Screening Of Anti-osteoporosis Drugs Targeting AXL And Study Of The Molecular Mechanism

Background:Osteoporosis has become a major social health problem in the background of aging.The current treatment of osteoporosis still focuses on inhibiting bone resorption,however,it cannot fundamentally reverse bone mass.Currently,only hormones and sclerostin inhibitors are clinically used to promote bone anabolism.Therefore,it is of great significance to develop drugs that can promote bone anabolism.High-throughput drug screening can discover new applications for compounds from existing drug libraries or natural compound libraries,as well as investigate the pharmacological mechanisms.Compared with traditional drug discovery and research,it has the advantages of a short period,a simplified method,and easy clinical application.TAM receptors(TYR03,AXL,MERTK)belong to the receptor protein tyrosine kinase family,which is widely expressed in bone tissue.It has been demonstrated that MERTK and TYR03 play key regulatory roles in bone anabolic metabolism,but the mechanism of AXL in bone metabolism is still unknown.The previous study found that AXL was lowly expressed in Ovariectomy(OVX)osteoporosis mice,and knocking out AXL significantly aggravated the osteoporosis of OVX mice.Therefore,it was inferred that AXL may also participate in the regulation of bone metabolism.Objective:(1)To observe the effect of AXL in OVX on osteoporosis and osteogenic differentiation in OVX mice;(2)Based on the drug library approved by the Food and Drug Administration(FDA)and natural compound library,drugs that can target AXL,promote osteogenic differentiation,and improve osteoporosis in OVX mice were screened;(3)To explore the molecular mechanism of the target drug promoting osteogenic differentiation and improving osteoporosis in vivo and in vitro.Methods:(1)The OVX osteoporotic mice were constructed to detect the differential expression of AXL in the femur.The primary osteoblasts were isolated to assess the dynamic expression of AXL during osteogenic differentiation.The effect of AXL on osteogenic differentiation was observed by upregulating and downregulating AXL in primary osteoblasts using the Growth arrest-specific protein 6(GAS6,ligand of AXL)and the specific inhibitor R428(Bemcentinib),respectively.AXL gene knockout mice(AXL-/-)were constructed and OVX was performed to stimulate osteoporosis.The effect of AXL knockout on osteoporosis in OVX mice was observed.The primary osteoblasts of AXL-/--OVX suckling mice and WILD-OVX suckling mice were extracted to observe the effect of AXL gene knockout on osteogenic differentiation.The femurs of AXL-/--OVX mice and WILD-OVX mice were resected,and the proteins differentially expressed in the bone of OVX osteoporotic mice after AXL gene knockout was analyzed by proteomic sequencing.(2)Screening the drugs that can up-regulate AXL from the compound library and observing the therapeutic effect.MC3T3-E1 cells were used to construct a drug screening model based on AXL fluorescent reporter gene.The high-throughput drug screening method was used.In the primary screening,Cell Counting Kit-8(CCK-8)and AXL fluorescence intensity was measured to screen compounds that can upregulate AXL and promote osteogenic proliferation.In the secondary screening system,CCK-8 was used to measure cell proliferation activity,and a high-content cell imaging system was used to measure the fluorescence intensity of AXL and Collagenl.Furthermore,literature was retrieved to eliminate compounds without research prospects and select candidate drugs.The molecular docking of AXL and candidate drugs were performed to observe the specific binding ability of the candidate drug to AXL.Different concentration and time gradient was set to observe the effects of different concentrations of drugs on the expression of AXL and Extracellular signal regulated kinase(ERK5),further to determine the optimal concentration of the drug to promote osteogenic differentiation.As one of the most obvious differentially expressed proteins after AXL knockout,ERK5 was selected and researched.The effects of candidate drugs on the expression of AXL,ERK5,osteogenic differentiation,and the therapeutic effect of osteoporosis in OVX mice were observed.