The Effects Of High Calorie Intake On Biological Characteristics Of Bone Marrow Derived Mesenchymal Stem Cells

Background:High-fat diet is one of the important reasons for the increasing incidence of metabolic diseases. The chronic complications related to metabolic disorders, including pathological changes and functional impairment in various organs and tissues, has received widespread attention. Mesenchymal stem cells (MSCs) are a class of stem cells that mainly exists in the connective tissue or organ stroma with highly self-renewal and multi-differentiation abilities. They play important roles in both normal tissue homeostasis and in damaged tissue regeneration. The pathophysiologic state changes caused by metabolic diseases could affect the function of MSCs and vice versa. Numerous studies have confirmed that chemokines and their receptors are not only involved in regulating glucose and lipid metabolism, but also affect the function of MSCs directly, so as to play an important role in the repair of the damaged tissue and organ. Therefore, investigate the effects of chemokines on MSCs and the underlying mechanisms during the progression of metabolic disease could assist us in developing a deeper understanding of these diseases. Aspirin may ameliorate the inflammatory microenvironment, so figure out the influence of amelioration of inflammatory microenvironment on the biological characteristics of MSCs will provide new insights into the mechanism of metabolic diseases and theoretical basis to clinical practice.Objective:1. To figure out the effects of high calorie intake on the biological characteristics of bone marrow derived MSCs (BMSCs)2. To further elucidate the influence of chemokine CINC-3 on the biological characteristics of BMSCs and the underlying mechanisms3. To figure out the effects of Aspirin on the inflammatory microenvironment induced by high calorie intake and the biological characteristics of BMSCsMethods:1. Establishment and identification of Sprague Dawley (SD) rat model with different dietary patternsSix-week-old SD rats were randomly divided into three groups, normal diet (ND, 100% basal diet) and customized high-fat diet (HFD,60% of basal diet,10% sucrose, 20% lard and 10% egg yolk powder) were used to feed control group and HFD group respectively. SD rats in reestablished diet group were fed HFD for 2 months followed by reestablishment of basal diet. Physiological and biochemical indicators (including body weight, body length, total serum cholesterol, fasting blood glucose, insulin levels, insulin tolerance levels) were monitored at different time points. Meanwhile, liver, fat and bone tissue were harvested for histopathological observation.2. The effects of high calorie intake on the biological characteristics of BMSCsRat BMSCs were isolated by whole bone marrow adherent method and cell morphology which obtained from rats received different dietary patterns was observed under inverted microscope. The proliferative capacities of BMSCs were detected by MTS assay, and the cell cycle analysis and apoptosis assay were performed utilizing Muse cell analyzer. The migration abilities of BMSCs were tested by transwell migration asssay. BMSCs at P3 were subjected to adipogenic and osteogenic differentiation in vitro.The gene expressions of specific markers were detected by real-time PCR and histochemical staining. To identify the impacts of high calorie intake on the differentiation abilities of BMSCs, senescence degree of BMSCs under different dietary patterns were evaluated by SA-β-Gal staining. To explore the mechanism associated with the accelerated aging process of BMSCs obstained from rats with high calorie intake, mitochondrial superoxide content was measured using MitoSOXTM staining and peroxynitrite content was quantified by flow cytometry. The mRNA levels of telomerase reverse transcriptase (TERT) and histone deacetylase (SIRT1) were detected by real-time PCR analysis.3. Impacts of chemokine CINC-3 induced by high calorie intake on the biological characteristics of BMSCsIn order to analyze the effects of high calorie intake on the inflammatory cytokines, the fasting serum was collected from tail vein of rats and the levels of interrelated factors were measured according to the protocol of the RayBio(?) Rat Cytokine Antibody Array G-Series 2.To study the effects of high calorie intake-induced CINC-3 on the biological characteristics of BMSCs, chemokine CINC-3 was added to culture medium and the proliferative capacity, cell