scholarly journals Baicalein inhibits inflammatory response and promotes osteogenic activity in periodontal ligament cells challenged with lipopolysaccharides

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Manman Ren ◽  
Ya Zhao ◽  
Zhiqi He ◽  
Jian Lin ◽  
Chuchu Xu ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Mapk Signaling ◽  
Periodontal Ligament Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Alizarin Red ◽  
Osteogenic Markers ◽  
Anti Inflammatory

Abstract Background Periodontitis is a chronic infection initiated by oral bacterial and their virulence factors, yet the severity of periodontitis is largely determined by the dysregulated host immuno-inflammatory response. Baicalein is a flavonoid extracted from Scutellaria baicalensis with promising anti-inflammatory properties. This study aims to clarify the anti-inflammatory and osteogenic effects of baicalein in periodontal ligament cells (PDLCs) treated with lipopolysaccharides (LPS). Methods Human PDLCs were incubated with baicalein (0–100 μM) for 2 h prior to LPS challenge for 24 h. MTT analysis was adopted to assess the cytoxicity of baicalein. The mRNA and protein expression of inflammatory and osteogenic markers were measured by real-time polymerase chain reaction (PCR), western blot and enzyme-linked immunosorbent assay (ELISA) as appropriate. Alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of PDLCs. The expression of Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling related proteins was assessed by western blot. Results MTT results showed that baicalein up to 100 μM had no cytotoxicity on PDLCs. Baicalein significantly attenuated the inflammatory factors induced by LPS, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), matrix metalloprotein-1 (MMP-1), MMP-2 and monocyte chemoattractant protein 1 (MCP-1) at both mRNA and protein level. Moreover, MAPK signaling (ERK, JNK and p38) was significantly inhibited by baicalein, which may account for the mitigated inflammatory response. Next, we found that baicalein effectively restored the osteogenic differentiation of LPS-treated PDLCs, as shown by the increased ALP and ARS staining. Accordingly, the protein and gene expression of osteogenic markers, namely runt-related transcription factor 2 (RUNX2), collagen-I, and osterix were markedly upregulated. Importantly, baicalein could function as the Wnt/β-catenin signaling activator, which may lead to the increased osteoblastic differentiation of PDLCs. Conclusions With the limitation of the study, we provide in vitro evidence that baicalein ameliorates inflammatory response and restores osteogenesis in PDLCs challenged with LPS, indicating its potential use as the host response modulator for the management of periodontitis.

scholarly journals Luteolin supports osteogenic differentiation of human periodontal ligament cells

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
He Quan ◽  
Xiaopeng Dai ◽  
Meiyan Liu ◽  
Chuanjun Wu ◽  
Dan Wang
Keyword(s):  
Cyclin D1 ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Cell Activity ◽  
Inhibitory Effect ◽  
Bone Morphogenetic Protein 2 ◽  
Periodontal Ligament Cells ◽  
Alizarin Red ◽  
Real Time Quantitative Pcr ◽  
Related Transcription Factor

Abstract Background Previous research revealed that luteolin could improve the activation of alkaline phosphatase (ALP) and osteocalcin in mouse osteoblasts. We aimed to determine the effect of luteolin on osteogenic differentiation of periodontal ligament cells (PDLCs). Methods Cultured human PDLCs (HPDLCs) were treated by luteolin at 0.01, 0.1, 1, 10, 100 μmol/L, Wnt/β-catenin pathway inhibitor (XAV939, 5 μmol/L) alone or in combination with 1 μmol/L luteolin. Immunohistochemical staining was performed to ensure cells source. Cell activity and the ability of osteogenic differentiation in HPDLCs were determined by MTT, ALP and Alizarin Red S staining. Real-time Quantitative PCR Detecting System (qPCR) and Western blot were performed to measure the expressions of osteogenic differentiation-related genes such as bone morphogenetic protein 2 (BMP2), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), Osterix (OSX) and Wnt/β-catenin pathway proteins members cyclin D1 and β-catenin. Results Luteolin at concentrations of 0.01, 0.1, 1, 10, 100 μmol/L promoted cell viability, ALP activity and increased calcified nodules content in HPDLCs. The expressions of BMP2, OCN, OSX, RUNX2, β-catenin and cyclin D1 were increased by luteolin at concentrations of 0.01, 0.1, 1 μmol/L, noticeably, 1 μmol/L luteolin produced the strongest effects. In addition, XAV939 inhibited the expressions of calcification and osteogenic differentiation-related genes in HPDLCs, and 1 μmol/L luteolin availably decreased the inhibitory effect. Conclusion 1 μmol/L luteolin accelerated osteogenic differentiation of HPDLCs via activating the Wnt/β-catenin pathway, which could be clinically applied to treat periodontal disease.

