The Role And Mechanism Of Protocatechualdehyde Inhibiting Osteoclast Differentiation And Maturation And Bone Loss In Glucocorticoid Osteoporosis

Osteoporosis(OP)is characterized by the decrease of bone mass,the damage of bone microstructure and the increase of bone brittleness,and its clinical manifestations are bone pain,bone deformation and fracture.OP affects 50%of postmenopausal women and 20%of men over 50 years of age,and its incidence tends to increase with aging.Most of the fractures of middle-aged and elderly people are caused by OP,which leads to the decline of the quality of life of the elderly and brings heavy economic burden to the patient's family and society.Therefore,OP has become an important social,health and economic problem that seriously affects human health.Under normal physiological conditions,the microenvironment of bone tissue is in dynamic balance to maintain bone reconstruction.Osteoclasts(OCs)mediate bone resorption by synthesizing and secreting various organic acids and proteases to dissolve bone salts and degrade organic matter.Osteoblasts(OBs)serve as bone tissue substrates for the formation and synthesis of receptor activator of nuclear factor?B ligand.RANKL,osteoprotegerin(OPG)and other bone metabolism regulatory cytokines are responsible for the formation of new bone.Osteoblasts are differentiated from osteoblasts,which synthesize and secrete sclerostin,RANKL and other cytokines to participate in the regulation of bone metabolism.RANKL,a key regulator of bone remodeling,induces OCs differentiation and maturation by binding to receptor activator of nuclear factor ?B(RANK)in monocyte macrophages.Abnormal bone remodeling leads to bone disease,and excessive activity of osteoclast differentiation and maturation is the main pathological sign of osteoporosis.Drugs are mainly used to treat osteoporosis.There are many kinds of drugs in clinical use,and bisphosphonates are the most commonly one.Bisphosphonates have a good therapeutic effect on osteoporosis by regulating the interaction between osteoclasts and osteoblasts.However,the safety of long-term use of bisphosphonates has become a concern problem.It has been reported that long-term use of bisphosphonates can cause osteonecrosis of the mandible,atypical subtrochanteric or femoral shaft fractures and other adverse reactions.Other osteoporosis drugs also have a variety of side effects,which limiting clinical use.Salvia Milorrhiza(SM)is a kind of Chinese herbal medicine with functions of promoting blood circulation and removing blood stasis and delaying aging,which is clinically used to treat a variety of diseases.Clinical trials show that SM has a good clinical effect in the treatment of osteoporosis.In vivo experiments have confirmed that SM has a good protective effect on osteoporosis induced by oophorectomy and diabetic osteoporosis.Tanshinone is a fat-soluble active component extracted from SM that can inhibit osteoclast activation.Salvianolic acid B is a water-soluble active component of salviorrhiza salviorrhiza that can inhibit glucocorticoid-induced osteoporosis,improve bone trabecular structure and increase bone density.Protocatechualdehyde(PCA)is a degradation product of salvianolic acid B,which has anti-inflammatory and antibacterial activities.PCA can significantly inhibit the destruction of femoral condylar cartilage caused by adjuvant arthritis in vivo.However,the effect of PCA on osteoclasts and bone tissue is still unclear,so the focus of this study is to explore the effect of PCA on osteoclast differentiation and maturation and its mechanism,and to explore the effect of PCA on osteoporosis based on the osteoporosis model.ObjectivesTo study the effect of PCA on the differentiation and maturation of osteoclast precursor cells(RAW264.7 cell line and primary BMMs cell line)induced by RANKL,and to explore the underlying mechanism.In addition,the osteoporosis model mice were established to observe the preventive effect of PCA on osteoporosis and to provide a theoretical basis for the study of new anti-osteoporosis drugs.Research methods1.Selection of the cell lineRAW264.7 is a mononuclear macrophage derived