RAGE pathway

Accepted Manuscript Irbesartan attenuates advanced glycation end products-mediated damage in diabetes-associated osteoporosis through the AGEs/ RAGE pathway

Yan-Zhen Cheng, Shuang-Li Yang, Ji-Yu Wang, Meng Ye, XiaoYun Zhuo, Li-Tao Wang, Hong Chen, Hua Zhang, Li Yang PII: DOI: Reference:

S0024-3205(18)30223-6 doi:10.1016/j.lfs.2018.04.042 LFS 15677

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

11 February 2018 22 April 2018 23 April 2018

Please cite this article as: Yan-Zhen Cheng, Shuang-Li Yang, Ji-Yu Wang, Meng Ye, Xiao-Yun Zhuo, Li-Tao Wang, Hong Chen, Hua Zhang, Li Yang , Irbesartan attenuates advanced glycation end products-mediated damage in diabetes-associated osteoporosis through the AGEs/RAGE pathway. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi:10.1016/ j.lfs.2018.04.042

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ACCEPTED MANUSCRIPT Irbesartan attenuates advanced glycation end products-mediated damage in diabetes-associated osteoporosis through the AGEs/RAGE pathway

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Yan-Zhen Chenga,&, Shuang-Li Yanga,b,&, Ji-Yu Wanga, Meng Yea,c·

These authors contributed equally to this work

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&

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* Corresponding author

Department of Endocrinology, Zhujiang Hospital of Southern Medical University,

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a

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Xiao-Yun Zhuoa, Li-Tao Wanga, Hong Chena , Hua Zhanga,*, Li Yanga,*

Guangzhou, Guangdong, P.R. China

Department of Endocrinology,Second Affiliated Hospital of GuiZhou Medical

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b

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University, Kaili , Guizhou, P.R. China Department of Endocrinology,Affiliated Hospital of GuiZhou Medical

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University,Guiyang,Guizhou, P.R. China

Correspondence to: Professor Li Yang, Department of Endocrinology, Zhujiang Hospital of Southern Medical University,253 Industrial Avenue, Guangzhou, Guangdong 510280, P.R. China E-mail: [email protected]

Telephone number: +86 13924207196

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ACCEPTED MANUSCRIPT Professor Hua Zhang, Department of Endocrinology, Zhujiang Hospital of Southern Medical University,253 Industrial Avenue, Guangzhou, Guangdong 510280, P.R. China Telephone number: +86 13711170617

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E-mail:[email protected]

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ACCEPTED MANUSCRIPT Abstract: Aims: Diabetes-associated osteoporosis are mainly caused by the formation and accumulation of advanced glycation endproducts(AGEs). Angiotensin II type 1 receptor blocker (ARB) has anabolic bone effects

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on the physicochemical properties of the bone in diabetes. We

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hypothesized that ARB could inhibit AGEs-induced deleterious effects.

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Main methods: In this study, we chose seven-week-old Leprdb/Lepr+ (db/+) and Leprdb/Leprdb (db/db) mice. After 12 week intervention by

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irbesartan, the microarchitecture and mechanical strength of the bone of

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seven-week-old db/db mice were investigated systematically. Meanwhile, the molecular mechanisms of the osteoblasts were analyzed, after AGEs

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or irbesartan were added to the culture. Also, intracellular formation of

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reactive oxygen species (ROS) was measured with DCF fluorescence. Key foundings: Results showed that 12-week irbesartan treatment could improve

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dramatically

trabecular

bone

microarchitecture

through

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increasing BV/TV (p=0.003, +46.7%), Tb.N (p=0.020, +52.0%), and decreasing that of Tb.Sp (p=0.005, -21.2%) and SMI (p=0.007, -26.4%), compareing with the db/db group. Irbesartan could also substantially raise biomechanical parameters including max load (p=0.013, +20.7%), fracture load (p=0.014, +70.5%), energy absorption (p=0.019, +99.4%). Besides, it could inhibit AGEs-induced damage of cell proliferation and osteogenic differentiation of osteoblasts, as well as suppressing the 3

ACCEPTED MANUSCRIPT activation of apoptosis caused by AGEs. Moreover, co-incubation with irbesartan could prevent the AGEs-induced increase of intracellular oxidative stress and RAGE expression in osteoblasts. Significance: In conclusion, this study suggested that irbesartan might

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play a protective role in diabetes-related bone damages by blocking the

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deleterious effects of AGEs/RAGE-mediated oxidative stress. This may

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provide a revolutionary benefits to therapy with irbesartan on diabetic osteoporosis.

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Key words: Irbesartan; AGEs;Diabetes mellitus; osteoporosis

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ACCEPTED MANUSCRIPT Introduction Diabetes mellitus is one of the most common diseases in the world. So far, there are 382 million people with diabetes worldwide and this number will rise to 592 million in the year 2035 [1]. Increasing number of

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evidence identifies diabetes as a risk factor of low bone mass and

characteristics

in

terms

of

collagen

posttranslational

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structural

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fractures[2]. Hyperglycemia deteriorates bone material properties and

modification such as enzymatic immature and mature cross-links and

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nonenzymatic advanced glycation end products (AGEs) formation.

