| Rhodiola rosea L, also known as "goden root", is a popular plant in traditional medicine in China and is reputable for improving depression, enhancing work performance, eliminating fatigue and treating symptoms of asthenia subsequent to intense physical and psychological stress. Due to these therapeutic properties, R. rosea L is considered to be one of the most active adaptogenic drugs. Salidroside and other salidroside-like compounds are considered to be most critical plant constitutes needed for the multiple therapeutic benefits of R. rosea L. Our previous study has shown that salidroside is able to extend the life span of drosophila and protect mouse against aging induced by D-galactose, showing considerable anti-aging effect. However, direct demonstration regarding the role of salidroside in anti-aging process in vitro and relevant molecular mechanisms is still deficient.In this study, we first selected the H2O2-induced premature senescence model in human fetal lung diploid fibroblasts (2BS cells) to investigate whether salidroside directly protects the cells against aging in vitro and to illustrate the associated molecular mechanisms. We found that salidroside considerably reverses the senescence-like phenotypes in the oxidant challenged model, which include morphological alterations, G1 phase arrest, positive SA-β-gal staining, as well as increase of senescence-associated molecules expression such as p53, p21 and p16. The protection occurs in a tightly dose-dependent manner, with 5μM offering best efficacy under most cases. The proposed antioxidant property of the compound was confirmed in this cellular system, evidenced by ROS scavenging, MDA decreasing as well as T-SOD increasing activity, and thus at least partially is accountable for the protection of the compound against cellular premature senescence. Meanwhile, similar protection of salidroside against replicative senescence model was observed as well and it was able to increase cumulative population doublings (CPDs) of 2BS by at least 8 PDs by the supplementation to the medium from early PD. Interestingly, the regulation of corresponding molecular markers by salidroside involves ROS-independent mechanisms when compared with NAC in above two models. One possibility for such phenomena could be that salidroside may directly regulate ROS-irrelevant DNA damage system that initiates the associated molecular changes resulting in senescence-like phenotypes. To confirm this, we used Lidamycin, a potent radiomimetic anti-tumor antibiotic and senescence inducer to induce DNA damaging response in 2BS cells and test the regulation of salidroside accordingly. Similarly, we observed the protection of salidroside against DNA damage response as evidenced by down-regulation of p53 protein and y-H2AX foci after Lidamycin exposure. As anticipated, the senescence-like phenotypes in young 2BS induced by Lidamycin are also partly reversed by the salidroside. Moreover, stimulation of mitochondria biogenesis is found to be another novel property of the compound. Notably, the stimulatory effect of salidroside on mitochondrial proliferation is comparable to that of resveratrol in late PD 2BS cells, while the mode of action for this compound may be different from that of resveratrol. These findings present salidroside as an attractive and bio-safe agent with the potential to retard aging process and more importantly, may attenuate age-related diseases in humans.In another part of work, we studied the protective role of salidroside against osteoporosis. Many observations clearly indicate the high potency of BMP2 as an inducer of osteogenesis which contributes to be a novel therapeutic target for diseases associated with bone loss, especially in menopausal and postmenopausal women. A considerable up-regulation of bmp2 expression by salidroside is observed in a high-throughput screening model in which a firefly luciferase reporter gene driven by the mouse bmp2 promoter has been transfected to clonal murine calvarial MC3T3-E1 cells, which indicates the possible stimulatory effect on bone formation of this compound. To confirm this effect, rat osteoblasts (ROB) and human osteoblast-like MG63 cells as well as an ovariectomized-osteoporosis (OVX-OP) rat model were used. Results in ROB indicate that salidroside increases the osteoblast viability, the alkaline phosphatase (ALP) activity and bone collagen synthesis by about 1.3,1.3 and 1.9 fold, respectively. Similar results were also observed in MG63 cells. Besides, the BMP2 expression at both mRNA and protein level is significantly elevated in response to salidroside in ROB and MG63 cells detected by real-time PCR as well as western blot analysis, repectively. Furthermore, our in vivo data demonstrates that salidroside significantly reverses the bone loss in ovariectomized rats, which is comparable to that of raloxifene, a clinical drug for prevention and therapy in menopausal and postmenopausal women. Interestingly, salidroside is observed to mildly inhibit the activity of osteoclasts at lower doses rather than higher doses, which was different from that of raloxifene. This finding indicates indirectly that salidroside elevated the bone mass in ovariectomized rats mainly due to its stimulatory effect on bone formation rather than the inhibition of bone resorption. In conclusion, salidroside is a potent BMP2 activator that stimulates bone formation and thus could be a potential anti-osteoporosis drug.Taken together, our work provide evidences supporting that salidroside, an active component of R. rosea L. extracts that has been generally recognized as bio-safe, exerts protective effects against cellular senescence in replicative and stess-induced models. The main mode of action for the anti-aging regulation of salidroside involves both ROS-dependent and ROS-independent mechanisms. The stimulatory effect of salidroside on mitochondria biogenesis, and on BMP2 expression as well, may be novel mechanisms for its benefit in delaying aging as well as age-related diseases such as osteoporosis.
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