(3)Research on the mechanism of the candidate drug in promoting osteogenic differentiation.The candidate drug that can significantly alleviate osteoporosis in OVX mice was selected for mechanism research.The concentration gradient was set up to observe the effects of the candidate drugs on the proliferation,apoptosis,and cell cycle of MC3T3-E1 cells.AXL and ERK5 in MC3T3-E1 cells were knocked down,respectively,and the protein levels of ERK5,and osteogenic differentiation markers OPN,OSX,and RUNX2 were observed.The mechanism of the candidate drug in regulating osteogenic differentiation was explored in Bone Marrow Stromal Cells(BMSC),and the effect of the candidate drug on the morphology of BMSC in the BMSC-MC3T3-E1 co-culture system was observed.The effect of the candidate drug on the phenotype of RAW264.7 macrophages and the secretion of osteogenic differentiation factors,and whether the target drug can regulate the osteogenic differentiation in the RAW264.7-MC3T3-E1 co-culture system was observed,as well as the effect of the candidate drug on the osteoclast differentiation of RAW264.7 cells.Finally,it was verified in vivo whether the candidate drug could improve osteoporosis in OVX mice by regulating the AXL-ERK5 axis.Results:(1)AXL was significantly downregulated in the OVX osteoporosis mouse model compared to the control group.AXL is dynamically down-regulated during the differentiation of primary osteoblasts,and it positively regulates osteogenic differentiation.Compared to the WILD-OVX group,the degree of osteoporosis in the AXL-/--OVX group was significantly aggravated.Proteomic analysis revealed that Mitogen-activated protein kinase(MAPK)is the key signaling pathway that is differentially expressed in the femurs of AXL-/--OVX mice.The results of proteomics were further verified in vitro.Based on the previous research,ERK5 was selected for mechanism research.(2)Twenty compounds were identified in the preliminary screening,and 4 compounds were identified in the secondary screening,which were Dabrafenib,pCoumaric Acid,Vildagliptin,and Mangiferin.Molecular docking results showed that the four compounds had specific binding sites with AXL.In vitro,the optimal concentrations of four compounds to promote the proliferation of MC3T3-E1 cells were identified.Mangiferin and Vildagliptin could significantly upregulate AXL/ERK5 in MC3T3-E1 cells and promote osteogenic differentiation.In vivo studies,both Mangiferin and Vildagliptin could improve osteoporosis in OVX mice,however,the effect of Vildagliptin was more obvious in up-regulating osteogenic differentiation markers in vivo.(3)Vildagliptin promotes osteoblast proliferation,inhibits apoptosis,and prolongs the S phase in a concentration-dependent manner when the concentration was lower than 50 μM,as well as upregulates AXL and ERK5,and promotes osteogenic differentiation in the MC3T3-E1 cells.The potential mechanism is by regulating the AXL-ERK5 axis.Vildagliptin promotes the osteogenic differentiation of BMSCs and the transformation of BMSCs into osteoblasts in the BMSC-Primary osteoblast coculture system.Vildagliptin promotes RAW264.7 macrophages to secrete Bone morphogenetic protein 2(BMP2)and Transforming growth factor beta 1(TGF-β1)in a concentration-dependent manner and polarize to M2 and promote osteoblas t differentiation in the RAW264.7-Primary osteoblast co-culture system.Besides,Vildagliptin inhibited the osteoclast differentiation of RAW264.7 macrophages in a concentration-dependent manner within the concentration range of 50 μM.In vivo studies,vildagliptin alleviated osteoporosis in OVX mice by up-regulating the AXL,and ERK5,and up-regulating the expression of osteogenic differentiation markers.Conclusions:(1)AXL positively regulated osteogenic differentiation in vitro and exerted osteoprotective effects in vivo.(2)Both Mangiferin and Vildagliptin can target AXL and promote osteogenic differentiation,and mitigate osteoporosis in OVX mice.(3)Vildagliptin promoted osteogenic differentiation of MC3T3-E1 cells through the AXL-ERK5 axis;promoted the osteogenic differentiation of BMSCs;promoted the secretion of pro-osteogenic differentiation factors of RAW264.7 cells and polarizes to M2;inhibited osteoclastic differentiation of RAW264.7 cells.(4)AXL-ERK5 axis is a promising target in anti-osteoporosis drug development.(5)Vildagliptin can be the first choice for people with type 2 diabetes and osteoporosis or a high risk of fracture.