Yuyu Liu

Salvianolic acid B prevents bone loss in prednisone-treated rats through stimulation of osteogenesis and bone marrow angiogenesis.

Glucocorticoid (GC) induced osteoporosis (GIO) is caused by the long-term use of GC for treatment of autoimmune and inflammatory diseases. The GC related disruption of bone marrow microcirculation and increased adipogenesis contribute to GIO development. However, neither currently available anti-osteoporosis agent is completely addressed to microcirculation and bone marrow adipogenesis. Salvianolic acid B (Sal B) is a polyphenolic compound from a Chinese herbal medicine, Salvia miltiorrhiza Bunge. The aim of this study was to determine the effects of Sal B on osteoblast bone formation, angiogenesis and adipogenesis-associated GIO by performing marrow adipogenesis and microcirculation dilation and bone histomorphometry analyses. (1) In vivo study: Bone loss in GC treated rats was confirmed by significantly decreased BMD, bone strength, cancellous bone mass and architecture, osteoblast distribution, bone formation, marrow microvessel density and diameter along with down-regulation of marrow BMPs expression and increased adipogenesis. Daily treatment with Sal B (40 mg/kg/d) for 12 weeks in GC male rats prevented GC-induced cancellous bone loss and increased adipogenesis while increasing cancellous bone formation rate with improved local microcirculation by capillary dilation. Treatment with Sal B at a higher dose (80 mg/kg/d) not only prevented GC-induced osteopenia, but also increased cancellous bone mass and thickness, associated with increase of marrow BMPs expression, inhibited adipogenesis and further increased microvessel diameters. (2) In vitro study: In concentration from 10−6 mol/L to 10−7 mol/L, Sal B stimulated bone marrow stromal cell (MSC) differentiation to osteoblast and increased osteoblast activities, decreased GC associated adipogenic differentiation by down-regulation of PPARγ mRNA expression, increased Runx2 mRNA expression without osteoblast inducement, and, furthermore, Sal B decreased Dickkopf-1 and increased β-catenin mRNA expression with or without adipocyte inducement in MSC. We conclude that Sal B prevented bone loss in GC-treated rats through stimulation of osteogenesis, bone marrow angiogenesis and inhibition of adipogenesis.

Meta-Analysis of Long-Term Vitamin D Supplementation on Overall Mortality

Introduction It has been suggested that vitamin D is effective to prevent mortality. However, there is no consistent conclusion that the effects of vitamin D supplementation on all-cause mortality are associated with duration of treatment. We conducted a meta-analysis regarding this issue in an effort to provide a more robust answer. Methods A comprehensive search in a number of databases, including MEDLINE, Embase and The Cochrane Central Register of Controlled Trials, was conducted for collecting randomized controlled trials (RCTs) on vitamin D supplementation preventing mortality. Two investigators independently screened the literature according to the inclusive and exclusive criteria and the relative data were extracted. Data analysis was performed by using Review Manager 5.0 software. Results Data from forty-two RCT s were included. Vitamin D therapy significantly decreased all-cause mortality with a duration of follow-up longer than 3 years with a RR (95% CI) of 0.94 (0.90–0.98). No benefit was seen in a shorter follow-up periods with a RR (95% CI) of 1.04 (0.97–1.12). Results remain robust after sensitivity analysis. The following subgroups of long-term follow-up had significantly fewer deaths: female only, participants with a mean age younger than 80, daily dose of 800 IU or less, participants with vitamin D insufficiency (baseline 25-hydroxyvitamin D level less than 50 nmol/L) and cholecalciferol therapy. In addition, the combination of vitamin D and calcium significantly reduced mortality and vitamin D alone also had a trend to decrease mortality in a longer time follow up. Conclusions The data suggest that supplementation of vitamin D is effective in preventing overall mortality in a long-term treatment, whereas it is not significantly effective in a treatment duration shorter than 3 years. Future studies are needed to identify the efficacy of vitamin D on specific mortality, such as cancer and cardiovascular disease mortality in a long-term treatment duration.

