Glucocorticoid (GC)-induced osteoporosis (GIO) is one of the most common secondary and iatrogenic forms of osteoporosis. GCs are widely used in clinical therapy and play a key role in the normal regulation of bone remodeling. However, the prolonged and high-dose administration of GCs results in the occurrence of osteoporosis, which is partially due to the dysfunction and apoptosis of osteoblasts and osteocytes. The aim of the present study was to investigate the effects of gastrodin, a natural bioactive compound isolated from the traditional Chinese herbal agent Gastrodia elata, on GC-treated MC3T3-E1 murine osteoblastic cells. MC3T3-E1 cells were exposed to dexamethasone (DEX), with or without gastrodin pretreatment, and cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. Quantitative polymerase chain reaction analysis was performed to evaluate osteogenic gene expression, and cellular alkaline phosphatase (ALP) activity was measured as well. Alizarin Red staining of calcium deposits was found to reflect the degree of osteoblast maturity. Western blotting was performed to determine the expression of osteogenic and adipogenic differentiation key proteins, as well as nuclear factor-like 2 (NRF2) pathway-related proteins. Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometric analysis was performed to determine osteoblast apoptosis. JC-1 staining was used to detect the changes of the mitochondrial membrane potential in cells. The results revealed that gastrodin prevented the decrease in cell viability caused by DEX-induced MC3T3-E1 cell dysfunction, and that groups pretreated with gastrodin exhibited higher mRNA levels of osteogenic genes, such as Runx2, osterix, bone morphogenetic protein-2 and osteocalcin. Furthermore, treatment with both DEX and gastrodin was associated with increased ALP activity in MC3T3-E1 cells, as well as more calcium deposits, compared with cells treated with DEX alone. In addition, gastrodin increased osteogenic key marker protein Runx2 while activating NRF2 and downstream effector protein expression. Therefore, gastrodin may have the potential to reduce DEX-induced cell apoptosis and increase the mitochondrial membrane potential against DEX. These results demonstrated that gastrodin was able to prevent and/ or delay DEX-induced osteoporosis by improving osteoblast function, and these protective effects were verified in an animal model.

PurposeTRIM29 overexpression has been reported in several human malignancies and showed correlation with cancer cell malignancy. The aim of the current study is to examine its clinical significance and biological roles in human bladder cancer tissues and cell lines.Materials and MethodsA total of 102 cases of bladder cancer tissues were examined for TRIM29 expression by immunohistochemistry. siRNA and plasmid transfection were performed in 5637 and BIU-87 cell lines. Cell Counting Kit-8, flow cytometry, western blot, and real-time polymerase chain reaction were performed to examine its biological roles and mechanism in bladder cancer cells.ResultsWe found that TRIM29 overexpression showed correlation with invading depth (p=0.0087). Knockdown of TRIM29 expression in bladder cancer cell line 5637 inhibited cell growth rate and cell cycle transition while its overexpression in BIU-87 cells accelerated cell proliferation and cell cycle progression. TRIM29 overexpression also inhibited cell apoptosis induced by cisplatin. In addition, we demonstrated that TRIM29 depletion decreased while its overexpression led to upregulated expression of cyclin D1, cyclin E, and Bcl-2. We also showed that TRIM29 knockdown inhibited protein kinase C (PKC) and nuclear factor κB (NF-κB) signaling while its overexpression stimulated the PKC and NF-κB pathways. BAY 11-7082 (NF-κB inhibitor) partly attenuated the effect of TRIM29 on expression of cyclin and Bcl-2. Treatment with PKC inhibitor staurosporine resulted in ameliorated TRIM29 induced activation of NF-κB.ConclusionThe current study demonstrated that TRIM29 upregulates cyclin and Bcl family proteins level to facilitate malignant cell growth and inhibit drug-induced apoptosis in bladder cancer, possibly through PKC–NF-κB signaling pathways.

Paeoniflorin, a natural product derived from Paeonia lactiflora, possesses diverse pharmacological activities such as anti-inflammatory, antitumor, and antidiabetic effects. It has been reported for promoting osteoblastogenesis and inhibiting osteoclastogenesis. This study investigates the therapeutic effects of paeoniflorin in glucocorticoid-induced osteoporosis (GIOP) in vitro and in vivo. MC3T3-E1 cells were incubated with dexamethasone (DEX; 200 μM) and/or paeoniflorin (10 μM), followed by the investigation of cell proliferation, differentiation, mineralization, apoptosis, and autophagy. The AKT activator SC79 was used for evaluating the involvement of the AKT/mTOR signaling pathway. After DEX pretreatments, paeoniflorin promoted osteoblast differentiation and mineralization characterized by increase in Runx2, ALP, beclin-1, and LC3-II/LC3-I ratio levels and a decrease in apoptosis. The autophagy-promoting effects of paeoniflorin were reversed by SC79. C57BL/6 mice were given DEX (1 mg/kg) once daily and paeoniflorin (15 mg/kg) 48 hours for a total of 8 weeks followed by the investigation of histological changes, the trabecular bone microarchitecture, and the levels of bone turnover markers. The results showed that paeoniflorin increased alkaline phosphatase (ALP) activity and upregulated the expression of osteocalcin and beclin-1 but reduced the levels of Bax and C-terminal telopeptide of type I collagen (CTX-1). Thus, paeoniflorin may alleviate DEX-induced osteoporosis by promoting osteogenic differentiation and autophagy via inhibition of the AKT/mTOR signaling pathway.

Background: Prolonged administration of overdoses of glucocorticoids results in increased bone remodeling, leading to glucocorticoid-induced osteoporosis (GIO), which is primarily due to the dysfunction and apoptosis of osteoblasts. The present study investigated the therapeutic effect and molecular mechanism of action of epigallocatechin-3-gallate (EGCG), a bioactive catechin in green tea, in high-dose dexamethasone-induced osteoblast differentiation in vivo and in vitro.Methods: The anti-dexamethasone (DEX) effects of EGCG on primary osteoblasts were determined on the basis of cell viability and alkaline phosphatase (ALP) and total cellular superoxide dismutase (SOD) activities. Flow cytometry and Western blot analysis were also used to evaluate the expression of related biomarkers in vitro, and bone microarchitecture was also extensively examined in a rat model in vivo.Results: The results showed that EGCG pretreatment significantly increased osteoblast viability and ALP and SOD activities when cells were exposed to DEX. Alizarin red staining indicated that there was more mineralization with EGCG pretreatment, countering DEX effects. EGCG reduced DEX-induced reactive oxygen species at both the mitochondrial and cellular levels in osteoblasts by activating the nuclear factor erythroid-derived 2-like-2 (Nrf2) pathway. In addition, EGCG protected osteoblasts from apoptosis. EGCG also regulated the formation of active glucocorticoid by 11β-hydroxysteroid dehydrogenase activity. Furthermore, femoral micro-computed tomography scans revealed that EGCG improved bone microstructure and mitigated DEX-induced deterioration of bone quality.Conclusion: These findings suggested that EGCG reversed GIO in rats by protecting osteoblasts by activating the Nrf2 signaling pathway.