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AGEs, the end product of Maillard process, are covalent compounds of macroprotein derivatives , which are formed through a non-enzymatic

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reaction among reducing sugars, amino groups of proteins, lipids and

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nucleic acids. The formation of AGEs are progressed at an accelerated rate under the diabetes condition and its accumulation contributes to the

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aging of macromolecules[3]. AGEs dramatically increase bone fragility

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and impair bone quality of diabetes[4]. It was reported that AGEs could impair bone formation by attenuating the differentiation of osteoblastic lineage[5], inducing osteoblasts' apoptosis[6], inhibiting the expression of osteoblast-specific transcription factors and decreasing mineralization[7] in vitro. Recently, academics have raised more concern about Renin-angiotensin system’s (RAS) correlation with bone health, structure and metabolism, 5

ACCEPTED MANUSCRIPT while various studies have been carried out to examine the role of RAS components on bone density and fractures risks. RAS acts on bone microenvironments both systemically and locally through classical angiotensin-converting enzyme (ACE)/angiotensin II (AngII)/angiotensin

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type-1 receptor (AT1R) axis [8, 9]. The relation between the RAS and

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bone metabolism is mainly based on the regulation of AngII on bone

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metabolism[10]. AngII could promote bone resorption via Ang II type I (AT1) and type II (AT2) receptors[11, 12] and the blockage of either of

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these receptors was supposed to ameliorate differentiation and bone

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formation in cell culture and in ovariectomized animal models[11, 13]. Interestingly, Donmez et al[14] reported that Losartan, an AT1 receptor

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blocker (ARB), had a therapeutic effect on the physicochemical

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properties of diabetic rat bone,leading to the improvement of bone strength at the material level. However, current findings about the effects

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of RAS on bone in diabetes are still limited and controversial. In other

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animal studies, ARB did not effectively reverse bone injuries in type I diabetic mice model[15]. Also, the mechanism of how ARB can affect the bone in type II diabetes is unclear. Thus, it is necessary to explore the ARB's effect on diabetes-associated osteoporosis and the possible pathophysiological mechanism further. Based on current evidence, which suggests that ARB could inhibit AGEs formation or accumulation in diabetic animal model [16, 17], we hypothesized that ARB attenuates 6

ACCEPTED MANUSCRIPT AGEs-mediated damage in diabetes-associated osteoporosis through its receptor(RAGE) pathway. The study was designed to assess the theory that irbesartan may be able to cancel the AGEs-induced deleterious effects on animal model and

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osteoblasts, by conducting a series of experiments and analyzing the bone

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mass and strength of db/db mice, a common-used animal model for type

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II diabetes. Materials and Methods

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Animal

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All experiments performed were endorsed by the Animal Ethics Committee of the Southern Medical University. Seven-week-old male

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Leprdb/Lepr+ (db/+) and Leprdb/Leprdb (db/db) mice were purchased

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from the Model Animal Research Center of Nanjing University. The previous research indicated that bone metabolism could be affected by

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estrogen[18]. Therefore, we excluded female mice in our original

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proposal to prevent the influence of estrogen. Mice were maintained in the Laboratory Animal Center of Southern Medical University under controlled temperature, light, and humidity. After one week of acclimatization, the mice were divided into 3 group, with 12 mice in each group: i) db/+ group (Vehicle-treated db/+ mice ); ii) db/db group (Vehicle-treated db/db mice); iii) Irbe group (db/db mice-treated with irbesartan in a concentration of 50 mg/kg/day). Mice were anesthetized at 7

ACCEPTED MANUSCRIPT twenty weeks of age by 2.5% pentobarbital sodium. The tibias and femurs of the mice were immediately harvested for a series of analyses. Analysis of bone structure All specimens were scanned by SHARP micro-CT scanner at a 20μm

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nominal resolution and analyzed by associated analysis software

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(ZKKS-MCT, SHARP, Japan). Images obtained were reconstructed based

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on Feldkamp convolution back-projection algorithm and segmented into binary images (8-bit BMP images) using adaptive local threshold.

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Analyses of the trabecular bone architecture were carried out in a

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2.5-mm-thick region, 1.2 mm distal to the growth plate of the knee joint. The same threshold setting was applied to all the samples to segment the

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trabecular bone from the background. The following parameters were BMD(Volumetric

Bone

Volume/Total

Volume,

BV/TV(Bone

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calculated:

Mineral BV/TV),

Density,

BMD),

Tb.Th(Trabecular

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Thickness, Tb.Th), Tb.Sp(Trabecular Spacing, Tb.Sp), Tb.N(Trabecular

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Number, Tb.N), SMI (Structure Model Index, SMI) and Ct.th (Cortical Thickness, Ct.th). Biomechanical testing Prior to biomechanical examination, the left femurs were thawed for 3 h at room temperature. The length between the inter-malleolar region and the inter-condylar region was measured to identify the midpoint of the diaphysis. The femur was then placed on two supports, which were 8

ACCEPTED MANUSCRIPT separated by a distance of 20 mm in the material testing machine (Instron ElectroPuls, E1000, USA), and a load was hanged at the midpoint of the diaphysis, creating a three-point bending test. The biomechanical quality of the right femoral diaphysis was examined at a loading speed of 2

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mm/min. When the central loading point was displacing, the load and

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displacement were recorded until the specimen was broken. Based on the

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load-deformation curve, the maximum load (ultimate strength), the maximum displacement, the stiffness (slope of the linear part of the curve,

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representing elastic deformation), and energy absorption (area under the

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curve) were obtained. Materials.

