High Caloric Intake On Rat Bone Marrow Mesenchymal Stem Cells Differentiation Capacity

Background:High calorie intake has been recognized as one of the important risk factors for a variety of diseases, including obesity, cardiovascular disease and metabolic syndrome. Bone marrow mesenchymal stem cells (BMSCs) are multipotent stromal cells that hold potentials to give rise to cells of diverse lineages, participating in the regeneration and reconstructing of multiple damaged tissues. A growing body of evidence suggests that high-fat-diet (HFD) can result in significant pathological changes in tissues or organs, but the influences of HFD on the biological characteristics of BMSCs remains to be elucidated. It is imperative to uncover the changes of BMSCs under the high calorie intake circumstances for the understanding and treatment of metabolic diseases.Objectives:1. To establish a high-calorie intake animal model by feeding rats with high-fat diet (HFD) and to define the effects of HFD on physiological, biochemical, and histopathology of tissues and organs in animal model, and to assess impacts of HFD on the biological characteristics of BMSCs.2. To study the cytokine profiles of peripheral blood of rats under HFD feeding, and to detect the effects of specified cytokines on the differentiation potentials of BMSCs.3. To explore the effects of HFD on gene expression profiles of rats BMSCs so as to provide a theoretical basis for understanding the effects of HFD on BMSCs which might give further impacts on regeneration and reconstructing of multiple damaged tissues in metabolic diseases.Methods:1. Construction of rats animal models with high-calorie intakeNormal diet (ND, basal diet) and customized HFD (60%of basal diet,20%sucrose,10%lard and10%egg yolk powder) were used to feed6-week-old female Sprague-Dawley (SD) rats respectively. Studies on rat physiological and biochemical indicators (including body weight, body length, fasting blood glucose, blood lipid and insulin levels, insulin tolerance levels) and histopathological changes in the corresponding organs were performed at different feeding time points.2. Isolation and characterization of rat BMSCs in vitroBMSCs were isolated by whole bone marrow adherent method and maintained in complete medium. Cells were cultured in the incubator with the atmosphere of5%CO2and100%humidity. Cells were digested and passaged when they reached80%to90%confluency. The expression of MSC cell surface markers, percentage of apoptotic cells and distribution of cells in different phases of cell cycle of harvested adherent cells were assessed by flow cytometry. In addition, proliferation ability of BMSCs was detected by MTT assay. Total mRNA of BMSCs at specified time points was collected using TRI Reagent and real-time PCR was used to detect the expressions of relevant marker genes. BMSCs at P3were subjected to osteogenic and adipogenic differentiation in vitro. Real-time PCR analysis, Alizarin Red S staining and Oil Red O staining were performed to detect the osteogenic and adipogenic abilities of BMSCs.3. Analysis of relevant cytokines in rat serumIn order to study the impacts of HFD on the inflammatory cytokines profiles, the fasting serum was collected from tail vein of rats after1,2or4months of feeding and the levels of the relevant cytokines were measured according to the protocol of the RayBio(?) Rat Cytokine Antibody Array G-Series2.4. Impacts of the specified cytokines on the differentiation ability of BMSCs in vitroIn vitro adipogenesis and osteogenesis of BMSCs isolated from4-month HFD or ND fed rats were performed. CINC-1or CINC-3was supplemented in differentiation medium of ND BMSCs while IL-1β was added to the induction medium of HFD BMSCs during adipogenic and osteogenic differentiation in vitro. After induced differentiation, expressions of marker genes were analyzed to assess the impacts of cytokines on the differentiation potentials of BMSCs.5. Detection and analysis of gene expression profiles of BMSCsThe total mRNA of BMSCs (P1) was purified and analyzed by the Rat Genome2302.0Array (Affymetrix). The targets were prepared and hybridization followed by cleaning, dyeing and scanning was operated according to manufacturers’ instructions. Data were collected by GeneChip(?) Scanner3000and analyzed by using Bio MAS (Molecule annotation system)3.0software (CapitalBio Corporation, China). Results1. Establishment of rat model with high-calorie intakeHFD changes the physiological and biochemical properties of rats. Body mass and Lee’s obesity index of the HFD rats were significantly higher than that of ND rats after3months of feeding. The fasting total cholesterol, fasting serum insulin levels and insulin tolerance of HFD rats were significantly increased since the1st month, and the difference between both groups increased in a time-dependent manner. Interestingly, the fasting blood glucose of rats in both groups didn’t show significant differences until feeding for4months.2. HFD changed the histopathology of ratMultiple tissues of HFD rats showed obvious pathological changes. Liver biopsy demonstrated that lipid droplets were presented in the hepatocytes of1month HFD fed rat and the hepatic steatosis became more serious at2nd month. Abnormal accumulation of fat in subcutaneous and abdominal cavity was observed. The diameters of omental fat cells were significantly increased than that of control group after feeding for1month onwards, and the difference between two groups was gradually increased in a time-dependent manner. The number of osteoclasts in the bone marrow of