Irbesartan Reverts Deleterious Effects Of Advanced Glycation End-products (AGEs) On Osteoblastic Cells

BackgroundOsteoporosis is a skeletal disorder characterized by reducing bone quantity and quality predisposing to an increased risk of fracture Additionally,it is critical for mortally and morbidity in patients with diabetes mellitus Several studies have demonstrated the deleterious effects of diabetes on bone.Epidemiological data underlines an increase fracture risk in patients with type 1 and type 2 diabetes Poor glycemic control over a prolonged period and the presence of vascular complications have been identified as potential determinants of low bone mass and an increased risk of fractures An in vitro study of osteoblast-like MG-63 cells revealed that a high glucose condition impaired osteoblast differentiation.The evidence unambiguously suggests that diabetes has effects on bone and it is necessary to differentiate the direct effect of diabetes on bone.Advanced glycation end products(AGEs)are formed by the Maillard process,a non-enzymatic reaction between etones or aldehydes,the amino groups of proteins,lipids nd nucleic acids that contributes to the aging of macromolecules.Additionally,AGEs excess is one of the most important mechanisms involved in the pathophysiology in the progression of diabetic complications.What's more,numerous studies have demonstrated that AGEs have detrimental effects on osteoblasts,which is responsible for bone formation.Under hyperglycemic conditions,the accumulation of AGEs may be partially involved in diabetic osteoporosis.The intensity of staining of AGEs in bone specimens of osteoporotic subjects was correlated with patient age and inversely associated with the percentage of bone surface covered with osteoblasts.In vitro studies have demonstrated that AGEs can attenuate the differentiation of osteoblastic lineage,inhibit the expression of osteoblast-specific transcription factors and decrease mineralization.Moreover,there is evidence that AGEs may induce osteoblasts' apoptosis,further impairing bone formation.We have reason to believe that AGEs products play a pivotal role in the progression of osteoporosis in diabetic.Renin angiotensin system(RAS)is operating not only systemically but also locally in bone microenvironments.Recent study suggests the pleiotropic effects of the RAS inhibitors,that is,beyond BP-lowering effects,on bone.Treatment with AT2 receptor blocker significantly enhanced the levels of bone mass,and this effect was based on the enhancement of osteoblastic activity as well as the suppression of osteoclastic activity in vivo.The hyperglycemia was detrimental to bone as compared to the control group,and the underlying mechanism was regulated through up-regulation of RAS activity in local bone.What's more,the blockade of RAS attenuated osteoporosis in a hypertensive rat.In recent years,the skeleton protective effects of the inhibitors of the RAS are largely ascribed.However,the pathophysiological crosstalk between the RAS and AGEs in bone remains unknown.In the present study,we have evaluated the hypothesis that irbesartan can abrogate AGEs-induced deleterious effects on cell and animal level.Osteoblasts in culture,to address this issue we investigated the effects of AGEs with or without irbesartan on osteoblastic differentiation,apoptosis and oxidative stress in two osteoblastic cell lines.Part 1 The effects of AGEs on the osteoporosisAimsWe amied to investigate the effects of AGEs in proliferation of osteoblast.Methods1.primary osteoblasts cultureNeonatal Sprague-Dawley rat calvaria were removed under sterile conditions and cleaned of attached connective tissues.The bones were cutted into 2-5mm2 pieces and digested with 0.25%trypsin-EDTA,then digestion was stopped by the addition of FBS and the supernatant was discarded.Subsequently the moderate fragments was inoculated into the bottom of a culture flask and the flask was incubated at 37 ? in a humidified atmosphere containing 5%CO2 for 6 hours.The DMEM/F12 with 10%FBS and 1%penicillin/streptomycin was added for covering the bones and cultured in the CO2 incubator.The medium was refreshed every 2 d and osteoblasts were purified by plastic adherence and expanded in culture flasks.At last,second or fifth passage cells were used for all experiments.2.GroupThe effects of different concentration