Annexin-V

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(DCFH-DA),

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Irbesartan, Trypsin-EDTA, 2',7'-dichlorodihydrofluorescein diacetate FITC

3-(4,5-dimethylthiazozyl)-2,5-diphenyl

Apoptosis

detection

tetrazolium bromide

kit, (MTT),

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Propidium Iodide, Ribonuclease A and Alizarin Red were all obtained

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from Sigma (St. Louis, MO, USA); JC-1 Dye-Mitochondrial Membrane Potential Probe was purchased from Thermo Fisher Scientific(Life Technologies, Grand Island, NY); PrimeScript® one-step RT-PCR kit and SYBR® Premix Ex Taq™ II were from Takara Biotechnology (Dalian, China); Mouse anti-receptor for advanced glycation end-products (RAGE) antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); Rabbit anti-β-actin antibody was from Cell Signaling Technology 9

ACCEPTED MANUSCRIPT (CST, Danvers, MA, USA); All other chemicals and reagents were purchased from commercial sources and were of analytical grade. AGEs-BSA preparation AGEs-BSA was produced by an incubation of 100mg/ml BSA

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with100mM ribose in 150mM PBS, under sterile conditions, pH 7.4 at

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37°C for 3 weeks. Unincorporated sugars were removed by PD-10

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column chromatography and dialysis against phosphate-buffered saline. Controlled non-glycated BSA was incubated in the same conditions

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except for the absence of reducing sugars. The AGEs-BSA was passed

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through a Detoxi-Gel column to wash off contaminated endotoxin. Endotoxin levels in the preparation were estimated using the amebocyte

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lysate assay kit, which were found to be below 0.25EU/ml. The formation

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of AGEs was assessed by their characteristic fluorescence-emission, maximum at 420 nm upon excitation at 340 nm.

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Primary osteoblasts culture

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Neonatal Sprague-Dawley rats’ calvaria were removed under sterile conditions. Attached connective tissues were shed off. The bones were sawed into pieces of 2 to 5 mm2, digesting within 0.25% trypsin-EDTA. By adding FBS to the solution, the digestion was stopped and then the supernatant was discarded. Subsequently, the moderate fragments were inoculated into the bottom of a culture flask, which was then incubated at 37°C in a humidified atmosphere containing 5% CO2. The medium was 10

ACCEPTED MANUSCRIPT refreshed every 2 days while the osteoblasts were purified by plastic adherence and expanded in culture flasks. Third or fourth passage cells were used for all experiments. MTT assay

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MTT assay was utilized to evaluate the proliferative ability of osteoblasts.

then

incubated

in

100μl

FBS-free

DMEM/F12

medium,

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and

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In a nutshell, osteoblasts in 96-well plates were washed twice with PBS

supplemented with 10μl of 5 mg/ml MTT solution at room temperature

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for 4h. Later on, the supernatant was removed and the crystals were

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dissolved by incubation with 150μl of DMSO for 20 min. The plates were shaken for 5 min. The cell viability was determined by measuring the

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absorbance at 490 nm on a 96-well plate-reader.

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Cell cycle distribution analysis

Osteoblasts (1x106 cells) with different treatments were trypsinized and

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collected by centrifugation at the speed of 900 rpm for 5 min. Then,

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osteoblasts were washed twice with cold PBS and fixed with cold 70% alcohol overnight. Cells were collected 12h later after centrifugation and washed once with cold PBS. Afterwards, the cells were resuspended in 500ul PBS and digested with 5ul 10 mg/mL RNase A for 1h at 4℃. Then, the osteoblasts were stained with 50 μg/mL PI, diluting with 0.2% Triton X-100 for 30 min at 4℃ in the dark. Of the incubation, cell cycle distribution was detected by a FACSCalibur flow cytometer (BD, 11

ACCEPTED MANUSCRIPT Franklin Lakes, NJ) and analyzed using the ModFit LT software (Verity Software House, USA). Measurement of apoptosis Apoptosis was detected with following the manufacturer’s instructions of

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the Annexin-V FITC Apoptosis Detection. Annexin V-FITC/PI stained

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cells were analyzed immediately at room temperature with a flow

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cytometer, using Cell Quest pro software (BD Biosciences, USA). Excitations of Annexin V-FITC and PI were done at 488 nm and 546nm

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respectively and emissions were detected through a 578/20-nm filter

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(FL-1) and a 647/20-nm filter (FL-3) respectively. Quadrant statistics were conducted to separate out healthy, early apoptotic, terminal

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apoptotic and dead cells from the total population of the cells. The

control group.

