title: Icariin stimulates the osteogenic differentiation of rat bone marrow stromal cells via activating the PI3K-AKT-eNOS-NO-cGMP-PKG.
abstract: Icariin, a prenylated flavonol glycoside isolated from Epimedii herba, has been found to be a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of icariin is related to the nitric oxide (NO) signal pathway and PI3K/AKT pathway in its upstream. Rat bone marrow stromal cells (rBMSCs) were cultured in osteogenic medium and treated with icariin or together with L-NAME, ODQ, PDE5, and/or LY294002 (the inhibitor of NOS, sGC, cGMP, and PI3K respectively), and effects were examined on the expression of signal messengers (NOS, NO, sGC, cGMP, PKG and PI3K) and the levels of osteogenic markers (alkaline phosphatase or ALP, osteocalcin and calcified nodules). It was found that icariin dose-dependently increased ALP activity, and treatment at the optimal concentration (10(-5)M) increased NOS activity, iNOS and eNOS expression, NO production, sGC and cGMP contents and PKG expression besides the phosphorylation of AKT. The addition of L-NAME, ODQ and PDE5 significantly inhibited the icariin effects on above markers respectively. The addition of LY294002 decreased the p-AKT level, NOS activity, eNOS expression and NO production significantly, but had no significant effect on iNOS expression. The addition of any of the four inhibitors also abolished the osteogenic effect of icariin on rBMSCs as indicated by ALP activity, osteocalcin synthesis, calcium deposition and the number and areas of calcified nodules. These results suggest that the osteogenic effect of icariin involves the PI3K-AKT-eNOS-NO-cGMP-PKG signal pathway. Furthermore, dosage response studies showed that icariin at 10(-6)M (a physiologically achievable concentration in vivo) also activated this signal pathway.
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou 730050, People's Republic of China. Electronic address: zhaiyk1984@163.com.
authors: Yuan-Kun
authors: YK
authors: Zhai
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou 730050, People's Republic of China. Electronic address: heihaguo@126.com.
authors: Xiao-Yu
authors: XY
authors: Guo
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou 730050, People's Republic of China. Electronic address: baofenggelz@126.com.
authors: Bao-Feng
authors: BF
authors: Ge
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou 730050, People's Republic of China. Electronic address: pingzhenlanzhou@126.com.
authors: Ping
authors: P
authors: Zhen
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou 730050, People's Republic of China. Electronic address: guxinyoulong@163.com.
authors: Xiao-Ni
authors: XN
authors: Ma
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou 730050, People's Republic of China. Electronic address: zhoujian715412@126.com.
authors: Jian
authors: J
authors: Zhou
authors: Department of Pharmacy, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou 730050, People's Republic of China. Electronic address: mahuipingcxr@aliyun.com.
authors: Hui-Ping
authors: HP
authors: Ma
authors: Sansom Institute for Health Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5001, Australia. Electronic address: cory.xian@unisa.edu.au.
authors: Cory J
authors: CJ
authors: Xian
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou 730050, People's Republic of China. Electronic address: chenkm@lut.cn.
authors: Ke-Ming
authors: KM
authors: Chen
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002867:Chromones / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009025:Morpholines; D015675:Osteocalcin; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D017869:Cyclic GMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester; C056599:icariin
keywords: Bone marrow stromal cells; Icariin; Nitric oxide; Osteogenic differentiation; Signal transduction