Tanshinol Attenuates Osteoporosis Mediated By Oxidative Stress Via FoxO/Wnt Signaling

BackgroundOxidative stress has currently been proposed as a risk factor associated with the development and progression of osteoporosis, and the underlying mechanism involved in regulation of FoxO/Wnt signaling pathway in osteoporosis mediated by oxidative stress (OS) has been studied extensively. There is increasing envidence that increase of reactive oxygen species (ROS) accumulation leads to oxidative stress under conditions of aging, or some illnesses, or use of medicions, and subsequently promotes induction of FoxO pathwy activation and suppression of Wnt signaling, which contributes to development and progession of osteoporosis. As a result, antioxidants may be adopted as a novel therapeutic approach in the prevention and treatment of osteoporosis. The previous eveidence in our research group has demonstrated that the aqueous component of Tanshinol isolated from Salvia miltiorrhiza Bunge is a natural antioxidant, shows a preventive influence on loss of bone mass induced by glucocorticoid owing to activation of Wnt pathway in bone marrow stromal cells. A hypothesis based on the arguments above was estanblished by our research group that Tanshinol exerts preventive influence on the pathological process of osteoporosis via a regulatory function of activation Wnt signaling and simultaneously suppression of FoxO pathway, owing to inhibition of oxidative stress triggered by ROS accumulation, as well as stimulation of activity of Tcf transcription factor responsible for bone formation. Accordingly, this study aims to elucidate that Tanshinol shows a potiential impact on prevention and treatment of osteoporosis midated by oxidative stress in vivo and in vitro, and that the underlying mechanism involves in manipulation FoxO/Wnt singaling pathway. Additionally, the study may be as a new research considerations and an experimental evidence for further investigatin of pharmacological effects of Danshen and therapeutic applications of natural antioxidant in prevention and treatment of osteoporosis.Objectives1. To study pathological mechanism that oxidative stress evokes osteoblasts injury and osteoporosis in vitro and in vivo.2. To elucidate and confirm that the inhibitory effect of Tanshinol on oxidative stress contributes to regulation of osteoblastic differentiation and bone formation, and to protection bone tissue against oxidative injury.3. To substantiate that Tanshinol exerts the therapeutic effect on osteoporosis via down-regulation of FoxO3a pathway and simultaneous up-regulation of Wnt signaling, and that the study may be taken as the basic theory and experimental evidence for natural antioxidant to treat osteoporosis.Contents and detection indices1. In vitroPluripotent mesenchymal precursor C2C12cells were treated with hydrogen peroxide (H2O2) and/or Tanshinol in the study. Measurements were made as following:cell viability was measured by MTT assay. Apoptotic morphology was detected using fluorescence microscopy and transmission electron microscopy. ROS level for the detection of oxidative stress was measured by DCFH-DA probe using Flow cytometry assay. Content of biomarkers for osteoblastic differentiation was detected by ELISA assay. Proteins expression and mRNAs level for Fox03a and Wnt pathway were detected by pRT-PCR and Western blot assays in cells. Cells were co-transfected transiently with indicated luciferase reporter constructs of FoxO3a-luc and Tcf-luc to monitor FoxO/Wnt pathway.The main contents were as following:1.1To observe the influences of oxidative stress on cell viability, proliferation and differentiation, and the effect of Tanshinol on cells under oxidative stress. 