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. 2016 Dec 29;11(12):e0166751.
doi: 10.1371/journal.pone.0166751. eCollection 2016.

Chlorogenic Acid Prevents Osteoporosis by Shp2/PI3K/Akt Pathway in Ovariectomized Rats

Rong Ping Zhou  1   2   3 , Si Jian Lin  2   4 , Wen Bing Wan  1   3 , Hui Ling Zuo  4 , Fen Fen Yao  4 , Hui Bing Ruan  1   3 , Jin Xu  1   3 , Wei Song  1   3 , Yi Cheng Zhou  1   3 , Shi Yao Wen  1   3 , Jiang Hua Dai  2   4 , Mei Lan Zhu  2   4 , Jun Luo  2   3   4
Affiliations

Chlorogenic Acid Prevents Osteoporosis by Shp2/PI3K/Akt Pathway in Ovariectomized Rats

Rong Ping Zhou et al. PLoS One. .

Abstract

Cortex Eucommiae is used worldwide in traditional medicine, various constituents of Cortex Eucommiae, such as chlorogenic acid (CGA), has been reported to exert anti-osteoporosis activity in China, but the mechanism about their contribution to the overall activity is limited. The aims of this study were to determine whether chlorogenic acid can prevent estrogen deficiency-induced osteoporosis and to analyze the mechanism of CGA bioactivity. The effect of CGA on estrogen deficiency-induced osteoporosis was performed in vivo. Sixty female Sprague-Dawley rats were divided randomly among a sham-operated group and five ovariectomy (OVX) plus treatment subgroups: saline vehicle, 17α-ethinylestradiol (E2), or CGA at 9, 27, or 45 mg/kg/d. The rats' femoral metaphyses were evaluated by micro-computed tomography (μCT). The mechanism of CGA bioactivity was investigated in vitro. Bone mesenchymal stem cells (BMSCs) were treated with CGA, with or without phosphoinositide 3-kinase (PI3K) inhibitor LY294002. BMSCs proliferation and osteoblast differentiation were assessed with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and alkaline phosphatase, with or without Shp2 interfering RNA (RNAi). The results display that CGA at 27 and 45 mg/kg/day inhibited the decrease of bone mineral density (BMD) that induced by OVX in femur (p< 0.01), significantly promoted the levels of bone turnover markers, and prevented bone volume fraction (BV/TV), connectivity density (CoonD), trabecular number (Tb.N), trabecular thickness (Tb.Th) (all p< 0.01) to decrease and prevented the trabecular separation (Tb.Sp), structure model index (SMI)(both p< 0.01) to increase. CGA at 1 or 10 μM enhanced BMSC proliferation in a dose-dependent manner. CGA at 0.1 to 10 μM increased phosphorylated Akt (p-Akt) and cyclin D1. These effects were reversed by LY294002. CGA at 1 or 10 μM increased BMSC differentiation to osteoblasts (p< 0.01), Shp2 RNAi suppressed CGA-induced osteoblast differentiation by decreasing Shp2, p-Akt, and cyclin D1. This study found that CGA improved the BMD and trabecular micro-architecture for the OVX-induced osteoporosis. Therefore, CGA might be an effective alternative treatment for postmenopausal osteoporosis. CGA promoted proliferation of osteoblast precursors and osteoblastic differentiation of BMSCs via the Shp2/PI3K/Akt/cyclin D1 pathway.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Effects of OVX, E2, and CGA on body and organ weights.
OVX rats were treated for a total of 12 wks. Each column represents the mean ± SD of 10 rats. *p < 0.01 vs. sham group. (A) Body weight at week 4 post-OVX or -sham procedure. (B) BW of OVX rats were treated with E2 or CGA for 12 wks. *p < 0.05 vs. sham group. (C) Uterine weight of OVX rats given E2 or CGA. *p < 0.01 vs. sham group.
Fig 2
Fig 2. Effects of E2 and CGA on biochemical markers of bone remodeling in OVX rats.
Rats were treated for a total of 12 wks. Each column represents the mean ± SD of 10 rats. *p < 0.01 vs. sham group. ▼decrease vs. OVX group (p < 0.01) ▲ increase vs. OVX group (p < 0.01). (A)S-Ca, S-P, U-Ca, and U-P of OVX rats in response to E2 and CGA. (B) Urinary DPD/Cr ratio of OVX rats in response to E2 and CGA. (C) Serum OC of OVX rats in response to E2 and CGA. (D) Serum ALP activity of OVX rats in response to E2 and CGA.
Fig 3
Fig 3. Effect of E2 and CGA on BMD in OVX rats.
Each column represents the mean ± SD of 10 rats. *p < 0.01 vs. sham group. ▲ increase vs. OVX group (p < 0.01).
Fig 4
Fig 4. Positive effects of E2 and CGA on parameters of bone microarchitecture in OVX rats.
*p < 0.01 vs. sham group. (A–F) μCT images showing representative trabecular bone microarchitecture of the femoral metaphysis for each treatment group. (G–K) Effects of E2 and CGA on BV/TV, Conn.D, Tb.N, and Tb.Th in OVX rats. ▲ increase vs. OVX group (p < 0.05). (L and M) Effects of E2 and CGA on SMI and Tb.Sp in OVX rats. ▼decrease vs. OVX group (p < 0.05).
Fig 5
Fig 5. Effect of CGA on osteoblast differentiation of rat BMSCs.
Each column represents the mean ± SD of optical density values. CON (+) cells were exposed to ODM without CGA. 0 μM group represents cells exposed to neither ODM nor CGA. *p < 0.01 vs.
Fig 6
Fig 6. Effect of CGA on proliferation of rat BMSCs.
(A) MTT assay of cell viability. Each column represents the mean ± SD of optical density values.*p < 0.05 vs. 0 μM group. (B) Western blots of p-Akt, Akt, and cyclin D1expression in cells exposed to CGA. β-Actin was used as the loading control. (C) Viability in cells exposed (+) to 10 μM CGA and 2 μM LY294002 (PI3K/Akt inhibitor). Each column represents the mean ± SD of the optical density values. *p < 0.05 vs. control (–/–) group. ★p < 0.05 vs. CGA+/ LY294002+ group. (D) Western blot of cyclin D1 expression in cells exposed (+) to 10 μM CGA and 2 μM LY294002. β-Actin was used as the loading control.
Fig 7
Fig 7. Effect of Shp2/PI3K/Akt/cyclin D1 pathway inhibition on CGA-induced upregulation of osteoblast differentiation in rat BMSCs.
(A) Viability in cells exposed to 10 μM Shp2 RNAi and 10 μM CGA. Each column represents the mean ± SD of the optical density values. *p < 0.01 vs. control (con) group. ▲ p < 0.01 vs. RNAi group. (B) Western blots of Shp2, p-Akt, cyclin D1, and Akt expression in cells exposed (+) to 10 μM CGA and RNAi. β-actin was used as the loading control.
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Grants and funding

This work was supported by National Natural Science Foundation of China (No: 81160508, 81260401, 81460342) and the Natural Science Foundation of Jiangxi Province (No: GZY0170).
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