enhanced by inflammation and oxidative stress. Zingerone is one of the active ingredients present in the ginger plant that has anti-inflammatory and antioxidant effects. Other functions include anti-obesity, anti-nausea effects. However, the functions of zingerone on vascular calcification has not yet been elucidated. This study investigated the effect of zingerone on vascular calcification and its molecular mechanism.
Methods
Reverse transcription-polymerase chain reaction (PCR), real-time PCR and Western blot analysis was used to measure expression levels of osteogenic marker genes and to investigate whether calcification was regulated by the expression of AMP-activated protein kinase (AMPK) and tissue inhibitor of metalloproteinase 4 (TIMP4). Alizarin red S staining was used to measure calcium deposition. Studies were carried out in VSMCs.
Results
Zingerone induced the expression of 2 markers of VSMCs differentiation (α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α)) and decreased the expression of core-binding factor α-1 (CBFA1). Additionally, zingerone decreased inorganic phosphate (Pi)-induced expression of distal-less homeobox 5 and CBFA1. AMPK phosphorylation and TIMP4 expression were increased by zingerone. Importantly, zingerone protected VSMCs from calcification, and this protective effect was confirmed by increased TIMP4 via overexpression of AMPK, and inhibition of TIMP4 by Compound C. Zingerone upregulated AMPK/TIMP4 expression and recovered Pi-induced inhibition of TIMP4.
Conclusions
Taken together, our results show that zingerone inhibits Pi-induced vascular calcification by regulating the AMPK/TIMP4 signaling cascade in VSMCs. These results suggest that the natural product zingerone could be useful for treating vascular and metabolic diseases.
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