| Background: Osteoporosis threatened many middle-aged and elderly patients,it can not only increase the risk of fracture,but also lead to a common postoperative complication of bone surgery.Many elderly people,especially post-menopausal women,are suffered from osteoporosis.Osteoporosis can significantly increase the risk of fracture,resulting in a direct reduction in the quality of life of patients,accompanied by increased morbidity and mortality in patients.Many studies had found that osteoporosis was associated with many physiological process,including gender,age,BMI(body mass index),changes of estrogen levels.In the past,we thought that osteoporosis was mainly due to the imbalance between osteogenesis and osteoclast in the bone microenvironment.In recent years,more and more studies focusd on lipid metabolism and osteoporosis.In the development of osteoporosis disorders,the degree of bone marrow fat is often increased.This also made the impact of adipose tissue metabolism on bone metabolism a hot topic.Bone marrow mesenchymal stem cells(BMMSCs)are not only a kind of adult stem cells with multi-directional differentiation and self-renewal,but also one of the main components that make up the bone marrow microenvironment.Among bone marrow stromal components,the main differentiation direction of bone marrow mesenchymal stem cells is osteoblasts and adipocytes.The abnormal differentiation of bone marrow mesenchymal stem cells,often lead to a significant impact on bone quality.Strontium ranelate(Sr RN)is a new drug to treat osteoporosis.Unlike previous antiosteoporosis drugs strontium ranelate can not only promote osteogenesis but also inhibit osteoclasts.However,it is still unknown whether strontium ranelate can modulate adipogenic differentiation of bone marrow mesenchymal stem cells.If it can modulate BMMSCs,What is the mechanism? Autophagy is a dynamic process of the recycling and recombination of subcellular structures and constituents.This process is influenced by a series of intracellular and extracellular signals and stimuli factors to ensure the homeostasis of cellular metabolism.Previous studies on autophagy have focused more on cell survival,death,metabolism and oncology.At present,given stem cells have the characteristics of multi-directional differentiation and autophagy plays an indispensable and important role in cell differentiation and maturation,autophagy and cell differentiation and maturation have gradually become new research perspectives and no definite conclusion has been reached yet.TOR kinase is a major regulator of autophagy and acts as a downstream node of growth factors and insulin signaling pathways and is influenced by a variety of factors including hypoxia and changes in ATP levels.TOR kinase acts downstream of the PI3 K / Akt and growth factor receptors and inhibits autophagy by modulating the Ulk1(Atg1)complex.Class III PI3 K complexes produce phosphatidylinositol 3 phosphate(PIP3)and induce the production of other Atg proteins(Including Atg12-Atg5-Atg16,and LC3(Atg8)-phosphatidylethanolamine complex.)Cleavage of the protease Atg4 hydrolyzes the LC3 precursor protein,producing LC3-I.The LC3-I protein produces LC3-II by highly conjugating lipophilic phosphatidylethanolamine(PE)to the Atg7,Atg3 and Atg12-Atg5-Atg16 L complexes.Finally,PE promotes LC3-II-mediated lipid membranemediated autophagosome formation.At present,it is found that the activation of Wnt / ?catenin signaling pathway plays an important regulatory role in the differentiation of stem cells in the study of differentiation characteristics of mesenchymal stem cells.Given the multidirectional nature of differentiation,Wnt / ?-catenin regulates the process of differentiation in a dual aspect.However,in the process of osteogenic differentiation,the classical and non-classical pathways of Wnt / ?-catenin play an absolutely positive role.In the past,we thought that ?-catenin degradation in the cytoplasm mainly depends on the proteasomal pathway,but in recent studies,it is found that autophagy may exist on ?-catenin protein degradation mechanism,and through this regulation closely affect the stem cells differentiation.Strontium ranelate is a new type of anti-osteoporosis drug with osteogenic and osteoclastic effects.Since strontium has good material integration properties,strontiumcontaining materials with its advantage of bone integration,has become another hot issue.In the further study of the mechanism of strontium,it was found that strontium plays an important role in the regulation of PI3 K / Akt signaling pathway and Wnt/?