cycle, apoptosis, migration abilities and the celluar senescence of BMSCs were detected.. In addition, to identify the impact of high calorie intake on the differentiation abilities of BMSCs, CINC-3 was added to osteogenic and adipogenic induction medium and the expression of key phenotypic markers were assessed by real-time PCR and histochemical staining.4. Effects and the mechanism of high calorie intake-induced CINC-3 on the migration and differentiation abilities of BMSCsDifferent concentrations of CINC-3 were added to the culture medium of BMSCs and the cells were harvested at different time points for proteins extraction. Western blot and G-LISA was applied to detect the expression of ELMO1 and Active Rac1 protein. CINC-3 and functional blocking antibody anti-CINC-3 were supplemented into culture medium to further verified the activation of ELMO1 and Active Racl induced by CINC-3. The effect of active Racl protein inhibitor NSC23766 on BMSCs proliferation was detected by MTS. Moreover, the mechanism by which CINC-3 inhibits adipogenic differentiation of BMSCs was illustrated by real-time PCR and histochemistry staining. Finally, the effect of CINC-3 on BMSCs migration ability was investigated by knockdown of ELMO 1.5. The impacts of Aspirin on the amelioration of inflammatory microenvironment and the biological characteristics of BMSCsSix-week-old SD rats were randomly divided into four groups; normal diet and customized high-fat diet were used to feed rats in ND group and HFD group respectively. Rats in reestablished diet group were fed HFD for 2 months followed by basal diet. Rats in reestablished diet and anti-inflammatory treatment group (2GC+Aspirin) were fed HFD for 2 months followed by reestablishment of basal diet concomitant with Aspirin treatment. Physiological and biochemical indicators were monitored at different time points and multiple tissues and organs were collected for histopathological observation. The levels of cytokines were measured according to the protocol of the RayBio(?) Rat Cytokine Antibody Array G-Series 2. In addition, the biological characteristics of BMSCs isolated from rats in different dietary pattern groups were assessed as described above.Results:1. Establishment and identification of SD rat model with different dietary patternsRats in HFD group experienced growth spurts during the diet course, and the body mass and Lee’s index was significantly increased. The levels of fasting cholesterol and blood glucose levels of HFD-fed rats were rised, while the insulin tolerance was decreased. The fasting cholesterol levels of rats in reestablished diet group were rapidly restored to normal after refeeding with basal diet. However, the insulin tolerance level was not returned to normal until refeeding basal diet for 4 months. Multiple tissues of rats showed obvious pathological changes after feeding with HFD for 6 months, such as liver, fat and bone tissue. Liver biopsy demonstrated a severe hepatic steatosis. The cross-sectional area of omental fat cells was significantly increased. Additionally, the number of osteoclasts in the bone marrow was increased evidently. However, there was no significant difference in the pathological changes in rats between normal diet group (6MC) and reestablish diet group (2G4C).2. The effects of high calorie intake on the biological characteristics of BMSCsResults of MTS assay showed that the proliferation ability of BMSCs in 6MG group was significantly decreased compared with the BMSCs in 6MC group. Cell cycle analysis indicated that the percentage of BMSCs in GOG1 phase was significantly increased in 6MG group. However, no significant difference was observed for the proliferation ability and cell cycle of BMSCs between 2G4C and 6MC groups. Meanwhile, the percentages of apopotic BMSCs were similar among these three groups. The results of transwell migration assay showed that the migration abilities of BMSCs in 6MG group and 2G4C group were significantly increased as compared with 6MC. Whereas; the differentiation capacities of BMSCs in 6MG and 2G4C groups were significantly inhibited. SA-P-Gal staining showed that BMSCs in HFD and 2G4C group had obvious aging phenomena. The contents of superoxide and peroxynitrite in mitochondria were also increased. Moreover, the expression levels of TERT and SIRT1 in BMSCs isolated from rats in HFD and 2G4C groups were decreased as compared with 6MC group.3. Impacts of chemokine CINC-3 induced by high calorie intake on biological characteristics of BMSCsResults of cytokine