Anti-osteoporosis Effect Of Paeoniflorin And Its GPR30-Related Mechanism

Osteoporosis(OP)is one of the common and frequently-occurring diseases that seriously threaten the health of middle-aged and elderly people.It is a systemic bone disease characterized by impairment of bone mass,strength,and microarchitecture.The balance of bone metabolic is an important prerequisite for maintaining bone mass.Bone-mass regulation depends on the dynamic balance of bone-resorbing osteoclasts(OCs)and bone-forming osteoblasts(OBs).OP develops when the bone resorption exceed those of bone formation.Danggui-Shaoyao-San(DSS),a traditional Chinese formula,is widely used in the treatment of various gynecological diseases.Our previous studies have confirmed that DSS exerted osteoprotective effects on ovariectomy-induced OP rats,but whether DSS can inhibit OCs differentiation or not is still unclear.Paeoniae radix alba,a main ingredient of DSS,is one of the most commonly used Chinese herbal medicine for the treatment of OP.Paeoniflorin(PF),the major bioactive component of the Paeoniae radix alba,which has been widely used in treatment of various autoimmune diseases such as rheumatoid arthritis and ankylosing spondylitis.However,there is lack of more research whether PF can inhibit OCs differentiation and anti-osteoporosis or not.Our previous research found that DSS significantly promotes the protein expression of the novel membrane estrogen receptor G protein-coupled receptor 30(G protein-coupled receptor 30,GPR30)and PF bind to the membrane estrogen receptor.The GPR30 was found to mediate OCs differentiation.Therefore,the aim of this study is to explore the effect of PF on OCs differentiation and the correlation between PF intervention OCs differentiation and GPR30.In addition,the effect of PF on apoptosis of OB precursor cells was exploredBased on the literature and the results of previous studies,the projest were mainly focused on the following aspects:1.The effects of DSS and PF on OCs differentiationA model of lipopolysaccharide(LPS)-induced differentiation of RAW 264.7 cells into OCs was established.The cells were grouped as follows:Control,LPS,LPS+DSS,LPS+PF.RAW 264.7 cells were treated with LPS for 4 days to induced OCs differentiation,and DSS or PF was administered on the model basis.Tartrate-resistant acid phosphatase(TRAP)staining and TRAP activity assay showed that DSS(10-7 g/L,10-6 g/L)and PF(0.1?25 ?M)significantly inhibited the production of TRAP-positive cells and TRAP activity.In addition,PF significantly inhibited the expression of OCs marker NFATcl,TRAP and CTSK in a dose-dependent manner.A model of RANKL-induced OCs differentiation was established.The cells were grouped as follows:Control,RANKL,RANKL+PF.TRAP staining and TRAP activity assays results showed that PF significantly reduced the number of TRAP positive cells and inhibited TRAP activity.RT-PCR results showed that PF significantly inhibited the mRNA expressions of OCs marker genes CTSK,NFATcl,c-Fos and DC-stamp.2.The effect of PF on bone resorptionTo evaluate the effect of PF on bone resorption,RAW 264.7 cells were seeded onto glass slide or bone-grinding slice,and incubated with various concentrations of PF for 6 days in the presence of LPS or RANKL.FITC-labeled phalloidin staining showed that PF significantly reduced the formation of F-actin ring in the typical skeletal structure of OCs in LPS or RANKL-induced OC differentiation.The result of toluidine blue staining showed that the number of bone resorption pits in the PF group was significantly decreased3.The effect of PF on ROS level and MAPKs and NF-?B signaling pathwaysReactive oxygen species(ROS)as a second messenger to promote OCs differentiation by activate several osteoclastogenesis signaling pathways including MAPKs(p38 MAPK,ERK and JNK)and NF-?B.RAW 264.7 cells were incubated with various concentrations of PF for 4 days in the presence of LPS.Flow cytometry showed that PF significantly inhibited LPS-induced elevated ROS level,especially in the 10 ?M.Compared with the LPS group,WB showed that the ratio of P-p3 8/p3 8 and P-p65/p65 were significantly reduced by PF.4.The relevance of GPR30 pathway in the inhibition effect of PF on OCs differentiationReferring back to the literature,studies found that the GPR30 participates in the process of OCs differentiation.The above results indicate that PF has a significant inhibitory effect on OCs differentiation.To further confirm the relevance of GPR30 pathway in the inhibition effect of PF on OCs differentiation,we performed the following experiments.