timosaponin-b-ii and Osteoporosis

Defective autophagy occurred in osteoblasts under stress induced by high glucose and played an essential role in the development of diabetic osteoporosis. Timosaponin BII, a steroidal saponin isolated from the rhizomes of Anemarrhena asphodeloides Bunge, possessed anti-osteoporosis properties. In this study, we investigated the efficacy and mechanism of timosaponin BII on diabetic osteoporosis. Timosaponin BII attenuated the deterioration in the microarchitecture of the tibias in diabetic rats. Furthermore, treatment with timosaponin BII dose-dependently reduced hyperglycemia-induced cell apoptosis in primary osteoblasts from rat calvaria. High glucose-exposed osteoblasts exhibited increased mitochondrial superoxide level, decreased mitochondrial membrane potential and impaired autophagic flux, which was attenuated by timosaponin BII, as evidenced by the upregulation of autophagosome numbers, LC3B puncta formation and Beclin1 expression. The antiapoptotic and antioxidative effect of timosaponin BII were repressed by the autophagy inhibitor 3-methyladenine and enhanced by the autophagy inducer rapamycin. Further studies showed that timosaponin BII suppressed the phosphorylation of mTOR and S6K, as well as the downstream factors NFκB and IκB, consequently activating autophagy and decreasing apoptosis. Of note, coincubation of timosaponin BII with MHY1485, a pharmacological activator of mTOR, diminished the protein expression of Bcl2 induced by timosaponin BII, which was in parallel with decreased autophagy and increased phosphorylation of NFκB and IκB. Overexpression of NFκB reduced timosaponin BII-evoked autophagy and promoted apoptosis. The in vivo results showed that oral administration of timosaponin BII downregulated the phosphorylation of mTOR and NFκB and upregulated Beclin1 expression in the proximal tibias of diabetic rats. These results suggested that timosaponin BII attenuated high glucose-induced oxidative stress and apoptosis through activating autophagy by inhibiting mTOR/NFκB signalling in osteoblasts.

Topics: Animals; Apoptosis; Autophagy; Diabetes Mellitus, Experimental; Hyperglycemia; Osteoblasts; Osteoporosis; Rats; Saponins; Signal Transduction; Steroids; TOR Serine-Threonine Kinases

Icariin (I), ferulic acid (F) and timosaponin B II (T) derived respectively from the leaf of Epimedium brevicornu Maxim (EBM, Berberidaceae), rhizome of Anemarrhena asphodeloides Bunge (AAB, Liliaceae) and root of Angelica sinensis (Oliv.) Diels (ASD, Umbelliferae) are included in several traditional Chinese medicine (TCM) formulas for the treatment of osteoporosis. In addition, the medicinal materials and chemical constituents in many traditional Chinese formulas have been shown to have potential synergistic, additive and antagonistic effects.. To explore the action mechanism and interactions between I, T and F as bone anabolic ingredients on osteoblasts, and fully understand their action mechanism and rationality of the formula design.. An osteoporotic model was established in bilaterally ovariectomized mice. Bone mineral density (BMD), bone mineral content (BMC) and serum biochemical parameters including alkaline phosphatase (ALP), tartrate resistant acid phosphatase (TRAP), osteoprotegerin (OPG) and deoxypyridinoline cross-links (DPD) were measured to evaluate the effects of I, T or F alone and their combinations on osteoporotic mice. UMR-106 osteoblastic cells and primary osteoblasts in neonatal rat calvarias were used to evaluate the osteogenesis effect. The immunohistochemical method and Western-blot analysis were used to detect the expression of critical proteins in the process of proliferation and differentiation of osteoblasts.. IFT combinations enhanced the therapeutic effect without increasing the adverse effects on osteoporotic mice, synergistically increased the osteoblast proliferation, ALP activity and mineralized nodule formation, and promoted the expression of bone matrix by regulating BMP and Wnt/β-catenin signaling pathways in osteoblasts.. IFT combinations reinforced the therapeutic effect on osteoporosis by modulating multi-signaling pathways and action targets.

Topics: Animals; Animals, Newborn; Calcium; Cell Line; Cell Proliferation; Coumaric Acids; Drugs, Chinese Herbal; Female; Flavonoids; Gene Expression Regulation; Mice; Molecular Structure; Osteoblasts; Osteogenesis; Osteoporosis; Rats; Saponins; Steroids