1.2To elucidate that the effects of oxidative stress on the expressions of genes and protein related to FoxO/Wnt pathway, and to study the regulation of FoxO/Wnt pathway in cells treated with tanshinol.1.3To explore the relationship between the effects of osteoblastic differentiation and inhibitory influence on oxidative stress and regulation of FoxO/Wnt signaling transduction, and to confirm the underlying mechanism of Tanshinol to regulate FoxO/Wnt pathway for osteoblastic differentiation under conditions of oxidative stress.2. In vivoGroups and administration:Thirty-two4-month-old female Sprague-Dawley rats were randomly divided in four groups with8rats per group:normal control group (Con), model group (GC), Tanshinol group (Tan) and Resveratrol group (Res). Model rats were orally administered prednisone acetate5mg/kg·d body weight (bw), which was dissolved in a vehicle (0.5%CMC-Na solution). Normal control rats was administered only0.5%CMC-Na solution. According to administration mentioned above, Tan treated and Res treated rats were fed additionally with Tanshinol and Resveratrol at a concentration of20mg/kg·d bw and5mg/kg·d bw, respectively. The rats in every group were treated with prednisone acetate in the morning and with other drugs in the afternoon by intragastric administration one day at a time during14weeks.Material collection and indices:Rats were sacrificed by way of which bleed were collected from right ventricle usingcardiac puncture method under anesthesia with peritoneal injection of3%mebumal sodium (1.5ml/kg). The serum was separated by centrifugation to detect bone turnover indices and anti-oxidative indices using ELISA assay and colorimetric method. The proximal ends and middle part of right tibia and the fourth lumbar vertebra were collected to analysis stationary and dynamical parameters of bone histomorphometry. The right femur and the fifth lumbar vertebra were prepared to determine bone mineral density (BMD), bone biomechanical characteristics and3-dimentional bone micro-architecture, using DXA technology, three-point bending method and Micro-CT assay, respectively. The left femur was used to measure cell apoptosis using TUNEL assay. Bone marrow cells flushed from the left tibia were prepared to measure oxidative stress level using DCFH-DA probe. The left tibia and the sixth lumbar vertebra were collected to detect genes expression and proteins level related to osteogenic differentiation, adipogenic differentiation and/or FoxO/Wnt signaling pathway using pRT-PCR method and Western blot assay, respectively.The main contents werw as following:2.1To study impacts of Tanshinol on capacity of osteogenic differentiation, the level of bone turnover, bone architecture, BMD and bone quality in GIO rats.2.2To detect oxidative stress level, cell apoptotic status and biomarkers involved in anti-oxidative capacity in bone tissue, and to observe effects of Tanshinol on regulation of FoxO/Wnt pathway of bone tissue in GIO rats under oxidative stress.2.3To elucidate the effects of Tanshinol on the treatment of osteoporosis, the protective impact on bone tissue from oxidative stress, and the relationship between the beneficial influence of Tanshinol and alterations of FoxO/Wnt signaling transduction in GIO rat, and to reveal the underlying mechanism that Tanshinol exerts therapeutic effect on osteoporosis mediated by oxidative stress via regulation of FoxO/Wnt signaling pathway.3. Statistical analysisData were reported as the means±standard deviation (x±SD) and analysed statistically with SPSS software version20.0. One-way ANOVA was used to detect the differences in changes between the groups of various treatments. P<0.05was defined as statistically significant difference. Fisher least significant difference (LSD) test was used to perform appropriate pairwise comparisons of treatment groups after establishing that the data were normally distributed and homogeneity of variances. Otherwise, Dunn’s method for post hoc test was used to perform pairwise comparisons of treatment groups.Results1. In vitro 1.1Tanshinol with the concentrations varied from0.0001to1000μM exerted no inhibitory effects on the growth of C2C12cells, and that an increase in viability was observed in C2C12cells treated with Tanshinol, especially at the concentration of1μM (P<0.05). Cell viability in C2C12cells treated with increasing concentrations of H2O2ranging from0.025mM to1.2mM was measured, and showed a significant decrease at the dose of0.2mM (optimal experimental dose)(P<0.05).1.2Cell death and ROS accumulation triggered by H2O2in C2C12cells were repressed by Tanshinol(P<0.05), and the underlying mechanism of Tanshinol in attenuating the deleterious effects of oxidative stress may be involved in antagonizing