Tanshinol Rescues the Impaired Bone Formation Elicited by Glucocorticoid Involved in KLF15 Pathway

Decreased bone formation is responsible for the pathogenesis of glucocorticoid- (GC-) induced osteoporosis (GIO), while the mechanism remains to be elucidated. The aim was to investigate how natural antioxidant tanshinol attenuates oxidative stress and rescues impaired bone formation elicited by GC in Sprague-Dawley rats and in C2C12 cells and/or MC3T3-E1 cells. The results showed that tanshinol prevented bone loss and decreased biomechanical characteristics and suppressed reduction of biomarkers related to osteogenesis in GIO rats. Further study revealed that tanshinol reversed decrease of transcription activity of Osterix-luc and rescued impairment of osteoblastic differentiation and bone formation involved in induction of KLF15 mRNA. Meanwhile, tanshinol diminished inhibition of protein expression of β-catenin and Tcf4 and transcription activity of Tcf4-luc induced by GC, especially under conditions of KLF siRNA in vitro. Additionally, tanshinol attenuated increase of reactive oxygen species (ROS) generation, phosphorylation of p66Shc expression, TUNEL-positive cells, and caspase-3 activity elicited by KLF15 under conditions of GC. Taken together, the present findings suggest that tanshinol attenuated the decrease of bone formation and bone mass and bone quality elicited by GC involved in KLF15/Wnt signaling transduction and counteracted GC-evoked oxidative stress and subsequent cell apoptosis involved in KLF15/p66Shc pathway cascade.

Tetrahydroxystilbene glucoside isolated from Polygonum multiflorum Thunb. demonstrates osteoblast differentiation promoting activity

Polygonum multiflorum Thunb. is a traditional Chinese medicinal herb that has been widely used to treat age-associated diseases. Tetrahydroxystilbene glucoside (TSG), also known as 2,3,5,4-tetrahydroxystilbene-2-O-β-D-glucoside, is a major component of this herb. The present study was designed to investigate the osteogenic differentiation promoting activity of TSG in rat mesenchymal stem cells (MSCs) and in zebrafish. Preliminary experiments using MTT assay and ALP methods indicate that the high potential activity for promoting osteogenic differentiation was observed when 50% ethanol eluate was used. Further isolation and purification of TSG from the 50% ethanol eluate was performed by bioassay-guided fractionation, and its structure was confirmed using nuclear magnetic resonance and mass spectrometry analyses. In addition, the relative content of TSG with the highest potential activity in the promotion of osteogenic differentiation was identified as 14.34% by reversed-phase high performance liquid chromatography. Subsequently, the osteogenic differentiation promoting abilities of TSG in MSCs were examined. The results demonstrated that TSG promoted the alkaline phosphatase activity at concentrations of 1.56–25 µg/ml, while it increased the content of osteocalcin 7 days after treatment with 6.25–25 µg/ml in MSCs. Furthermore, experiments in zebrafish indicated that different concentrations of TSG (3.12–12.5 µg/ml) protected against further bone loss induced by 10 µmol/l dexamethasone (Dex), simulating an osteoporosis (OP) model. TSG treatment (12.5 µg/ml) in Dex-induced zebrafish significantly increased the area of nodules by 50.14% compared with the untreated model group. In conclusion, TSG, as a major component of P. multiflorum Thunb. exhibited an osteogenic promoting activity in MSCs and in zebrafish. The results provided scientific evidence to support the potential use of TSG for protecting the bone in degenerative diseases, such as OP.

Cycloastragenol: An exciting novel candidate for age‑associated diseases (Review)

Cycloastragenol (CAG) is a triterpenoid saponin compound and a hydrolysis product of the main active ingredient in Astragalus membranaceus (Fisch.) Bunge. An increasing body of evidence has indicated that CAG has a wide spectrum of pharmacological functions, which are attracting attention in the research community. The aim of the present review paper was to review and elucidate the advanced study of CAG. The focus was on advanced studies of CAG in English and Chinese databases; the literature was collected and reviewed to summarize the latest efficacy, pharmacokinetics and adverse reactions of CAG. Extensive pharmacological effects have been attributed to CAG, including telomerase activation, telomere elongation, anti-inflammatory and anti-oxidative properties; CAG has also been reported to improve lipid metabolism. Clinical research has demonstrated that CAG activates telomerase in humans and ameliorates various biomarkers. CAG is absorbed through the intestinal epithelium via passive diffusion and undergoes first-pass hepatic metabolism. Within a certain dose range, oral CAG is relatively safe; however, underlying mechanisms associated with CAG are not clear, and thus, we should be aware of potential adverse reactions associated with CAG. According to existing studies and clinical trials, CAG is safe and has broad application prospects. However, further studies are required to fully understand its efficacy and potential adverse reactions, and to ensure the proper use of CAG is applied to treat diseases clinically.

