| Background: Periodontitis is a chronic inflammatory disease, which has high incidence and plaque is as the initiating factor, damaging alveolar bone and other periodontal tissues invasively. Not only can cause tooth mobility, it can induce systemic diseases, such as diabetes or cardiovascular disease, and serious damage to the oral and general health. Type 2 diabetes mellitus(T2DM) is a systemic metabolic disease which is caused by insulin resistance and islet β-cell dysfunction and interacts with periodontitis. T2DM not only participates in the change of inflammation microenvironment in periodontal tissue, still can cause metabolic abnormalities of alveolar bone, imbalances between bone formation and resorption, so as to accelerate periodontal bone loss.Bone marrow mesenchymal stem cells(BMSCs) differentiate in suitable proportion to induce stable balance between bone formation and resorption and maintain the normal bone strength and integrity. Therefore, BMSCs is cytological basis of bone metabolism or reconstruction. T2DM significantly inhibits the osteogenic differentiation of BMSCs to deteriorate diabetic bone defect and periodontitis. Therefore, to explore and regulate the influence mechanism of BMSCs osteogenesis on T2DM, next delaying the degenerative changes of bone tissue, and reconstructing normal morphology of bone may be an effective method for the treatment of diabetic periodontitis.Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1(BMAL1) is one of the core components of body’s circadian gene families, linking blood glucose metabolism and BMSCs osteogenic differentiation, and may positively affect the recovery of BMSCs osteogenic ability in T2DM. Wnt/β-catenin pathway is a well-characterized mechanism that promoted osteogenic differentiation of BMSCs and is associated with BMAL1, however, the mechanism by which BMAL1 regulates Wnt/β-catenin pathway is uncertain and warrants further exploration.Glycogen synthase kinase 3β(GSK-3β) negatively correlates with whole-body glucose disposal and plays an important role in bone regeneration by inhibiting the Wnt/β-catenin pathway. Our previous study has also confirmed that GSK-3β influenced the migration of BMSCs in a diabetic microenvironment. Although the interaction between BMAL1 and GSK-3β has been identified, the results are inconsistent. GSK-3β can phosphorylate BMAL1, which controls the stability of the protein and the amplitude of circadian oscillation. In addition, activation of the PI3K/Akt pathway that phosphorylated GSK-3βwas directly regulated by BMAL1. Therefore, in this study, we demonstrated that BMAL1 was insufficient to stimulate activity of Wnt/β-catenin pathway and BMSCs osteogenesis in T2DM via interaction with GSK-3p.Objective: We aimed to determine the mechanism that BMAL1 regulated osteogenic differentiation of BMSCs and may have a potential use in repairing diabetic bone metabolic disorders. Our findings would provide new insights into the etiological study of periodontitis, and lay the foundation for delaying bone damage and reconstruction of periodontal bone tissue morphology and function in T2DM.Methods:Part 1: BMSCs was obtained from T2DM GK rats and controlled Wistar rats after detection of body weight, random blood glucose, fasting blood glucose and insulin, and oral glucose tolerance tests. We tested the expression of mesenchymal stem-cell markers by flow cytometry of GK and Wistar BMSCs. To investigate the potential of BMSCs to undergo osteogenic/adipogenic differentiation, GK and Wistar BMSCs were cultured in adipogenic or osteogenic medium. Alizarin red and oil red O staining were used and the mRNA expression of RUNX2, OCN, PPAR-γ and LPL was tested by qRT-PCR. SA-β-Gal staining, telomere length, and telomerase activity were tested to detect cell senescence of GK and Wistar BMSCs. Colony forming efficiency, flow cytometry, MTT, and CCND1,CDK4, IL-β and TNF-α mRNA expression were detected to compare the proliferation,apoptosis and inflammation index of the GK and Wistar BMSCs.Part 2: As the core of the circadian rhythm genes, BMAL1 plays a vital role in T2DM and stem cell differentiation. Therefore, we aimed to determine whether BMAL1 regulates BMSCs osteogenesis in T2DM. Thus, Western blot analyses were used to compare BMAL1 expression level in nucleus of the GK and Wistar BMSCs. The protein and mRNA levels of BMAL1 were also detected in BMSCs before and during osteo-induction. ALP staining, the quantitative evaluation of ALP activity and qRT-PCR were used for determining ALP, OCN, RUNX2 level in Wistar and GK BMSC after 7 days in osteogenic medium culture.Part 3: To determine whether BMAL1 participated in the regulation of BMSCs osteogenesis in T2DM through interaction with GSK-3β and activation of the Wnt/β-catenin pathway, an immunofluorescence assay and Western blot were performed to confirm the expression of GSK-3β in GK and Wistar BMSCs, Then expression of cytoplasmic act-β-catenin and nuclear β-catenin, NLK, and TCF were measured by Western blot before and during osteo-induction. For verification that T2DM affected BMAL1 to regulate GSK-3β expression and Wnt/β-catenin pathway activity, BMSCs were transfected by BMAL1-overexpressing lentiviral vector. The transfection efficiency of BMAL1 lentivirus was determined via