Identification of potential diagnostic biomarkers associated with periodontitis by comprehensive bioinformatics analysis.

Periodontitis is a chronic inflammatory disease that affects the tissues surrounding the teeth, including the gums and the bones supporting the teeth. Early detection and intervention are crucial for effective management of periodontitis. Our study aims to identify a diagnostic biomarker for periodontitis and explore the pathways associated with the occurrence and development of periodontitis. The expression of gingival tissue from periodontitis and healthy control were downloaded from the Gene Expression Omnibus. The weighted gene co-expression network analysis (WGCNA) were used to analyze module genes associated with periodontitis and DESeq2 were performed to identify differently expressed genes (DEGs) between periodontitis and healthy control. Then the candidate genes were obtained by intersecting the genes from interest modules and DEGs. Functional enrichment analysis was performed using gene ontology and kyoto encyclopedia of gene and genomes, followed by the protein-protein interaction (PPI) network analysis. The hub genes were identified by the cytoCNA plugin in Cytoscape. Finally, immunohistochemical staining of the hub genes was performed to validate the findings. WGCNA analysis found that the expression of the MEblack module was significantly higher in individuals with periodontitis compared to those in the healthy control group. A total of 888 DEGs, including 750 upregulated and 138 downregulated genes, were identified. Finally, 427 candidate genes were identified potentially associated with periodontitis after intersecting the DEGs and the black module genes. Several critical signaling pathways were identified associated with periodontitis by functional enrichment analysis, including cytokine-cytokine receptor interaction, neutrophil extracellular trap formation, Staphylococcus aureus infection, and Interleukin-17 signaling pathway. The PPI network analysis revealed that C-X-C motif chemokine ligand 5 (CXCL5) and C-X-C motif chemokine ligand 6 (CXCL6) could play an important role in the process of periodontitis. The gene expression level of CXCL5 and CXCL6 detected using immunohistochemical verified the findings. In conclusion, we found that CXCL5 and CXCL6 are closely associated with the occurrence of periodontitis. Our present pilot study suggests that CXCL5 and CXCL6 have the potential to be used as a diagnostic biomarker of periodontitis.

Effects of recombinant human growth hormone on proliferation and differentiation of cementoblast and ERK1 / 2, JNK / SAPK and p38MAPK signaling pathway.

To investigate the effects of recombinant human growth hormone on the proliferation and differentiation of cementoblast and the signal pathways of ERK1 / 2, JNK / SAPK and p38MAPK, osteoblasts (OCCM-30) were cultured in vitro. The OCCM-30 was treated with different concentrations of recombinant human growth hormone (rhGH) (0, 10, 50 ng/mL) for 1 d and 2 d, respectively. MTT assay was used to test the proliferation of OCCM-30 cells by rhGH. The effect of BSP, OPN, OCN and ALP genes was detected by RT-PCR after one day. The activity of alkaline phosphatase (ALP) was detected after treatment with OCCM-30 for five days at different concentrations of rhGH. After treatment with OCCM-30 at 100 ng/mL rhGH for 0 min, 5 min, 10 min, 15 min, 30 min and 60 min, the phosphorylation levels of ERK1/2, JNK/SAPK and p38MAPK were detected by Western blot. Results showed that rhGH could promote the proliferation of OCCM-30 cells, and the proliferation of OCCM-30 cells increases with the increase of rhGH concentration. After one day of culture, the levels of the BSP and ALP genes increased with the increase of rhGH concentration (P<0.05); the OPN gene level in the 10 ng/mL group was significantly higher than that in the blank group, and the 50 ng/mL group was significantly lower than the blank group. (P<0.05); OCN gene levels in the 10 ng/mL group and 50 ng/mL group were not significantly different from those in the blank group (P>0.05). Compared with the blank group, the ERK 1/2 phosphorylation level increased at 5 min in the 100 ng/mL group, reached the maximum at 10 min, decreased significantly at 15 min, decreased to the original level at 30 min, and had no significant change in ERK 1/2 total protein level; 100 ng/mL rhGH had no significant effect on SAPK/JNK, p38MAPK phosphorylation and total protein levels in OCCM-30 cells (P>0.05). It was concluded that 10ng/mL and 50ng/mL rhGH could promote the proliferation of OCCM-30 cells and promote the expression of the BSP gene and ALP gene. Low-dose rhGH is beneficial to OPN gene expression, and high-dose rhGH inhibits OPN gene expression. 100 ng/mL rhGH promoted the ERK 1/2 pathway and had no effect on the SAPK/JNK and p38 MAPK pathways.

