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OBJECTIVE@#To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ).@*RESULTS@#Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area.@*CONCLUSION@#After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.
Subject(s)
Female , Mice , Animals , Core Binding Factor Alpha 1 Subunit/pharmacology , PPAR gamma/metabolism , Steroid 12-alpha-Hydroxylase/metabolism , Mice, Inbred C57BL , Cell Differentiation , Osteogenesis , Mesenchymal Stem Cells , Bile Acids and Salts/pharmacology , Bone Marrow Cells , Cells, Cultured , Azo CompoundsABSTRACT
ObjectiveTo explore the underlying mechanism of Bushen Huatan prescription in alleviating postmenopausal osteoporosis （PMOP） by maintaining the balance of osteogenesis and adipogenic differentiation in ovariectomized rats with osteoporosis. MethodSeventy-five 6-month-old non-pregnant female SD rats were randomly divided into sham-operation group， model group， atorvastatin group， liviol group， and Bushen Huatan prescription group. Bilateral ovaries were removed in the four groups except the sham-operation group， while only the same mass of adipose tissue around the ovaries was removed in the sham-operation group. On the 5th week after surgery， drugs were consecutively administrated for 8 weeks. Rats in the Bushen Huatan prescription group received 9.4 mg·kg-1 of the prescription， rats in the atorvastatin group received 0.92 mg·kg-1 of atorvastatin， rats in the Liviol group received 0.23 mg·kg-1 of liviol， and rats in the model group and the sham-operation group received saline once a day. Micro-computed tomography （Micro CT） was used to detect bone mineral density （BMD） of rat tibia in each group. Hematoxylin-eosin （HE） staining was used to detect the relative area of rat bone marrow adipose tissue （BMAT） in each group. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the relative expression levels of Runt-related transcription factor 2 （Runx2）， peroxisome proliferator-activated receptor （PPARγ）， leptin （LPN）， and leptin receptor （OBR） in bone tissues. ResultAs compared with the sham operation group， the BMD of rats in the model group decreased （P<0.05）， while the relative area of BMAT increased （P<0.05）. In addition， the expression levels of LPN， OBR， and Runx2 decreased in the model group （P<0.05）， while the level of PPARγ increased （P<0.05）. As compared with the model group， the BMD of rats in the atorvastatin group， the Livial group， and the Bushen Huatan prescription group increased （P<0.05）， and the relative area of BMAT decreased （P<0.05）. The expression levels of LPN， OBR， and Runx2 in these groups increased （P<0.05）， while the expression level of PPARγ decreased （P<0.05）. ConclusionBushen Huatan prescription plays the anti-osteoporosis role in the rat model of PMOP through up-regulating LPN and OBR in bone tissues and maintaining the balance of osteogenesis and adipogenic differentiation， thereby reducing postmenopausal bone loss and playing a role in the prevention and treatment of PMOP.
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Circular RNA (circRNA), as a competitive endogenous RNA (ceRNA), plays an importantrole in the regulation of cell differentiation. The purpose of this study was to identify and analyze porcinecircular RNA insulin-like growth factor 1 receptor (circIGF1R), explore its expression patterns, construct a ceRNA regulatory network related to circIGF1R, and explore the regulation of its ectopicexpression on adipogenic differentiation of mouse mesenchymal stem cells (C3H10T1 / 2) effect. Forwardand reverse PCR, Sanger sequencing, RNase R enzyme digestion tests, and qRT-PCR were used toverify that circIGF1R is a circRNA formed by the second exon of insulin-like growth factor 1 receptor(IGF1R). It was expressed in all tissues of pigs, and its expression level increased with age in adiposetissues. miRDB, TargetScan and miRWalk online software were used to predict circIGF1R target genes. RNAhybrid software was used for binding site prediction. DAVID bioinformatics functional analysissoftware was used to perform GO and KEGG enrichment analysis on candidate target genes. Cytoscapesoftware was used to construct the ceRNA network diagram. Based on the gene expression correlation andpredicted target relationship, the GO and KEGG enrichment analysis was drawn and the ceRNA networkwas constructed; the dual luciferase reporter gene test was used, and we found that circIGF1R andFABP4 can bind to ssc (Sus scrofa chromosome) -miR-133a-5p. The circIGF1R overexpression vectorwas successfully constructed and expressed in C3H10T1/ 2 cells. It was found that after overexpression ofcircIGF1R, the expression of key adipogenic regulatory factors CEBPa, CEBPß, FABP4 and PPAR? increased significantly(P<0. 01), and the number of lipid droplets increased significantly. The results ofthis study show that circIGF1R exists in pig adipose tissues, and may positively regulate the adipogenicdifferentiation of C3H10T1/ 2 cells through the ceRNA mechanism, which lays a theoretical foundation forfurther research on circIGF1R regulating the adipogenic differentiation of pig precursor intramuscularadipocytes.
