Analysis of anti-osteoporosis function of chlorogenic acid by gene microarray profiling in ovariectomy rat model

Postmenopausal osteoporosis is a worldwide problem that affects more than 200 million people [1]. The main cause of the postmenopausal osteoporosis is the change in hormonal levels. Low estrogen can reduce bone resorption by inhibiting osteoclast development as well as inducing osteoclast apoptosis [2,3]. In addition, estrogen deficiency is also involved in bone remodeling and then leads to bone mineral density (BMD) loss, a phenomenon which associates with higher risks of fractures and disability [4–6]. Hormone replacement therapy (HRT) as well as antiresorptive or anabolic drugs have been widely used in the prevention and treatment of the postmenopausal osteoporosis. However, long-term HRT might give rise to severe side effects, such as impairment of the normal bone metabolism and atypical fractures [7].

Therefore, more researchers focussed on exploiting natural compounds which had fewer side effects to treat the osteoporosis [8,9]. Previous studies reported that Soy foods could partially reverse ovariectomy (OVX)-induced osteopenia in both rats and women, which had a similar effect to the traditional drug raloxifene [10,11]. The plant-derived compounds with antioxidant activities could prevent bone loss by promoting osteoclastic differentiation [12,13].

Chlorogenic acid (CGA) was the most abundant polyphenol in Chinese herb Du zhong and these polyphenolic compounds had exhibited osteoprotective activity [14–16]. CGA could be widely found in many other plants and had numerous biological effects, such as antiapoptosis, antiobesity, and antitumor activities [17]. In our previous study, we found that CGA could effectively promote the BMD via the Shp2/PI3K/Akt pathway in OVX rat models [18]. However, the potential mechanism of the effective genes or functional pathways remained uncertain.

In the present study, we verified that CGA could prevent OVX-induced osteoporosis in rats’ models based on our previous study. Gene microarray profile was used for searching different gene expression and effective signal pathway from BMCs samples of CGA-treated OVX rats. The mitogen-activated protein kinase (MAPK) pathway was found by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, which might be worthy of study in the future.

The study protocol was approved by the Institutional Animal Care and Use Committee of Nanchang University. Eighteen six-week-old Sprague–Dawley, specific-pathogen free, female rats (body weight (BW): 243.66 ± 3.89 g) were housed at 23–25°C (air conditioned) under a 12-h light/dark cycle, housed with the multiple standard rodent cages (545 × 395 × 200 mm) in a cage. Ethical approval for the use of animals in the present study was granted by the Animal Research Ethics Committee of China. During the experimental period, all rats were allowed free access to distilled water and standard rat chow. The stable baseline and circadian variation in temperature and activity were monitored everyday. Acclimatized rats underwent either bilateral laparotomy (sham, n=6) or bilateral OVX (n=12) after being anesthetized with an intraperitoneal injection with 3% sodium pentobarbital (5 mg/kg/day). After the surgical procedures, sham rats were fed similar to the OVX rats to minimize differences in BW between the two surgical groups. Next, the OVX rats were divided randomly into two treatment subgroups (n=6 per group): OVX plus saline vehicle (CMC-Na) and OVX plus 45 mg/kg/day CGA (≥gical groups), Sigma–Aldrich, U.S.A.), respectively (OVXT). CGA was administered orally through a commerce-made gastric tube and treatments started on day 2 post-OVX and continued for 12 weeks. The BWs of animals were recorded weekly during the experimental period.

