Exendin-4 Improves Osteoporosis Through Bone Marrow-derived Macrophage Polarization

ObjectivesBone remodeling is to keep the integrity of skeleton.Osteoblasts and osteoclasts participate in the bone modeling and the balance of them play an important role to maintain bone mass.The age,hormone,gravity,cytokines,angiogenesis and microenvironment can affect the bone remodeling process and the microenvironment attract more attention.It has been aproved that,the local cytokine?IL-1?IL-4 and TGF-?are essential in bone remodeling.Thus,the different bone microenvironment and the various mechanisms that affect the bone remodeling will be the target,especially in finding the potential target in osteoporosis.Macrophages have important roles in development,homeostasis and both innate and adaptive immunity.Recent studies found that,bone marrow-derived macrophage?BMDM?could regulate the bone remodeling.Depletion of BMDM resulted in suppression of new bone formation,but the mechanisms are unknown.Our previous study demonstrated that glucagon-like peptide-1 receptor agonist Exendin-4 could promote bone formation by inducing BMSC differentiation into osteoblasts,while inhibiting the osteoclasts resorption.It is reported that microglia express the GLP-1 receptor and promote the secretion of alkaline phosphatase and collagen I when coculture with BMSC.But microglia also could secret TNF-?to induce osteoclastgenesis which leading to osteoarthritis.Thus,we proposed that Exendin-4 stimulates the receptor to active the BMDM to modulate the secretion of inflammatory and promote bone formation to improve the osteoporosis.In this study,we hypothesized the effect of BMDM on bone formation by eliminating BMDM in vivo and investigated the effect of Exendin-4 in BMDM polarization in vitro.Methods1.The determination of mice bone morphology and protein expressionFemale C57BL/6 mice?3 months?were divided into control group?OVX group and OVX+Exendin-4 group.There are 8 mice in each group.Mice were given Exendin-4through intraperitoneal injection for 12 weeks.Micro-CT was used to observe the changes of trabecular bone and bone structure.immunohistochemical tests?HE staining?TRAP staining were used to determinate the expression of CD29,Sca-1,TGF-?1 and CD68;calcein and tetracycline double-labeled fluorescence were used to observe the new bone formation;Western blot was used to determinate the protein expression of Osterix,Runx-2and TGF-?1.To observe the effect of BMDM on bone formation,clodronate liposome was used to eliminate BMDM in vivo.Female C57BL/6 mice?3 months?were randomly devided into OVX group?OVX+Exendin-4 group?OVX+clodronate liposome group?OVX+clodronate liposome+Exendin-4 group.Each group has 8 mice.Clodronate liposome was injected through intraperitoneal for 12 weeks.Micro-CT was used to observe the changes of trabecular bone and bone structure.Immunohistochemical tests were adopted to test the expression of CD68 and TGF-?1.2.The culture?identification and polarization of BMDM?1?BMDM isolation?culture and identificationThe femurs and tibias were separated from the 6 weeks C57BL/6 female mice,and cells were collected in H-DMEM culture medium supplemented with 10%fetal bovine serum?FBS,Gibco?,100 U/ml penicillin,100 mg/ml streptomycin in a humidified atmosphere of 95%air and 5%CO₂ at 37°C for 6h.Then suspended cells were cultured in H-DMEM with 10%fetal bovine serum and MCSF?25 ng/ml?.The BMDM were a relatively pure population that were positive for F4/80 and CD11b.?2?The polarization of BMDM and idetificationBMDM were cultured in H-DMEM with 10%fetal bovine serum,LPS?100 ng/ml?and IFN-??20 ng/ml?for 3 days to induce M1 polarization.BMDM were cultured in H-DMEM with 10%fetal bovine serum and IL-4?25 ng/ml?for 3 days to induce M2polarization.ELISA was used to determine the expression of TNF-?and IL-1ra,RT-PCR was used to determine the expression of inducible nitric oxide synthase?iNOS?and Arg-1.3.The effect of Exendin-4 on the polarization of BMDMBMDM were devided into control group?Exendin-4 group?M1 induced group and M2 induced group.Different stimuli?LPS+IFN-?,IL-4,Exendin-4?were given to BMDM for polarization analysis.TNF-?and IL-1ra were determined by ELISA,iNOS and Arg-1were determined by Western blot and RT-PCR.4.The effect of BMDM on the migration of BMSCTo observe the effect of Exendin-4 on BMDM to induce the migration of BMSC by transwell experiment.BMDM cultured in the lower compartments with 10⁶/ml and BMSC with 2×10⁵/ml in the upper compartments.Different stimuli?LPS+IFN-?,LPS+IFN-?