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healthy osteoblasts treated with serum-free media were considered as

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Determination of ROS generation

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Intracellular reactive oxygen species (ROS) generation was measured by flow cytometry with the DCFH-DA probe. Osteoblasts grown in 10-cm culture plates were subjected to various culture conditions as described above for 2 h. The medium was replaced by control medium with 10μM DCFH-DA probe for 30 min in the dark. Intracellular ROS generation was visualized under a fluorescent microscope (Olympus, Tokyo, Japan). DCF fluorescence was measured by a flow cytometer. Data were 12

ACCEPTED MANUSCRIPT standardized to the control values. MMP Measurement Mitochondrial membrane potential (MMP) was evaluated by JC-1 probe. The cells were incubated with 1ml 10ug/ml JC-1 for 20 min at 37°C. To

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estimate the relative MMP, samples were analyzed by flow cytometry and

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fluorescent microscope. Excitations of JC-1 monomers and JC-1

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aggregation were done at 514 nm and 585nm respectively and emissions were detected at 529-nm filter and 590-nm filter respectively. Compared

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with the normal condition when the mitochondrial membrane shows red

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fluorescence, the red fluorescence will decrease while the green fluorescence will increase if MMP is changing. The intensity ratio of red

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Alizarin red staining

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to green fluorescence represents the change of MMP.

Osteoblasts were plated in differentiation medium at a concentration of 1

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× 105 per well in 6-well plates. After 21 days, the medium was removed

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and alizarin red staining was performed. Next, osteoblasts were fixed with 4% paraformaldehyde after washing twice with sterile PBS. Subsequently, the cells were washed three times with 1× PBS and stained with 0.1% Alizarin Red (PH 8.3) diluting with Tris-HCL for 30 min at room temperature. Bone nodule formation was observed under a light microscopy (Olympus, Tokyo, Japan).

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ACCEPTED MANUSCRIPT Real-time PCR analysis Osteoblasts were stimulated with osteogenic differentiation medium for 1 week. They were then serum-deprived overnight and subjected to various culture conditions as described above for 3 days. After this period, total

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RNA was extracted using TRI zol reagent according to the manufacturer's

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instructions. cDNA was reverse-transcribed from 0.8 μg of total RNA

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using the PrimeScript one-step RT-PCR kit. Real-time PCR was carried out in a Real-Time PCRSystem (Stratagene/Agilent Technologies,

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Wilmington, DE, USA) using SYBR Premix Ex Taq II. The cycling

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conditions were as follows: 95˚C for 2 min and 40 cycles of 95˚C for 5 sec, 60˚C for 30 sec. The value of 2-ΔΔCt represents the relative level of

GTGGAAACCTGATGTATGCTT

and

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Forward

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target gene expression. The primers used were as follows: Collagen I: Reverse

GACTTCTGCGTCTGCGTCTGGTGATA; Alkaline phosphatase(ALP): AGATGGACAAGTTCCCCTTTG

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Forward

Runt-related

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ACACAAGTAGGCAGTGGCA;

and

Reverse

transcription

factor

2(RUNX2): Forward TGGTTACTGTCATGGCGGGTA and Reverse TCTCAGATCGTTGAACCTTGCTA;

Osteocalcin(OC):

TCACACAGGAGCTGATGACC

and

CCACAATGAACAAGTGGCTG;

RAGE:

Forward

Forward Reverse ACTCACA

GCCAAT GTCCCTAA and Reverse CTTT GCCATCA GGAATCA GAG; β-actin: Forward TTCTACAATGAGCTGCGTGTGGC and Reverse 14

ACCEPTED MANUSCRIPT CTCATAGCTCTTCTCCAGGGAGGA.

Western blot analysis The osteoblasts were stimulated with various culture conditions as

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described above for 3 days following starvation overnight in serum-free

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medium. Proteins were then extracted from the osteoblasts using RIPA

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lysis buffer followed by centrifugation at 4˚C, 12,000 rpm for 20 min. Protein concentration was determined by BCA assay. Proteins were then

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separated by SDS-PAGE and electrotransferred onto a PVDF membrane,

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which was incubated with mouse anti-RAGE (1:500) and mouse anti-β-actin antibodies (1:500) overnight at 4˚C. Then the membrane was

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washed 3 times with TBST and incubated with HRP-conjugated

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secondary IgG for 50 min. The bands were detected using the ECL chemiluminescence detection system.

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Statistical analysis

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Data were reported as mean ± SD and analyzed using SPSS for Windows version 15.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA followed by the Student-Newman-Keuls t-test or Dunnett's T3 (equal variances not assumed) was performed for multiple comparisons. Differences with a P value under 0.05 were considered to be significant statistically.

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ACCEPTED MANUSCRIPT Results Irbesartan improved bone microarchitecture of db/db mice Representative data of two-dimensional images of femoral metaphysis were shown in Figure 1A. Compared with the db/+ group, mice in the

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db/db group displayed a less, thinner and more broken trabecular bone

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microarchitecture. Irbesartan treatment dramatically alleviated these

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damages of trabecular in diabetic mice. Trabecular bone parameters including BV/TV, Tb.N, and BMD in the db/db group were significantly

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lower than those in the control db/+ group，while the values of Tb.Sp. and

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SMI were strikingly higher (Figure 1B). Irbesartan treatment statistically increased the levels of BV/TV (p=0.003, +46.7%), Tb.N (p=0.020,

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+52.0%), and decreased that of Tb.Sp (p=0.005, -21.2%) and SMI

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(p=0.007, -26.4%) compared with the db/db group that did not receive the treatment. Although the db/db mice exhibited a decrease in cortical BMD,

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irbesartan exposure did not reverse cortical bone parameters including

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Ct.Th. and cortical BMD.