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Aim To explore the role of angiotensin U type 1 a reeeptor ( AT 1 aR ) , an important component of HAS, in obesity-induced insulin resistance.Methods Wild type ( WT) and ATlaR gene knockout (ATlaR ) SD rats were fed with normal diet and 60% high-fat diet for 12 weeks, respectively.After 12 weeks, blood was collected from the abdominal aorta of rats to obtain serum, and the serum insulin level was measured by ELISA.The epididvmal adipose tissue was obtained, and gene expressions of peroxisome pro- liferator-activated receptor -y ( PPAR7) and sterol reg¬ulator}' element binding protein lc (SREBP-lc) in ad¬ipose tissue were detected by RT-PCR method.The protein expressions of insulin signaling pathway and protein kinase C (PKC) in adipose tissue were detec¬ted by Western blot.Results ATI aR knockout signif¬icantly reduced HOMA-IR and improved insulin resist¬ance induced by high-fat diet.In ATlaR rats fed with high-fat, the protein expressions of insulin signa¬ling pathway were much higher than those of WT rats, indicating that ATlaR gene knockout improved the in¬sulin signaling pathway in high-fat diet.In addition, the PKCa, PKCe and PKCr| expressions of ATlaR rats were significantly lower than those of WT rats.And the gene expressions of PPAR-y and SREBP-lc, which promoted adipogenic differentiation, significantly increased in ATlaR rats fed with a high-fat diet, demonstrating that ATlaR knockout promoted adipo¬genic differentiation.Conclusions ATlaR knockout significantly improves high-fat diet induced 1R by en¬hancing protein expressions of insulin signaling path¬way, inhibiting PKC expression and promoting adipo¬genic differentiation.
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OBJECTIVE@#To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs.@*RESULTS@#The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P < 0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P < 0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs.@*CONCLUSION@#lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.
Subject(s)
Adipogenesis/genetics , Bone Marrow Cells/metabolism , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells , Osteogenesis , PPAR gamma/pharmacology , RNA, Long Noncoding/genetics , RNA, Messenger/metabolismABSTRACT
The objective of this study was to investigate the expression profile of the myozenin2 (MYOZ2) gene and elucidate its effect on adipogenic differentiation of C3H10T1 / 2 cells and its possible mechanism∙ The longissimus dorsi‚ subcutaneous fat and liver tissue was collected from 180-day-old Mashen pigs‚ 60-day-old ICR mice‚ 35-day-old Ross broiler and 12-month-old Small tail han sheep‚ and the expression profile of the MYOZ2 gene mRNA was detected∙ The results showed that the MYOZ2 gene has similar patterns of tissue expression in examined species‚ with the highest expression level in longissimus dorsi‚ and a small amount of expression in the subcutaneous fat and liver tissue∙ After the MYOZ2 gene was silenced in C3H10T1 / 2 cells‚ qRT-PCR results showed that the expression levels of key adipogenic genes PPARγ and FABP4 were significantly down-regulated compared with the control group (P < 0∙ 01) ; Western Blotting results showed that the PPARγ protein content was significantly decreased (P < 0∙ 05) ; Oil red O staining showed that the number of lipid droplets and the content of triglyceride were significantly decreased after silencing MYOZ2 (P < 