Ages Inhibit The Osteogenic Differentiation Of BMSCs Through The Wnt/β-catenin Pathway And The Intervention Of PTH

Diabetes and osteoporotic fractures both are important healthy problems,of which can affect the quality of life who suffer the disease and even increase the mortality.Advanced glycation end products(AGEs)are a variety of structurally stable and irreversible compounds formed by non-enzymatic Maillard reactions of proteins in the body.Accelerated formation and accumulation of AGEs during high glucose is the main cause of many chronic complications of diabetes.Under the conditions,bone marrow mesenchymal stem cells(MSCs)can be induced to differentiate into osteoblasts,chondrocytes,and adipocytes.The bone marrow-derived mesenchymal stem cells derived from streptozotocin-induced diabetic mice are more senescent,and the proliferation and differentiation abilities are weakened.In vitro,AGEs inhibit proliferation,self-renewal,and differentiation of MSCs.AGEs increase the expression of advanced glycation endproduct receptors(RAGE)and inhibit the mineralization and maturation of MSCs.In vivo and in vitro experiments have shown that AGEs-RAGE can affect bone quality in many ways and is one of the main causes of osteoporosis caused by diabetes.Wnt/β-catenin signaling pathway regulates bone formation and bone differentiation,promotes bone formation of bone marrow mesenchymal stem cells,and inhibits adipogenesis.The extracellular Wnt glycated protein binds to cell surface receptors and stimulates intracellular events.The most representative one is the classical Wnt/β-catenin signaling pathway.Previous studies have mainly focused on the mechanism of AGEs or RAGE acting on bone marrow mesenchymal stem cells or osteoblasts.However,there have been few reports on the role and mechanism of AGEs in the proliferation of cultured bone marrow mesenchymal stem cells.This study is aimed at investigating the mechanism of the inhibition of osteogenic differentiation of advanced glycation end products on the primary cultured bone marrow mesenchymal stem cells and the intervening action and mechanism of parathyroid hormone(PTH),providing a theoretical basis for the prevention and treatment of osteoporos induced by diabetes.Part One: Isolation,Culture and Identification of Rat BMSCsObjective: To master the methods of inducing and purifying BMSCs,observe the differentiation ability of osteoblasts and adipocytes in BMSCs,and identify the surface markers of BMSCs.Methods: Four-week-old healthy male Sprague Dawley(SD)rats were sacrificed after cervical dislocation.Bone marrow mesenchymal stem cells were isolated from both lower extremities,inoculated into culture flasks,and incubated at 37°C in a 5% CO2 incubator.The 1:2 ratio was subcultured to the third generation.After cultivation,the morphological changes and growth of adherent cells were observed under an inverted microscope;continuous observation for 1 week,cell growth curve was drawn by counting blood cell counts;third generation BMSCs cell phenotypes were identified by Rhodhosin staining;alizarin red staining And oil red O staining observed BMSCs osteoblasts,adipogenic cells induced differentiation.Results: The primary forms of BMSCs are mainly spindle cells arranged in radial colony cells.As the channels increase,cell morphology becomes shorter and there are foot protrusions.The cell growth curve shows that the bone marrow mesenchymal stem cells are consistent with the growth characteristics of normal cells and thrive.CD29,CD44 and CD90 but negative for CD34 and CD45 were positive in the third generation of bone marrow mesenchymal stem cells,but CD34 and CD45 were negative.After osteogenic and adipogenic differentiation,alizarin red staining was positive for both Von Kossa staining and oil red O staining.Conclusion: Whole marrow adherent culture method is simple,a large number of mesenchymal stem cells can be isolated,purified and expanded,as well as the obtained cells have obtained the biological characteristics of bone marrow mesenchymal stem cells。