-catenin,a key signaling pathway of osteogenic / adipogenic differentiation of mesenchymal stem cells,significantly improve the osteogenic osteointegration in bones.Objetive: To explore the effect of strontium ranelate on adipogenic differentiation of mesenchymal stem cells under adipogenic conditions.To provide the evidence for further study on the possible mechanism of strontium ranelate.To investigate the regulatory effect of strontium ranelate on autophagy during BMMSCs adipogenic differentiation.To explore the underlying mechanism of autophagy regulation of strontium ranelateMethods:(1)Rat bone marrow mesenchymal stem cells were purchased from Cyagen Biosciences Inc.;BMMSCs were obtained through passage.(2)The adipogenic ability of stem cells were tested by adipogenic differentiation experiment.(3)The effects of different concentrations of strontium ranelate(0.00 m M,0.05 m M,0.10 m M,0.50 m M,1.00 Mm,2.00 m M)on the proliferation and apoptosis of BMMSCs were detected.(4)The differentiation ability of BMMSCs was analyzed by oil red O staining and triglyceride assay kit under the condition of different concentration(0.00 m M,0.10 m M,0.50 m M,1.00 m M)of strontium ranelate;Immunofluorescence and Western-blot was used to detect the protein expression of PPAR-? and C / EBP-?.According to the basis of the previous test,select the appropriate strontium ranelate drug concentration(1.00 m M).(5)To detect the change of autophagy of strontium ranelate under adipogenic regulation of BMMSCs by immunofluorescence and Western blotting.At the same time,Akt signaling pathway inhibitor API-2 and inhibitors(chloroquine,CQ)were used to detect the content of ?-catenin.(6)Adiponectin-PPAR-? was detected by immunofluorescence technique through the intervention of autophagy and Akt signaling pathway inhibitors.(7)To investigate the changes of Akt / m TOR and Akt / GSK3-? signaling pathway induced by strontium ranelate in adipogenic BMMSCs.Results:(1)There was no significant difference in the proliferation and apoptosis of BMMSCs between different concentrations of strontium ranelate(0.00 m M,0.05 m M,0.10 m M,0.50 m M,1.00 Mm,2.00 m M))detected by CCK-8 kit.(2)After adipogenic differentiation for 14 days,the results of oil red O staining showed that there was a significant difference between groups.The differentiation of BMMSCs was inhibited under the action of Sr RN of 0.5 and 1.0 m M,(P <0.05).By detecting the concentration of triglyceride,we found that the triglyceride level of induction groups were significantly higher than blank group(P <0.05),Among induction groups,the level of triglyceride content reduced with the increase of the concentration of strontium ranelate.(3)Strontium ranelate can significantly reduce the protein content of PPAR-? and C / EBP-? in adipogenic differentiation(P <0.05).(4)Immunofluorescence and Western-blot results suggested that strontium ranelate negatively regulates the autophagic activation of BMMSCs during adipogenic differentiation.(5)After addition of CQ,the adipogenic differentiation of BMMSCs induced a marked decrease in PPAR-?(3)The expression of ?-catenin detected by western-blot was found to be regulated by autophagy in the early stage of adipogenic differentiation(6)The regulation of strontium ranelate on adipogenic differentiation of BMMSCs may depend on the activation of Akt / m TOR,Akt / GSK3-? signaling pathway.Conclusion:(1)The lipid droplets and triglyceride content of BMMSCs were significantly increased during adipogenic differentiation.At the same time,the contents of PPAR-? and C/EBP-? in adipogenic differentiation increased significantly.(2)With the intervention of strontium ranelate,the differentiation of BMMSCs was inhibited in a dose-dependent manner.(3)Within a certain range,the regulation of adipogenic differentiation intensified as the concentration of strontium ranelate drug increased.(4)Under the conditions of adipogenic induction,BMMSCs significantly increased the level of autophagy in early differentiation.In the intervention of strontium ranelate,its autophagy level was inhibited to a certain extent.(5)The level of autophagy during adipogenic differentiation has a role in regulating the content of ?-catenin in cells.(6)Strontium ranelate can activate Akt / m TOR and Akt / GSK3-? in adipogenic differentiation of BMMSCs Signaling pathway to play a role. |
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