Anti-bone Resorption Effect Of Ligustilide And Its GPER Associated Mechanism

Bone homeostasis depends on the relative balance between bone-resorption osteoclast(OCs)and bone-forming osteoblasts.Osteoclasts are the only cells involved in bone resorption.Excessive activation of osteoclasts leads to higher bone resorption than bone formation,which leads to osteoporosis(OP).Regulating the differentiation and maturation of OCs is the main treatment strategy for prevention and treatment of OP.Angelica sinensis(Oliv.)Diels(A.sinensis)is a traditional Chinese medicine widely used in the treatment of OP and its frequency of use is the fourth.Its therapeutic effect on OP has been confirmed by clinical and experimental studies.Ligustilide(LIG)is considered to be the most effective biologically active ingredient in A.sinensis and its content can reach 45-70%.It has been found that LIG has a wide range of pharmacological activities,However,little is known regarding its effects on osteoclasts differentiation and anti-bone resorption.It has been reported that LIG has a protective effect on osteoblasts and promotes the protein expression of G protein-coupled estrogen receptor(GPER).Therefore,the aims of this study were to explore the effect of LIG on bone resorption and the related mechanism between LIG intervention on OCs differentiation and GPER.1.Effects of LIG on osteoclasts differentiation(1)A model of receptor activator of nuclear factor-κB ligand(RANKL)-induced differentiation of RAW 264.7 cells into OCs was established.Different concentrations of LIG were added and cultured for 4 days.When the concentration was not higher than 10 μmol·L-1,LIG had no effect on OCs activity.Tartrate-resistant acid phosphatase(TRAP)staining and TRAP activity assay showed that LIG significantly inhibited the production of TRAP-positive cells and TRAP activity.qRT-PCR results showed that LIG significantly reduced the expression of OCs differentiation marker Dc-stamp,Nfatcl,Ctsk and Rank;Western Blot results showed that LIG significantly reduced the protein expression of NFATc1,CTSK and TRAP.(2)In order to evaluate the effect of LIG on bone resorption,RAW264.7 cells were seeded onto glass slides and cortical bones,and induced by RANKL.At the same time,different concentrations of LIG were added,and cultured for 4 days.The results of FITC-labeled phalloidin staining showed that LIG inhibited F-actin ring formation.Toluidine blue staining was performed on cortical bones,and the results showed that LIG could significantly reduce the number of bone resorption pits.(3)The zebrafish osteoporotic phenotype induced by prednisolone(Pred)was used to verify the efficacy of LIG.The zebrafish were grouped as follows:Control group,Model group(25 μmol·L-1 Pred),Etidronate Disodium group(25 μg/ml)and LIG group(0.1,1,10 μmol·L-1).TRAP staining and TRAP activity assay showed that the reduction of TRAP formation in zebrafish scales was dose-dependent after treatment with LIG.2.The role of GPER as a potential target of LIG in osteoclast differentiationTo explore the role of GPER in OCss,we performed the following experiments:(1)The effect of G1 on RANKL-induced OCs differentiation.On the basis of RANKL-induced RAW264.7 OCs differentiation model,different concentrations of GPER agonist G1 were added and cultured for 4 days.MTT assay showed that G1 had no effect on OCs activity.TRAP staining and TRAP activity assay showed that G1 inhibited the formation of TRAP-positive cells in a dose-dependent manner.qRT-PCR results showed that 10 nmol·L-1 G1 significantly decreased the gene expression of Nfatcl and Ctsk.(2)GPER specific antagonist G36 was added to observe the effect of G1 on OCs differentiation.MTT assay showed that G36 had no effect on the activity of precursor OCs and RANKL-induced OCs.TRAP activity assay showed that 100 and 250 nmol·L-1 G36 significantly increased TRAP activity in OCs.TRAP staining showed that the number of TRAP-positive cells increased after G1 combined with 100 nmol·L-1 G36.qRT-PCR results also showed that co-treatment with G36 increased the gene expression of Nfatcl and Ctsk.