The obtained cells possessed the differentiation potential after osteogenic differentiation.Part Two Wnt/β-catenin signaling pathway mediates AGEs inhibiting osteogenic differentiation of BMSCsPurpose: AGEs inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells through the Wnt/β-catenin signaling pathway.Methods: After bone marrow mesenchymal stem cells were acted by different concentrations of AGEs(0.01 mg/ml,0.02 mg/ml,0.05 mg/ml,0.1 mg/ml,0.2 mg/ml)for 72 h,then we compared cell proliferation with CKK-8 assays.0.2mg/ml AGEs and corresponding concentration of bovine serum albumin were used to measure the proliferation of cells at 3-7 days.On the 7th day after osteogenic induction,the expression of Col-I and LRP5 mRNA was measured by RT-PCR.The effect of AGEs on the expression of B-cateninm,OSX,RUNX2 and RAGE during the osteogenic differentiation of BMSCs was detected by Western-blot.Compare the formation of mineralized nodules.The effect of AGEs on the expression of ALP,LRP5,RUNX2,OSX and RAGE mRNA and the expression of B-cateninm,OSX,RUNX2 and RAGE proteins were observed after RAGE signaling antibody was blocked by RAGE neutralizing antibody.Expression of β-catenin,OSX and RAGE protein after Wnt3 a administration of Wnt/β-catenin pathway enhancer.Results: After adding different concentrations of AGEs in BMSCs medium,compared with the corresponding concentrations of BSA group,the OD value of BMSCs was reduced,and it was in the range of 0.05 mg/ml to 0.2 mg/ml(P<0.05).The effects of 0.2mg/ml AGEs and corresponding concentrations of BSA on the proliferation of BMSCs in the AGEs group compared with BSA group were different(P<0.05).The number of mineralized nodules in the AGEs group compared with the control group after 28 days of BMSCs was reduced.The osteoblasts containing 0.2 mg/ml AGEs were used to culture cells for 7 days.Western-blot have showed that AGEs increased RAGE protein expression and inhibited the expression of β-catenin,OSX,and RUNX2 proteins(P<0.05).Administration of RAGE neutralizing antibody blocked RAGE signaling pathway and decreased RAGE expression and increased ALP,Runx2,and OSX mRNA expression(P<0.05).Results of Western-blot have showed that AGEs could increase RAGE protein expression and inhibite the expression of β-catenin protein(P<0.05).Administration of Wnt3 a,a promoter of Wnt/β-catenin pathway,could increase the expression of β-catenin and OSX protein and inhibited the expression of RAGE protein(P<0.05).28 Days of staining with alizarin red mineralized nodules suggested that AGEs significantly inhibited the formation of calcium nodules,while intermittent addition of Wnt3 a partially counteracted the inhibition of osteogenic differentiation of AGEs.Conclusion: Wnt/ β-catenin signaling pathway mediates that AGEs inhibit osteogenic differentiation of rat bone marrow mesenchymal stem cells.Anti-RAGE neutralizing antibodies block the binding of AGEs to RAGE,and the ability of bone marrow mesenchymal stem cells to differentiate into osteoblasts increases.Wnt3 a promotes the Wnt/β-catenin pathway and partially antagonizes the osteogenic differentiation inhibitory effects of AGEs.Part Three Effect of AGEs on BMSCs proliferation,osteogenic differentiation and PTH interventionObjective: On the basis of the AGEs stimulating factors,exogenous PTH was added to detect cell proliferation and differentiation,and it was observed whether PTH could antagonize the effects of AGEs.Methods: After 7 days of AGEs acting on BMSCs,ALP and COL-I mRNA were detected by RT-PCR and β-catenin,OSX,RUNX2 and RAGE proteins were detected by Western Blot.28 days after BMSCs were treated with AGEs,the effects of AGEs on the mineralization of BMSCs were detected by alizarin red staining and Von Kossa staining.After pretreatment of BMSC with PTH,AGEs were given as an intervention factor.ALP and COL-I mRNA were detected by RT-PCR and B-cateninm,OSX,RUNX2 and RAGE proteins were detected by Western Blot.On day 28,alizarin red staining was performed to observe the formation of mineralized nodules.Results: After intervention with PTH,it was found to inhibit the effects of AGEs.RAGE protein expression was reduced compared to AGEs.The expression of β-catenin,OSX and RUNX2 increased(P<0.05).After partial intervention with 10-8 mmol/L PTH,it partially reversed the osteogenic differentiation of AGEs.Quantitative analysis of the formation of alizarin-stained calcium nodules indicated that 28 days of AGEs inhibited 88% of cell mineralization(P<0.05).PTH can repair the negative effect of 23% AGEs on bone marrow mesenchymal stem cells(P<0.05).Conclusion: PTH partially reversed the negative effects of AGEs on proliferation and osteogenic differentiation of BMSCs through the Wnt/β-catenin signaling pathway.