from mice,which can be induced to osteoclasts.So we selected this cell line as an experimental model.2.BMMs of bone marrow derived macrophages were obtained Femurs from male BALB/c mice at 6-8 weeks were irrigated to obtain BMMs.3.Establishment of animal modelsMale BALB/c mice were injected with dexamethasone(5 mg/kg/d)for 10 weeks to establish a hormone-induced osteoporosis model.4.Induction of osteoclastsRANKL induced RAW264.7 into osteoclasts,and RANKL and MCS-F induced BMMs into osteoclasts.5.Detection of the effect of PCA on cell viabilityBMMs and RAW264.7 were inoculated in 96-well plates,and the effects of PCA on cells were detected by CCK-8 kit and Go1510 microplate.Search for a safe concentration of PCA.6.Count of osteoclastsThe TRAP detection kit was used to stain the osteoclasts.Cells that were TRAP-positive and had more than three nuclei were considered osteoclasts.7.Absorption Pit AssayOsteoclast precursor cells were seeded in 24-well Corning osteoblast culture plates.After cultivating with RANKL and PCA,the area of absorption pit was analyzed by microscope and Image J software.8.F-actin immunofluorescence ring assayBMMs were cultured in 24-well plates with sliders and treated with different concentrations of PCA,30 ng/mL of M-CSF and 50 ng/mL of RANKL for 4 days.Cells were stained with Phalloidin after fixation and permeation.Fluorescent microscopes capture fluorescent images.9.NF-?B activated nuclear translocation testBMMs were placed in a 24-well plate,treated with 5 ?g/mL of PCA and 50 ng/mL of RANKL.The cells were stained with fluorescence at the end of culture.The expression of NF-?B was observed by fluorescence microscopy.10.Measurement of mRNA levelsThe mRNA levels of CTSK,NFATC1,C-FOS,MMP9 and DC-stamp were detected by real-time PCR11.Detection of protein levelsAt the end of cell culture,cells were collected and total protein was extracted.Western blot was used to detect the changes of protein levels.12.Immunofluorescence assayLC3 expression within cells was detected by immunofluorescence assay13.MicroCT determinationMicroCT was used to detect the changes of femur microstructure in model mice,to observe the bone density(BMD),bone volume(BV),bone volume fraction(BV/TV),and bone trabecular number(Tb.N).14.H?E stainingThe histological changes of femur were observed by H?E staining.15.Assesment of calcium and phosphorus levelsCalcium and phosphorus detection kit was used to detect the levels of ions in peripheral blood of model miceResults1.PC A had no effect on cell activityThe effect of PCA on RAW264.7 cells and BMMs activity was evaluated by CCK-8 method.The results showed that the highest level of PCA used in the experiment(5?g/mL)had no effect on osteoclast precursor cell activity.2.PCA attenuates RANKL-induced osteoclast differentiationRANKL induced osteoclast precursor cells to differentiate into osteoclasts.TRAP staining showed the osteoclasts were large and full,with vacuoles forming in the middle and additional nuclear accumulation throughout the cells.However,when PCA was present,the size of the osteoclasts decreased,the vacuoles disappeared,and the number of surrounding nuclei decreased.The test results indicated that PCA prevented osteoclastic differentiation3.PCA has a more obvious inhibitory effect on osteoclast differentiation in the early stageFive ?g/mL of PCA was added into osteoclast induction medium on day 1 and day 2(early stage),day 3 and day 4(late stage),and day 1 and day 5(early stage+late stage),respectively.The results showed that early intervention of PCA had greater inhibitory effect than later intervention.4.PCA reduced RANKL-induced bone absorption in vitroTo investigate the effect of PCA on bone absorption function of osteoclasts,osteoclast precursor cells were inoculated on Corning osteoblast plates.Bone absorption activity stimulated by RANKL was monitored for 4-8 days.Bone absorption pits were obvious in the RANKL group,but decreased in the presence of PCA.The results showed that PCA could inhibit the bone absorption function of osteoclasts.5.PCA inhibits the formation of F-actin,We studied the effect of PCA on the formation of F-actin,and the