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Introduction: Osteoblasts depend on a constant supply of prosurvival signals from their microenvironment. When trophic factors become limited by injury or disease, cells undergo apoptosis. This study establishes the regulation and function of Bim, Bak, and Bax in this response., Materials and Methods: MBA-15.4 murine osteoblasts and primary human bone marrow stromal cells (hBMSCs) were subjected to growth factor depletion by serum starvation (1% FCS or serum withdrawal). Protein phosphorylation, activation, or expression was quantified by Western blotting and gene expression by real-time PCR. Regulation of apoptosis in response to serum depletion was determined using siRNA specific for Bim, Bak, or Bax, followed by TUNEL staining. Statistical significance was determined by one-way ANOVA after multiple experimental repeats., Results: Serum depletion strongly induced expression of the proapoptotic protein Bim in both hBMSC and MBA-15.4 osteoblasts. Detailed analysis of the mouse line showed that both mRNA and protein levels rose from 2 h to peak between 16 and 24 h, in conjunction with activation of caspase 3 and rising levels of apoptosis. Both actinomycin D and cycloheximide prevented this increase in Bim, indicating transcriptional regulation. Serum deprivation caused immediate and sustained decreases in phosphorylation of prosurvival kinases, ERK and PKB, preceding upregulation of Bim. Pathway inhibitors, U0126 or LY294002, strongly increased both Bim mRNA and protein, confirming that both kinases regulate Bim. These inhibitors also induced osteoblast apoptosis within 24-72 h. JC-1 tracer detected mitochondrial potential disruption after serum deprivation, indicating involvement of the intrinsic pathway. Moreover, activation-associated conformational changes were detected in the channel-formers, Bax and Bak. Selective knockdown of Bim or Bak by siRNA protected osteoblasts from serum depletion-induced apoptosis by 50%, whereas knockdown of Bax alone or Bak and Bax together reduced apoptosis by 90%., Conclusions: Our data indicate that Bim, Bak, and Bax actively mediate osteoblast apoptosis induced by trophic factor withdrawal. The complex upstream regulation of Bim may provide targets for therapeutic enhancement of osteoblast viability.

Periodontitis is a chronic inflammatory disease that results in the destruction of periodontal soft tissue and the resorption of alveolar bone. Evidence indicates that in diabetic patients, hyperglycemia suppresses periodontal ligament stem cell (PDLSC) functions and leads to difficulties in periodontal repair. The present study aimed to explore the mechanisms by which high‐glucose concentrations aggravate cell viability reduction in human CD146‐positive PDLCs (CD146+PDLCs) under tumor necrosis factor‐alpha (TNF‐alpha) induction. CD146+PDLCs were isolated from periodontal ligament tissues and treated in the absence or presence of 10 ng/ml of TNF‐alpha and 30 mM glucose. Cell viability was detected using Cell Counting Kit‐8 assays and Luminescent Cell Viability Assays. Western blotting and real‐time polymerase chain reaction were performed to determine tumor necrosis factor‐alpha receptor‐1 (TNFR‐1) protein and messenger RNA expression. Bisulfite and MassArray methylation analyses were used to analyze the methylation status of the TNFR‐1 gene. Our results indicated that cell viability was reduced after treatment with a combination of both high‐glucose concentration and TNF‐alpha. Treatment with 30 mM glucose suppressed DNA methyltransferase (DNMT) activities and DNMT1 protein expression, and this was accompanied by the upregulation of TNFR‐1. Additionally, we found that the CpG island located within the TNFR‐1 gene was hypomethylated under 30 mM glucose conditions. S‐adenosylmethionine, an established methyl donor, reversed TNFR‐1 upregulation and restored cell viability against high‐glucose concentration and TNF‐alpha. In conclusion, the present findings suggest that high‐glucose‐induced CpG island hypomethylation within the TNFR‐1 gene plays an essential role in TNFR‐1 upregulation, and this further enhances the cell viability reduction of CD146+PDLCs caused by TNF‐alpha. [ABSTRACT FROM AUTHOR]

Shenqi Maixintong capsule (SMC), a traditional Chinese medicine compound, exerts various therapeutic effects, including nourishing one's vitality, improving blood circulation, regulating vital energy, and promoting coronary circulation. Studies have demonstrated that SMC could effectively alleviate atherosclerosis (AS) and delay its development. However, the effect of SMC on lipid metabolism remains unclear. Herein, we investigated the mechanism of SMC on lipid metabolism of RAW264.7 cells induced by oxidized low-density lipoprotein (ox-LDL). Drug-containing serum was prepared by intragastric administration of SD (Sprague Dawley) rats. RAW264.7 cells were transformed into foam cells with 25 mg/L ox-LDL. SMC-treated rat serum increased the survival rate of ox-LDL-induced RAW264.7 cells. Oil Red staining, total cholesterol, and free cholesterol detection data showed that the intermediated dose of SMC had the best effect on reducing the lipid accumulation, lipid droplets, intracellular cholesterol, and total cholesterol content of RAW264.7 cells. The results of western blotting showed that the expression of ABCA1, ABCG1, LXRα, PPARγ, and peroxisome proliferation-activated receptor alpha (PPARα) was increased by SMC, and proprotein convertase subtilisin/kexin type 9 (PCSK9) was reduced, which promoted reverse cholesterol transport (RCT) of RAW264.7 cells and inhibited foam cell formation. Furthermore, SOD and MDA data indicated that SMC could reduce the senescence of RAW264.7 cells. These findings suggest that SMC might prevent ox-LDL-induced damage of macrophages in AS patients by improving cell viability and slowing down lipid accumulation and senescence. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Periodontal ligament stem cells (PDLSCs) can be used in periodontal regeneration. Tumour necrosis factor-alpha (TNF-α) participates in the regulation of cell proliferation, apoptosis, differentiation, and migration. However, whether TNF-α can affect the biological features of PDLSCs is still unclear. The objective of this study was to illustrate the biological effects (proliferation, apoptosis, osteogenesis and migration) of TNF-α on human CD146 positive periodontal ligament cells (CD146+PLDCs) and CD146 negative periodontal ligament cells (CD146−PDLCs). Methods: CD146±PDLCs were isolated from human PDLCs and analyzed using a fluorescence-activated cell sorter. The biological effects of TNF-α on CD146±PDLCs were evaluated by CCK-8 assay (proliferation), DAPI staining (apoptosis), alizarin red staining and alkaline phosphatase activities assay (osteogenesis), and wounding assay and transwell assay (migration). Results: CD146+PDLCs, which expressed MSC surface markers CD105, CD90, CD73, CD44, and Stro-1, showed higher proliferative and osteogenic potential than CD146−PDLCs. TNF-α at a dose of 2.5ng/ml was found to enhance both proliferation and osteogenesis in CD146+PDLCs. At 5ng/ml, TNF-α promoted proliferation, osteogenesis, and apoptosis in CD146+PDLCs and enhanced osteogenesis in CD146−PDLCs. At 10ng/ml, TNF-α only aggravated apoptosis in CD146+PDLCs. The migratory ability of both CD146+PDLCs and CD146−PDLCs was not altered by TNF-α. Conclusions: CD146+PDLCs were subpopulation of MSC. It showed greater proliferative and osteogenic potential than CD146−PDLCs. At low concentration, TNF-α was beneficial to CD146+PDLCs on proliferation and osteogenesis, and at high concentration it was detrimental. CD146−PDLCs were found to be less sensitive to TNF-α. [Copyright &y& Elsevier]