The Prevention Of2,3,5,4-tetrahydroxystilbene--o-β-D-glucoside On Osteoporosis And The Related Mechanism

Osteoporosis is a systemic, degenerative and metabolic bone disease, which ischaracterized by deterioration of bone mass, microarchitecture, bone mineral density andbone biomechanics and leads to an increased risk of fracture. It is seriously threatening thephysical and mental health of the elderly. Oxidative stress is caused by excessivegeneration of reactive oxygen species （ROS） beyond the body’s scavenging capacity andresults into a state of intracellular reduction–oxidation imbalance in the body. Highconcentrations of ROS could oxidize the nucleic acids, proteins and lipids, and damage thestructure and function of the cells, leading to the development of a disease. Many degenerative diseases have been shown to be closely related to oxidative stress. In recentyears, a large number of studies have confirmed that oxidative stress plays an importantrole in the development of osteoporosis. Excessive generation of ROS can stimulateapoptosis and inhibit osteogenic differentiation of osteoblasts, stimulate formation andactivity of osteoclasts and affect the bone formation and resorption coupling. As a result,bone formation decreases and bone resorption increases, leading to bone mass loss.Meanwhile, the elevated oxidative stress level in plasma has a correlation with deceasedbone density in osteoporotic patients. Therefore, using antioxidant to prevent and treatosteoporosis has become research focus. Epidemiological studies have provided evidenceof a link between nutrient, antioxidant intake and bone health, and have led toinvestigations of the antioxidant properties of nutrients.2,3,5,4-tetrahydroxystilbene-2-o-β-D-glucoside（TSG） is a kind of extracts of Polygonum, which is a traditional Chinesemedicine, has antioxidant effect. It is unclear that whether TSG could be applied to thetreatment of OP until now.This study was divided into three parts: firstly, we tested the protective effect of TSGon H₂O₂induced cytotoxicity in MC3T3-E1cells and clarified part of the relatedmechanism; then, we evaluated the protective effect of TSG on osteoblast dysfunctioninduced by H₂O₂; finally, we demonstrated the protective effect of TSG against bone lossin OVX mice. We confirmed a new use of TSG to the treatment of OP in this study andprovided a theoretical basis for the treatment of OP with the traditional Chinese medicine.1. Protective effect of TSG on oxidative stress induced cytotoxicity in MC3T3-E1cells and the related mechanismObjective: the purpose of this study was to investigate the protective effect of TSG onapoptosis of MC3T3-E1cells induced by oxidative stress and clarify the relatedmechanism. Methods: At first, we built oxidative stress model in vitro. In this study,MC3T3-E1cells were treated with different concentrations of H₂O₂（0,100,200,300and400μM/L） for2,6,12and24h and cell viability was evaluated by MTT assay. In this way,we chose the appropriate concentration of H₂O₂. Meanwhile, cells were treated with different concentrations of TSG （0,0.1,1and10μM/L） for24and72h in order toinvestigate the toxicity of TSG. The selected concentrations of H₂O₂and TSG were usedin the following studies. The experiment was divided into6groups, including the controlgroup, H₂O₂, H₂O₂+TSG（0.1μM）, H₂O₂+TSG（1μM）, H₂O₂+TSG（10μM） and H₂O₂+NAC（1mM）. The protective effect of TSG on apoptosis of the cells induced by oxidative stresswas demonstrated by MTT assay, general observation under microscope, Hoechst33258staining and flow cytometry. The level of ROS and MDA were tested by fluorescencemicroplate reader and MDA assay kit to evaluate the cellular oxidative stress status. Inorder to further clarify the related mechanism, we examined the production of Bcl-2andBax protein using Western-blotting and the gene expression of Bcl-2and Bax by RT-PCR.Results: The suitable concentration and duration of action for H₂O₂was300μM/L for24hwhich was used in the following study. TSG alone was non-toxic to MC3T3-E1cells atthe concentrations used in this study （P>0.05）. Compared with the control group, H₂O₂alone led to obvious cell death which was affirmed by observation under the microscopeand Hoechst33258staining. After being pretreated with different concentrations of TSG,the rate of apoptosis was reduced to some degree. The results of flow cytometrydemonstrated that: the rate of apoptosis in each group was5.8%、30.1%、22.1%、15.3%、14.3%、9.2%. We can deduced that treated with H₂O₂alone can lead to a great degree ofapoptosis in MC3T3-E1cells and pretreated with different concentrations of TSG coulddecrease the rate of apoptosis （P<0.05）. At the same time, H₂O₂alone can increase theproduction of cellular ROS and lipid oxidation products MDA and pretreated with TSG（1-10μM） can significantly reduce the cellular ROS and MDA （P<0.05） and improve theoxidative stress status in MC3T3-E1cells. The results from western-blotting and real-timequantitative RT-PCR suggested that pretreatment with TSG can decrease the expression ofpro-apoptosis protein Bax and increase the expression of anti-apoptosis protein Bcl-2inthe level of protein and mRNA. Conclusions: Oxidative stress could lead to increasedapoptosis of MC3T3-E1cells and TSG play a protective role during this process byreducing the oxidative stress level and improving the Bcl-2/Bax signal pathway. 