Inhibition of ER stress-activated JNK pathway attenuates TNF-α-induced inflammatory response in bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMMSCs) are characterized by their pluripotent differentiation and self-renewal capability and have been widely applied in regenerative medicine, gene therapy, and tissue repair. However, inflammatory response after BMMSCs transplantation was found to impair the osteogenic differentiation of BMMSCs. Thus, understanding the mechanisms underlying inflammation response will benefit the clinical use of BMMSCs. In this study, using a cell model of TNF-α-induced inflammatory response, we found that TNF-α treatment greatly elevated intracellular oxidative stress and induced endoplasmic reticulum (ER) stress by elevating the expression levels of ER sensors, such as PERK, ATF6 and IRE1A. Oxidative stress and ER stress formed a feedback loop to mediate TNF-α-induced inflammation response in BMMSCs. Moreover, c-Jun N-terminal kinase (JNK) signal pathway that coupled to the ER stress was significantly activated by increasing its phosphorylation upon TNF-α treatment. Importantly, pharmacological inhibition of ER stress effectively eliminated the phosphorylation of JNK and attenuated the TNF-α-induced inflammation response. In conclusion, our results indicated that TNF-α induced oxidative and ER stress, thereby leading to JNK activation, and generating inflammation response in BMMSCs. This pathway underlying TNF-α-induced inflammation response may provide new strategies to improve BMMSCs osteogenesis and other inflammation-associated bone diseases.

Intracellular Ca2+ signaling mediates IGF-1-induced osteogenic differentiation in bone marrow mesenchymal stem cells.

Insulin-like growth factor 1 (IGF-1), a multifunctional peptide that involves in cell proliferation and differentiation, can induce strong osteogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs). However, it remains unknown whether intracellular Ca2+ signal contributes to the IGF-1-induced osteogenic differentiation of BMMSCs. In this study, we attempted to investigate the effect of IGF-1 on the gene expression of intracellular Ca2+-handling proteins and figure out whether the intracellular Ca2+ signal affects IGF-1-induced osteogenic differentiation. We found that IGF-1 treatment significantly increased cell proliferation and induced cell morphological changes with an increase of cell surface area. Quantitative PCR and Western blot analysis showed that osteoblast marker proteins, including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) were significantly upregulated by IGF-1 treatment, indicating IGF-1 induced osteogenic differentiation in BMMSCs. Interestingly, the expression levels of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 3 and inositol-1,4,5-triphosphate receptor (IP3R) 2 were dramatically elevated during the IGF-1-induced osteogenic differentiation. Consistently, IGF-1-treated cells exhibited greater Ca2+ response to ATP. Importantly, blocking SERCA by thapsigargin markedly impaired IGF-1-induced osteogenic differentiation, indicating that intracellular Ca2+ mediated IGF-1-induced osteogenic differentiation in BMMSCs, probably via Akt signal pathway, which may provide new insight for the treatment of osteoporosis.

MiR-128-3p mediates TNF-α-induced inflammatory responses by regulating Sirt1 expression in bone marrow mesenchymal stem cells.

