Effects And Mechanism Of Adiponectin On The Differentiation And Maturation Of Osteoclasts In Patients With Multiple Myeloma

Objective:To investigate the correlation of visfatin,leptin,and adiponectin with multiple myeloma bone disease(MBD)and its influence in MBD.Osteoclasts were isolated and cultured in vitro and were used to study the effects and mechanism of adiponectin on the differentiation and maturation of osteoclasts in Multiple Myeloma(MM).Methods:Part 1.The levels of serum and bone marrow visfatin,leptin and adiponectin were measured in 20 newly diagnosed multiple myeloma patients and 20 healthy control group.Their relationships between the index of myeloma tumor load and bone disease such as carboxy-terminal cross-linking telopeptide of type I collagen(CTX),osteocalcin(OCN)and procollagen I amino-terminal propeptide(PINP)were analysed.Part 2.Thirty-two patients with newly diagnosed multiple myeloma were selected from the Department of Hematology of General Hospital of Tianjin Medical University from August 2017 to March 2018.Bone marrow mononuclear cells(BMMCs)were extracted and used to inducing into osteoclasts in vitro with nuclear factor ?B receptor activating ligand(RANKL)and macrophage colony-stimulating factor(M-CSF).The BMMCs were divided into two groups: blank control group(group A)and 10 ?g/ml adiponectin group(group B).Osteoclasts were identified by staining with tartrate-resistant acid phosphatase(TRAP).The number of mature osteoclasts were also examined by TRAP positive staining.RT-PCR was used to detect the expressions of RANKL,OSCAR,TRAP and Cathepsin K genes.Flow cytometry(FCM)was used to detect the expressions of AdipoR1 on the surface of osteoclast precursors(CD14+ monocytes)and phosphorylation of mTOR pathway related proteins mTOR and 4EBP1.Results:Part 1.There were no significant linear correlation between leptin,visfatin and the indexes of myeloma tumor load and bone disease.Serum adiponectin levels were significantly higher in newly diagnosed multiple myeloma patients(n=39)(12.37±3.13?g/ml)than that in control group(n=20)(13.75±1.02 ?g/ml),but the levels of adiponectin in the newly diagnosed multiple myeloma patients had no significant difference between bone marrow group(n=20)(12.66±2.37?g/ml)and serum group(n=39)(12.37±3.13?g/ml).A significant linear correlation relationship between the serum adiponectin and ?2-microglobulin(r=-0.421,P=0.008),the percentage of plasma cell of bone marrow(r=-0.495,P=0.001),serum creatinine(r=-0.437,P=0.005)and osteocalcin(r=0.456,P=0.007)in newly diagnosed multiple myeloma patients.A significant linear correlation was seen among adiponectin and lactic dehydrogenase(r=-0.725,P=0.001),?2-microglobulin(r=-0.757,P=0.001),the percentage of plasma cell(r=-0.645,P=0.002),serum creatinine(r=-0.568,P=0.009),osteocalcin(r=0.463,P=0.040),carboxy-terminal cross-linking telopeptide of type I collagen(r=-0.699,P=0.001)in bone marrow in newly diagnosed multiple myeloma patients.Part 2.BMMCs was cultured in vitro for 14 days and then identified by TRAP staining.Mature osteoclasts were obtained in both the control group and the adiponectin group.Compared to the control group,adiponectin inhabits the osteoclasts differentiation and maturation.The number of maturation osteoclasts was less than the control(control group versus adiponectin group: 25.4±4.51 vs 18.6±4.03,p<0.05).Adiponectin also inhabit the mRNA expression of osteoclast differentiation and maturation related factor-RANKL(0.96(0.38,2.27)vs 0.92(0.28,1.63),p<0.05)?OSCAR(2.18(0.18,3.72)vs 0.73(0.10,2.29),p<0.05),TRAP(1.32 ± 0.91 vs 0.90 ± 0.81,p< 0.05),Cathepsin K(1.60±1.24 vs 0.99±0.81,p<0.05).Flow cytometry showed that adiponectin increased the expression of AdipoR1 on the surface of osteoclast precursor((26.21±4.27)% vs(29.86±6.23)%,p < 0.05)and reduced the expression of p-mTOR((7.89±1.00)% vs(5.91±1.26)%,p<0.05)and p-4EBP1((26.78±5.00)% vs(22.49±4.24)%,p<0.05).Conclusions:The level of serum adiponectin were correlated with multiple myeloma and multiple myeloma bone disease,adipokines may play a crucial role in the development of multiple myeloma.Bone marrow mononuclear cells can be induced into osteoclasts with RANKL and M-CSF in vitro.Adiponectin inhibits the differentiation and maturation of osteoclasts in patients with multiple myeloma.Adiponectin can increase the expression of AdipoR1 on the surface of osteoclast precursor cells,and then reduce the phosphorylation levels of mTOR and 4EBP1 proteins which is involved in mTOR signal pathway,resulting in inhibiting the differentiation and maturation of osteoclasts in patients with multiple myeloma.