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. 2016;54(6):954-63.
doi: 10.3109/13880209.2015.1079224. Epub 2015 Oct 9.

Flavonoids of Herba Epimedii stimulate osteogenic differentiation and suppress adipogenic differentiation of primary mesenchymal stem cells via estrogen receptor pathway

Dawei Zhang  1   2   3 , Li Liu  1 , Zhenbin Jia  1 , Xinsheng Yao  4 , Mengsu Yang  3
Affiliations

Flavonoids of Herba Epimedii stimulate osteogenic differentiation and suppress adipogenic differentiation of primary mesenchymal stem cells via estrogen receptor pathway

Dawei Zhang et al. Pharm Biol. 2016.

Abstract

Context: Accumulating evidence indicates that Herba Epimedii [Epimedii folium (Berberidaceae)] has anti-osteoporotic effect by stimulating osteoblastic bone formation and reducing osteoclastic bone resorption VSports手机版. However, the effect of Herba Epimedii in regulating the cross-talk between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) remains unclear. .

Objective: The present study investigates the effect of total flavonoids of Herba Epimedii (HETF) on the osteogenesis and adipogenesis of primary MSCs V体育安卓版. .

Materials and methods: HETF were prepared and identified by HPLC-fingerprinting, primary mouse MSCs in the presence of 0. 006-6 μg/mL HETF for 2-10 d were subject to morphological, biochemical, and quantitative real-time PCR analysis V体育ios版. .

Results: Sixteen chemical components were identified in HETF by HPLC-fingerprinting and account for over 95% of the total area of HPLC peaks. During osteogenesis of MSCs, 0. 006-6 μg/mL HETF promoted the proliferation of MSCs from 17% to 22%, increased alkaline phosphatase activity up to 3. 7-fold (0. 6 μg/mL), and extracellular calcium deposits from 1. 2- to 1 VSports最新版本. 4-folds by up-regulating the expression of runt-related transcription factor-2 (Runx-2) and bone morphogenetic protein-2 (BMP-2). Meanwhile, HETF suppressed the adipogenesis of MSCs by reducing the formation of adipocyte-like cells and accumulation of fat droplets by down-regulating the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ). The above biological activities of HETF were mainly through estrogen receptor-mediated pathway, which were blocked by estrogen receptor antagonist, ICI 182,780. .

Conclusion: HETF could regulate Runx-2-mediated osteogenesis and PPAR-γ-mediated adipogenesis in MSCs and thus exhibit beneficial effects to bone health, which suggests a new strategy for treating patients with osteoporosis and obesity V体育平台登录. .

Keywords: Adipogenesis; PPAR-γ; Runx-2; osteogenesis. VSports注册入口.

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Conflict of interest statement

The authors report that they have no conflicts of interest.

This work was supported by the National Natural Science Foundation of China (No VSports在线直播. 81402933), Natural Science Foundation of Guangdong Province (No. 2014A030313539), the Scientific Research Foundation for the Returned Overseas Chinese Scholars[(2014)-1685], State Education Ministry, Research Foundation for Building Strong Province of Chinese Medicine-Traditional Chinese Medicine Bureau of Guangdong Province (No. 20131260), the Project for the Development of Social Science Research of Dongguan (2013108101054), Medical Scientific Research Foundation of Guangdong Province (B2014302), 2014 Project of Teaching Quality and Teaching Reform of Undergraduate Course in Colleges and Universities in Guangdong Province, and the Doctor Scientific Start-up Fund Projects of Guangdong Medical College (B2012008).

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Figure 1.
Figure 1.
HPLC-fingerprint analysis of HETF from E. koreanum Nakai. Chromatographic analyses were performed on the Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA) and a Gemini C18 column, flow rate: 1 mL/min, detection wavelength: 254 nm, mobile phase (MeOH:H2O) and elution time: 30–40% (0–10 min), 40–50% (10–20 min), 50–60% (20–40 min), 60–70% (40–60 min), 70–80% (60–70 min), 80–90% (70–80 min), and 90–100% (80–90 min). The data shown are from three independent experiments. Peaks: icariin (1), baohuoside-I (2), sagittatoside B (3), korepimedoside C (4), caohuoside E (5), baohuoside-II (6), astragalin (7), epimedin B (8), 3,5,7-trihydroxyl-4′-methoxyl-8-prenylflavone-3-O-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranoside (9), sagittatoside A (10), acuminatin (11), epimedin C (12), icariside A5 (13), epimedoicarisoside A (14), epimedoside A (15), hexandraside F (16).
Figure 2.
Figure 2.
HETF enhanced the cell viability and the proliferative capacity of MSCs. Values are presented as means ± SD (n = 6 per group). *p < 0.05 compared with control.
Figure 3.
Figure 3.
HETF stimulated the osteogenic differentiation of MSCs by increasing the ALP activity. Values are presented as means ± SD (n = 6 per group). *p < 0.05 compared with control, #p < 0.05 compared with ES (17β-estradiol).
Figure 4.
Figure 4.
HETF stimulated the osteogenic differentiation of MSCs by increasing bone nodules formation. In vitro matrix mineralization in the presence of HETF and 17β-estradiol stained by alizarin red S (40×, A–C). (A) Mineralization supplement (MS); (B) MS + 17β-estradiol; (C) MS + HETF; scale bar = 20 μm; (D) Quantification of alizarin red S staining. Values are presented as means ± SD (n = 6 per group). *p < 0.05 compared with MS.
Figure 5.
Figure 5.
HETF suppressed the adipogenic differentiation of MSCs by decreasing adipocytic-like cells formation. Fat droplets within differentiated adipocytes stained by oil red O method (100×, A–C). (A) Adipogenic supplement (AS); (B) AS + 17β-estradiol; (C) AS + HETF; scale bar = 40 μm. (D) Quantification of oil red O staining. Values are presented as means ± SD (n = 6 per group). *p < 0.05 compared with AS.
Figure 6.
Figure 6.
The two-way regulation of HETF on the expression of key factors during osteogenic and adipogenic differentiation of MSCs. The gene expression level of Runx-2 and BMP-2 was significantly up-regulated by HETF and ES, while the gene expression level of PPAR-γ was significantly down-regulated by HETF. The antiestrogen ICI 182,780 completely blocked HETF and ES action on the gene expression level of Runx-2, BMP-2, and PPAR-γ. Values are presented as means ± SD (n = 4 per group). #p < 0.05 compared with control, *p < 0.05 compared with OS (osteogenic supplement) or AS (adipogenic supplement).
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