the activation of FoxO3a transcription factor contributing to rescue cell cycle arrest and decreased cell apoptosis in caspase3-manner (P<0.05).1.3H2O2shows an inhibitory effect on related indices of osteogenic differentiation in C2C12cells stimulated by BMP-2(100ng/ml), including the decrease of ALP activity(P<0.05), OCN content (P<0.05)and the capacity of mineralization, and the underlying mechanism of H2O2involved in repression of Wnt/β-catenin/Tcf pathway responsible for osteogenic differentiation. Tanshinol attenuates the inhibitory of H2O2on osteoblastic differentiation to a certain extent (P <0.05).1.4To consolidate previous results, alterations of target genes of Wnt signaling and FoxO3a pathway were measured in C2C12cells treated with H2O2(0.2mM) or Tanshinol. The evidence revealed that H2O2exerts an obvious influence on the suppression of Axin2、ALP and OPG mRN A of Wnt downstream (P<0.05) and on activation of Gadd45a and CAT mRNA of FoxO3a downstream simultaneously (P<0.05), while Tanshinol show a capacity to counteract the alterations triggered with H2O2treatment (P<0.05).In summary, these results strongly support the viewpoint that Tanshinol attenuates oxidative stress via down-regulation of FoxO3a signal, and simultaneously up-regulates Wnt signal cascade for osteoblastic differentiation.2. In vivo 2.1Analysis of dynamic changes of body weight:body weight per week of all rats in every group was recorded during14weeks. The evidence showed that mean body weight of rats in three experimental groups decreased significantly compared to that of normal control group, and the decline was significant statistically since3nd week in GC group (P<0.05). Furthermore, the rats in the Tan group and Res group exhibited a trend toward increase in the body weights since11st week, compared to that in GC group (P<0.05).2.2Preventive effects of Tanshinol on osteoporosis in GIO rats:(1) The evidence of biomarkers of bone turnover in serum demonstrated that rats treated with prednisone acetate exhibited a significant decrease of OCN level (P<0.05), OPG content (P<0.05)and OPG/sRANKL ratio(P<0.05), and an obvious increase of sRANKL (P<0.05) and TRAP5b activity(P<0.05), while BAP activity showed a trend toward decline in GIO rats compared to that in Con group(P>0.05). The data revealed that prednisone acetate caused deleterious consequences of bone metabolism, including a decrease in bone formation and an increase in bone resorption, and which was reversed by Tanshinol and Resveratrol (P<0.05).(2) The results of detection of BMD showed that there was a trend toward decreased BMD of rats in GC group, but has no statistic significance compared to that in Con group(P>0.05), while BMD of rats in Tan group and Res group was closed to that in Con group(P>0.05).(3) Parameters of bone histomorphometery indicated that GC treatment in rats contributes to a series of deleterious effects, including reduction of trabecular area and number(P<0.05), decrease of trabecular thickness and spacing(P<0.05), increase of trabecular separation(P<0.05), which leads to impaired structure of cancellous bone, decreased activity of bone formation, increased activity of bone resportion, resulting in loss of bone mass. However, GC treatment exerts minor influence on cortical structure and periosteal activity(P>0.05), and but on increase of endosteal activity related to osteoclasts and of bone surface formation(P<0.05), contribution to high bone turnover, and subsequently inducing loss of bone mass. Tanshinol and Resveratrol, however, exhibit a significant positive influence on the prevention of the alterations of bone tissue in GIO rats induced by glucocorticoids(P <0.05).(4) Micro-architecture determined by Micro-CT showed that prednisone acetate treatment contributes to the decline of BV/TV (P<0.05)and Tb.Th(P<0.05), and the increase of Tb.SP(P<0.05), SMI (P<0.05)and DA(P<0.05). The result discovered that GC treatment leads to decline of interconnection of trabecular of cancellous bone and trabecular thickness, while Tanshinol and Resveratrol can attenuate the changes induced by prednisone acetate (P<0.05).