Tanshinol alleviates impaired bone formation by inhibiting adipogenesis via KLF15/PPARγ2 signaling in GIO rats

Glucocorticoid (GC)-induced osteoporosis (GIO) is characterized by impaired bone formation, which can be alleviated by tanshinol, an aqueous polyphenol isolated from Salvia miltiorrhiza Bunge. In this study we investigated the molecular mechanisms underlying GC-induced modulation of osteogenesis as well as the possibility of using tanshinol to interfere with GIO. Female SD rats aged 4 months were orally administered distilled water (Con), prednisone (GC, 5 mg·kg−1·d−1), GC plus tanshinol (Tan, 16 mg·kg−1·d−1) or GC plus resveratrol (Res, 5 mg·kg−1·d−1) for 14 weeks. After the rats were sacrificed, samples of bone tissues were collected. The changes in bone formation were assessed using Micro-CT, histomorphometry, and biomechanical assays. Expression of Kruppel-like factor 15 (KLF15), peroxisome proliferator-activated receptor γ 2 (PPARγ 2) and other signaling proteins in skeletal tissue was measured with Western blotting and quantitative RT-PCR. GC treatment markedly increased the expression of KLF15, PPARγ2, C/EBPα and aP2, which were related to adipogenesis, upregulated FoxO3a pathway proteins (FoxO3a and Gadd45a), and suppressed the canonical Wnt signaling (β-catenin and Axin2), which was required for osteogenesis. Thus, GC significantly decreased bone mass and bone quality. Co-treatment with Tan or Res effectively counteracted GC-impaired bone formation, suppressed GC-induced adipogenesis, and restored abnormal expression of the signaling molecules in GIO rats. We conclude that tanshinol counteracts GC-decreased bone formation by inhibiting marrow adiposity via the KLF15/PPARγ2/FoxO3a/Wnt pathway.

Polygonum multiflorm alleviates glucocorticoid‑induced osteoporosis and Wnt signaling pathway

It is known that long-term excessive administration of glucocorticoid (GC) results in osteoporosis. The present study aimed to evaluate the protective effects of Polygonum multiflorm (PM) on the bone tissue of rats with GC-induced osteoporosis (GIO). A total of 90 6-month-old female Sprague Dawley rats (weight range, 190–210 g) were randomly divided into nine groups: Control (normal saline); prednisone (GC; 6 mg·kg−1·d−1; Model); GC plus PMR30 (the 30% ethanol eluent fraction of PM) (H) (400 mg·kg−1·d−1); GC plus PMR30 (M) (200 mg·kg−1·d−1); GC plus PMR30 (L) (100 mg·kg−1·d−1); GC plus PMRF (fat-soluble fraction of PM) (H) (400 mg·kg−1·d−1); GC plus PMRF (M) (200 mg·kg−1·d−1); GC plus PMRF (L) (100 mg·kg−1·d−1); GC plus calcitriol (CAL; 0.045 µg·kg−1·d−1; positive). Rats were administered intragastrically with prednisone and/or the aforementioned extracts for 120 days, and weighed once/week. The serum was collected for detection of biochemical markers. The left tibia was used for bone histomorphometry analysis. The right tibia was prepared for hematoxylin and eosin staining. The left femur was used to analyze the protein expression of dickkopf-1 (DKK1), WNT inhibitory factor 1 (WIF1) and secreted frizzled related protein 4 using western blotting. Long-term excessive treatment of prednisone inhibited the bone formation rate accompanied with a decrease in bone mass, growth plate, body weight, and the level of bone-specific alkaline phosphatase and hydroxyl-terminal propeptide of type I procollagen in the serum. Furthermore, a simultaneously increase in the level of tartrate resistant acid phosphatase-5b and cross-linked carboxy-terminal telopeptide of type I collagen in the serum, in addition to DKK1, and WIF1 protein expression, was observed. PMR30 (M and L) and PMRF (H) groups were able to reduce the negative effects of GC on the bones. PMR30 (M and L) and PMRF (H) dose demonstrated a protective effect of PM on bone tissue in GIO rats. The mechanism underlying the preventive effect of PM for the treatment of GIO may be associated with direct upregulation of the canonical Wnt/β-catenin signaling pathway.

