Comparative proteomic and metabolomic analysis reveal the antiosteoporotic molecular mechanism of icariin from Epimedium brevicornu maxim
Graphical abstract
Introduction
Bone metabolism, which is normally maintained by osteoclast-mediated bone resorption and osteoblast-mediated bone formation, is a lifelong dynamic remodeling process (Rachner et al., 2011). Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) are central regulators of osteoclasts (OC) differentiation for activation of RANK in the osteoclast precursors. Osteoclasts adhere to bone surface through αvβ integrin, while creating F-actin sealing zone to resorb bone matrix. Osteoclasts are capable of generating an acidic microenvironment necessary for bone resorption. Carbonic anhydrase II generates H+ and HCO3− by the hydration of CO2, and the hydrogen ions are transported through the apical ruffled border of the osteoclasts to the resorption zone by a vacuolar H+ adenosine triphosphatase (H+ V-ATPase). The H+ V-ATPase also provides the proton source for extracellular acidification by exchange of bicarbonate to chloride by HCO3/ Cl− exchanger (Laitala and Väänänen, 1993, Teitelbaum, 2000). Intracellular vesicles are fused to bone facing plasma membrane inside actin ring, release acid into the resorption space through ruffled border membrane, and starts dissolution of apatite crystals, exposing its organic matrix, with subsequent degradation by acidic proteases, such as cathepsin K (CatK), matrix metalloprotein 9 (MMP-9), and matrix metalloprotein 13 (MMP-13) ( Teitelbaum, 2000). Meanwhile, osteoblasts secrete new bone matrixs (layers of oriented collagen fibers), and form dense crosslinked ropes. An essential event in osteoblast (OB) differentiation is the activation of runt-related transcription factor 2 (Runx2), a master switch can positively regulate mesenchymal stem cells (MSCs) to immature osteoblast (IO) and regulates expression of osterix (OSX). OSX, which are regulated by the Ca2+-calcineurin pathway, interact with the nuclear factor for activated T cells 2 (NFAT2) to control the transcription of target genes, such as collagen 1 and osteocalcin (Nakashima et al., 2002). Bone morphogenetic proteins (BMP) in skeletal tissue belong to transforming growth factor-β (TGF-β) superfamily, play an important role in fracture healing. BMP and TGF-β signaling are via binding to specific receptors and activation of Smad effector proteins, such as Smad-1,−4 and −5, which also can interact with Runx2 and cooperates in controlling BMP signaling, ultimately stimulating bone formation (Feng and Derynck, 2005). The underlying mechanism of osteoporosis is an imbalance between bone resorption and bone formation during bone remodeling, characterized as excessive bone resorption, and inadequate formation of new bone.
Epimedii Folium (Chinese name as Yinyanghuo), the leaf of Epimedium brevicornu Maxim, E. sagittatum (Sieb. et Zucc.) Maxim, E. pubescens Maxim, E. wushanense T. S. Ying and E. koreanum Nakai (Chinese Pharmacopoeia Commission, 2015), has been clinically used for more than 2000 years. This herb, firstly recorded in the Shennong’s Herbal Atlas (Shennong Bencao Jing, written around 221BCE to 220CE), then following recored in Chinese medicine books, was characterized as tonifying kidney, strengthening tendons and bones, treating impotence and rheumatoid arthritis. In recent decades, Epimedii Folium is frequently applied as the principal ingredients in many new Chinese formulas treating osteoporosis clinically in China (Wang et al., 2016, Zhang et al., 2016), and in experiment study, such as Er-xian Decoction(Xue et al., 2012a), Xianlingubao (Wang et al., 2015). Icariin (ICA, pubchem CID: 5,318,997), the most abundant active component of Epimedii Folium, possess anti-osteoporosis, anti-oxidation, anti-tumor, anti-aging, anti-depression and anti-atherosclerosis activity in pharmacological studies (Jia et al., 2012, Kang et al., 2012, Mao et al., 2000, Pan et al., 2013, Yang et al., 2013, Zhang et al., 2010). As a bone protective agent, ICA modulate bone remodeling by stimulating osteoblastic cell proliferation and differentiation through regulating the signaling of BMP, TGF-β, and insulin-like growth factors (IGFs), Wnt and Runx2/Osterix (Ma et al., 2011, Ma et al., 2013), and mediate bone resorption by inhibition of osteoclastic differentiation and bone resorption enzymes, such as CatK (Sun et al., 2013), MMP-9 (Zhang et al., 2015) and CAII. In our previous study, the bone protective effects of ICA has been demonstrated by the enhancement in bone microstructure of ovariectomized (OVX) rats, inhibition of osteoclasts differenation mainly through OPG/RANKL signal and MAPK signal. ICA also can reduce the number and activity of osteoclasts, show significantly inhibitory effects on tartrate-resistant acid phosphatase (TRAP) activity, CatK activity and the formation of F-actin ring (Nian et al., 2009, Xue et al., 2012a, Xue et al., 2012b). However, the mechanism involved in the bone protective effects of ICA is still not clear.