0∙ 05) ∙ The expression of fatty acid metabolism related genes SCD‚ FASN‚ SREBP1‚ NR1H3‚ DGAT1‚ PNPLA2‚ HSL‚ CES1‚ CPT1 after MYOZ2 silencing were detected by qRT-PCR∙ The results showed that SCD‚ FASN‚ SREBP1‚ PNPLA2 and HSL were significantly down-regulated (P < 0∙ 01) ‚ NR1H3 was significantly reduced (P < 0∙ 05) ‚ DGAT1 expression was down-regulated but the difference was not significant‚ CES1 and CPT1 were significantly up-regulated (P < 0∙ 05) ∙ The STRING database was used to construct a MYOZ2-related protein interaction network map‚ and it was found that MYOZ2 may affect the adipogenic differentiation through the interaction of titin-cap (TCAP) and PPARγ∙ After silencing TCAP‚ qRT-PCR results showed that compared with the control group‚ the expression of key adipogenic genes PPARγ and FABP4 were significantly up-regulated (P < 0∙ 01) ; Western Blotting results showed that PPARγ protein was significantly increased (P< 0∙ 05) ; Oil red O staining showed that the number of lipid droplets and the content of triglyceride were significantly increased after TCAP silencing (P < 0∙ 05) ∙ qRT-PCR was used to detect the expression of TCAP after silencing MYOZ2‚ and the results showed that the expression of TCAP was significantly increased (P<0∙ 01) ∙ In summary‚ MYOZ2 was highly expressed in longissimus dorsi and lower expressed in subcutaneous fat and liver tissues∙ In addition‚ MYOZ2 may regulate the expression of key adipogenic genes PPARγ and FABP4 through the interaction of MYOZ2-TCAP -PPARγ‚ and to further regulate the expression of fatty acid metabolism related genes SCD‚ FASN‚ SREBP1‚ NR1H3‚ DGAT1‚ PNPLA2‚ HSL‚ CES1 and CPT1‚ thus playing an important role in the process of adipogenic differentiation∙
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The aim of this study was to explore the regulatory mechanism of Type Ⅲ domain-containing protein5 (FNDC5) on adipogenic differentiation in C3H10T1/2 cells. qRT-PCR and Western blot were used to detect the expression of FNDC5 during adipogenic differentiation of C3H10T1/2 cells. The lentivirus-coated overexpression and interference vector of FNDC5 were constructed and transfected into C3H10T1/2 cells. qRT-PCR was used to detect the expression of the key genes of adipogenic differentiation. Oil red O staining was used to detect the formation of lipid droplets; Western blot was used to detect the content of ERK1/2 and ERK1/2 phosphorylated protein (P-ERK1/2). After 8 days of adipogenic differentiation of C3H10T1/2 cells, the expression of Fndc5 increased significantly. After overexpression of FNDC5 in C3H10T1/2 cells, the expression of key genes for adipogenic differentiation, including peroxisome proliferator-activated receptor-酌 (PPAR酌), CCAAT enhancer binding protein beta (C/EBP茁), fatty acid binding protein 4 (FABP4) and CCAAT enhancer binding protein alpha (C/EBPα), all decreased significantly. The content of lipid droplets and P-ERK1/2 also decreased significantly. On the contrary, after interference of FNDC5 in C3H10T1/2 cells, the expression of key genes for adipogenic differentiation, including PPARγ, C/EBP茁, FABP4 and C/EBPα were significantly increased. Meanwhile, the content of lipid droplets and P-ERK1/2 also increased significantly. This study found that FNDC5 can inhibit the adipogenic differentiation of C3H10T1/2 cells by inhibiting the phosphorylation level of ERK1/2, which can provide reference data for the mechanism of FNDC5 in regulating fat deposition.
