MiR-155 inhibition alleviates suppression of osteoblastic differentiation by high glucose and free fatty acids in human bone marrow stromal cells by upregulating SIRT1.

Diabetic osteoporosis is a severe and chronic complication of diabetes in the bone and joint system, and its pathogenesis is needed to be explored. In the present study, we examined the effect and underlying mechanism of miR-155 on osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs) under high glucose and free fatty acids (HG-FFA) conditions. It was shown that miR-155 levels in hBMSCs increased corresponding to the time of exposure to HG-FFA treatment. MiR-155 expression was altered by transfecting miR-155 mimic or miR-155 inhibitor. HG-FFA exposure resulted in an obviously decrease in cell viability and alkaline phosphatase (ALP) activity, and downregulated the expressionof runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) in hBMSCs. Transfection of miR-155 mimic further exacerbated HG-FFA-induced inhibitory effect on osteogenic differentiation, and miR-155 inhibitor neutralized this inhibitory effect. Luciferase assays confirmed that SIRT1 was a direct target of miR-155 and can be negatively modulated by miR-155. Furthermore, SIRT1 siRNA partially counteracted miR-155 inhibitor-induced upregulation of SIRT1in HG-FFA-treated hBMSCs. SIRT1 siRNA also reversed the promotional effect of the miR-155 inhibitor on ALP activity and expression of the Runx2 and OCN proteins under HG-FFA conditions. In conclusion, the results suggest that miR-155 suppression promoted osteogenic differentiation of hBMSCs under HG-FFA conditions by targeting SIRT1. Inhibition of MiR-155 may provide a new therapeutic method for the prevention and treatment of diabetic osteoporosis.

MiR-449 overexpression inhibits osteogenic differentiation of bone marrow mesenchymal stem cells via suppressing Sirt1/Fra-1 pathway in high glucose and free fatty acids microenvironment.

Diabetic osteoporosis is a chronic complication caused by diabetes mellitus, and However, the exact mechanism of diabetes mellitus-induced osteoporosis is still unknown. In this study, we investigate the effect of miR-449 on osteogenic differentiation and its underlying mechanism in human bone marrow-derived mesenchymal stem cells (hBMSCs) with high glucose (HG) and free fatty acids (FFA) treatment. Results showed that after culturing for 14 days, high glucose (HG) and free fatty acids (FFA) treatment dramatically decreased mineralization of human bone marrow-derived mesenchymal stem cells (hBMSCs) compared with cells treated with osteogenic medium (OM) alone. We also found that miR-449 expression was up-regulated during osteogenic differentiation of hBMSCs with HG and FFA treatment. Moreover, during osteogenic differentiation of hBMSCs with HG and FFA treatment, miR-449 mimics notably decreased the alkaline phosphatase (ALP) activity and the mRNA and protein expression levels of runt-related transcription factor 2 (Runx2), ALP, collagen I, osteocalcin (OCN), and bone sialoprotein (BSP), which was remarkably increased by miR-449 inhibitors. Furthermore, miR-449 directly targets Sirt1 by binding to its 3'-UTR. Sirt1 overexpression reverses the suppressive effect of miR-449 mimics on Fra-1 mRNA and protein expression, which was also alleviated by Fra-1 overexpression. In addition, Fra-1 overexpression alleviates the inhibitory effect of miR-449 mimics on the ALP activity and the mRNA and protein of Runx2, collagen I, OCN and BSP. Taken together, our results indicated that miR-449 overexpression inhibited osteogenic differentiation of HG-FFA-treated hBMSCs through the Sirt1/Fra-1 signal pathway. It is conceivable that modulating miR-449 might provide a new therapy for intervention in diabetic osteoporosis.

Bone Turnover Markers and Bone Mineral Density to Predict Osteoporotic Fractures in Older Women: A Retrospective Comparative Study.

