Effect Of Glucose On The Differentiation Of RAW264.7Cells To Osteoclast Induced By RANKL

Diabetes mellitus is a chronic endocrine disease associated withhyperglycemia and altered bone metabolism which may lead to complicationsincluding osteopenia, increased risk of fracture and osteoporosis.However，the biological mechanism of diabetic osteoporosis is stillunclear. Recently，the research of diabetic osteoporosis has become theworldwide research highlight. Hyperglycaemia is one of the main factorsthat cause bone homeostasis imbalance, long-term hyperglycaemia canproduce large amounts of advanced glycation end products. Highconcentration of AGE allows osteoclasts acid phosphatase (ACP) andtartrate resistant acid phosphatase (TRAP) activity to increasesignificantly. However,whether glucose influences osteoclast directlyor not has not been reported recently. Osteoclasts are the cells involvedin bone homeostasis mainly of osteoclast resorption and osteoblastic boneformation function of regulating the interaction, a special couplingmechanism is formed, maintaining bone homeostasis. Thus, the couplingmolecular mechanisms recently become the focal point of bone physiologyand treatment of bone disease research or drug development. Bonehomeostasis imbalance can lead to osteoporosis and other bone diseases,including bone sclerosis caused by abnormal regulation. Early studiesof the molecular mechanisms concentrate on the RANKL/RANK/OPG signalingsystem which American and Japanese scholars found in1997. In recentyears, the researchers found that Eph/ephrin bidirectional signaltransduction pathway can promote bone formation and inhibit boneresorption while promoting the formation of bone resorption inhibitionby regulating osteoblast and osteoclast function. Since Ephrin ligand hasreceptor-like function, the Eph/ephrin systerm has bidirectional signaltransduction. The bond between membrane protein Eph and Ephrin ligand,called forward signal, leads to a cell intracellular signal transductionby Eph; The bond between membrane protein ephrin ligand and Eph, calledforward signal, leads to a cell intracellular signal transduction byephrin. The passway which explains the key word of bone homeostasisrelease function has become the worldwide research highlight.Purpose: In this study, RANKL induces monocyte-macrophage RAW264.7osteoclast differentiation, while giving different concentrations ofglucose intervention in order to observe the effect of glucose onosteoclast differentiation and provide further theoretical basis on the pathogenesis of diabetic osteopathy.Methods: Under conditions of the different concentrations of glucose(5mmol/L,15mmol/L,30mmol/L), RANKL stimulates murine macrophagecell-Raw264.7cells and induces them to differentation. In thisstudy,take5mmol/L glucose as the low-glucose control group, highconcentration of hypertonic mannitol as the hypertonic control group anddifferent concentrations of glucose as the experimental group. Inducingfor5days,the activity change in each group of cells is analyzed by TRAPstaining kit of tartrate-resistant acid phosphatase.And the expressionchange of ACP5、ctsk、c-Fos, NFATc1、EphA2gene in each group of cellsis analyzed by Real-time PCR technique. RAW264.7cells were stained bytoluidine blue calculation and evaluated by lacuna number afterco-cultured with bone slices.Results: After the1st,3rdand5thdays cultures were detected usingReal-time PCR on ACP5, ctsk, c-Fos, NFATc1, EphA2gene expression changes.After the1stand3rddays, c-Fos and NFATc1expression of the experimentalgroup was significantly higher than low-glucose control group (P<0.05)and hypertonic control group (P<0.05); after the5thday, c-Fos and NFATc1expression of the experimental group was significantly lower than sugarin the control group (P<0.05) and hypertonic group (P<0.05); and theexpression gradually decreased with increasing concentrations ofglucose. After the1st,3rdand5thdays, EphA2expression of the experimentalgroup was significantly lower than the low-glucose control group (P<0.05)and hypertonic control group (P <0.05); and the expression decreased withincreasing concentrations of glucose. After the1stand3rddays, ACP5andctsk expression of the experimental group had no significant changecompared to the expression of control group; after the5thday, ACP5andctsk expression of the experimental group was significantly lower thanlow-glucose control group (P<0.05) and hypertonic control group (P<0.05);and the expression of ACP5and ctsk gradually decreased with increasingconcentrations of glucose. After toluidine blue staining，bone lacuna wasround, oval or irregularly shaped with blue tinct. After the5thday, thenumber of bone lacuna in the experimental group was significantlylower than low-glucose control group (P<0.05) and hypertonic controlgroup (P<0.05); and the number of bone lacuna decreased withincreasing concentrations of glucose.Conclusions: The ability of RANKL induced differentiation andosteoclastic bone-resorption decrease in high glucose conditions.