(1)The effect of PF on the expression of GPR30 in LPS-induced OC differentiation.WB results showed that PF treatment promoted the expression of GPR30 in a concentration-dependent manner.(2)The effects of PF on LPS-induced increased ROS level and OCs differentiation after GPR30-specific antagonist G15 treatment.The results showed that co-treatment with G15 reversed the eliminative effect of PF on the ROS levels.In addition,the ability of PF to inhibit LPS-induced increased TRAP-positive cells.enhanced TRAP activity,and elevated osteoclast marker gene levels was reversed by G15.(3)The effect of PF on LPS-induced OCs differentiation was observed after GPR30 gene silencing.RT-PCR results showed that the inhibition effect of PF on OCs marker gene expression disappeared after gene silencing of GPR305.The effect of PF on prednisolone-induced OP in zebrafish(1)Zebrafish larvae OP model was induced by prednisolone.Healthy zebrafish with 3 days post fertilization(dpf)were selected and divided into control group,prednisolone group(25 ?M Pred)and PF group(1 ?M and 10 ?M).In addition to the control group,each group was pre-administered with prednisolone for 2 days prior to administration,followed by continuous administration of PF for 4 days.Calcein staining showed that PF significantly promoted the formation of the first vertebrae of prednisolone-induced zebrafish larvae.RT-PCR results showed that PF promoted the expressions of OB marker genes and inhibited the expressions of the marker genes.(2)The zebrafish fishtail fin regeneration model was established to observe the effect of PF on the regeneration of zebrafish caudal fin bone.The zebrafish were grouped as follows:Control group,Model group(25 ?M Pred),Model+PF group(1 ?M and 10 ?M).The photograph were collected on days 4,7,11 and 14,the result showed that PF significantly promoted the regeneration of zebrafish tail fins.RT-PCR analysis of the the tail fins showed that PF inhibited the expressions of OCs marker genes and promoted the expressions of OBs marker genes6.The effect of PF on apoptosis of OBs precursorsThe above results suggest that PF promotes OBs-mediated bone formation.A model of dexamethasone(DEX)-induced MC3T3-E1 cells apoptosis was established to observe the effect of PF on OBs precursors apoptosis.MTT showed that PF can alleviate the inhibitory effect of DEX on MC3T3-E1 cell viability.Flow cytometry showed PF significantly reduced the rate of apoptosis.Immunofluorescence showed that PF significantly inhibited the nuclear transfer of Fox03a protein,and WB showed that PF significantly promoted the protein expression of Bcl-2.Conclusion:(1)DSS could inhibit OCs differentiation.PF exhibits significant inhibition effect on OCs differentiation and bone resorption.These results suggest that PF may an effective component of DSS for anti-osteoporosis(2)G15 reverses the inhibitory effect of PF on ROS level,and resistants the effect of PF on LPS-induced OCs differentiation.After GPR30 silencing,the inhibition effect of PF on OCs differentiation is eliminated.The above suggests that PF inhibits OCs differentiation may through a GPR30/ROS-dependent pathway.(3)PF improves the bone area of the first vertebrae of OP zebrafish larvae and the regeneration of the tail fin of adult zebrafish,meanwhile inhibits the expressions of OCs marker genes and promotes the expressions of the OBs marker genes.These results suggest that PF has an anti-OP effect by promoting bone formation and inhibiting bone resorption.(4)PF restrains the preosteoblast apoptosis by inhibiting the nuclear translocation of Fox03aIn summary,PF suppresses OCs differentiation through promoting the GPR30 protein expression to and restrains the pre-OBs apoptosis through inhibiting the nuclear transfer of FOXO3 a protein.Then inhibiting bone resorption and promoting bone formation,maintaining bone homeostasis,exerting therapeutic effects on OP.