(3)Transfect RAW264.7 cells with siGPER to observe the effect of G1 on the differentiation of OCs.qRT-PCR results showed that after siGPER was transfected,the effect of G1 on inhibiting the expression of OCs marker genes was weakened.(4)In order to explore the relevant mechanism in the process of activating GPER to inhibit the differentiation of OCs,we used Western Blot experiments to detect the activation of MAPKs and NF-κB pathways.The results showed that G1 inhibited the phosphorylation levels of ERK,JNK and p65 in OCs;compared with the G1 group,the co-treatment with G36 increased the phosphorylation levels of ERK,JNK and p65 in OCs.(5)A zebrafish osteoporosis model induced by prednisone(Pred)was established.The Zebrafish were grouped as follows:Control group,Model group(25 μmol·L-1 Pred),Etidronate Disodium group(25 μg/ml)and G1 group(50,100 and 200 nmol·L-1).TRAP staining and TRAP activity assay showed that the formation of TRAP in zebrafish scales was significantly increased after 25 μmol·L-1 Pred and G1 reduced TRAP formation in zebrafish scales.In order to further detect the effects of GPER,G36 was added for the study.The results showed that TRAP formation in zebrafish scales increased significantly after co-treatment with G36.3.Ligustilide inhibits osteoclast differentiation and its mechanism with GPERThe above experimental results show that LIG has a significant inhibitory effect on OCs differentiation,and GPER plays an important role in OCs differentiation.To further confirm the relevance of GPER pathway in the inhibition effect of LIG on OCs differentiation,we performed the following experiments:(1)To observe the effect of LIG on OCs differentiation after GPER antagonist G36 treatment.The results showed that co-treatment with G36 increased the number of TRAP-positive cells;TRAP activity and the expression of OCs marker genes Dc-stamp,Nfatcl,Ctsk and Rank.(2)Effects of LIG on the expression of GPER in RANKL-induced OCs differentiation.qRT-PCR results showed that LIG treatment promoted the gene expression of GPER in a dose-dependent manner.(3)To further evaluate the effect of LIG on bone resorption after GPER antagonist G36 treatment.The results showed that after co-treatment with G36,the number of F-actin ring and bone resorption pits increased significantly.(4)Transfect RAW264.7 cells with siGPER to observe the effect of LIG on the differentiation of OCs.qRT-PCR results showed that after siGPER was transfected,the effect of LIG on inhibiting the expression of OCs marker genes was weakened.(5)In order to explore the relevant mechanism of LIG inhibiting the differentiation of OCs,we used Western Blot experiment to detect the activation of MAPKs and NF-κB pathways.The results showed that LIG inhibited the phosphorylation levels of ERK and p65 in OCs;compared with the LIG group,the the co-treatment with G36 increased the phosphorylation levels of ERK and p65 in OCs.(6)The zebrafish osteoporotic phenotype induced by prednisolone(Pred)was used to further observe the effect of LIG in the formation of OCs in zebrafish scales afterco-treatment with G36.The results showed that TRAP formation in zebrafish scales increased significantly after co-treatment with G36.Conclusions:(1)LIG inhibits OCs differentiation and bone resorption by inhibiting the expression of RANK and NFATc1 and reducing the secretion of TRAP and CTSK.(2)LIG inhibits OCs differentiation and bone resorption in vivo and in vitro.(3)Activating GPER inhibits the activation of MAPKs and NF-κB signaling pathways and inhibits the formation and differentiation of OCs,suggesting that activation of GPER may inhibit the MAPKs and NF-κB signaling pathways,reduce the expression of TRAP and CTSK in OCs,and exert anti-bone resorption effect.(4)LIG inhibits the differentiation of OCs by interfering with GPER to play an anti-bone resorption effect.In summary,LIG inhibits OCs differentiation and reduces bone resorption.Further studies have founded that activation of GPER inhibits the MAPKs and NF-κB signaling pathway,regulates OCs differentiation.LIG inhibits the MAPKs and NF-κB signaling pathways,reduces the expression of TRAP and CTSK in OCs may be by increasing GPER level.This study provides a pharmacological basis for LIG in the treatment of osteoporosis and provides new clues for the discovery of anti-osteoporosis drugs.