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Figure 1. Effect of 12-week irbesartan treatment on (A) two-dimensional images of

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metaphysis and (B) the distal femur of structural bone parameters(BV/TV, Tb.Th., Tb.Sp., Tb.N, SMI, Ct.th., trabecular BMD, cortical BMD and total BMD) analyzed

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by mioro-CT.

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db/+, the db/+ mice group; db/db, the db/db mice group; Irbe, the db/db mice treated with irbesartan.

Values are expressed as means±SD (n=12).

Irbesartan increased bone strength of db/db mice In comparison with the db/+ group, significant decreases were observed in biomechanical parameters including max load, fracture load, stiffness 17

ACCEPTED MANUSCRIPT and energy absorption in the db/db group (Figure 2). Adoption of Irbesartan statistically enhanced femoral biomechanical structural properties, which include max load (p=0.013, +20.7%), fracture load (p=0.014, +70.5%), energy absorption (p=0.019, +99.4%). Nevertheless,

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the values of the stiffness, total displacement and yield displacement

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between the irbesartan group and the db/db group did not have significant

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difference statistically (Figure 2).

Figure 2. Effects of 12-week irbesartan treatment on femoral biomechanical structural properties in db/db mice via three-point bending test, including max load, fracture load, total displacement, yield displacement, stiffness, and energy absorption.

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ACCEPTED MANUSCRIPT db/+, the db/+ mice group; db/db, the db/db mice group; Irbe, the db/db mice treated with irbesartan. Values are expressed as means±SD (n=12).

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Irbesartan attenuated AGEs-mediated damage in proliferation

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Osteoblasts proliferation was evaluated by MTT assay. As shown in

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Figure 3A, osteoblasts were cultured in control medium, BSA (100μg/mL) and AGEs(100μg/mL) respectively for seven days, while 2μM Irbesartan

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was added in AGEs group on the fifth day. AGEs significantly inhibited

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the proliferation of osteoblasts compared with the control medium and the unmodified BSA preparation. No significant difference was found in cell

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proliferation on the first day after the Irbesartan treatment. However, as

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the duration of intervention increased, Irbesartan showed a stimulative effect on the proliferation of osteoblasts.

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Irbesartan affected cell cycle of osteoblasts

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Cell cycle distribution of osteoblasts was evaluated by flow cytometry. AGEs-treated osteoblasts had a 15.36% and 11.62% increase at both the G1 and the S phases respectivel compared with the control group, while at concomitant 26.98% decrease in the G2/M stage. On the other hand, the proportion of G0/G1 stage cells in the irbesartan+AGEs group was similar to that in control group (56.39% vs. 49.11%), which implied that irbesartan may be able to attenuate the cell cycle retardation brought by 19

ACCEPTED MANUSCRIPT AGEs. It suggested that AGEs arrested the cell cycle at the G1 stage, thus leading to the inhibition of osteoblasts proliferation， and irbesartan may ameliorate that inhibition(Figure 3B). Irbesartan attenuated AGEs-mediated osteoblasts apoptosis

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Annexin-V FITC apoptosis detection kit was used to investigate the

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apoptosis of osteoblasts (Figure 3C, 3D). Flow cytometry analysis

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showed that both the proportion of early apoptosis and the late apoptosis declined substantially under irbesartan+AGEs treatment compared to

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those of AGEs group. Only 5.62% of the cells were apoptotic in the

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irbesartan+AGEs group, which was 21.25% lower than the AGEs group. MMP was examined by JC-1 probe under a fluorescent microscope and

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through flow-cytometry analysis. As shown in Figure 3F, osteoblasts in

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AGEs group was significantly increased in green fluorescence (monomers) accompanied by a loss of red fluorescence (aggregates) in

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merged pictures. However, osteoblasts treated with irbesartan showed

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normal level of JC-1 staining. Consistently, quantitative analysis using flow cytometry also demonstrated the reversible role of irbesartan in the disruption of membrane potential (ratio of red and green fluorescence) induced by AGEs (Figure 3E).

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Figure 3. Irbesartan attenuated AGEs-induced osteoblast proliferation and apoptosis. (A) Cell proliferation was measured by the absorbance at 490 with a MTT assay.

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Osteoblasts were treated with different medium for 3 days(A).

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(B) Cell cycle was evaluated by flow cytomety. AGEs induced osteogenic cell cycle arrest in G1 stage and irbesartan improved it.

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(C,D) Osteoblast apoptosis was assessed by Annexin-V FITC apoptosis detection. different medium.

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Osteoblasts were treated with

(E,F) MMP was examined by JC-1 probe under fluorescent microscope(D) and with flow-cytometry analysis(C). The intensity ratio of red to green fluorescence represents the change in MMP. In fluorescent microscopic images, red dot-like images reflecting JC-1 aggregation within the mitochondria were observed in cells with high ΔΨ , and in cells with low ΔΨ, the red color turned into diffuse green fluorescence, reflecting the monomeric state of JC-1. 21

ACCEPTED MANUSCRIPT Ctrl: control group; BSA: Cell treated with BSA (100μg/ml); AGEs: Cell treated with AGEs(100μg/ml); Irbe: Cell treated with irbesartan(2.0 μM)+ AGEs(100μg/ml). Values are means ± SD from three independent experiments.

