A Study On The Suppressive Effect And Mechanism Of Paris Phyllin Ⅶ On Osteoclast Differentiation

Background:Osteoporosis(OP)is a metabolic bone disease characterized by increased trabecular bone loss and bone fragility.Epidemiological data have confirmed that bone loss,bone mineral density decline is inevitable with the growth of age,followed by the increasing of risk of fracture.It is estimated that about 40% of white women over 50 years of age and 13% of white men over 50 years of age will suffer from osteoporotic fracture.In 2005,the cost of osteoporotic-related fractures in the United States reached about $ 13 billion to $ 20 billion a year,and the cost is expected to rise to$ 25 billion by 2025 due to the rapid growth of fractures.In China,about 210 million people whose bone mass are below the normal standard,at least nearly 70 million people are suffering from osteoporosis.With the arrival of an aging society,osteoporosis and its associated fractures have become a major worldwide health problem that has attracted wide attention.The integrity of the skeletal system needs to be maintained by the balance of bone resorption and bone formation,whereas osteoclasts are derived from bone marrow macrophages and have important implications for bone resorption during bone balance.So,it’s important to understand differentiation and activity of osteoclasts so as to achieve the purpose of anti-osteoporosis.Reactive oxygen species(ROS)is a class of small particles containing oxygen,produced in the normal bio-aerobic process.Aging and inflammationcan increase the production of ROS,and ROS may lead to imbalance of oxidants and antioxidants.Over the past few years,it has been found that ROS-induced oxidative stress increases with age and inflammation which creating an environment that promotes osteoclasticity.There is growing evidence that ROS is a key factor in the regulation of osteoclast differentiation.A variety of antioxidants are likely to reduce the production of ROS,especially a variety of natural antioxidants which does not add more side effects,so the antioxidant plants have a goodpotential to treat osteoporosis.Phyllin Ⅶ is a plant extract,which is a saponin compound and a natural antioxidant,with the effect of reduction of inflammatory response,anti-tumor and protecting nervous system.But its inhibition of osteoporosis has not been reported.In this study,we conducted a series of experiments to confirm whether the Paris Phyllin Ⅶ inhibited RANKL-induced osteoclast differentiation and explored its mechanism.Purposes:1.Establishing a model of BMM cells differentiating into osteoclasts2.In vitro osteoclast differentiation model,observing inhibiting effect of Paris Phyllin Ⅶ on osteoclast differentiation.,3.The osteoclast differentiation model was used to study the mechanism of inhibiting effect of Paris Phyllin Ⅶ on osteoclast differentiation.,4.Establishing an animal model of osteoporosis and observing inhibiting effect of Paris Phyllin Ⅶ on osteoporosis.Methods:1.BMM cells were isolated and cultured in DMEM medium,and the model of cells differentiating into osteoclasts was established.2..CCK-8 was used to detect the proliferation of BMM cells.TRAP staining,TRAP enzyme activity assay,bone lacunae were used to observe the inhibition effect of Paris Phyllin Ⅶ on osteoclast differentiation.The effects of Paris Phyllin Ⅶ on osteoclast-specific m RNA expression during osteoclast differentiation were observed by fluorescence quantitative PCR.3.The changes of ROS in RANKL-induced osteoclast differentiation were observed by flow cytometry,and westem blot was used to oberve the role of Nox1,TRAF6-c Src-PI3 K,MAPK And NF-κB in the inhibition of Paris Phyllin Ⅶ on osteoclast differentiation.Pre-mi R-100-5p was transfected into BMM cells to make sure its effect on osteoclasts,and RT-q PCR was used to observe the relationship of Paris Phyllin Ⅶ and micro RNA-100-5p.4.Micro-CT was used at the animal model to observe the inhibition effect Paris Phyllin Ⅶ on osteoporosis.Results:1.Raw264.7 was proved not to be a good choice to