Study On The Mechanism Related To The Regulation Of Osteoclastogenesis By Protopine

Background: Osteoporosis（osteoporosis）is a systemic skeletal disease caused by multiple causes characterized by decreased bone density and bone mass and destruction of bone microarchitecture,predisposing patients to fractures of the hip,spine and other skeletal areas.As a major public health problem worldwide,osteoporosis is becoming increasingly prevalent as the world’s population ages.The social and economic burden of osteoporosis is steadily increasing due to the aging of the population.The main drugs used clinically for the treatment of osteoporosis are anti-bone resorption drugs such as bisphosphonates,bone-forming drugs such as parathyroid hormone PTH（1-34）and dualaction drugs such as strontium ranelate.However,each drug has its limitations,such as certain side effects or high price.For example,bisphosphonates,which are the first-line drugs,have side effects of causing osteonecrosis of the jaw,severe musculoskeletal pain,esophageal cancer and renal failure.In addition,these drugs are less effective in agerelated,hormone-related,and other types of osteoporosis.Therefore,it is imperative to develop safe and effective drugs for the treatment of osteoporosis.Natural plant extracts have received increasing attention in recent years as potential drugs for the prevention and treatment of human diseases due to their wide availability and low side effects.Protopine is an isoquinoline alkaloid that acts as a plant monomer,its numerous pharmacological effects have been explored,such as: anti-inflammatory,anti-platelet aggregation,anti-cancer,analgesic,vasodilator,anticholinesterase,anti-addictive,anticonvulsant,anti-pathogenic,antioxidant,hepatoprotective,neuroprotective,cytotoxic and anti-proliferative activities,but the pharmacological effects of protopine in bone reconstruction and osteoclasts are still unexplored.Objectives: RAW264.7 cells were induced to differentiate into osteoclasts using nuclear factor-κB receptor activator ligand（RANKL）.This model was used to explore the possible effects and specific mechanisms of protopine on osteoclastogenesis in vitro,as well as to provide new possible therapeutic ideas for diseases such as osteoporosis caused by excessive osteoclast differentiation,and to provide some basis and reference for further in vitro and in vivo studies of plant monomeric protopine.Methods: Osteoclast-specific staining-TRAP staining assay was used to identify RANKL（50ng/ml）induced osteoclast formation in RAW264.7 cells and to evaluate the effect of different concentrations of protopine（0,5,10,20μM）on RANKL（50ng/ml）induced osteoclasts.Protopine（20μM）was added to RAW264.7 cells containing RANKL（50ng/ml）at the indicated time points,and the specific time points at which protopine exerted its inhibitory effect on osteoclast differentiation were investigated by TRAP staining.The effect of different concentrations of protopine（0,5,10,20μM）on the viability of RAW264.7 cells was investigated by CCK-8 assay.The effect of different concentrations of protopine（0,5,10,20μM）on RANKL（50ng/ml）induced F-actin ring in osteoclasts was investigated by Rhodamine Phalloidin staining assay.Real-time PCR assays were used to genetically analyze the effects of different concentrations of protopine（0,5,10,20μM）on osteoclast-formation-essential transcription factors NFATc1 and c-Fos and osteoclast-specific genes TRAP,Cathepsin K（Cts K）,ATP6V0d2 and MMP-9.The effects of different concentrations of protopine（0,5,10,20μM）on the expression of key transcription factors NFATc1 and c-Fos and osteoclast-specific markers TRAP,Cathepsin K（Cts K）,ATP6V0d2 and MMP-9 protein were analyzed using western blotting assays.The effect of protopine（20μM）on the protein expression of key signaling molecules NF-κB（IκB,p65）and MAPKs（JNK,ERK,p38）during osteoclastogenesis was analyzed by western blotting assay.All experiments were repeated three times individually,and all results are expressed as mean ± standard deviation.The obtained experimental data were subjected to one-way ANOVA and Student’s t-test using Graph Pad Prism（version 8）software,and *p<0.05 was considered statistically significant.Results: A model of RANKL-induced differentiation of RAW264.7 cells into osteoblasts was successfully constructed.Protopine inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner（0,5,10,20μM）and had no effect on the viability of RAW264.7 cells,and its inhibitory effect was mainly in the early stage of osteoclastogenesis.Protopine also inhibited RANKL-induced osteoclast F-actin rings formation in a dose-dependent manner.At the gene and protein expression levels,protopine inhibited RANKL-induced expression of key osteoclast transcription factors NFATc1 and c-Fos and osteoclast-specific markers TRAP,Cathepsin K（Cts K）,ATP6V0d2 and MMP-9 in a dose-dependent manner.Finally,protopine inhibited the phosphorylation of key signaling molecules IκB,NF-κB p65,JNK,ERK,and p38 during osteoclastogenesis at different time points.Conclusions: Our study showed that protopine,one of the major components of Corydalis yanhusuo W.T.Wang,inhibited the expression of osteoclast-specific markers TRAP,Cts K,MMP-9 and ATP6V0d2 by suppressing NF-κB and MAPKs signaling pathways as well as osteoclast-specific transcription factors NFATc1 and c-Fos,and ultimately inhibited RANKL-induced differentiation of RAW264.7 cells to osteoclasts.Therefore,protopine has potential therapeutic promise as an inhibitor of osteoclast differentiation for diseases caused by overactive osteoclasts,such as osteoporosis.