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Irbesartan attenuated AGEs-mediated damage in differentiation

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Alizarin staining was performed after 21 days of osteogenic culture to

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assess mineral deposition, which is considered as the late marker of functional mature osteoblast. Calcium node was not spotted in the

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AGEs-treated osteoblasts. However, enhanced alizarin deposition

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presented after adding irbesartan (Figure 4A). Similar results were obtained when studying transcription levels of osteoblast differentiation

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markers including ALP, collagen I, RUNX2, and OC (Figure 4B).

Figure 4 Irbesartan attenuated AGEs-mediated damage in osteoblast differentiation (A) Alizarin red staining, which represented the mineralization levels of later stages of osteogenesis, was performed at the indicated time points. (B) The mRNA expression of osteogenic differentiation markers (Runx2, ALP, Col-1,OC) of osteoblasts in different group assessed by real-time PCR. 22

ACCEPTED MANUSCRIPT Ctrl: control group; BSA: Cell treated with BSA (100μg/ml); AGEs: Cell treated with AGEs(100μg/ml); Irbe: Cell treated with irbesartan(2.0 μM)+ AGEs(100μg/ml). Values are means ± SD from three independent experiments.

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Irbesartan attenuated AGEs-mediated oxidative stress damage in

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osteoblasts

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Oxidative stress was examined by the detection of DCF fluorescence. As Figure 5A and B show, DCF fluorescence of AGEs group strikingly

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increased compared with the control group. The flow cytometry analysis

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results suggested that AGEs enormously increased the ROS content in osteoblasts, while irbesartan protected osteoblast from ROS increasing.

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in osteoblasts

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Irbesartan attenuated AGEs-mediated increase of RAGE expression

RAGE protein and mRNA levels of osteoblasts in AGEs group were

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significantly higher than those in control group (Figure 5C,D). After

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treatment with irbesartan, the relative RAGE mRNA level was 1.99±0.12, significantly lower than those in AGEs group (P=0.044). Irbesartan decreased the RAGE expression in the protein and mRNA levels of diabetic mice femur As shown in Figure 5E and F, the db/db mice group showed a significant rise in RAGE expression both at protein and mRNA levels, On the contrary, the level of RAGE expression dropped after 12-week treatment 23

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with irbesartan.

Figure 5. Effect of irbesartan on the AGEs-mediated ROS and RAGE expression. different medium. Intracellular ROS

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(A,B) Osteoblasts were incubated with

generation was measured with the probe DCFH-DA, and visualized using a

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fluorescent microscope.

(C) RAGE mRNA levels were detected by PCR in osteoblasts incubated with

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different medium.

(D) RAGE Protein levels were detected by western blot analysis in osteoblasts incubated with different medium. (E) RAGE mRNA levels of the femur from mice were detected by PCR. (F) RAGE Protein levels of the femur from mice were detected by western blot analysis. Ctrl: control group; BSA: Cell treated with BSA (100μg/ml); AGEs: Cell treated with 24

ACCEPTED MANUSCRIPT AGEs(100μg/ml); Irbe: Cell treated with irbesartan(2.0 μM)+ AGEs(100μg/ml). db/+, the db/+ mice group; db/db, the db/db mice group; Irbe, the db/db mice treated with irbesartan.

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Values are means ± SD from three independent experiments.

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Discussion

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Various studies have been carried out to examine the role of RAS components on bone density and fractures risks, and the RAS-induced

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osteoporosis which was demonstrated independently of hypertension[19].

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Moreover, plentiful evidence from epidemiology gives proofs that ARB is able to decrease the risk of fractures and enhance the bone mass[11, 20].

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ARB could inversely influence osteoclastic activity by suppressing the

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AngII–induced up-regulation of Receptor Activator for Nuclear Factor-κ B Ligand(RANKL) [12]. Growing evidence indicates that ARB could

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also attenuate diabetes-induced osteoporosis, for example, the losartan, a

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kind of ARB, was proofed to be capable to improve the physicochemical properties of the diabetic rats through increasing bone strength at the material level [14]. Yet ARB’s effects on bone in diabetes are still limited and controversial, as well as the mechanism of ARB ameliorated bone formation in diabetes-related osteoporosis remains unknown. This study reported that blockade of the RAS by irbesartan might play a protective role against bone injury in diabetes through attenuating the deleterious 25

ACCEPTED MANUSCRIPT effects of AGEs via down-regulation of RAGE. The mutation of leptin receptors in db/db mice leads to the break down of satiety and insulin-sensitizing signals from leptin, thus the deficient mice are hyperphagic and suffer from obesity and type 2 diabetes[21, 22].

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Furthermore, the db/db mouse has a decreased peripheral bone volume in

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both the trabeculae and cortices of the tibiae and vertebral bones[23],

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making it an excellent model for osteoporosis. Through this study, accordant findings were presented that there were a significant

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deterioration of bone microarchitecture and a decrease of bone strength of

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the hind limbs of the db/db mice [23]. Moreover, the cancellous bone microarchitecture, rather than the cortical bone thickness, was improved

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after 12-week irbesartan treatment for the db/db mice, which was

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discovered for the first time.