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BACKGROUND: The method of promoting osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions to inhibit adipogenic differentiation can provide prevention and treatment ideas for the treatment of bone metabolic diseases such as diabetic osteoporosis. OBJECTIVE: To explore the effects of uncarboxylated osteocalcin on adipogenic and osteogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions so as to reveal the action mechanism of uncarboxylated osteocalcin on the differentiation of bone marrow mesenchymal stem cells. METHODS: Mouse bone marrow mesenchymal stem cells were cultured by whole bone marrow culture and adherent purification. Cells were treated with uncarboxylated osteocalcin at different concentrations (0, 1, 3, 10, and 30 μg/L). Cell proliferation was detected by cell counting kit-8 to determine the best mass concentration. Passage 3 bone marrow mesenchymal stem cells were incubated with adipogenic (or osteogenic) differentiation medium, and assigned to four groups: control group, high glucose group, uncarboxylated osteocalci n group, and high glucose + uncarboxylated osteocalcin group. Corresponding groups received the addition of 25.5 mmol/L exogenous glucose and 3 μg/L uncarboxylated osteocalcin. Lipid droplets and calcium nodules were detected by oil red and alizarin red staining. Quantitati ve reverse transcription-polymerase chain reaction was used to detect the relative expression levels of adipogenic marker genes (Fabp4, PPARγ, Adipsin and FAS) and osteogenic differentiation marker genes (Runx2, Osx, alkaline phosphatase, and type I collagen). Kits were used to detect alkaline phosphatase activity and type I collagen levels. The relative expression levels of P-Erk and P-AMPKα were detected using signal pathway specific inhibitors (PD98059 and BML) and western blot assay. RESULTS AND CONCLUSION: (1) Uncarboxylated osteocalcin 3 μg/L promoted cell proliferation (P < 0.01). (2) Uncarboxylated osteocalcin promoted the formation of calcium nodules (P < 0.01) in bone marrow mesenchymal stem cells under high-glucose conditions but inhibited the formation of lipid droplets (P < 0.05), down-regulating the relative expression levels of adipogenic marker genes (PFabp4 < 0.01; PPPARγ < 0.05; PAdipsin < 0.01; PFAS < 0.01), but increasing the relative expression levels of osteogenic differentiation marker genes (PRunx2 < 0.05; POsx < 0.05; PALP < 0.01; PCOLI < 0.01). Uncarboxylated osteocalcin increased alkaline phosphatase activity (P < 0.01) and type I collagen level (P < 0.05). (3) Uncarboxylated osteocalcin up-regulated the expression levels of P-Erk (P < 0.01) and P-AMPKα (P < 0.01) under high-glucose conditions. (4) These results indicate that uncarboxylated osteocalcin promoted osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions through Erk/AMPKα signaling pathway and inhibited adipogenic differentiation.
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Objective: To study the effects of astragalus polysaccharide (APS) on adipogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) irradiated by X-ray. Methods: Human bone marrow mesenchymal stem cells irradiated by 2 Gy X-ray were intervened with 50 μg/mL of APS. The number and size of lipid droplets of stem cells were observed by oil red O staining and the expressions of CEBPα and PPAR-γ were detected by Western blot after induction with special medium. Results: The number of adipocytes differentiated from bone marrow mesenchymal stem cells decreased, and the area of lipid droplets in adipocytes decreased significantly after irradiation (P<0.05). The area of lipid droplets in the bone marrow mesenchymal stem cells treated with APS in advance increased compared with that in radiation alone (P<0.05). Similarly, the expressions of PPAR-γ and recombinant human CCAAT enhancer binding protein (CEBPα ) decreased after X-ray irradiation, while the expressions of PPAR-γ and CEBPα increased after the intervention of APS (P<0.05). Conclusion: X-ray can damage the directional adipogenic differentiation of human bone marrow mesenchymal stem cells, and APS has a protective effect on the adipogenic differentiation of human bone marrow mesenchymal stem cells after X-ray irradiation.