According to the previous method, BMD was calculated using the 2D total bone mineral content (t-BMC) of the measured area and reported as g/cm³ [19]. Each rat’s right femur was measured by dual-energy X-ray absorptiometry (DXA) with the Lunar Prodigy Advance system (GE Healthcare, U.S.A.) equipped with appropriate software for bone density assessment in small animal’s laboratory. After that, we selected three representative right femurs from each group to evaluate the trabecular micro-architecture of the femoral metaphysis by micro-computed tomography (micro-CT) (Scanco Medical, Zu¨rich, Switzerland). Selection of the representative samples was based on the median value of t-BMD of the respective group [20]. Because the trabecular bone is rich in the distal femur compared with the proximal and middle regions, scan was performed from the proximal growth plate in the distal direction (18 μm/slice) for every selected femur sample. The volume of interest (VOI) was selected as a cross-sectional area spanning 100 slices from the proximal growth plate. This scanning generated 3D images of the micro-architecture that were examined and displayed. Bone morphometric parameters obtained from the CT, including the bone volume fraction (bone volume/tissue volume (BV/TV)), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), and structure model index (SMI), were analyzed for the VOI. The operator conducting CT analysis was blinded to the treatments associated with the specimens. All examinations were conducted according to the principles and procedures described in the most recent National Research Council publication of the Guide for the Care and Use of Laboratory Animals and referred to the ARRIVE guidelines [21].

All animal experiments complied with the animal protocols approved by the Institutional Review Board of the Second Affiliated Hospital of Nanchang University. Both femurs and tibias were dissected. Bone marrow cells were flushed out with PBS in a 5-ml syringe fitted with a gauge needle. Mononucleated cells were isolated by density gradient centrifugation in rat lymphocyte separation medium (Solarbio, Beijing, China) at a concentration of 1.091 g/ml. Isolated cells were seeded in standard low-glucose Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Shanghai Bioleaf Biotech Co., Ltd., China), supplemented with 15% FBS (HyClone, Shanghai, China), and then cultured in a humidified incubator with 5% CO₂ at 37°C. Then CD33 and CD44 marker was used to identify the bone marrow mesenchymal stem cells (BMSCs) and the specimens were sent to gene assay profile to find the differentially expressed genes.

Microarray hybridization, scanning, and analysis were performed by Shanghai Biotechnology Corporation (Shanghai, People’s Republic of China). Briefly, each slide was hybridized with 1.65 μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Catalog# 5188-5242, Agilent Technologies, Santa Clara, CA, U.S.A.) in Hybridization Oven (Catalog# G2545A, Agilent Technologies, Santa Clara, CA, U.S.A.), according to the manufacturer’s instructions. After 17 h of hybridization, slides were washed in staining dishes (Catalog# 121, Thermo Shandon, Waltham, MA, U.S.A.) with Gene Expression Wash Buffer Kit (Catalog# 5188-5327, Agilent Technologies, Santa Clara, CA, U.S.A.), following the manufacturer’s instructions. Slides were scanned by Agilent Microarray Scanner (Catalog# G2565CA, Agilent Technologies, Santa Clara, CA, U.S.A.) with default settings, dye channel: green, scan resolution = 3 μm, PMT 100%, 20 bit. Data were extracted with Feature Extraction software 10.7 (Agilent Technologies, Santa Clara, CA, U.S.A.). Raw data were normalized by Quantile algorithm, limma packages in R. For comparison analysis, a two-class unpaired method in the Significant Analysis of Microarray software was applied to identify significantly differentially expressed genes between the two selected groups.

Differentially expressed genes were identified by statistically significant differences (P<0.05) in the microarray probe sets between the groups using the Mann–Whitney nonparametric test. Hierarchical cluster analysis was performed on each comparison to assess correlations amongst samples for each identified gene set. Initially identified candidate genes from the microarray were further assayed by RT-PCR analysis. Briefly, total RNA was extracted using TRIzol reagent (Catalog# 15596-018, Life Technologies, Carlsbad, CA, U.S.A.) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, U.S.A.). Qualified total RNA was further purified by RNeasy mini kit (Catalog# 74106, Qiagen, GmBH, Germany) and RNase-free DNase set (Catalog# 79254, Qiagen, GmBH, Germany). All PCR assays were repeated three times. For RT-PCR results, the relative amount of expressed mRNA was calculated by comparison with the expression of the housekeeping gene in the same sample. Using the 2^−ΔΔC_t method, we present the data as the fold change in gene expression normalized and relative to the expression in the control group.