The study illustrated the extrinsic whole-bone structural parameters of the

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db/db mice, including maximum load, fracture load, stiffness, and energy

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absorption, which were enhanced after 12-week irbesartan exposure, implying an improved mechanical integrity and enhanced fracture resistance capacity. According to evidence, the risk factors for bone fracture includes BMD, bone structure, porosity and physicochemical properties of bone components (e.g., mineral and collagen)[24]. The therapeutic effect of irbesartan reducing the risk of bone fracture might be associated with recovering bone strength at tissue level given that no 26

ACCEPTED MANUSCRIPT beneficial effect was observed in the experiment. The formation and accumulation of AGEs are well known as one of the main reasons for the suppression of osteoblast activity and involvement of bone quality caused by diabetes. Since irbesartan can block damage in

proximal

tubular cell[17], glomerular

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AGE-induced

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microvascular and glomerular endothelial cells[25], we tried to evaluate

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the pathophysiological crosstalk between irbesartan and AGEs in osteoporosis. MTT and flow cytometry analysis were conducted to

Data demonstrated that irbesartan dramatically attenuated

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arrest.

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analyze the effects of irbesartan on osteoblast proliferation and cell cycle

AGEs-mediated osteoblasts proliferation in a time-dependent manner. In

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addition, AGEs increased the cell cycle retardation of osteoblasts at the

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G1 stage, while irbesartan ameliorated cell cycle composition. Apoptosis is also involved in the inhibition of osteogenic differentiation

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and bone loss[26, 27]. The present data showed that irbesartan was able

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to prevent AGEs-induced apoptosis in osteoblasts. MMP level was assessed in order to further investigate the relationship between irbesartan and the intrinsic apoptotic pathway. MMP is an indicator of mitochondrial membrane permeability, which idecreases during early apoptosis[27]. In the study, the decreased of MMP reflected the activation of the mitochondrial apoptotic pathway by exposing to AGEs, while irbesartan could notably block cell apoptosis via the intrinsic apoptotic pathway. 27

ACCEPTED MANUSCRIPT Biochemical markers were detected to explore the irbesartan's effects on osteogenic differentiation. The osteoblast differentiation can be devided into three stages according to their characteristics, including cell proliferation, extracellular matrix production, and mineralization. The

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production of collagen I occurs during proliferation, followed by an

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increased expression of ALP and final calcium deposition[28]. Collagen I

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comprises approximately 90% of the organic material and was considered as the most abundant protein component of the bone matrix. The

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enhancement of the expression of ALP metabolizes calcium phosphate

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into insoluble phosphate salts, thus mediating calcification[29]. RUNX2 is a key transcriptional factor that can stimulate the expression of other

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osteoblast-specific genes during the early stage of osteogenesis[30]. OC

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is the most advanced mark product of osteoblastic differentiation, involving in maintaining the normal bone mineralization, suppressing

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abnormal hydroxyapatite formation, and slowing down the growth of

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cartilage mineralization[31]. In this study, irbesartan significantly increased the transcription level of ALP, OC and collagen I. Also, after exposing to AGEs, dystrophic and ameliorative mineralizations of osteoblasts were detected using the alizarin red staining while irbesartan could reverse these effects. These data indicated that irbesartan could inhibit AGEs-induced damages during the differentitation of osteogenic. Oxidative stress and overproduction of ROS induced by AGEs could 28

ACCEPTED MANUSCRIPT disrupt the balance between oxidation and antioxidant defense systems, leading to bone loss by facilitating lipid peroxidation and lowering antioxidant enzymes, inhibiting bone formation and inducing apoptosis of osteoblasts[32-34]. It is suggested that oxidative stress could also

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decrease osteoblast differentiation[35], while a reduction in ROS permits

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the restoration of osteoblastic markers, specifically the induction of

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osteoprotegerin and osteocalcin[36]. ROS is also believed to function as critical regulator of apoptosis[37, 38]. Either exogenously administered or

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endogenously produced ROS can induce the opening of the mitochondrial

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permeability transition pore and cause apoptosis[37, 39]. ROS levels were examined to investigate the potential mechanisms behind the

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protective effects of irbesartan on osteoblasts. The study reported that

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co-incubation with irbesartan prevented the alterations in intracellular oxidative stress induced by AGEs in osteoblast.

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Accumulating evidence suggests that the engagement of AGEs on its

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specific receptors RAGE could trigger several deleterious effects[40]. The interaction of AGEs-RAGE induces the generation of ROS through NADPH oxidase, resulting in the inhibition of the proliferation, differentiation, as well as the apoptosis of osteoblasts[41, 42]. RAGE overexpression inhibits osteoblast proliferation via impeding Wnt, PI3K and ERK signals, which provides novel mechanisms by which RAGE regulates osteoblast growth[43]. These data suggest that RAGE 29

ACCEPTED MANUSCRIPT suppression is the primary target for the anti-oxidative, anti-apoptotic, anti-inflammatory effects on osteoblasts. It is reported that ARB inhibits AGEs-induced cell damages in vitro by suppressing RAGE in proximal tubular cell injury[17], and human endothelial cells[44]. Consistently, the

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findings of the study indicated that irbesartan could inhibit AGEs

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actions on osteoblasts via ROS generation.