femur in HFD rats was significantly increased when compared with ND rats after feeding for4months.3. Isolation and characterization of rat BMSCsThe adherent cells were obtained after adherent culture of12to14days and the morphologies of BMSCs isolated from HFD and ND rats were similar after two passages. Percentages of CD31-/CD90+cells in HFD and ND rats decreased along with the feeding time while the percentage of CD31-/CD90+cells in HFD rats was significantly lower than in ND rats after one month feeding. After feeding for4months, there was no significant difference in the apoptosis and cell cycle profiles of HFD and ND BMSCs, but the proliferation abilities of HFD BMSCs were dramatically decreased than ND BMSCs.4. Comparison of the differentiation abilities of rat BMSCs in vitroDuring the adipogenic differentiation, the expression of adipogenic markers in BMSCs in HFD group were significantly higher than those from ND group after1month of feeding, but rapidly decreased after2months. For osteogenic differentiation, there was no significant difference in the expressions of Runx2mRNA between HFD and ND BMSCs after feeding for2months. After feeding for4months, both the expressions of osteogenic genes expression were significantly decreased in HFD BMSCs than in ND BMSCs, suggesting that the capacities of differentiation potentials of rat BMSCs were significantly inhibited.5. Evaluation of cytokines levels in peripheral blood serum of ratsResults of cytokine antibody microarray showed that after HFD feeding for1month, levels of MIP-3a, VEGF, TIMP1, MMP-8, beta-NGF, Fractalkine, L-Selectin and LIX in HFD rats showed20%increase than in ND rats while the levels of IL-1R6and PDGF-AA were significantly reduced. After feeding for2months, levels of CINC-1, leptin, TNF-alpha, CINC-3and Prolactin-R in HFD rats were increased by20%or more when compared to ND rats while serum levels of IL-1R6and PDGF-AA went back to the same levels as ND rats. Levels of IL-1β, MIP-3a and TIMP1in HFD rats were20%lower than that in ND rats; the serum levels of beta-NGF, Fractalkine and L-Selectin in HFD rats were decreased by about15%while MMP-8and VEGF levels returned to the same level as control group. After feeding for4months, levels of VEGF, CINC-1, leptin, TNF-alpha, CINC-3and Prolactin-R in serum of HFD rat were higher than that in ND rats for more than30%while level of IL-1β continued to decrease to less than65%of ND rats.6. Effect of CINC-1, CINC-3and IL-1β on the differentiation ability of rat BMSCs in vitroSupplement of CINC-3in the differentiation medium could significantly reduce the mRNA expression of adipogenic markers of ND BMSCs, but showed no influence in the expressions of osteogenic differentiation marker genes. No significant effect of CINC-1on the expressions of adipogenic or osteogenic marker gene in ND BMSCs was observed. Supplementation of IL-1β could rescue the inhibited mRNA expressions of adipogenic markers but further inhibited the expressions of osteogenic markers in HFD BMSCs.7. Evaluation and analysis of the gene expression profiles of BMSCsAfter1month of feeding, the gene expression profiles of HFD BMSCs were significantly different from ND BMSCs. The differences of BMSCs between two groups were more pronounced after feeding for2months. Genes with significant changes were mainly involved in metabolism and inflammation signal pathways, including cytokines and cytokines receptor interaction, JAK-STAT signaling and MAPK signaling; as well as the pathways that regulated the functions of BMSCs, such as Toll-like signaling, Wnt signaling and TGF-1β signaling. The results of microarray analysis further confirmed that the changes in the serum levels of inflammatory cytokines could change the biological characteristics of BMSCs.Conclusion1. After1month of HFD intervention, blood lipids and insulin resistance were significantly increased and the histopathologies of corresponding organs were also changes. However, the blood glucose level of HFD rats did not change until feeding for4months, suggesting that the pathological changes of corresponding organs were synchronous with the changes in the blood lipids and insulin tolerance, and occurred earlier than the changes of blood glucose. These findings indicated that multiple clinical scenarios rather than blood glucose alone should be tested for early diagnosis of metabolic diseases in future.2. The BMSCs isolated from HFD rats showed reduced percentages in adherent cell population, decreased proliferation and differentiation abilities in vitro, suggesting that HFD may affect the biological characteristics of BMSCs, and thus caused impacts on the regeneration and reconstruction ability of tissues and organs in organism.3. HFD modified serum levels of inflammatory cytokines in rats, of which leptin, CINC-1and CINC-3were significantly elevated while IL-1β were reduced. Supplementation of CINC-3and IL-1β in induced differentiation culture of BMSCs in vitro significantly changed the expression of differentiation-related marker genes, indicating that changes of inflammatory cytokines in the microenvironment may be one of the factors that resulted in the change of the differentiation abilities of BMSCs. The results of gene expression microarray analysis demonstrated that a large number of genes involved in pathways that regulate cytokine signaling or stem cell functions were down or up-regulated, suggesting a correlation between cytokines and biological properties of BMSCs. In this study, we firstly reported that CINC-3could inhibit the adipogenic differentiation ability of BMSCs.