OBJECTIVE: To investigate the clinical significance of the detection of bone mineral density (BMD) and bone turnover markers (BTM) in older women with osteoporosis, and to compare their predictive power for osteoporotic fractures (OF). METHODS: In this retrospective study, 96 patients with OF and 107 patients with osteoporosis who were hospitalized in the Department of Orthopedics at the First Affiliated Hospital of Chengdu Medical College were examined from October 2017 to February 2019. All selected patients were divided into either the fracture group (96 cases, 47.3%) or the non-fracture group (107 cases, 52.7%). BMD was measured by dual-energy X-ray absorptiometry (DXA). BTM were detected by electrochemical luminescence: aminoterminal propeptide of type I procollagen (PINP), ß-cross-linked C-telopeptide of type I collagen (ß-CTX), and molecular fragment of osteocalcin N terminal (N-MID). Bone metabolism-related indicators were detected, including alkaline phosphatase (ALP), calcium (Ca), and phosphorus (P). Independent-samples t-tests were used to compare the measurement data between the two groups, one-way ANOVA to compare the gaps between groups, and binary logistic regression to analyze the correlation of BMD and BTM with OF. RESULTS: There were no significant differences in age, weight, height, body mass index, age, and time of menopause between the two groups. There were a total of 71 cases (35.0%) in group A (60-70 years), 80 cases (39.4%) in group B (71-80 years), and 52 cases (25.6%) in group C (81-90 years). The fracture group was compared with the non-fracture group for BMD in the lumbar (0.75 ± 0.05 vs 0.88 ± 0.13, 0.75 ± 0.16 vs 0.87 ± 0.09, 0.74 ± 0.21 vs 0.87 ± 0.12 g/cm2 ; P < 0.05), BMD in the hip (0.62 ± 0.16 vs 0.74 ± 0.14, 0.61 ± 0.15 vs 0.73 ± 0.0, 0.58 ± 0.13 vs 0.73 ± 0.08 g/cm2 ; P < 0.05), PINP (83.7 ± 5.7 vs 74.8 ± 5.0, 80.7 ± 4.1 vs 72.1 ± 5.1, 81.2 ± 7.0 vs 68.7 ± 6.3 ng/mL, P < 0.05), and ß-CTX (829.7 ± 91.5 vs 798.8 ± 52.2, 848.1 ± 71.2 vs 812.4 ± 79.0, 867.3 ± 53.1 vs 849.1 ± 67.2 pg./mL, P < 0.05). N-MID (19.0 ± 6.7 vs 21.3 ± 9.7, 16.2 ± 7.0 vs 18.0 ± 5.3 ng/mL, P < 0.05) in the fracture cases was lower than in the non-fracture cases for groups B and C, and there was statistical significance. Among the fracture cases, PINP in group A was higher than in group B and C, and ß-CTX in group C was higher than in group A and B (P < 0.05). There was no significant difference in the ALP, P, and Ca between the two groups (P > 0.05). Binary logistic regression analysis showed that for BMD in the lumbar and hip, ß-CTX and OF were significantly correlated (respectively, odds ratio [OR] = -4.182, 95% confidence interval [CI] 1.672-3.448; OR = 6.929, 95% CI 2.586-12.106; OR = 7.572, 95% CI 1.441-3.059), and the differences were statistically significant. PINP and N-MID were correlated with OF (respectively, OR = 4.213, 95% CI 0.978-1.005; OR = 2.510, 95% CI 1.070-1.134, P > 0.05), the difference was not statistically significant. CONCLUSION: Osteoporotic older women, with lower bone density and higher ß-CTX, are more likely to incur OF. ß-CTX is better than BMD at predicting OF and can help in its management and in implementing interventions in high-risk populations.

SIRT1 suppresses high glucose and palmitate-induced osteoclast differentiation via deacetylating p66Shc.

Findings concerning the role of diabetes mellitus (DM) in osteoclast differentiation are contradictory in vivo and in vitro. Sirtuin 1 (SIRT1) can inhibit RANKL-induced osteoclastogenesis and deacetylate p66Shc suppress its phosphorylation in high glucose (HG)-stimulated human umbilical vein endothelial cells. This study aimed to investigate the role and mechanism of SIRT1 in DM-related osteoclast differentiation. Osteoclast precursors were cultured with HG and palmitate (PA), with or without resveratrol/sirtinol. TRAP staining was used to evaluate osteoclast formation. The expression of SIRT1, RANK, RANKL, OPG, NFATc1, TRAP, c-fos, p66Shc, phospho-p66Shc (S36), phospho-NF-κBp65 (p-p65), and IκB was determined by real-time PCR or western blotting. Lysine acetylation of p66Shc was assayed by immunoprecipitation. Reactive oxygen species (ROS) production was analyzed by DCFH-DA fluorescence. p66Shc siRNA and PDTC were used to confirm the mechanism of SIRT1 in osteoclastogenesis. We found HG and PA enhanced osteoclast differentiation, decreased SIRT1 and OPG expression, and increased levels of RANK, RANKL, NFATc1, TRAP, and c-fos. Upregulation of SIRT1 by resveratrol inhibited HG- and PA-induced osteoclast differentiation, whereas sirtinol further enhanced it. Resveratrol suppressed lysine acetylation and S36 phosphorylation of p66Shc, ROS production, and NF-κB activation induced by HG and PA, while sirtinol boosted these processes. p66Shc siRNA abrogated HG- and PA-induced ROS production and NF-κB activation. In addition, p66Shc siRNA and PDTC greatly suppressed the expression of RANK and RANKL induced by HG and PA. In conclusion, this study confirms the role of DM in osteoclast differentiation in vitro. SIRT1 suppresses HG- and PA-induced osteoclast differentiation via p66Shc/ROS/NF-κB signaling.