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-enhanced RAGE expression and the AGEs/RAGE-mediated pleiotropic

In conclusion, this study demonstrates that reduction of RAGE expression

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by irbesartan may block the AGEs-signaling to osteoblasts' damages by

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inhibiting the ROS generation. This may provide a revolutionary benefits to therapy with irbesartan on diabetic osteoporosis as it could work as an

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agent against the AGEs–RAGE axis and may play a protective role

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against osteoporosis in diabetes.

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Conflict of interest

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The authors declare that there are no conflicts of interest.

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ACCEPTED MANUSCRIPT Acknowledgments: The study was supported by grants from the National Natural Science Foundation of China (Grant No. 81270966, No.81500679), the Natural Science Foundation of Guangdong Province, China (Grant No. 2014A030310036

,2014A030310472

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S2012010009494,

and

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2017A030313519), Science and technology plan of Guangdong province

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(Grant No. 2016A020215097，2017A020215045) and the Natural Science

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China(Grant No. PY2013N050).

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Foundation of Southern Medical University, Guangdong Province,

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ACCEPTED MANUSCRIPT Figure 1. Effect of 12-week irbesartan treatment on (A) two-dimensional images of metaphysis and (B) the distal femur of structural bone parameters(BV/TV, Tb.Th., Tb.Sp., Tb.N, SMI, Ct.th., trabecular BMD, cortical BMD and total BMD) analyzed by mioro-CT.

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db/+, the db/+ mice group; db/db, the db/db mice group; Irbe, the db/db

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Values are expressed as means±SD (n=12).

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mice treated with irbesartan.

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ACCEPTED MANUSCRIPT Figure 2. Effects of 12-week irbesartan treatment on femoral biomechanical structural properties in db/db mice via three-point bending test, including max load, fracture load, total displacement, yield displacement, stiffness, and energy absorption.

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db/+, the db/+ mice group; db/db, the db/db mice group; Irbe, the db/db

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Values are expressed as means±SD (n=12).

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mice treated with irbesartan.

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ACCEPTED MANUSCRIPT Figure 3. Irbesartan attenuated AGEs-induced osteoblast proliferation and apoptosis. (A) Cell proliferation was measured by the absorbance at 490 with a MTT assay. Osteoblasts were treated with different medium for 3

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days(A).

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(B) Cell cycle was evaluated by flow cytomety. AGEs induced osteogenic

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cell cycle arrest in G1 stage and irbesartan improved it.

(C,D) Osteoblast apoptosis was assessed by Annexin-V FITC apoptosis

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detection. Osteoblasts were treated with different medium.

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(E,F) MMP was examined by JC-1 probe under fluorescent microscope(D) and with flow-cytometry analysis(C). The intensity ratio of red to green

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fluorescence represents the change in MMP. In fluorescent microscopic

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images, red dot-like images reflecting JC-1 aggregation within the mitochondria were observed in cells with high ΔΨ , and in cells with low

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ΔΨ, the red color turned into diffuse green fluorescence, reflecting the

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monomeric state of JC-1. Ctrl: control group; BSA: Cell treated with BSA (100μg/ml); AGEs: Cell treated with AGEs(100μg/ml); Irbe: Cell treated with irbesartan(2.0 μM)+ AGEs(100μg/ml). Values are means ± SD from three independent experiments.

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ACCEPTED MANUSCRIPT Figure 4 Irbesartan attenuated AGEs-mediated damage in osteoblast differentiation (A) Alizarin red staining, which represented the mineralization levels of later stages of osteogenesis, was performed at the indicated time points.

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(B) The mRNA expression of osteogenic differentiation markers (Runx2,

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ALP, Col-1,OC) of osteoblasts in different group assessed by real-time

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PCR.

Ctrl: control group; BSA: Cell treated with BSA (100μg/ml); AGEs: Cell

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treated with AGEs(100μg/ml); Irbe: Cell treated with irbesartan(2.0 μM)+

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AGEs(100μg/ml).

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Values are means ± SD from three independent experiments.

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ACCEPTED MANUSCRIPT Figure 5. Effect of irbesartan on the AGEs-mediated ROS and RAGE expression. (A,B) Osteoblasts were incubated with different medium. Intracellular ROS generation was measured with the probe DCFH-DA, and visualized

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using a fluorescent microscope.

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(C) RAGE mRNA levels were detected by PCR in osteoblasts incubated

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with different medium.

(D) RAGE Protein levels were detected by western blot analysis in

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osteoblasts incubated with different medium.

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(E) RAGE mRNA levels of the femur from mice were detected by PCR. (F) RAGE Protein levels of the femur from mice were detected by

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western blot analysis.

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Ctrl: control group; BSA: Cell treated with BSA (100μg/ml); AGEs: Cell treated with AGEs(100μg/ml); Irbe: Cell treated with irbesartan(2.0 μM)+

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AGEs(100μg/ml).

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db/+, the db/+ mice group; db/db, the db/db mice group; Irbe, the db/db mice treated with irbesartan. Values are means ± SD from three independent experiments.

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ACCEPTED MANUSCRIPT

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Graphical abstract

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