Kaempferol protects MC3T3-E1 cells through antioxidant effect and regulation of mitochondrial function

Abstract

Kaempferol, a natural flavonoid present in fruits, vegetables, and teas, provides beneficial effects for human health. In this study, we investigated the protective effects of kaempferol on antimycin A (AMA)-induced toxicity in osteoblast-like MC3T3-E1 cells. Exposure of MC3T3-E1 cells to AMA caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, intracellular calcium $([{\text{Ca}}^{2+}{]}_{\text{i}})$ elevation, and reactive oxygen species (ROS) production. Pretreatment with kaempferol prior to AMA exposure significantly reduced AMA-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, $[{\text{Ca}}^{2+}{]}_{\text{i}}$ elevation, and ROS production. Kaempferol also induced the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B), and CREB (cAMP-response element-binding protein) inhibited by AMA, which result demonstrates that kaempferol utilizes the PI3K/Akt/CREB pathway to augment metabolic activity inhibited by AMA. All these data indicate that kaempferol may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders.

Introduction

Aging is a multi-faceted process associated with several functional and structural deficits, and also often associated with major defects on the skeleton itself. Aging-related bone diseases are comprised of three major classes: the erosion of the joints or osteoarthritis; an increase in bone resorption leading to osteoporosis, a low bone mass disease with high risk of fracture; and metastatic bone disease that relates to breast cancer in women or prostate cancer in men. Osteoarthritis and osteoporosis are not only the most frequent degenerative diseases of the skeleton, they are also the most frequent degenerative diseases in developed countries (Cooper and Melton, 1996). In aging, there are alterations in Ca²⁺ handling that may affect the bioenergetic and mitochondrial functions and may contribute to cell death process. The production of reactive oxygen species (ROS) may cause a leak of Ca²⁺ from stores in the intracellular compartments. Transient increase in Ca²⁺ is essential element in the control of many physiological processes. However, sustained increases in Ca²⁺ may contribute to oxidative stress and cell death. It is well known that intracellular Ca²⁺ overload causes mitochondrial Ca²⁺ accumulation, change in the mitochondrial pH, increase of the ROS production, decrease or complete loss of mitochondrial membrane potential and opening of the mitochondrial permeability transition pore.

Several reports showed that inhibitors of the mitochondrial electron transport chain induce cell injuries in vitro and in vivo (Brouillet et al., 1993, Smith and Bennett, 1997). Mitochondria energy metabolism is extremely sensitive to impairment by free radicals and that mitochondrial oxidative stress limits metabolic recovery (Fiskum et al., 2004). Damage of mitochondria leads to cell death, because mitochondria are involved in energy metabolism and calcium homeostasis. Chemicals generating ROS, such as rotenone and antimycin A (AMA), decrease cell viability and reduce mitochondrial membrane potential. AMA is an inhibitor of mitochondrial electron transport via its binding to complex III. Though it is generally accepted that mitochondrial complexes have the potential to generate superoxide anions leading to oxidative stress, complex III is a major site for ROS generation (Dawson et al., 1993) since inhibition of complex III by antimycin A in isolated liver mitochondria results in the accumulation of an ubisemiquinone intermediate with subsequent superoxide anion production (Garcia-Ruiz et al., 1995).

Kaempferol belongs to the flavonol group and is present at high levels in broccoli, chives, and kale. Recently, food flavonoids have attracted great interest owing to their apparent beneficial effects on human health. The health-promoting effects of flavonoids have been attributed to their antioxidant activities. Wattel et al. (2003) demonstrated that flavonols such as kaempferol decrease osteoclastic bone resorption in vitro by targeting directly the mature osteoclast by a mechanism involving, at least in part, the estrogen receptor (ER). Miyake et al. (2003) reported the promoting effect of kaempferol on the differentiation and mineralization of murine pre-osteoblastic cell line. Prouillet et al. (2004) showed that the kaempferol increases the alkaline phosphatase activity, and that this effect is dependent upon both the ERK pathway and the ER activation. A model commonly used to study osteogenic development is the MC3T3-E1 osteoblast-like cell line (Quarles et al., 1992). The MC3T3-E1 cell culture system represents a very useful model for studying the process of osteoblast function. Therefore, we investigated the protective effects of kaempferol against AMA-induced cytotoxicity using osteoblast-like MC3T3-E1 cells.