Zingerone Attenuates Pi-induced Vascular Calcification via AMPK-mediated TIMP4 Expression

Abstract

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.

Zingerone ameliorates non‐alcoholic fatty liver disease in rats by activating AMPK

“…Interestingly and very recently, Zingerone was shown to reverse asthma complications in mice and to prevent Pi‐induced vascular calcification, in vitro, via the activation of AMPK (Lim et al, 2021) these data were very encouraging to us who highlight potential stimulatory effects of Zingerone on the expression of hepatic AMPK.…”

Policosanol Stimulates Osteoblast Differentiation via Adenosine Monophosphate-Activated Protein Kinase-Mediated Expression of Insulin-Induced Genes 1 and 2

“…In general, mineralization means that calcium is deposited and hardened in hard tissues such as teeth and bones, and calcification refers to abnormal calcium deposition in soft tissues, resulting in a pathological phenotype [37]. In addition to this study, we have reported the results of studies in which other natural products increased bone mineralization but inhibited vascular smooth muscle cell calcification [38,39]. Thus, our findings suggest that policosanol acts by increasing mineralization in hard tissue bone and inhibiting calcification in soft tissue.…”

Vascular calcification: Molecular mechanisms and therapeutic interventions

“…For example, AMPK/tissue inhibitors of metalloproteinases 4 signaling cascade in VSMCs were involved in the progress of VC. 186 Interestingly, the knockdown of AMPK by siRNA significantly reversed the anti‐calcification effects of metformin, resveratrol, and exendin‐4 and the reduction of RANKL in the calcified VSMCs. 187 Inhibition of AMPK‐mediated cell deaths was observed in the regulation of aldosterone on high phosphate‐induced VC.…”

“…The AMP‐activated protein kinase (AMPK)‐mediated signaling pathway plays a very important role in VC. For example, AMPK/tissue inhibitors of metalloproteinases 4 signaling cascade in VSMCs were involved in the progress of VC 186 . Interestingly, the knockdown of AMPK by siRNA significantly reversed the anti‐calcification effects of metformin, resveratrol, and exendin‐4 and the reduction of RANKL in the calcified VSMCs 187 .…”