flow cytometric analysis by calculation of the percentage of transfected fluorescent cells in Wistar and GK BMSCs. And then over-expression of BMAL1 protein and BMAL1 mRNA were measured by Western blot and qRT-PCR after transfection. To further elucidate the influence of an elevated BMAL1 level on activation of the Wnt/β-catenin pathway, expression of cytoplasmic GSK-3β,β-catenin, and act-β-catenin were measured by an immunofluorescence assay and Western blot after BMAL1 transfection in Wistar and GK BMSCs. In order to deny that inhibition of GSK-3(3 could affected BMAL1 expression and activated the Wnt/β-catenin pathway solely, Western blot shows changes in the expression of GSK-3p, BMAL1, act-p-catenin,NLK and TCF in BMSCs using CHIR99021. And Western blot was also used to show changes in the expression of BMAL1, act-β-catenin and TCF under the influence of BMAL1 transfection and GSK-3β inhibition in GK BMSCs.Part 4: To further estimate the effects of BMAL1 over-expression on the osteogenic differentiation capacity of BMSCs in T2DM, osteogenic differentiation of BMSCs after BMAL1 over-expression were determined by OCN fluorescence-positive cells staining and ALP staining. OCN and RUNX2 mRNA expression analysed by qRT-PCR as well. In the study, TNF-α and IL-β mRNA expression were analyzed by qRT-PCR in each group after BMAL1 over-expression.Results:Part 1: Wistar and GK BMSCs displayed different biological characteristics1. GK BMSCs expressed cellular morphology and stem cell markers similar to those of Wistar BMSCs. All cells were strongly positive for CD90, CD 105 and CD 146, moderately positive for Stro-1, but negative for CD 14 and CD31.2. Both the osteogenic and adipogenic differentiation capacities were more profound in Wistar BMSCs than in GK BMSCs.3. T2DM would not cause statistical differences in the features of senescence between GK and Wistar BMSCs.4. GK BMSCs displayed suppressed proliferation capacity and increased IL-1 and TNF-a level.Part 2: Decreased BMAL1 expression suppressed osteogenic differentiation of BMSCs in a T2DM microenvironment1. The expression of BMAL1 in GK BMSCs decreased significantly than those in Wistar BMSCs.2. The protein and mRNA levels of BMAL1 increased significantly after osteogenesis in both GK and Wistar BMSCs, but BMAL1 levels in GK BMSCs were lower than those in the control group before and after osteogenesis.3. Markedly lower number of positively stained cells were found among the GK BMSCs than among the Wistar BMSCs. Similar results in the ALP quantitative analysis.4. ALP, OCN, and RUNX2 mRNA level was obviously lower in GK BMSCs compared with the respective control Wistar BMSCs after 7 days of culture in osteogenic medium.Part 3: Decreased BMAL1 expression suppressed BMSCs osteogenesis through transcriptional regulation of GSK-3β in the Wnt/β-catenin pathway in T2DM.1. The number of GSK-3β fluorescence-positive cells was high in GK BMSCs, suggesting an inhibitory effect of BMAL1 on GSK-3β in BMSCs.2. Although decreased after osteogenesis, GSK-3β protein expression in GK BMSCs was greater than that in the controls.3. Increased GSK-3β expression in GK BMSCs inhibited the Wnt/p-catenin pathway during osteogenic induction.4. After the highly efficient and steady transfection of BMAL1 confirmed, over-expression of BMAL1 mRNA and protein was successfully induced in the Wistar and GK groups.5. GSK-3β fluorescence-positive cells decreased significantly in the lenti-BMAL1 GK BMSCs compared with lenti-EGFP controls, and were similar with those in lenti-EGFP Wistar BMSCs.6. The expression of GSK-3β decreased due to BMAL1 over-expression, while the expression in GK BMSCs was higher than that in the controls, inducing expression patterns opposing those of act-β-catenin and β-catenin.7. BMAL1 and act-β-catenin were not regulated by GSK-3β inhibition in GK BMSCs.8. GSK-3β inhibition led to a mild increase in the levels of act-β-catenin and TCF rather than BMAL1 compared with lenti-BMAL1 only.Part 4: Over-expression of BMAL1 enhanced osteogenic differentiation of GK BMSCs1. OCN fluorescence-positive cells staining and ALP staining showed that osteogenic differentiation capacity of GK BMSCs recovered to a certain degree after BMAL1 transfection.2. OCN, RUNX2 mRNA expression level was significantly enhanced after BMAL1 over-expression in GK BMSCs.3. The IL-1 level decreased slightly, whereas the TNF-α level reduced significantly after BMAL1 over-expression.Conclusion:1. Proliferation and multipotential differentiation of BMSCs were inhibited by T2DM significantly.2. Decreased BMAL1 expression level was one of the important reasons for suppressed osteogenic differentiation of BMSCs in T2DM.3. BMAL1 expression was suppressed by T2DM, which its negative regulation for GSK-3β abated, resulting in upregulation of GSK-3β,inhibited Wnt/β-catenin pathway activity and eventually impaired BMSCs osteogenesis.4. By increasing BMAL1 expression, followed by GSK-3β inhibition and activation of the Wnt/β-catenin pathway, BMSCs-driven bone regeneration was considerably improved.This study highlights the crucial role of stabilized BMAL1 in modulating the osteogenesis capacity of BMSCs and the importance of effective recovery of impaired bone regenerative strategies caused by T2DM. |
PDF Full Text Request