(5) Bone biomechanical parameters of femur in GIO rats showed that there is a significant decrease of structural mechanical properties, such as elastic load (P<0.05)and bending energy(P<0.05), and a trend toward decline of fracture load and material mechanical parameter stiffness, but had no statistical significance, compared to that of Con group(P>0.05). The results implied that the capacity to resist deformation was hampered by GC in rats, as well as the biomechanical properties, which leaded to the increase in bone fracture risk, while Tanshinol and Resveratrol may counteract the deleterious effects of prednisone acetate on biomechanics(P<0.05).2.3Iinfluence of Tanshinol on oxidative stress and cell apoptosis of bone tissue in GIO rats:The findings demonstrated that GIO rats exhibited a trend toward increase of phosphorylated p66shc protein expression (P<0.05)and ROS accumulation(P<0.05), and a decreased GSR activity(P<0.05), which might be responsible for oxidative stress of skeletal tissue. Meanwhile, acitivities of SOD and CAT of serum were decreased (P<0.05)and MDA level was elevated in rats treated with prednisone acetate(P<0.05). Tanshinol and Resveratrol could attenuate the deleterious consequence initiated by oxidative stress(P<0.05). Furthermore, the number of TUNEL-positive stained cells of bone tissue was increased significantly in rats treated with prednisone acetate, and that cell apoptosis of skeletal tissue induced by prednisone acetate could be blocked by Tanshinol and positive control drug, In brief, GC exerted a significant influence on acitivition of p66Shc/ROS signaling, contributing to the increase of oxidative stress status and cell apoptosis, and to the decline of anti-oxidative capacity. Tanshinol and Resveratrol could reverse the increase of oxidative stress and cell apoptosis in GIO rats.2.4Impacts of Tanshinol on osteogenic differentiation and adipogenic differentiation:Induction of PPARγ2(a main regulatory factor for adipogenic differentiation) mRNA level and corresponding protein expression were activated by GC treatment(P<0.05), and the mRNA expression level of the main regulatory factors for osteogenic differentiation (Runx2gene and Osteorix gene) and functional proteins of bone tissue (OCN gene and Collα gene) was hampered by the treatment of GC(P<0.05), with the exception of Alpl mRNA(P>0.05). The results demonstrated that GC exerted dual deleterious influence on skeletal tissue, including increase of adipogenic differentiation and decline of osteogenic differentiation, which might be responsible for osteoporosis in GIO rats, and while Tanshinol and Resveratrol exert therapeutic effects on osteoporosis via adjustment of regulatory switch from adipogenic differentiation to osteogenic differentiation(P<0.05).2.5Mechanism of Tanshinol on regulation of FoxO3a/Wnt pathway in GIO rats: The evidence confirmed that the induction of Gadd45a (a susceptive effector of FoxO3a transcription factor) mRNA and FoxO3a protein were activated by GC(P<0.05), and while the expressions of Axin2(indicator of Wnt pathway) gene and β-catenin protein were hampered by GC(P<0.05). The results demonstrated that GC exerted a positive impact on activation of FoxO3a transcription factor, and an inhibitory effect on regulation of Wnt/β-catenin signaling transduction, as well as an indirect influence on suppression of Wnt/β-catenin singaling via p66Shc/ROS/FoxO3a pathway. The evidence further confirmed that oxidative stress induced by GC in skeletal tissue ameliorated the pathway responsible for bone formation, resulting in the development and osteoporosis, and Tanshinol and Resveratrol could attenuate the pathology process of osteoporosis via regulation of FoxO3a/Wnt signaling pathway (P <0.05).ConclusionTanshinol exerts therapeutic influence on osteoporosis mediated by oxidative stress, and the underlying mechanism was as followed:Tanshinol promotes decrease of FoxO3a transcription activity, contributing to attenuation of oxidative stress via down-regulation of p66Shc/ROS/FoxO3a/caspase3signaling pathway. Simultaneously, Tanshinol exhibits potential applications for the treatment of osteoporosis via either direct up-regulation of canonical Wnt/β-catenin/Tcf pathway, or indirect induction to rescue block of Wnt signaling transcuction depending on suppression of activiation of FoxO3a pathway.