2. Protective effect of TSG on osteoblast dysfunction induced by oxidative stress andthe related mechanismObjective: The purpose of this study was to investigate the protective effect of TSG onosteoblast dysfunction induced by oxidative stress and the related mechanism, in order tolay a solid foundation for the in vivo study. Methods: Murine osteoblastic MC3T3-E1cells were divided equally into6well plates and cultured in osteogenic induction mediumat about80%confluency. The induction culture medium contains10mMβ-glycerophosphate and50μg/ml ascorbic acid to initiate differentiation. In thisexperiment, we divided into6groups according to the way of treatment, including thecontrol group, H₂O₂, H₂O₂+TSG（0.1μM）, H₂O₂+TSG（1μM）, H₂O₂+TSG（10μM） andH₂O₂+NAC（1mM）. After induction for6,14or21days, MC3T3-E1cells werepre-incubated with different concentrations of TSG for24h before treatment with300μMH₂O₂for another24h. Alkaline phosphatase （ALP） staining and activity were used toevaluate the expression of osteogenic marker ALP; Alizarin Red S staining was used toassess calcium deposition; Real-time RT-PCR was used to test the expression level ofostegenic differentiation genes ALP, COL-I and OCN. In order to further clarify therelated mechanism, we tested the MDA and ROS levels in the cells to evaluate theoxidative stress and measured the production of bone-resorbing mediators: RANKL andIL-6by sandwich ELISA assay kit. Results: Cellular ALP staining and activity, calciumdeposition and osteogenic differentiation genes were significantly decreased in H₂O₂group when compared with the control group （P<0.05）. Pre-incubated for24h, TSG（0.1-10μM） could significantly increase ALP activity in a dose-dependent manner（P<0.05）, improve ALP staining and calcium deposition and significantly increase theexpression level of osteogenic differentiation genes （P<0.05）. Meanwhile, the cellularROS and MDA levels were increased after H₂O₂treatment （P<0.05） and this effect wasobviously reversed by pre-incubated with TSG （1-10μM）. The results from ELISA assayindicated that H₂O₂raised the production of RANKL and IL-6and these was reduced bypre-incubated with different concentrations of TSG,0.1-10μM and10μM respectively （P<0.05）. Conclusions: Oxidative stress could inhibit the osteogenic differentiation andTSG could reverse this effect partially by its antioxidant ability and reducingbone-resorbing mediators.3. Protective effect of TSG against bone loss in OVX miceObjective: The purpose of this study was to investigate the protective effect of TSGagainst bone loss in a murine ovariectomized （OVX） osteoporosis model. Methods:Thirty-two9-week-old and weighing19.73±2.011g BALB/c female mice were randomlydivided into4groups:（1） untreated （Sham: sham-operated controls）;（2） untreated （OVXcontrols）;（3） OVX administered intraperitoneally with TSG （5mg/kg body weight） daily;（4） OVX administered intraperitoneally with TSG （20mg/kg body weight） daily.According to the groups, both of the ovaries were removed under anesthesia bypentobarbital sodium （50mg/kg body weight, i.p.） with dorsum approach. TSG weredissolved in distilled water. The administration of TSG started1week after the surgeryand lasted for3months. In order to evaluate the oxidative stress level in serum, we tookblood samples from the heart in anesthetized mice and prepared it by centrifugation to testthe content of MDA and GSH. The4th lumbar vertebra （L4） and the left femur from eachmouse were removed, cleaned of adherent tissue and fixed in4%paraformaldehyde for48hours. The effects of TSG on bone mineral density, bone mass and bone microarchitecturewere assessed by Micro-CT, H&E staining, Van Gieson staining and Von Kossa staining.Results: Compared with the sham group, serum MDA level was increased and GSH levelwas decreased （P<0.05） in the OVX group. TSG supplementation （20mg/kg） significantlydecreased serum MDA level （P<0.05）, without significantly increased the GSH level（P>0.05）. There was no difference in MDA and GSH levels in OVX mice treated withhigh or low concentration of TSG （P>0.05）. The results from Micro-CT scanningindicated that ovariectomy induced deterioration of the trabecular bone microarchitecturein distal femoral metaphyses and the fourth lumber vertebrate （L4）, as demonstrated by thereduction in BV/TV, Conn.D, Tb.N, Tb.Th and BMD when compared with the shamgroup （P<0.01）. In contrast, SMI and Tb.Sp were significantly increased in response to OVX （P<0.05）. However, supplementation of TSG with a high dose at20mg/kg couldsignificantly （P<0.05） reverse the changes induced by ovariectomy and could maintain themicroarchitecture of trabecular bone in distal femoral metaphyses and the fourth lumbervertebrate （L4）. TSG at5mg/kg could improve the trabecular bone parameters, but withoutsignificant difference when compared with the OVX group （P>0.05）. There was nodifference between TSG treated groups （P>0.05）. Undecalcified histological examinationwith Van Gieson and Von Kossa staining demonstrated that ovariectomy could lead toreduced trabecular number and broader spaces between trabecules compared with thesham group. TSG supplementation reversed these deleterious effects, as demonstrated byan increase in trabecular number and a decrease in the trabecular space in the TSG treatedgroups. H&E staining obtained the similar result. Conclusions: TSG could reduce theserum oxidative stress level in murine ovariectomized osteoporosis model and reverse thereduced bone mineral density, bone mass loss and deleterious bone microarchitecture.