Tumor Necrosis Factor α (TNF-α), a multifunctional pro-inflammatory cytokine, is produced by macrophages/monocytes during acute inflammation, and plays a critical role in orchestrating the cytokine cascade in various inflammatory diseases. Previous studies demonstrated that TNF-α induces inflammatory responses in bone marrow mesenchymal stem cells (BMSCs) transplantation, leading to unsatisfactory effects and limit the clinical use of BMSCs. MicroRNAs are reported to involve in inflammation by regulating the expression of their targets in inflammatory response pathway. However, whether microRNAs mediate TNF-α-induced inflammatory responses in BMSCs remains elusive. Here, we found that TNF-α treatment induced an inflammatory response by increasing the levels of key inflammatory mediators, including IL-6, IL-1ß, matrix metalloproteinase 9 (MMP9) and monocyte chemotactic protein-1 (MCP-1) in BMSCs. Moreover, real-time PCR result showed dramatically up-regulation of miR-128-3p after exposure to TNF-α. Interestingly, miR-128-3p over-expression exacerbated the TNF-α-induced inflammatory response, while suppression of miR-128-3p effectively eliminated the inflammatory response in BMSCs. Bioinformatic analysis identified sirtuin 1 is a direct target of miR-128-3p. Up-regulation of sirtuin 1 induced by resveratrol also diminished the TNF-α-induced inflammatory response in BMSCs. Altogether, our results indicated that miR-128-3p targets sirtuin 1 to mediate the TNF-α-induced inflammatory response in BMSCs, which may provide new strategies to protect against inflammatory-dependent impairments in BMSCs.

Reactive oxygen species mediate TNF-α-induced inflammatory response in bone marrow mesenchymal cells.

OBJECTIVES: It is generally believed that the inflammatory response in bone marrow mesenchymal stem cells (BMSCs) transplantation leads to poor survival and unsatisfactory effects, and is mainly mediated by cytokines, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α). In this study, we explored the mechanisms underlying the TNF-α-induced inflammatory response in BMSCs. MATERIALS AND METHODS: We treated BMSCs with TNF-α (1 and 10 ng/ml) for 5 days. The expression levels of key inflammatory mediators were evaluated by Real-time PCR. Intracellular ROS level was measured by using a 2, 7-dichlorofluorescein diacetate (DCF-DA). RESULTS: We found that TNF-α treatment dramatically increased the expression levels of some key inflammatory mediators, including IL-6, IL-1ß, IFN-γ and transforming growth factor ß (TGF-ß). Moreover, TNF-α induced intracellular oxidative stress by elevating intracellular reactive oxygen species (ROS) level, which is due to the increase of lipid peroxidation, the reduction of antioxidant Glutathione (GSH) levels and the inhibition of many antioxidant enzyme activities in BMSCs. Interestingly, 5 µM curcumin, a ROS scavenger, dramatically lowered the TNF-α-induced inflammatory response in BMSCs. In addition, TNF-α induced the activation of extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), p38 and their down-stream transcription factors nuclear factor kappa B (NF-κB) pathway. CONCLUSION: ROS mediated the TNF-α-induced inflammatory response via MAPK and NF-κB pathway, and may provide a novel strategy to prevent the inflammatory-dependent impairments in BMSCs.

MicroRNA-320 involves in the cardioprotective effect of insulin against myocardial ischemia by targeting survivin.

It is generally accepted that insulin exerts an antiapoptotic effect against ischemia/reperfusion through the activation of PI3K/Akt/mTOR pathway. MicroRNAs involve in multiple cardiac pathophysiological processes, including ischemia/reperfusion-induced cardiac injury. However, the regulation of microRNAs in the cardioprotective effect of insulin is rarely discussed. In this study, using a cell model of ischemia through culturing H9C2 cardiac myocytes in serum-free medium with hypoxia, we demonstrated that pretreatment with insulin significantly inhibited cell apoptosis and downregulated microRNA-320 (miR-320) expression. Interestingly, miR-320 mimic impaired the cardioprotective effect of insulin against myocardial ischemia injury by targeting survivin, which is a member of the family of inhibitor of apoptosis proteins. Suppression miR-320 expression by miR-320 inhibitor in H9C2 cells with myocardial ischemia mimics the cardioprotective effect of insulin by maintaining survivin expression. Taken together, miR-320-mediated survivin expression involves in cardioprotective effect of insulin against myocardial ischemia injury. SIGNIFICANCE OF THIS STUDY: Myocardial ischemia/reperfusion (I/R) injury remains an important clinical problem with extremely deficient clinical therapies. Insulin exerts an antiapoptotic effect against I/R through the activation of PI3K/Akt/mTOR pathway. Here, we provided evidences to show that microRNA-320 involves in the cardioprotective effect of insulin by targeting survivin, which is an inhibitor of apoptosis protein and functions as a key regulator in cell apoptosis and involves in the tumour genesis and progression. Our findings may provide a new potential therapeutic strategy for I/R injury and ischemic heart disease.