results showed that PCA reduced F-actin.6.PCA inhibited NF-?B P65 translocationThe translocation of NF-?B P65 in the presence of PCA was studied.The results showed that the fluorescence intensity of P65 in the nucleus of RANKL-induced group increased.However,the presence of PCA reduced this trend,suggesting that PCA inhibited NF-?B P65 translocation.7.PCA attenuates the mRNA expression of RANKL-induced osteoclastic markersThe RT-qPCR results showed the mRNA levels of factors produced by RANKL-stimulated osteoclasts.The RANKL-induced group exhibited substantially increased expression of these mRNAs.However,PCA significantly reduced the mRNA levels of these related factors.8.PCA suppresses the activation of the MAPK pathwayThe MAPK signaling pathway exerts an irreplaceable effect on the generation of osteoclasts.Primary members of this signaling pathway are ERK,JNK,and p38.Phosphorylation of these proteins is the main indicator of activation.We measured the levels of these proteins produced during the formation of osteoclasts in cells treated with or without PCA.The RANKL-induced group exhibited substantially increased phosphorylation of these proteins.However,the changes were inhibited by PCA.9.PCA inhibits NF-?B pathway activationWe detected the phosphorylation of P65 during RANKL stimulation.Phosphorylation of P65 increased significantly after RANKL induction.However,phosphorylation of P65 decreased in the presence of PCA,suggesting that PCA inhibited NF-?B activation.When Bayl1-7082(NF-?B inhibitor)was added into the osteoclast induction system,the number of osteoclasts decreased in the presence of Bay 11-7082,indicating that NF-?B plays an important role in osteoclast maturation.10.PCA down-regulates the autophagy level of osteoclastsThe results showed that autophagy activator(Rap)could significantly promote the formation of osteoclasts and reverse the inhibitory effect of PCA osteoclast formation,suggesting that PCA inhibiting of osteoclast differentiation by regulating the autophagy level of OC precursor cells.Meanwhile,we detected the expression of autophagy related protein LC3 during osteoclast formation by the cell immunofluorescence method,and the results showed that the immunofluorescence of cytoplasm and membrane of osteoclast induced by RANKL was significantly enhanced,but the fluorescence was weakened in the presence of PCA.These results showed that PCA inhibited the expression of autophagy related protein LC3,which further proved that PCA may inhibit OC cell differentiation and maturation by regulating the autophagy level of OC precursor cells.11.PCA can reduce the bone microstructure index of hormone-induced osteoporosisMicroCT 3D showed that the trabecular in the proximal femur control group was thicker,with good continuity,no fracture,and lamellar arrangement;the Dex group had less trabecular,more fracture discontinuity,and almost no trabecular in the bone marrow cavity;the Dex+PCA group had continuous trabecular bone,but the number was less,rod-shaped,and the trabecular was thin.At the distal end of femur,the trabecular in the control group was dense above the epiphysis line,extending to the proximal bone marrow cavity.In Dex group,bone trabeculae above epiphyseal line decreased significantly,and there was almost no extension of trabeculae in medullary cavity.In Dex+PCA group,trabecular bone was distributed above the epiphysis line.It is showed that PCA can ameliorate the changes of bone microstructure induced by GIO and reduce bone loss.12.PCA improves the histopathological changes of osteoporosisThe results showed that the trabecular bone was more continuous and wider in the control group,while the trabecular bone in the Dex group was less than that in the control group,with fracture and discontinuity.The condition of trabecular bone improved in the PCA group,suggesting that PCA can reverse and ameliorate the histological changes in glucocorticoid osteoporosis.Conclusion:PCA inhibited RANKL-induced osteoclast formation by down-regulating MAPK,NF-?B and autophagy signaling pathways.At the same time,PCA can aslo play a certain role in the prevention of glucocorticoid osteoporosis.