+M?,IL-4,IL-4+M?,Exendin-4,Exendin-4+M?,IL-10?10 ng/ml?,IL-10+M?,IL-10+M?+TGF-?1 Ab?0.5?g/ml?,Exendin-4+M?+TGF-?1 Ab,TGF-?1?1 ng/ml?,TGF-?1+TGF-?1 Ab?were given to BMDM to determine the migration of BMSC.After incubation at 37?for 24 h,the filters were fixed in 4%paraformaldehyde for 20 min and stained with 0.1%crystal violet for 15 min.The number of migrated cells was counted in five fields under the Olympus BX53 microscope?Olympus,Tokyo,Japan?.5.The effect of Exendin-4 on angiogenesis?1?The examine the effect of Exendin-4 on angiogenesis in vivoFemale C57BL/6 mice?3 months?were devided into control group?OVX group and OVX+Exendin-4 group.Mice were given Exendin-4 through intraperitoneal injection for12 weeks and femur were collected after the treatment.Immunohistochemical tests were adopted to test the expression of CD31;RT-PCR and Western blot were used to determinate the expression of VEGF,MMP-2 and CD31.?2?The examine the effect of Exendin-4 on BMDM in angiogenic factors secretionDifferent stimuli?LPS+IFN-?,IL-4,Exendin-4?were given to BMDM to examine the expression of angiogenesis factors.RT-PCR and Western blot were used to determine the expression of VEGF and MMP-2.6.The detection of signaling molecules of Exendin-4 on BMDM differentiationThe BMDM were treated with H-89?PKA inhibitor?,Rapamycin?mTOR inhibitor?and wortmannin?PI3K inhibitor?to observe the effect of Exendin-4 on the expression of Arg-1.The protein levels of P-PKA,P-STAT3 and TGF-?1 were detected under H-89stimulation to observe the effect of Exendin-4.Forskolin,H-89 and Exendin?9-39?were given to BMDM to detect the expression of P-STAT3 by Western blot.Co-immunoprecipitation was used to detect the relationship between cAMP/PKA signaling pathway and JAK2/STAT3.Results1.Exendin-4 ameliorated osteoporosis in OVX mice through BMDMThe bone trabecular number and thickness were significantly improved after treatment.HE staning showed that the number of osteoblasts was significantly increased in bone surface and the expression of Runx-2 and Osterix were also significantly increased.TRAP staining showed that Exendin-4 could inhibit the activity of osteoclasts.IHC showed that Exendin-4 could increase the levels of TGF-?1 in bone.Immunohistochemical results showed that the number of BMDM was increased in OVX mice and clodronate liposome could eliminate BMDM,which destroyed the effect of Exendin-4 and decreased the expression of TGF-?1 in bone marrow.The above results indicated that Exendin-4 promoted bone formation.2.Exendin-4 induced migration of BMSC through BMDM polarization and TGF-?1 secretionBMDM expressed GLP-1 receptor and Exendin-4 could promote Arg-1 and CD206expression,inhibit iNOS?TNF-?expression.In vivo,Exendin-4 increased Arg-1 and CD206 expression,inhibit iNOS?TNF-?expression,which indicated that Exendin-4promoted M2 polarization of BMDM.Transwell results showed that Exendin-4 could induce the migration of BMSC and promote BMDM secrete TGF-?1.Pre-treated with TGF-?1 Ab could inhibit the migration of BMSC.The above results indicated that Exendin-4 promoted BMDM secrete TGF-?1to induce BMSC migration.3.Exendin-4 induced polarization of BMDM through PKA-STAT3 signaling pathwayWith the treatment of H-89?PKA inhibitor?,the effect of Exendin-4 on the expression of Arg-1?P-STAT3 and TGF-?1 was reduced.And further experiments found that Exendin?9-39?could inhibit the P-STAT3 expression and Forskolin could increase the expression of P-STAT3.The above results indicated that Exendin-4 promoted the polarization of BMDM and secretion of TGF-?1 through cAMP/PKA/STAT3 signaling pathway to induce the migration of BMSC.4.Exendin-4 promoted angiogenesis through BMDM secretion of VEGF and MMP-2After the treatment,the expression of VEGF?MMP-2 and CD31 were increased in bone.IHC showed that Exendin-4 increased the number of CD31 positive cells in bone.Besides,Exendin-4 increased the expression of VEGF and MMP-2 in BMDM.The above results indicated that Exendin-4 promoted H type vessel formation in bone through inducing BMDM secretion of angiogenic factors.Conclusion1.Exendin-4 ameliorated the OVX osteoporosis which has relation to BMDM.2.Exendin-4 could induce BMDM polarize into M2 subtype to secret TGF-?1which induced migration of BMSC into bone surface to promote bone formation.3.Exendin-4activatedGLP-1receptorandsubsequentlyactivated cAMP/PKA/STAT3 signaling pathway,which leading to M2 polarization of BMDM.4.Exendin-4 induced M2 BMDM to secrete VEGF and MMP-2 to promote angiogenesis and bone formation.