of AGEs on proliferation of osteoporosis:the control group,the BSA group,the 50,100,200,400?g/ml AGEs group,which were cultured for 3d.The effects of AGEs on proliferation of osteoporosis for different time:100?g/ml AGEs incubated with cells for 1,3,5,7,9d.3.Assessment of cell proliferation by CCK-8 assay.Statistical analysesThe results were expressed as the means ± SEM.Statistical analyses were conducted using SPSS 20.0 software The one-way ANOVA was conducted to compare multiple groups.Differences were considered statistically significant at P<0.05.Results1.The effects of AGEs on proliferation of osteoblas.t1.1 The effects of different concentrations of AGEs on osteogenic proliferation:After treatment with 50,100,200 and 400p,g/ml AGEs for 3d,the 100?g/ml AGEs significantly inhibited the proliferation of the osteoblasts compared with the control gourp.1.2 The effects of AGEs on osteogenic proliferation for different timeAfter treatment with 100?g/ml AGEs for 1,3,5,7,9d.At third day,the osteogenic proliferation were significantly inhibited compared with the control gourp.ConclusionsOur studys shows that treatment with 100?g/ml AGEs for three days significantly inhibited the proliferation of the osteoblasts.Part 2 Irbesartan reverts deleterious effects of Advanced Glycation End-Products(AGEs)on osteoblastic cellsAimsWe aimed to investigate the effects of AGEs with or without irbesarn on osteoblastic differentiation,apoptosis and oxidative stress.We also assessed the expression of AGE receptors as a possible mechanism could modulate the action of AGEs.Methods1.Proliferation assayMTT assay was used to evaluate the proliferative ability the osteoblasts.In a nutshell,osteoblasts in 96-well plates were washed twice with PBS,then incubated in 100?l of FBS-free DMEM/F12 supplemented with 10 ?l of 5 mg/ml MTT solution at room temperature for 4h.Subsequently,the supernatant was removed and the crystals were dissolved by incubation with 150?l of DMSO for 20 min.The plates were shaken for 5 min and the cell viability was determined by measuring the absorbance at 570 nm on a 96-well plate-reader.Experiments were performed in triplicate with three independent experiments for each condition.2.Cell cycle distribution analysisAt the end of the treatment period,1x106 cells were trypsinized and collected by centrifugation at 900 rpm for 5 min,then washed twice with cold PBS and were fixed with cold 70%alcohol overnight.After 12h,the cells were collected by centrifugation and washed once with cold PBS.Subsequently,the cells were resuspended with 500?l PBS and digested with 5?l 10 mg/mL RNase A for 1h at 37?.Then,the osteoblasts were stained with 50 ?g/mL PI diluting with 0.2%Triton X-100 for 30 min in the dark at 4?.At the end of incubation,cell cycle distribution were detected by a FACSCalibur flow cytometer(BD,Franklin Lakes,NJ)and analyzed on ModFit LT software(Verity Software House,USA).3.Measurement of apoptosis in osteoblastsApoptosis was detected with Annexin-V FITC Apoptosis Detection Kitaccording to instruction given by the manufacturer.Annexin V-FITC/PI stained cells were analyzed immediately at room temperature with a flow cytometer,using Cell Quest pro software(BD Biosciences,USA).Excitation of Annexin V-FITC and PI was done at 488 nm and 546nm respectively and emission was detected at 578/20-nm filter(FL-1)and 647/20-nm filter(FL-3)respectively.Quadrant statistics were performed to determine healthy,early apoptotic,terminal apoptotic and dead cells from the total population of the cells.The healthy osteoblasts treated with serum free media was considered as control.4.MMP MeasurementMitochondrial membrane potential(MMP)was evaluated by JC-1 probe.After treatment,the cells were incubated with 1ml 10?g/ml JC-1 for 20 min at 37?.To estimate the relative MMP,samples were analyzed by flow cytometry and fluorescent microscope.Excitation of JC-1 monomers and JC-1 aggregation were done at 514 nm and 585nm respectively and emission was detected at 529-nm filter and 590-nm filter respectively.Under normal condition,the mitochondrial membrane showed red fluorescence;when MMP is lost,red fluorescence decreases and green fluorescence increases.The intensity ratio of red to green fluorescence represents the change in MMP.5.Alizarin red S stainingAfter treatment,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-HCLfor 