The access to KEGG is through the GenomeNet website at http://www.genome.ad.jp/kegg/ [22]. The SOAP server is used for computerized access to KEGG at http://www.genome.ad.jp/kegg/soap/.

Data were analyzed using SPSS software from IBM (version 13.0, U.S.A.). Student’s ttest and Fisher’s exact test were used, as appropriate. Results were expressed as mean ± S.D. and a P-value <0.05 was considered to indicate statistical significance.

Three-dimensional images of femoral metaphyses generated by micro-CT showed differences in trabecular micro-architecture amongst the various treatment groups (Figure 1). Analysis of data from the representative samples indicated that OVX decreased trabecular BV/TV (Sham: 19.97 ± 3.40%; OVX: 2.70 ± 0.33%), Tb.N (Sham: 2.32 ± 0.44 mm; OVX: 0.27 ± 0.01 mm), and Tb.Th (Sham: 0.08 ± 0.01 mm; OVX: 0.02 ± 0.01 mm) (all P<0.05), compared with the parameters obtained for the sham group. By contrast, SMI (Sham: 1.73 ± 0.22; OVX: 2.66 ± 0.08, P<0.05) in the proximal femur was increased in response to OVX. In addition, BMD was decreased (Sham: 0.28 ± 0.04 g/mm³; OVX: 0.07 ± 0.01 g/mm³; P<0.05). Then, all of these OVX effects were partially reversed in OVXT groups ((BV/TV (OVX: 2.70 ± 0.33%; OVXT: 7.20 ± 0.22%), Tb.N (OVX: 0.27 ± 0.01 mm; OVXT: 0.50 ± 0.09 mm), and Tb.Th (OVX: 0.02 ± 0.01 mm; OVXT: 0.04 ± 0.01 mm), SMI (OVX: 2.66 ± 0.08; OVXT: 2.19 ± 0.04), BMD (OVX: 0.07 ± 0.01 g/mm³; OVXT: 0.14 ± 0.02 g/mm³), all P<0.05 compared with OVX group) (Table 1).

Using Affymetrix GeneChip HG-U133 Plus 2.0, we analyzed global gene expression changes in BMSCs samples from six rats (three from OVX group and three from OVTX group). Genes were identified as being significantly regulated between groups when the unpaired ttests corrected for multiple comparisons had a false discovery rate ≤5%. A total of 121 genes were identified as differentially expressed between OVX and OVXT groups (fold change >2 and P<0.05) (Figure 2 and Supplementary Table S1). Amongst them, the top five most differential mRNAs were RGD1564664, Pdlim3, Myh11, Ear11, and Nnat Four genes (Acss2, Cd24, Nnat, and Pkp2) were further screened by fluorescence quantitative PCR to validate the microarray result. For all the four genes, changes in expression observed by RT-PCR agreed favorably with the microarray data (Figure 3).

KEGG is a bioinformatics resource for understanding higher order functional meanings and utilities of the cell or the organism from its genome information. According to general gene expression profiling in the present study and network searching in official KEGG websites, five genes including Hspa1a, Fgfr2, Gadd45a, Tgfb3, and Hspa1b were mapped on MAPK pathway and considered worth to further analyze potential functional effects on bone remodeling (Supplementary Figure S1 and Table S2). Next, we chose the classical proteins of MARK pathway to validate the bioinformatics results and we found that phosphorylations of ERK and p38 was increased in the OVXT group compared with the OVX group (Figure 5).

In the present study, we evaluated the specific parameters of femur micro-architecture to identify the CGA’s osteogenic effects in rats [21]. As expected, OVX resulted in a decreased femur BMD after 12 weeks and loss of bone mass was accompanied by a significant increase in bone remodeling. Although inhibition of bone remodeling would be considered beneficial, the bone function might be decreased if bone remodeling was inhibited for an excessive period of time. For instance, treatment with pamidronate (14 mg/kg/day for 25 days) could decrease intrinsic diaphyseal bone strength in rats [24]. Preservation of the trabecular micro-architecture contributed to bone strength and might reduce fracture risk [25–27]. The SMI was used to distinguish between rods and plates in trabecular bone. Analysis of our results indicated that there was a significant transition of trabecular structures from rods to a mixed plates-and-rods by OVX. However, CGA treatment could partially restore the trabecular bone [28].

