H.G. Kim

Porphyromonas gingivalis-induced miR-132 regulates TNFα expression in THP-1 derived macrophages

BackgroundPeriodontitis is a chronic inflammatory disease induced by periodontopathogens such as Porphyromonas gingivalis (P. gingivalis). MicroRNAs (miRNAs) are small single-stranded noncoding RNAs that regulate gene expression at the level of translation. MiRNAs have been reported to be involved in inflammatory processes. In this study, we examined the effects of P. gingivalis-induced inflammatory miRNAs expression on TNFα production in THP-1 derived macrophages.ResultsPorphyromonas gingivalis induced the expression of miR-132. P. gingivalis-induced miR-132 expression was significantly inhibited by TLR2/4 knock-down and NF-κB inhibitor. Additionally, miR-132 antagomir strongly repressed production of TNFα. The expression of NFE2L2 and NFAT5, the putative target genes of miR-132 involved in regulation of TNFα, decreased in response to P. gingivalis. Furthermore, miR-132 antagomir rescued P. gingivalis-induced suppression of NFE2L2 and NFAT5.ConclusionsThese results suggest that the induction of miR-132 by P. gingivalis can modulate the pathogenesis of periodontitis induced via regulatory expression of TNFα.

Elevated MicroRNA-128 in Periodontitis Mitigates Tumor Necrosis Factor-α Response via p38 Signaling Pathway in Macrophages.

BACKGROUND Periodontitis is a chronic inflammatory disease resulting from an inflammatory response to subgingival plaque bacteria, including Porphyromonas gingivalis. MicroRNA (miRNA) is a current focus in regulating the inflammatory processes. In this study, the inflammatory miRNA expression in gingival tissues of patients with periodontitis and of healthy individuals is compared, and its role in regulating the inflammatory response is examined. METHODS Gingival tissues from patients with periodontitis and healthy individuals were collected for miRNA microarray. THP-1 and CA9-22 cells were challenged with P. gingivalis, and miRNA expression was determined by real-time polymerase chain reaction. Target genes for miRNA were predicted using TargetScanHuman database, and miRNA gene expressions were reviewed using public databases. For the functional study, THP-1 cells were transfected with a miRNA-128 mimic, and target gene expression was compared with THP-1 cells challenged with P. gingivalis. For the tolerance test, THP-1 cells transfected with miRNA-128 mimic were treated with phorbol 12-myristate 13-acetate (PMA) or paraformaldehyde (PFA)-fixed Escherichia coli. Tumor necrosis factor (TNF)-α production was determined by enzyme-linked immunosorbent assay, and mitogen-activated protein kinase (MAPK) protein phosphorylation was determined by Western blot. RESULTS Gingival tissues from patients with periodontitis showed increased expression of miRNA-128, miRNA-34a, and miRNA-381 and decreased expression of miRNA-15b, miRNA-211, miRNA-372, and miRNA-656. THP-1 cells and CA9-22 cells challenged with P. gingivalis showed increased miRNA-128 expression. Among the predicted miRNA-128 target genes, several genes that are involved in MAPK signaling pathway showed similar gene expression pattern between P. gingivalis challenge and miRNA-128 mimic transfection. In THP-1 cells transfected with miRNA-128 mimic, TNF-α production was lower, and phosphorylation of p38 was inhibited when challenged with PMA or PFA-fixed E. coli. CONCLUSION miRNA-128 may be involved in mitigating the inflammatory response induced by P. gingivalis in periodontitis.

Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein

Purpose The aim of this study was to evaluate the ability of Porphyromonas gingivalis (P. gingivalis) to induce oxidation of high-density lipoprotein (HDL) and to determine whether the oxidized HDL induced by P. gingivalis exhibited altered antiatherogenic function or became proatherogenic. Methods P. gingivalis and THP-1 monocytes were cultured, and the extent of HDL oxidation induced by P. gingivalis was evaluated by a thiobarbituric acid-reactive substances (TBARS) assay. To evaluate the altered antiatherogenic and proatherogenic properties of P. gingivalis-treated HDL, lipid oxidation was quantified by the TBARS assay, and tumor necrosis factor alpha (TNF-α) levels and the gelatinolytic activity of matrix metalloproteinase (MMP)-9 were also measured. After incubating macrophages with HDL and P. gingivalis, Oil Red O staining was performed to examine foam cells. Results P. gingivalis induced HDL oxidation. The HDL treated by P. gingivalis did not reduce lipid oxidation and may have enhanced the formation of MMP-9 and TNF-α. P. gingivalis-treated macrophages exhibited more lipid aggregates than untreated macrophages. Conclusions P. gingivalis induced HDL oxidation, impairing the atheroprotective function of HDL and making it proatherogenic by eliciting a proinflammatory response through its interaction with monocytes/macrophages.