Acute arsenic exposure induces inflammatory responses and CD4+ T cell subpopulations differentiation in spleen and thymus with the involvement of MAPK, NF-kB, and Nrf2.

Increasing lines of evidence indicate that arsenic may be associated with immune related problems, but its detailed effects on immune organs are poorly understood. The objective of this study was to explore inflammatory responses and T cell differentiation of arsenic exposure in spleen and thymus. Female C57BL/6 mice were used as a model to systemically administration 2.5, 5 and 10mg/kg NaAsO2 intra-gastrically for 24h. We found that arsenic significantly decreased the spleen and thymus weights and indices, and flow cytometry revealed that arsenic decreased the relative frequency of CD4+ T cell subpopulation and the ratios of CD4/CD8 in spleen. In contrast, serum concentration of tumor necrosis factor α (TNF-α), IL-1ß and IL-6 as well as the mRNA of key inflammatory mediators in spleen and thymus, including transforming growth factor ß (Tgf-ß), Tnf-α, Il-12, Il-1ß and Il-6 were significantly increased in arsenic-treated mice compared to the control as assayed by ELISA and real time PCR, respectively. In addition, arsenic increased the expression of T helper cell 1 (Th1), Th2 and regulatory T cell (Treg) -associated transcription factors and cytokines as well as decreased Th17-associated transcription factors and cytokines. Moreover, arsenic enhanced oxidative stress and induced the activation of extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 and their downstream transcription factors nuclear factor kappa B (NF-kB) and nuclear factor E2-related factor 2 (Nrf2), which comprise important mechanistic pathways involved in immune-inflammatory manifestations. Together, these results provide a novel strategy to block the arsenic-dependent impairments in immune responses.

The mediating role of general self-efficacy in the association between perceived social support and oral health-related quality of life after initial periodontal therapy.

BACKGROUND: Although initial periodontal therapy can ease some physical and psychological discomforts from periodontitis and improve patients' oral health-related quality of life (OHRQoL), it is also vital to find positive resources from psychological and social aspects to promote the overall OHRQoL. This study aims to explore the associations of perceived social support (PSS) and general self-efficacy with OHRQoL and the mediating role of general self-efficacy in PSS-OHRQoL association after initial periodontal therapy. METHODS: A prospective case series study was conducted among consecutive outpatients with chronic periodontitis during the period of July 2014-April 2015. A total of 145 eligible patients responded to OHRQoL questionnaire and periodontal examination at baseline. About 4 to 5 weeks after initial periodontal therapy, 120 patients completed the second OHRQoL measurement and periodontal examination, along with PSS and general self-efficacy measurement. The Wilcoxon matched-pairs signed-rank test was used to determine the difference between baseline and post-treatment OHRQoL scores and periodontal parameters. Hierarchical linear regression analysis was used to explore the associations of PSS and general self-efficacy with post-treatment OHRQoL after adjusting for some demographic and periodontal variables. Asymptotic and resampling strategies were performed to explore the mediating role of general self-efficacy. RESULTS: Initial periodontal therapy resulted in a significant improvement in the mean total score and all domains of OHRQoL and all periodontal parameters measured. In hierarchical linear regression analysis, clinical attachment loss (CAL) was significantly and positively associated with post-treatment OHRQoL score (ß = 0.265, p < 0.01), while PSS (ß = -0.303, p < 0.01) and general self-efficacy (ß = -0.221, p < 0.01) were significantly and negatively associated with post-treatment OHRQL score, respectively. A significant mediating role of general self-efficacy (a*b = -0.139, BCa 95 % CI: -0.298, -0.011) in the association between PSS and post-treatment OHRQoL was revealed, and the proportion of the mediating role of general self-efficacy was 31.4 %. CONCLUSIONS: Initial periodontal therapy could significantly improve all aspects of OHRQoL. PSS and general self-efficacy could be the positive resources for improving OHRQoL after initial periodontal therapy among patients with periodontitis. General self-efficacy partly mediated the association between PSS and post-treatment OHRQoL.