30 min at room temperature.Bone nodule formation was observed under a light microscopy(Olympus,Tokyo,Japan).6.Determination of ROS generation.Intracellular ROS generation was measured by flow cytometry with the probe DCFH-DA.After treatment,cells were washed twice with PBS and then treated with lml 10?g/ml DCFH-DA at 37? for 30 min.Subsequently,the stained cells were resuspended in PBS after washing three times in PBS.To estimate the relative ROS accumulation,the fluorescence intensity was determined by flow cytometry and fluorescence microscopy(Leica,wetzlar,Germany).Data were normalized to the control values.7.Real-time PCR(qPCR)analysis.The expression of ALP,collagen I,RUNX2 and RAGE in mRNA level was analyzed by qPCR.After treatment,the osteoblastic total RNA was extracted using Trizol reagent according to the manufacturer's instructions.Then cDNA was reverse-transcribed from 0.8 pg of total RNA using the PrimeScript one step RT-PCR kit as soon as possible.All cycle hreshold(Ct)values were collected at the exponential phase of the qPCR.Real-time PCR was carried out in a 7500 Real-Time PCR System(Thermo Fisher Scientific,Waltham,USA)using SYBR Premix Ex Taq II.The cycling conditions were as follows:95? for 2 min and 40 cycles of 95? for 5 sec,60? for 34 sec.The value of 2-??Ct represents the relative level of target gene expression.Statistical analysesUse the same method as that of part 1.Results1.AGEs inhibited osteogenic proliferation and irbesartan reversed it.Osteogenic proliferation was evaluated by MTT assay.The AGEs significantly inhibited the proliferation of the osteoblasts compared with the control medium and the unmodified BSA preparation.Amazing is that irbesartan can reverse it.what's more the different concentration of irbesartan had a significantly protective action for the cell and the positive function rely on "linear" growth with the increase of the concentration.There a non-significant increase in cell proliferation when the irbesartan was added on the seventh day comparing with AGEs group.However irbesartan showed a significant effect on osteoblasts by increasing intervening time.These results indicated that AGEs inhibited osteoblasts proliferation and irbesartan changed the situation in a dose-and time-dependent manner.2.AGEs induces osteogenic cell cycle arrest and irbesartan reversed it.In order to examine the possible mechanism of proliferation and anti-apoptosis activity of irbesartan,the cell cycle distribution of osteoblasts was evaluated by flow cytometry.Cultivating osteoblasts with AGEs resulted in 15.36%increases in the percentage of cells in the G1 phase compared with the control group and BSA group,which was accompanied by a concomitant increase in the percentage of cells in the S phase.However irbesartan significantly reversed the changes of mentioned.It suggested that AGEs induces cell cycle arrest,and irbesartan can reverse it.3.AGEs promoted osteogenic apoptosis and irbesartan reversed it.To investigate the effect of AGEs and irbesartan on osteoblasts apoptosis,the samples were detected with Annexin-V FITC apoptosis detection kit.The cells were stimulated as described above.Pre-incubation of AGEs induced cells a significant increase in osteoblasts apoptosis and the influence changed when irbesartan was added.These data indicate that AGEs increased the osteogenic apoptosis and irbesartan had a protective effect against AGEs.4.AGEs decreased osteogenic MMP and irbesartan reversed it.MMP was examined by JC-1 probe under fluorescent microscope and with flow-cytometry analysis.When MMP is lost,red fluorescence decreases and green fluorescence increases.The cells with AGEs significantly increase in green fluorescence(monomers)accompanied by loss of red fluorescence(aggregates)and irbesartan had a significantly protective effect against AGEs.Quantitative analysis using flow cytometry revealed the disruption of membrane potential(ratio of red and green fluorescence).These data indicate that AGEs decreased the osteogenic MMP and irbesartan had a protective effect against AGEs.5.AEs inhibited osteogenic differentiation and irbesartan reversed it.The calcium deposition in osteoblasts were revealed by Alizarin Red S staining under a light microscopy.The cells were stimulated as described above.We hardly saw any calcium nodes in the osteoblasts with AGEs.However some bone