antibody microarray showed that levels of CINC-3、 CX3CL1 、 LIX in 6MG group were 20% higher than in 6MC group, while the levels of CINC-3 and LIX in 2G4C group remained high and did not return to normal levels. The results of ELISA analysis further confirmed that the serum levels of CINC-3 in 2G4C group were significantly higher than in 6MC group. Supplement of CINC-3 in culture medium showed no obvious influences on the proliferation of BMSCs, whereas the migration abilities and aging degree of BMSCs were significantly promoted. Moreover, expression of adipogenic markers in BMSCs was down-regulated after exposure to CINC-3, which indicated that CINC-3 could inhibit adipogenic differentiation of BMSCs.4. Effects and the mechanism of high calorie intake-induced CINC-3 on the migration and differentiation abilities of BMSCsChemokine signal pathway in BMSCs was quickly activated by CINC-3, and the expression levels of ELMO1 and active Rac1 were increased. Functional anti-CINC-3 antibody could specifically and efficiently inhibit the activation of chemokine pathway induced by CINC-3.The proliferation of BMSCs was not affected by active Racl inhibitor NSC23766; nevertheless, it could rescue the suppression of adipogenic differentiation induced by activation of active Racl. Meanwhile, the migration capacities of BMSCs were decreased by knocking down ELMO1.5. The impacts of Aspirin on the amelioration of inflammatory microenvironment and the biological characteristics of BMSCsThe weight, Lee’s index and fasting glucose level of rats were maintained in a relatively stable level in "2G4C+Aspirin" group. Nevertheless, the fasting serum cholesterol levels in rats were returned to normal quickly after reestablishing to basal diet together with anti-inflammatory treatment for a month while insulin tolerance was back to normal after 3 months. Results of cytokine antibody microarray showed that the vast majority of cytokines in " 2G4C+Aspirin" rats returned to normal except CINC-3 and LIX, which were still significantly higher than in ND rats.The adipogenic differentiation potential and degree of cell aging had not yet returned to normal in "2G4C+Aspirin" group, but showed substantial improvement compared with 2G4C group.Conclusions:1. This research established two kinds of animal models using SD rats fed with high-calorie intake and reestablished diet. After 4 months of reestablished normal diet intervention, blood cholesterol and insulin tolerance of rats returned to normal in 2G4C group. However, BMSCs isolated from rats after diet reestabliehment showed distinct aging phenomena, increased migration ability and decreased differentiative capacities as compared with BMSCs isolated from rats in control group. Results of cytokine antibody array and ELISA analysis showed that the levels of CICN-3 could not go back to normal, suggesting that the elevated CINC-3 induced by high calirie intake may be associated with the changes of the biological characteristics of BMSCs.2. Addition of CINC-3 in the culture medium did not show distinct effect on the proliferation of BMSCs. However, supplementation of CINC-3 could promote migration and aging but down-regulate expression of adipogenic genes of BMSCs. All these findings suggested that CINC-3 is closely related to the biological characteristics of BMSCs.3. Chemokine signal pathway in BMSCs were quickly activated by CINC-3 together with the increased expressions of ELMO1 and active Rac1. Functional antibody anti-CINC-3 could specifically and efficiently inhibit the activation of chemokine pathway in BMSCs induced by CINC-3. Exposing BMSCs to active Rac1 inhibitor NSC23766 could rescue the suppression of adipogenic differentiation induced by activation of Active Rac1. Meanwhile, the migration capacities of BMSCs were decreased by knocking down ELMO1. In conclusion, these findings suggested that ELMO1 and active Rac1 were involved in the regulation of migration and differentiation capacities of BMSCs.4. Results of cytokine antibody microarray showed that the levels of vast majority of cytokines in rats in "2G4C+Aspirin" group were returned to normal while levels of CINC-3 were still significantly higher than in rats from ND group. Nevertheless, the adipogenic differentiation potentials and degrees of cell aging of BMSCs from rats in "2G4C+Aspirin" group were significantly improved as compared with BMSCs in 2G4C group. In conclusion, Aspirin could ameliorate the inflammatory microenvironment and biological characteristics of BMSCs to some extent.