Evaluation of the Changes in microRNA Expression Profiles in Rat Primary Osteoblastic Cells Stimulated with Cotinine.

Objective: Smoking stands as a significant factor contributing to aberrations in bone metabolism, while microRNAs are intricately linked to the regulation of bone metabolism. This study aimed to identify cotinine-responsive microRNAs (miRNAs) and downstream regulatory pathways of their target genes involved in the regulation of osteoblastic cells, providing a foundation for new treatments targeting miRNAs for the bone metabolism imbalance induced by smoking. Methods: Primary osteoblastic cells of Sprague-Dawley rats were cultured through a modified enzymatic digestion method from the cranial bone of neonatal rats and stimulated with a high concentration of cotinine (1000 ng/mL) for 7 days. Then, miRNA gene chip technology was utilized to detect the changes in miRNA expression profiles in cotinine-stimulated osteoblastic cells, and differential expression profiles of cotinine-responsive miRNAs in osteoblastic cells were identified. Real-time polymerase chain reaction was used to detect the levels of significantly differentially expressed miRNAs in rat osteoblastic cells. Gene ontology (GO) and Kyoto encyclopedia of Genes and Genomes (KEGG) pathway analyses were utilized to predict target genes of these miRNAs to reveal the potential biological functions and pathways. Results: We identified 6 statistically differentially expressed miRNAs in the miRNA microarray analysis, of which 3 were upregulated and 3 were downregulated. We chose bone metabolism-related miRNAs as the miRNAs of interest. Quantitative real-time polymerase chain reaction was used to detect the expression levels of the differentially expressed miRNAs, and only miR-210 was significantly upregulated (3.34-fold), consistent with the microarray data. GO and KEGG pathway analyses of predicted miR-210 target genes revealed that miR-210 might participate in numerous signaling pathways, such as the RAS, Rap, PI3K-Akt, and calcium signaling pathways. Conclusion: We found that the strongly upregulated miR-210 may play an important regulatory role in osteoblast cells' biological behavior and bone formation function. The GO analysis results showed that miR-210 mainly involved protein binding, transporter activity, growth factor binding, and ion channel activity. According to the results of the KEGG analysis, miR-210 might negatively regulate the PI3K-Akt signaling pathway, thus affecting the proliferation of osteoblastic cells. These findings suggest that miR-210 may be a potential target for regulating the imbalance of bone metabolism caused by smoking, offering a new direction for clinical treatment of patients with bone metabolism-related diseases.

Effects of Porphyromonas gingivalis and Its Underlying Mechanisms on Alzheimer-Like Tau Hyperphosphorylation in Sprague-Dawley Rats.

Hyperphosphorylated tau is the main component of neurofibrillary tangles and involved in the pathogenesis of Alzheimer's disease (AD). Increasing evidences suggest close associations between Porphyromonas gingivalis (P. gingivalis) and AD, but the relationship between P. gingivalis and tau hyperphosphorylation is still unclear. In this study, we investigated whether peripheral infection with P. gingivalis caused tau hyperphosphorylation by using wild Sprague-Dawley (SD) rats and HT-22 cells. The rats were injected with P. gingivalis suspension or phosphate-buffered saline 3 times per week. After 4 weeks or 12 weeks, the rats were sacrificed for analyzing systemic inflammation, neuroinflammation, and tau hyperphosphorylation. The results showed that the severity of phosphorylated tau at the AD-related sites Thr181 and Thr231 and the number of activated astrocytes were notably greater in the hippocampus of rats with P. gingivalis injection. And the levels of the inflammatory cytokines interleukin (IL)-1ß and IL-6 and tumor necrosis factor-α in serum and hippocampus were also increased in the rats with P. gingivalis injection. In addition, the activity of protein phosphatase 2A (PP2A) was significantly inhibited in the hippocampus of rats with P. gingivalis injection. In vitro, IL-1ß induced tau hyperphosphorylation by inhibiting the activity of PP2A in HT-22 cells and application of the PP2A promoter efficiently attenuated IL-1ß-induced tau hyperphosphorylation in HT-22 cells. These results indicated that P. gingivalis could induce tau hyperphosphorylation via, in part, attenuating the activity of PP2A through triggering systemic inflammation and neuroinflammation in wild-type SD rats.

Effects of nicotine on the metabolism and gene expression profile of Sprague­Dawley rat primary osteoblasts.

Smoking is a risk factor associated with bone and oral diseases, particularly periodontitis. Nicotine, the major toxic component of tobacco, is able to affect the quality and quantity of bone. Osteoblasts serve an important role in bone formation. Thus far, the effects of nicotine on metabolism­associated gene and protein expression in osteoblasts have been controversial and the mechanisms remain unclear. The present study assessed alterations in osteogenic activity by performing a Cell Counting kit­8 assay to investigate proliferation, Annexin V­fluorescein isothiocyanate/propidium iodide staining to investigate apoptosis, alizarin red staining to investigate the formation of mineralized nodules, reverse transcription­quantitative polymerase chain reaction and western blotting to investigate the mRNA and protein levels of collagen I, alkaline phosphatase, bone osteocalcin, bone sialoprotein and osteopontin; and mRNA microarray expression analysis, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis to investigate the whole genome expression profile of Sprague­Dawley (SD) rat primary osteoblasts following treatment with different concentrations of nicotine. The results demonstrated that nicotine inhibited proliferation, promoted early apoptosis and inhibited mineralized nodule formation in a dose­dependent manner by regulating alkaline phosphatase activity and the expression of osteoblast metabolism­associated genes and proteins. According to microarray analysis, several genes associated with bone metabolism and genes in the Hedgehog and Notch signaling pathways were downregulated significantly in nicotine­treated osteoblasts. The results of the present study indicated that nicotine may serve an inhibitory, dose­dependent role in SD rat primary osteoblasts that may be caused by the perturbation of genes and signaling pathways associated with bone formation. These results may provide a theoretical basis for future research regarding bone metabolism and targeted treatment of oral diseases associated with smoking.