Protective effects of remifentanil against H2O2-induced oxidative stress in human osteoblasts

Background Bone injury is common in many clinical situations, such as surgery or trauma. During surgery, excessive reactive oxygen species (ROS) production decreases the quality and quantity of osteoblasts. Remifentanil decreases ROS production, reducing oxidative stress and the inflammatory response. We investigated remifentanils protective effects against H2O2-induced oxidative stress in osteoblasts. Methods To investigate the effect of remifentanil on human fetal osteoblast (hFOB) cells, the cells were incubated with 1 ng/ml of remifentanil for 2 h before exposure to H2O2. For induction of oxidative stress, hFOB cells were then treated with 200 µM H2O2 for 2 h. To evaluate the effect on autophagy, a separate group of cells were incubated with 1 mM 3-methyladenine (3-MA) before treatment with remifentanil and H2O2. Cell viability and apoptotic cell death were determined via MTT assay and Hoechst staining, respectively. Mineralized matrix formation was visualized using alizarin red S staining. Western blot analysis was used to determine the expression levels of bone-related genes. Results Cell viability and mineralized matrix formation increased on remifentanil pretreatment before exposure to H2O2-induced oxidative stress. As determined via western blot analysis, remifentanil pretreatment increased the expression of bone-related genes (Col I, BMP-2, osterix, and TGF-β). However , pretreatment with 3-MA before exposure to remifentanil and H2O2 inhibited remifentanils protective effects on hFOB cells during oxidative stress. Conclusions We showed that remifentanil prevents oxidative damage in hFOB cells via a mechanism that may be highly related to autophagy. Further clinical studies are required to investigate its potential as a therapeutic agent.

Effect of remifentanil on pre-osteoclast cell differentiation in vitro

Background The structure and function of bone tissue is maintained through a constant remodeling process, which is maintained by the balance between osteoblasts and osteoclasts. The failure of bone remodeling can lead to pathological conditions of bone structure and function. Remifentanil is currently used as a narcotic analgesic agent in general anesthesia and sedation. However, the effect of remifentanil on osteoclasts has not been studied. Therefore, we investigated the effect of remifentanil on pre-osteoclast (pre-OCs) differentiation and the mechanism of osteoclast differentiation in the absence of specific stimulus. Methods Pre-OCs were obtained by culturing bone marrow-derived macrophages (BMMs) in osteoclastogenic medium for 2 days and then treated with various concentration of remifentanil. The mRNA expression of NFATc1 and c-fos was examined by using real-time PCR. We also examined the effect of remifentanil on the osteoclast-specific genes TRAP, cathepsin K, calcitonin receptor, and DC-STAMP. Finally, we examined the influence of remifentanil on the migration of pre-OCs by using the Boyden chamber assay. Results Remifentanil increased pre-OC differentiation and osteoclast size, but did not affect the mRNA expression of NFATc1 and c-fos or significantly affect the expression of TRAP, cathepsin K, calcitonin receptor, and DC-STAMP. However, remifentanil increased the migration of pre-OCs. Conclusions This study suggested that remifentanil promotes the differentiation of pre-OCs and induces maturation, such as increasing osteoclast size. In addition, the increase in osteoclast size was mediated by the enhancement of pre-OC migration and cell fusion.

Rutin induces autophagy in cancer cells

Rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside) is a bioactive flavonoid from the plant kingdom. Rutin has been studied as potential anticancer agent due to its wide range of pharmacological properties including antioxidative, anti-inflammatory and anticancer. Autophagy is a conserved intracellular catabolic pathway to maintain cell homeostasis by formation of autophagosome. Processing of autophagy involves various molecules including ULK1 protein kinase complex, Beclin-1–Vps34 lipid kinase complex, ATG5, ATG12, and LC3 (light chain 3). Cargo-carried autophagosomes fuse with lysosomes resulting in autophagolysosome to eliminate vesicles and degrade cargo. However, the actions of rutin on autophagy are not clearly understood. In this study, we analyzed the effect of rutin on autophagy and inflammation in cancer cell lines. Interestingly, rutin induced autophagy in leukemia (THP-1), oral (CA9-22), and lung (A549) cell lines. TNF-α, key modulator of inflammation, was upregulated by inhibition of rutin-induced autophagy. Taken together, these data indicated that rutin induced autophagy and consequently suppressed TNF-α production.