Currently, there is a great deal of scientific interest and debate concerning the possible advantages that transcriptomic, proteomic and metabonomic technologies (‘Omics’) might have over traditional biomarkers. Up to now, there is only the combined transcriptomic and proteomic approaches were applied to evalue antiosteoporostic of ICA in zebrafish larvae (Li et al., 2011). The detailed proteomic on bone tissue and serum metabonomic data in animal are still lacking. We propose modulating protein expression and serum metabolites may reveal more insights on bone protective effects of ICA. A comparative proteomic approach was used for identifying the proteins with altered expression levels in ICA-treated ovarictomized mice (classic osteoporostic model). A 1H nuclear magnetic resonance spectroscopy (1HNMR) -based metabonomics method was applied to obtain a systematic view of the serum metabolites. Based on proteomic and metabonomic findings, a hypothetical mechanism for antiosteoporotic activity of ICA were put forward. Furthermore, the effects of ICA on osteoblast and osteoclast were analyzed to confirm the implication of proteomic and metabonmic results. The global insights of effects of ICA in protein and metabolite level will be given in our study and the possible signal pathway involved in antiosteoporotic of ICA will be clarified.
Section snippets
Reagents
ICA, purity 98%, was purchased from ChemFaces (CFN99554, China). Reagents used in this study included α-Modified minimum essential medium (α-MEM) and fetal bovine serum (FBS) (Gibco, US); trypsin and coomassie brilliant blue G-250 (Sigma, US); recombinant Rat macrophage colony-stimulating factor (M-CSF, 400–28) and RANKL (400-30) (Peprotech EC, US); β-actin (sc81178) antibody (Santa Cruz, US); anti-phospho-JNK (9251), anti-phospho-ERK (3371), anti-phospho-p38 (9211), anti-αvβ integrin
Protein expression profiles and identification of differentially expressed proteins
2-DE maps between sham and OVX mice, or OVX mice and ICA-treated OVX mice with protein differential expression areas were shown in Supplement Fig. 1 and Fig. 2, respectively. The results were reproducible due to the repeated results of the protein samples from independent femur extracts of mice. A protein score greater than 70 means confidence limits >95% in the present study and was considered statistically significant. Total twenty-four spots were absolutely differentially expressed in sham,
Discussion
The present study systematically evaluated the mechanism of osteoporosis and actions of ICA in protein and metabolites level in Sham, OVX and ICA-treated OVX mice. The results clearly demonstrated that significantly changed metabolites and differentially expressed proteins among three groups were involved in multiple biochemical functions, mainly including energy metabolism, cytoskeleton organization, lipid metabolism, regulation of bone remodeling, choline metabolism, and oxdative stress
Conclusion
Based on our study, ICA was considered as multi-targeting on osteoporosis (Scheme 1), The results demonstrated that ICA treatment decrease the bone loss in ovariectomized mice, promote bone formation of osteoblast, and inhibit osteoclastic bone resorption mainly by regulating three different metabolic process: energy homoeostasis, cytoskeleton formation and lipids metabolism. In addition, ICA also regulate proteins and metabolites involved in bone metabolism, such as microtubule actin
Authors’ roles
Study design by Liming Xue ([email protected]), Luping Qin ([email protected]) and Qiaoyan Zhang ([email protected]), study conduct by Liming Xue and Yiping Jiang([email protected]), data analysis by Liming Xue and Yiping Jiang. Data interpretation: Hailiang Xin([email protected]), Naidan Zhang ([email protected]) and Ting Han ([email protected]). Drafting manuscript: Liming Xue and Yiping Jiang. All authors approving final version of manuscript.
Acknowledgment
This work supported by the National Natural Science Foundation of China (90709023, 81403162 and 81503493).
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The two authors contribute equally to this paper.