Salvianolic acid B and danshensu induce osteogenic differentiation of rat bone marrow stromal stem cells by upregulating the nitric oxide pathway

The aim of the present study was to investigate the effect of salvianolic acid B (Sal B) and danshensu (DSU) on the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) and the mechanisms of the effects. The osteogenic differentiation of MSCs in culture was assessed by measuring alkaline phosphatase (ALP) activity, osteocalcin (OCN) production, nitric oxide (NO) production and the mRNA expression levels of osteoprotegerin (OPG) and its ligand by MSCs. MSCs were successfully induced to differentiate into osteoblasts and adipocytes. Sal B and DSU increased the ALP activity and the production of OCN in the absence of an ossification inducer. The increase in ALP activity was more pronounced when induction was combined with the osteogenic inducer, Sal B, which enhanced the expression of OPG; however, Sal B reduced the expression of receptor activator of nuclear factor-κB ligand (RANKL) by MSCs. Sal B reversed the inhibitory effect of N-nitro L-arginine methylester on the MSCs and increased ALP activity, OCN content and the OPG/RANKL ratio. Based on these results, it was concluded that Sal B increases the osteogenic differentiation of MSCs, most likely by regulating the nitric oxide pathway.

Preventive effects of Polygonum multiflorum on glucocorticoid‑induced osteoporosis in rats

In Traditional Chinese Medicine, Polygonum multiflorum (PM) is known for its anti-aging properties. A previous study by our group showed that extracts of PM were able to prevent and treat bone loss in vivo, and the active components emodin and 2,3,5,4,-tetrahydroxystilbene-2-O-β-glucoside (TSG) promoted the osteogenic differentiation of mesenchymal stem cells in vitro. The aim of the present study was to investigate the preventive effects of PM on glucocorticoid-induced osteoporosis (GIO) in rats. A crude extract of PM was prepared with 75% ethanol, purified and enriched using a D-101 macroresin column and elution with 30% ethanol, and the material obtained was assessed by high-performance liquid chromatography. Male or female Sprague Dawley rats (n=180) were randomly divided into nine groups: Control, prednisone, prednisone plus calcitriol (CAL), prednisone plus 30% ethanolic eluate of PM [high (H), medium (M) and low (L) dose] and prednisone plus crude extract of PM (H, M and L dose). Prednisone was orally administered to the osteoporosis model rats for 21 weeks, alongside which they received PM extracts. The weight of the viscera, anterior tibial muscle and other tissues was recorded at the end of the experiment. The femur and lumbar vertebra were collected for the measurement of three-dimensional microarchitecture by micro-computed tomography scanning, assessment of biomechanical properties and determination of bone mineral density (BMD). In the 30% ethanolic eluate of the PM extract, the content of TSG and combined anthraquinone was 9.20 and 0.15%, respectively, and that in the crude extract of PM was 2.23 and 0.03%, respectively. Over 6 weeks, the weight of the rats the in prednisone group decreased (P<0.05), while the weight of rats treated with M and H doses of 30% ethanolic eluate was increased compared with that in the prednisone group (P<0.05). Rats exposed to prednisone exhibited a deteriorated bone microarchitecture, low BMD, decreased bone volume/total volume and poor biomechanical properties. Furthermore, the weight of the adrenal gland and the anterior tibial muscle was decreased. 30% ethanolic eluate of PM at M and L doses and crude extract of PM at the H dose counteracted the alterations of skeletal and other characteristics induced by prednisone in rats, as did CAL. In conclusion, extracts of PM exerted a protective effect on bone tissue in GIO rats.