nodule formation was observed when irbesartan was added.These data indicate that AGEs inhibit the osteogenic differentiation and irbesartan had a significantly protective effect against AGEs.6.Effects of AGEs on ALP,collagen I,and RUNX? of osteoblasts.The ALP,collagen I and RUNX ? are important indicators of osteogenesis differentiation.We found that the exposure of osteoblasts to AGEs resulted in a significant decrease in the mRNA expression of ALP,collagen I and RUNX2,however the changes can reverse when irbesartan was added.These data indicate that AGEs inhibit the osteogenic differentiation and irbesartan can significantly ameliorate the changes.7.AGEs induces osteogenic oxidative stress and irbesartan reversed it.Oxidative stress was examined by the detection of DCF fluorescence under fluorescent microscope and with flow-cytometry analysis.Treatment with AGEs increased the ROS level(as visualized by green fluorescence)compared with the control group and BSA group.However,the ROS levels were decreased when irbesartan were added.When the DCF fluorescence intensity was determined by flow cytometry we came to the conclusion.These results indicated that the increase in the ROS level was associated with AGEs.Collectively,these data suggested that irbesartan alleviated AGEs-induced oxidative stress in osteoblasts.8.Effects of AGEs on RAGE mRNA of osteoblasts.RAGE is considered a receptor of AGEs.Exposure to AGEs,as opposed to exposure to the control group and BSA group,increased the mRNA expression of RAGE and decreased when irbesartan was added.ConclusionsOur data showed that AGEs inhibited osteoblasts proliferation and irbesartan changed the situation in a dose-and time-dependent manner.What's more,we found that AGEs increased cell apoptosis,induces cell cycle arrest,generated oxidative stress and decreased MMP.Fortunately,irbesartan had a protective effect against AGEs.Following,we assessed the effect of irbesartan on osteoblastic differentiation by evaluating the specific marker.We found that AGEs inhibit the osteogenic differentiation and irbesartan had a significantly protective effect against AGEs.Using alizarin red staining,we observed that protective mineralization occurred in the osteoblasts exposed to irbesartan compared with AGEs group.In this study,AGEs significantly decreased ALP,collagen I and Runx-II mRNA levels and irbesartan attenuated AGEs-elicited effects.The potential mechanisms involved in the effects of AGEs and irbesartan are not completely known.AGEs have been shown to bind with specific receptors RAGE.AGEs-RAGE interaction induces the generation of ROS through NADPH oxidase,resulting in the apoptosis of osteoblasts and in the inhibition of the proliferation and differentiation of osteoblasts.RAGE overexpression inhibits osteoblast proliferation via suppression of Wnt,PI3K and ERK signaling,which provides novel mechanisms by which RAGE regulates osteoblast growth.Schurman et al reported up-regulation of the mRNA expression of RAGE induced proliferation and the osteogenic differentiation.A reduction in ROS permits the restoration of osteoblastic markers,specifically the induction of osteoprotegerin and osteocalcin.These data suggest that RAGE and ROS suppression is the primary target for the anti-oxidative,anti-apoptotic,anti-inflammatory effects on osteoblasts.In the present study,we found that irbesartan treatment significantly inhibited the AGEs-induced up-regulation of RAGE mRNA levels and ROS generation in osteoblasts.Although we did not clarify the precise molecular mechanisms by which irbesartan reduced RAGE gene expression,irbesartan may reduce RAGE mRNA levels in AGE-exposed osteoblasts by blocking the endogenous angiotensin II because previous study found that angiotensin II itself up-regulates RAGE mRNA levels in endothelial cells and vascular pericytes,at the same time induces mitochondrial oxidative stress.In conclusion,since AGEs-RAGE interaction exerts pleiotropic actions on various cells via ROS generation,Our present study proved that reduction of RAGE expression by irbesartan may block the AGEs-signaling to tubular cell apoptosis and damage by inhibiting the ROS generation.This provides a novel beneficial aspect of irbesartan on bone;it could work as an agent against the AGEs-RAGE axis and may play a protective role against diabetes-associated osteoporosis.