Global gene expression profiling can be used to explore functional gene pathways. In the current study, we used global gene transcription analysis to identify gene changes in BMSCs derived from OVX and OVXT groups. A total of 36 genes with a threshold of a two-fold change and P<0.05 have been identified. We further validated four genes (Acss2, Cd24, Nnat, and Pkp2) by RT-qPCR and the similar trends suggested the results of global gene expression profiling were reliable.

Next, we found that Hspa1a/Fgfr2/Gadd45a/Tgfb3/Hspa1b genes were mapped on MAPK pathway by KEGG analysis. Previous study reported that CGA could affect the expression of apoptosis-related genes that were part of oxidative stress and p38 MAP-dependent pathways [29]. CGA could also counteract AP-induced liver injury by preventing apoptosis and oxidative stress damage, and more specifically, the MAPK signaling cascade appeared to be engaged in this protective mechanism [30]. Bone remodeling is also an important course during antiosteosis process which may be regulated by MAPK pathway. Strontium could promote osteogenic differentiation of MSCs through activating the Ras/MAPK signaling pathway and the downstream transcription factor Runx2 [31]. A similar study pointed out that sesamin had the ability to trigger osteoblast differentiation by activation of the p38 and ERK-MAPK signaling pathway and possibly indirectly regulated osteoclast development via the expression of OPG and RANKL in osteoblasts [32]. MAPK signaling pathway also might be involved during bone mechanism process. Researchers indicated strain-induced osteogenic differentiation of OVX BMSCs might be regulated by ERK1/2-MAPK signaling pathway [33]. In this study, we found that CGA administration in the OVX group could involve in the MARK signaling pathway by regulating the phosphorylation of p38 and ERK.

In our previous study, we induced the BMSCs to differentiate to the osteoblast and we found that CGA could prevent osteoporosis by Shp2/PI3K/Akt pathway [18]. However, we only used BMSCs as our models and our goal was to identify the gene changes in BMSCs. The limitation of our work was that we assumed that the changes of gene expression in BMSCs could directly regulate the anti-osteoporosis function, which needed to be validated by more experiments in the further investigations.

In summary, we found that CGA could effectively counteract osteoporosis in OVX rat models. Microarray results combined with bioinformatics analysis suggested that the MAPK pathway might be valuable for the mechanism research in future.

The authors declare that there are no competing interests associated with the manuscript.

J.M. performed the research and wrote the paper. Z.Y. and Q.Z. performed the research. S.L. and K.W. analyzed the data. J.L. designed and supervised the experiment process.

This work was supported by the National Natural Science Foundation of China [grant numbers 81160508, 81260401, 81460342]; and the Natural Science Foundation of Jiangxi Province [grant number GZY0170].

Akt

protein kinase B

AP

acetaminophen

BMD

bone mineral density

BMSC

bone marrow mesenchymal stem cell

BV/TV

bone volume/tissue volume

BW

body weight

CGA

chlorogenic acid

CMC-Na

saline vehicle

HRT

hormone replacement therapy

KEGG

Kyoto Encyclopedia of Genes and Genomes

MARK

mitogen-activated protein kinase

micro-CT

micro-computed tomography

OPG

osteoclastogenesis inhibitory factor

OVX

ovariectomy

OVXT

ovariectomy plus CGA treatment

PI3K

phosphoinositide 3-kinase

PMT

a parameter in the microarray scanner

RANKL

receptor activator of nuclear factor kappa-B ligand

RIN

RNA integrity number

RT-PCR

reverse transcription-polymerase chain reaction

shp2

SH domain-containing protein tyrosine phosphatase 2

SMI

structure model index

Tb.N

trabecular number

Tb.Th

trabecular thickness

VOI

volume of interest