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PURPOSE: Adipogenic differentiation of adipose tissue-derived mesenchymal stem cells (AMSCs) is critical to many disease-related disorders, such as obesity and diabetes. Studies have demonstrated that miRNA-138 (miR-138) is closely involved in adipogenesis. However, the mechanisms affected by miR-138 remain unclear. This work aimed to investigate interactions between miR-138 and lipoprotein lipase (LPL), a key lipogenic enzyme, in AMSCs. MATERIALS AND METHODS: Human AMSCs (hAMSCs) isolated from human abdomen tissue were subjected to adipogenic differentiation medium. Quantitative real-time polymerase chain reaction and Western blot assay were applied to measure the expressions of miR-138, LPL, and the two adipogenic transcription factors cytidine-cytidine-adenosine-adenosine-thymidine enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). The relationship between miR-138 and LPL was predicted utilizing the miRTarBase database and validated by dual luciferase reporter assay. RESULTS: Showing increases in C/EBPα and PPARγ expression levels, hAMSCs were induced into adipogenic differentiation. During adipogenesis of hAMSCs, miR-138 expression was significantly downregulated. Overexpression of miR-138 by transfection inhibited hAMSCs adipogenic differentiation in vitro. Mechanically, LPL was a target of miR-138. LPL expression was upregulated during adipogenesis of hAMSCs, and this upregulation was reversed by miR-138 overexpression. Functionally, silencing of LPL by transfection exerted similar inhibition of the expressions of C/EBPα and PPARγ. Meanwhile, LPL ectopic expression was able to partly abolish the suppressive effect of miR-138 overexpression on adipogenic differentiation of hAMSCs. CONCLUSION: Upregulation of miR-138 inhibits adipogenic differentiation of hAMSCs by directly downregulating LPL.
Subject(s)
Humans , Abdomen , Adipogenesis , Blotting, Western , Carrier Proteins , Ectopic Gene Expression , In Vitro Techniques , Lipoprotein Lipase , Lipoproteins , Luciferases , Mesenchymal Stem Cells , Obesity , PPAR gamma , Real-Time Polymerase Chain Reaction , Transcription Factors , Transfection , Up-RegulationABSTRACT
Aim To study the relationship between WntlOb and bone morphogenetic protein-9 (BMP9)-induced osteogenic differentiation of mesenchymal stem cells (MSCs), and the molecular mechanisms underlying this process. Methods PCR, Western blot and histochemical staining were used to detect the effect of BMP9 on WntlOb and the effect of WntlOb on BMP9-induced osteogenic differentiation in MSCs. Meanwhile, real-time PCR, Western blot, oil red O staining, and flow cytometry assay were used to analyze the potential mechanism of Wnt10b affecting the function of BMP9. Results Wnt10b could be detected in C3H10T1/2, C2C12, MEFs and MC3T3-E1 cells. BMP9 up-regulated the expression of WntlOb in C3H10T1/2 cells. WntlOb enhanced the capability of BMP9 to increase the level of OCN and mineralization in C3H10T1/2 cells, and silencing Wnt10b attenuated these effects of BMP9. Wnt10b exhibited no substantial effect on cell cycle affected by BMP9, but it enhanced the effect of BMP9 on inducing phosphorylation of Smadl/5/8. While silencing Wnt10b attenuated this effect of BMP9. In addition, Wnt10b inhibited BMP9-induced adipogenic differentiation in C3H10T1/2 cells, and silencing Wnt10b promoted this effect of BMP9. Conclusions Wnt10b can promote BMP9-induced osteogenic differentiation of MSCs, which may be mediated through enhancing BMP/Smad signaling and reducing adipogenic differentiation.
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Osteoporosis (OP), a systemic bone metabolism disease, mainly manifested in the decrease of bone mass, the increase of bone fragility and the microstructure degeneration of the bone. Along with the in-depth research of the pathogenesis of osteoporosis, the imbalance differentiation of bone marrow mesenchymal stem cell (BMSCs) (Osteogenic differentiation decrease and adipogenic differentiation increase) is the main reason that causes osteoporosis. In this paper, we summarize the signal pathways of osteogenic differentiation and adipogenic differentiation of BMSCs. Better understand these signal pathways is conducive to elucidate and treat osteoporosis.
