title: The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells.
abstract: BACKGROUND
  Kaempferol, a kind of flavonol, has been reported to possess various osteogenic biological activities, such as inhibiting bone resorption of osteoclasts and promoting the differentiation and mineralization of preosteoblasts. However, the precise cellular mechanism of action of kaempferol in osteogenesis is elusive. Autophagy is a major intracellular degradation system, which plays an important role in cell growth, survival, differentiation and homeostasis in mammals. Recent studies showed that autophagy appeared to be involved in the degradation of osteoclasts, osteoblasts and osteocytes, potentially pointing to a new pathogenic mechanism of bone homeostasis and bone marrow disease. The potential correlation between autophagy, osteogenesis and flavonoids is unclear.
  
  
  METHODS
  The present study verified that kaempferol promoted osteogenic differentiation and mineralization and that it elevated osteogenic gene expression based on alkaline phosphatase (ALP) activity, alizarin red staining and quantitative PCR. And then we found that kaempferol induced autophagy by acridine orange (AO) and monodansylcadaverine (MDC) staining and autophagy-related protein expression. The correlation between kaempferol-induced autophagy and the osteogenic process was confirmed by the autophagy inhibitor 3-methyladenine (3-MA).
  
  
  RESULTS
  Kaempferol promoted the proliferation, differentiation and mineralization of osteoblasts at a concentration of 10 μM. Kaempferol showed cytotoxic properties at concentrations above 50 μM. Concentrations above 10 μM decreased ALP activity, whereas those up to 10 μM increased ALP activity. Kaempferol at concentrations up to 10 μM also increased the expression of the osteoblast- activated factors RUNX-2, osterix, BMP-2 and collagen I according to RT-PCR analyses. 10 μM or less, the higher of the concentration and over time, kaempferol promoted the activity of osteoblasts. Kaempferol induced autophagy. It also increased the expression of the autophagy-related factors beclin-1, SQSTM1/p62 and the conversion of LC3-II from LC3-I. The application of 3-MA decreased the activity of ALP and the autophagy induced by kaempferol. In the RT-PCR analysis, the expression of RUNX-2, osterix, BMP-2 and collagen I was decreased.
  
  
  CONCLUSIONS
  The present study showed that kaempferol stimulated the osteogenic differentiation of cultured osteoblasts by inducing autophagy.
authors: Department of Oral Anatomy, School of Dentistry, Pusan National University, Busandaehak-ro, 49, Mulguem-eup, Yangsan-si, Gyeongsangnam-do, 626-870, South Korea.
authors: In-Ryoung
authors: http://orcid.org/0000-0003-0232-0385
authors: IR
authors: Kim
authors: Department of Oral and Maxillofacial Surgery, Pusan National University Dental Hospital, 20, Geumo-ro, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do, 626-770, South Korea.
authors: Seong-Eon
authors: SE
authors: Kim
authors: Department of Oral and Maxillofacial Surgery, Pusan National University Dental Hospital, 20, Geumo-ro, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do, 626-770, South Korea.
authors: Hyun-Su
authors: HS
authors: Baek
authors: Deptment of Oral and Maxillofacial Surgery, Medical center, Dong-A University, 26, Daesingongwon-ro, Seo-gu, Busan, 602-715, South Korea.
authors: Bok-Joo
authors: BJ
authors: Kim
authors: Deptment of Oral and Maxillofacial Surgery, Medical center, Dong-A University, 26, Daesingongwon-ro, Seo-gu, Busan, 602-715, South Korea.
authors: Chul-Hoon
authors: CH
authors: Kim
authors: Department of Oral and Maxillofacial Surgery, Pusan National University Dental Hospital, 20, Geumo-ro, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do, 626-770, South Korea.
authors: In-Kyo
authors: IK
authors: Chung
authors: Department of Oral Anatomy, School of Dentistry, Pusan National University, Busandaehak-ro, 49, Mulguem-eup, Yangsan-si, Gyeongsangnam-do, 626-870, South Korea. parkbs@pusan.ac.kr.
authors: Bong-Soo
authors: BS
authors: Park
authors: Department of Oral and Maxillofacial Surgery, Pusan National University Dental Hospital, 20, Geumo-ro, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do, 626-770, South Korea. ssh8080@pusan.ac.kr.
authors: Sang-Hun
authors: SH
authors: Shin
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D005419:Flavonoids / Q000494:pharmacology*; D044949:Kaempferols / Q000494:pharmacology*; D051379:Mice; D010012:Osteogenesis / Q000187:drug effects
chemical_list: D005419:Flavonoids; D044949:Kaempferols; C025946:3-methyladenine; C006552:kaempferol; D000225:Adenine
keywords: Autophagy; Flavonoids; Kaemferol; Osteogenesis