differentiate into osteoclasts and BMM cells was at last selected to establish the model of cells differentiating into osteoclasts2.By CCK-8 detection,we found 1u M to 30 u M concentration of Paris Phyllin Ⅶ on BMM cells cause no significant toxic effects.3.TRAP staining,TRAP activity assay,bone lacunar experiment confirmed that:compared to the control group,lu M to 30 u M concentration of Paris Phyllin Ⅶ could significantly inhibit RANKL induced BMM cells to osteoclast differentiation.Real-time quantitative PCR showed that lu M to 30 u M concentration could inhibit the expression of osteoclast-related genes in osteoclast differentiation.4.Flow cytometry was used to confirm the production of ROS in the process of osteoclast differentiation.The results showed that RANKL could increase the production of ROS,and Paris Phyllin Ⅶ could inhibit the production of ROS induced by RANKL.5.The effect of Paris Phyllin Ⅶ on Nox1 and TRAF6-c Src-PI3 K pathway in osteoclast differentiation was detected by westem blot.The results showed that RANKL could increase the expression of Nox1,TRAF6 and c Src and increase the phosphorylation of PI3 K,while Paris Phyllin Ⅶ could decrease the expression of Nox1,TRAF6 and c Src induced by RANKL and inhibit the phosphorylation of PI3 K.6.The effect of Paris Phyllin Ⅶ on MAPK pathway in osteoclast differentiation process was tested by western blot.The results showed that RANKL could increase the phosphorylation of p38,ERK and JNK in MAPK pathway,and Paris Phyllin Ⅶ could inhibit the phosphorylation of p38,ERK and JNK induced by RANKL.7.The effect of Paris Phyllin Ⅶ on NF-κB pathway in osteoclast differentiation process was tested by western blot.RANKL significantly increased phosphorylation and degradation of IκB,while Paris Phyllin Ⅶ significantly inhibited the phosphorylation and degradation of IκB,and at the same time it could inhibit the phosphorylation of p65.Immunofluorescence also showed that RANKL could promote the transfer of p65 from the cytoplasm to the nucleus,whereas the Paris Phyllin Ⅶ significantly inhibited the translocation.8.RT-q PCR confirmed that mir-100-5p was down-regulated during osteoclast differentiation.Pre-mir-100-5p and its control were transfected into BMM cells,and it was found that the expression levels of TRAP,Ctsk,and NFATc1 of osteoclast specific genes were significantly down-regulated after the transfection of BMM cells,and the TRAP-positive polykaryotic giant cells were significantly reduced.All these indicated that the overexpression of mir-100-5p could inhibit osteoclast differentiation.Subsequently,RT-q PCR confirmed that Paris Phyllin Ⅶ can up-regulate the expression of mir-100-5p,and Paris Phyllin Ⅶ may also inhibit osteoclast differentiation through this pathway9.The effect of Paris Phyllin Ⅶ on osteoporosis in vivo was detected by Micro-CT,The results indicated that compared with the control group,the ovariectomized +PP7groups have higher bone trabeculae thickness(Tb.Th),bone volume fraction(BV/TV),bone density(BMD)and number of bone trabeculae(Tb.N)but lower Trabecular Separation(Tb.Sp).Micro-CT also indicated that compared with control group,the number of bone trabeculae,the trabecular thickness increased significantly and the fracture decreased significantly in the ovariectomized +PP7 group.Conclusion:1.We successfully establised RANKL-induced osteoclast differentiation model which made a good bais for the follow-up observation of inhibition effect of Paris Phyllin Ⅶ on osteoclast differentiation and related mechanisms.2.Paris Phyllin Ⅶ significantly inhibit RANKL-induced osteoclast differentiation3.The effect of inhibition of Paris Phyllin Ⅶ on osteoclast differentiation was associated with its ability of reducing ROS production and its inhibitory effect on Nox1,TRAF6-c Src-PI3 K,MAPK and NF-κB signaling pathway.Mir-100-5p can inhibit osteoclast differentiation,and the inhibitory effect of Paris Phyllin Ⅶ on osteoclast differentiation is also related to the up-regulation of mir-100-5p4.Paris Phyllin Ⅶ also significantly inhibited osteoporosis in vivo...