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Objective: The study was designed to investigate the molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rBMSCs. Methods: rBMSCs were harvested from SD rats, and determination of alkaline phosphatase (ALP) activity, quantification of mineralization by Alizarin Red S staining, and the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) by RT-PCR after rBMSCs stimulated by osteogenic induction with (0.1–10) µg/mL of quercitrin, quantification of Lipid droplet by Oil Red O staining and the mRNA expression of adipogenic differentiation marker (PPARγ C/EBPα and aP2) by RT-PCR after rBMSCs stimulated by adipogenic induction with (0.1-10) µg/mL of quercitrin. Results: Quercitrin can up-regulate the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) and increase ALP activity and mineralization after osteogenic induction, on the other hand quercitrin can suppress the mRNA expression of adipogenic differentiation markers (PPARγ C/EBPα and aP2) and decrease lipid droplet after adipogenic induction. Conclusion: This study suggested that quercitrin not only stimulated osteogenic differentiation but also inhibited adipogenic differentiation of rBMSCs, which was associated with the up-regulation of Runx2, BMP-2, and OSX mRNA expression and the down-regulation of PPARγ C/EBPα and aP2 mRNA expression.
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AIM:To investigate the effects of sinapine,an effective monomer of Chinese medicine,on hydro-gen peroxide(H2O2)-induced adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).METHODS:The undifferentiated rat BMSCs were identified and screened by flow cytometry.The adipogenic differentiation of BMSCs was induced by H2O2,and the toxicity of sinapine on BMSCs was tested by CCK-8 assay.After the modeling method and the concentration range of sinapine were determined,the lipid droplets in the cells were detected by Oil Red O semi-quanti-tative assay,and the optimal drug concentration was selected.Finally,Oil Red O assay was observed 24 h after drug inter-vention,and the expression of adipogenic differentiation-related proteins,adipocyte protein 2(aP2),peroxisome prolifera-tor-activated receptor γ(PPARγ)and glucose transporter 4(Glut4),at mRNA and protein levels in the BMSCs was deter-mined by qPCR and Western blot.RESULTS:Treatment with H2O2at 200 μmol/L for 1 h induced BMSCs to differentiate into adipocytes.Below the concentration of 40 μmol/L,sinapine had no toxicity to BMSCs.The best inhibitory concentra-tion of sinapine on adipogenic differentiation was at 15 μmol/L.The number of lipid droplets in sinapine(15 μmol/L) group was significantly lower than that in model group.In sinapine group,the expression of aP2,PPARγand Glut4 at mR-NA and protein levels was lower than that in model group(P<0.01).CONCLUSION: Sinapine inhibits H2O2-induced adipogenic differentiation of rat BMSCs.The mechanism may be related to the PPARγ/AMPK signaling pathway.
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Objective To investiagte the adipogenic differentiative ability of adipose tissue-derived stromal cells(ADSCs) between the patients with type 2 diabetes mellitus(T2DM) and healthy persons.Methods The adipose tissues were taken from the adipose tissue in T2DM patients and healthy persons for separating and culturing ADSCs.The cells of third generation were taken for inoculation.The difference in cellular phenotype and growth speed were compared between the two groups.Adding adipogenesis inducing fluid,the adipogenic differentiative situation was observed in the two groups.The oil red O was added on 14 d for conducting the cell staining and observation.The oil red O was extracted by isopropanol,and the cellular absorbances were compared between two groups.Meanwhile,the expression of PPAR-γ,C/EBP-α and C/EBP-β on 14 d of adipogenic differentiation were compared between two groups by using qPCR method.Results The cellular phenotype and growth speed of ADSCs had no statisticat difference between T2DM patients and healthy persons.On 14 d of adipogenic differentiation,the oil red O absorbance value of AD-SCs in T2DM patients was significantly higher than that in the healthy persons,and the expression of PPAR-γ,C/EBP-α and C/EBP-β were significantly higher than those in the healthy persons.Conclusion The adipogenic differentiative ability of ADSCs in T2DM patients is obviously higher than that in healthy persons,which may be one of causes easy to be obese in T2DM patients.
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Chalcones is a flavonoid wildly presented in many herbs. It has the effect to inhibit cells adipogenic differentiation. In order to study the effect of pinostrobin chalcone extracted and isolated from leaves of hickoryes on the adipogenic differentiation of murine embryonic mesenchymal stem cell (C3H10T1/2), MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium] method was used to detect the cell proliferation; adipogenic differentiation was characterized by oil red O staining and isopropanol extraction; the triglyceride content was detected by GAP-PAP enzyme method; and the C3H10T1/2 cell differentiation into adipocytes was also examined by the mRNA and protein expression of PPARγ, C/EBPα and FABP4 by RT-PCR and Western blot respectively. Results indicated that pinostrobin chalcone almost had no effect on cell proliferation activity when the concentration was less than or equal to 50 μmol•L⁻¹; the oil red O staining, isopropanol extraction and GAP-PAP enzyme method showed that pinostrobin chalcone significantly decreased the C3H10T1/2 adipogenic differentiation and triglyceride content in the cytoplasm of adipocytes; the RT-PCR and Western blot analysis showed that pinostrobin chalcone can down-regulate the mRNA and protein levels of FABP4, PPARγ and C/EBPα in C3H10T1/2 cells(P<0.05 or P<0.01). The experiment results suggest that pinostrobin chalcone can inhibit C3H10T1/2 adipogenic differentiation.
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To study the effect of Astragalus polysaccharide (APS) on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) cultured in hypoxic environment. The optimal APS concentration, which could promote the proliferation of BMSCs, was screened by methyl thiazolyl tetrazolium method. The concentration was used to intervene in BMSCs-induced by adipogenic differentiation fluid growing in different oxygen concentrations (3%, 6%, 10% and 20%). The formation of lipid droplets in the BMSCs-intervened was observed by oil red O staining under the optical microscope. The mRNA and protein levels of the lipid relating genes peroxisome proliferator activated receptor gamma 2 (PPAR-γ₂) and lipoprotein lipase (LPL) were detected by Real-time PCR and Western blotting, respectively. The results showed that, comparing with the control group, 40 μg/mL APS could significantly promote the proliferation of BMSCs under low oxygen concentration. A large amount of lipid droplets existed in BMSCs growing in the adipogenic inducing fluid containing 40 μg/mL APS and the hypoxic environment, and the protein and mRNA levels of PPAR-γ₂ and LPL also raised. It was worth noting that the phenomenon was more significant in 10% oxygen concentration, and the difference was statistically significant (P<0.05). 40 μg/mL APS had effect on promoting the proliferation and adipogenic differentiation of BMSCs cultured in hypoxic environment, and the effect was related to the concentration of oxygen of BMSCs-cultured.
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Objective:To culture canine dental pulp stem cells(cDPSCs)in vitro.Methods:Canine pulp cells were isolated and cultured by enzyme digestion and explanted tissue culture respectively.Cell morphology was observed under phase-contrast micro-scope.The clone forming unit(CFU)of the cells was examined by plate clone formation assay.Cell markers and protein-expression were examined by flow cytometry(FC)and immunofluorescence.Odontogenic and adipogenic potential were evaluated by alizarin red staining and oil red O staining.Results:Short spindle fibroblast-like and steadily growing cells were obtained by both methods.The clone assay showed that CFU was 1 5.1 7% ±2.79%.FC observasion showed that the CD90,STRO-1 and CD24 positive cells were 24.43% ±7.1 0%,20.67% ±1 .42% and 2.03% ±0.06% respectively,but CD34 was negative.Immunofluorescence analysis showed positive expression of Nestin,Vimentin,weak expression of ALP and negative expression of DSP of the cells.Differentiation ex-periment confirmed the odontogenic and adipogenic differentiation potential of the cells.Conclusion:cDPSCs can be cultured in vitro.
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Objective To investigate the the multi-directional differentiation potential between pluripotent of human gingival fibroblasts (HGFs) and human periodontal ligament cells (HPDLCs). Methods HPDLCs and HGFs were obtained from the primary culture. HPDLCs and HGFs at 3rd-4th passage were cultured in osteogenic, adipogenic or chondrogenic me-dium. Cells without differentiation were taken as control. Alizarin red, Alcian blue and oil red O staining were performed to detect osteogenic differentiation, chondrogenic and adipogenic differentiation in vitro, respectively. Reverse transcription polymerase chain reaction (RT-PCR) was applied to examine the expression of osteocalcin (OCN), runt-related transcription factor 2 (RUNX2) and collagen 1 (Col 1), peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and collagen 10 (Col 10). Results HPDLCs and HGFs cultured in osteogenic medium showed massive calicium nodulus at day 28, but HP-DLCs formed more calicium nodulus than those of HGFs. The expressions of OCN, RUNX2 and Col 1 were significantly high-er in HPDLCs than those in HGFs (P<0.05). In chondrogenic medium both cells were found blue deposit at day 14, and the expression of Col 10 was significantly higher in HGFs than that of HPDLCs (P<0.01). Furthermore, in adipogenic medium HGFs showed more lipid-filled droplets stained with oil red O than HPDLCs at day 21. The expression of PPARγ2 was sig-nificantly higher in HGFs than that of HPDLCs (P<0.01). Conclusion HPDLCs has the better potency of osteogenic differ-etiation than HGFs, however, HGFs has the better potency of adipogenic and chondrogenic differentiation.
ABSTRACT
Objective To observe the effect of different concentrations of parathyroid hormone 1-34 on the adipogenic potential of rat bone marrow mesenchymal stem cells (BMSCs).Methods ① Rat bone marrow mesenchymal cells were separated and expanded by adherent culture.The morphology of cells was observed and cell surface markers were examined by flow cytometry.② The multi-lineage differentiation capability of cells was examined by culturing cells under conditions favorable for adipogenic and osteogenic differentiation.③ Taken P3 of BMSCs for test,different concentrations of PTH1-34 (0,10-10,10-9,10-8 mol/L) were used to stimulate BMSCs respectively,14 days later,lipoprotein lipase (LPL) activity were measured by enzyme linked immuno-sorbent assay (ELISA),mRNA expression of alkaline phosphatase (ALP) and PPARγ-2 were measured by realiime polymerase chain reaction (PCR).④ Statistical analysis:data were presented as x±s.All statistical analysis was performed with windows Statistical Praduct and Serice Solutions (SPSS) 13.0.One-way analysis of variance (ANOVA) was applied to determine the difference between groups.Least signisicant difference (LSD) was used to determine the difference between the two randomized groups.Differences were considered significant at a value of P＜0.05.Results The cells expressed CD44,CD29 but without expression of CD45.By culturing in adipogenic medium for 3 weeks and in osteogenic medium for 4 weeks respectively,and then identified by oil red O and Alizarin red,the cells were successfully induced to adipocytes and osteogenesis.Expressions of LPL were 11.20±0.16,7.62±0.48,5.84±0.57,5.32±0.52,mRNA expressions of PPARγ-2 were 2.80±0.05,1.36±0.23,0.94-±0.11,0.78±0.04,ALP activity were 0.191 ±0.016,0.333±0.024,0.549±0.025,0.684±0.021 respectively.Compared with the control group,different concentrations of PTH1-34 groups could decrease mRNA expression of LPL and PPARγ-2.ALP activity were increased(P＜0.05).Conclusion PTH1-34 inhibits BMSCs of adipogenic differentiation and promotes osteogenic differentiation in a dose-dependent manner.