scholarly journals The Role of Neutrophil Elastase in Aortic Valve Calcification

2022 ◽  
Author(s):  
Yan Liu ◽  
Peng Jiang ◽  
Liqin An ◽  
Mengying Zhu ◽  
Jin Li ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Neutrophil Elastase ◽  
Calcium Deposition ◽  
Enzyme Linked Immunosorbent Assay ◽  
Osteogenic Medium ◽  
Rt Pcr ◽  
Alizarin Red ◽  
Valve Calcification

Abstract Background: Calcific aortic valve disease (CAVD) is the most commonly valvular disease in the western countries initiated by inflammation and abnormal calcium deposition. Currently, there is no clinical drugs for CAVD. Neutrophil elastase(NE) plays a causal role in inflammation and participates actively in cardiovascular diseases. However, the effects of NE on valve calcification remains unclear. So we next explore whether it is involved in valve calcification and the molecular mechanisms involved.Methods: NE expression and activity in calcific aortic valve stenosis (CAVS) patients (n=58) and healthy patients (n=30) were measured by enzyme-linked immunosorbent assay (ELISA), western blot and immunohistochemistry (IHC). Porcine aortic valve interstitial cells (pVICs) were isolated and used in vitro expriments. The effects of NE on pVICs inflammation, apoptosis and calcification were detected by hochest 33258 staining, MTT assay, reverse transcription polymerase chain reaction (RT-PCR) and western blot. The effects of NE knockdown and NE activity inhibitor Alvelestat on pVICs inflammation, apoptosis and calcification under osteogenic medium induction were also detected by RT-PCR, western blot, alkaline phosphatase staining and alizarin red staining. Changes of Intracellular signaling pathways after NE treatment were measured by western blot.Results: The level and activity of NE were evaluated in patients with CAVS and calcified valve tissues. NE promoted inflammation, apoptosis and phenotype transition in pVICs in the presence or absence of osteogenic medium. Under osteogenic medium induction, NE silencing or NE inhibitor Alvelestat both suppressed the osteogenic differentiation of pVICs. Mechanically, NE played its role in promoting osteogenic differentiation of pVICs by activating the NF-κB and AKT signaling pathway.Conclusions: Collectively, NE is highly involved in the pathogenesis of valve calcification. Targeting NE such as Alvelestat may be a potential treatment for CAVD.

scholarly journals miRNA-125b Regulates Osteogenic Differentiation of Periodontal Ligament Cells Through NKIRAS2/NF-κB Pathway

2018 ◽  
Vol 48 (4) ◽  
pp. 1771-1781 ◽  
Author(s):  
Nan Xue ◽  
Lin Qi ◽  
Guorong Zhang ◽  
Yang Zhang
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Osteoblast Differentiation ◽  
Bone Repair ◽  
Alveolar Bone ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Periodontal Ligament Cells ◽  
Glycerol Phosphate

Background/Aims: Osteogenesis of periodontal ligament cells (PDLCS) is essential for alveolar bone repair. Varieties of factors have been found involved in the regulation of PDLCs osteoblast differentiation. Aim of this study was to identify microRNA as a regulator of the os-teogenic differentiation of PDLCs. Methods: The CD markers were analyzed by flow cytometry analysis. Osteoblast differentiation of PDLCs was induced by treatment with dexamethasone, β-glycerol phosphate and α-ascorbic acid. The expression of osteoblastic phenotype was evaluated after the induction by simultaneous monitoring of alkaline phosphatase activity, the expression of genes involved in osteoblastic differentiation by RT-qPCR and Western Blot, and mineralization at the same time. MicroRNA and NKIRAS2 expression was determined by RT-qPCR. Luciferase reporter assays were performed to test whether miR-125b is capable of interacting with the 3’UTR sequence of NKIRAS2. The possible signaling pathway was determined by Western Blot. Results: In this study, we found that the expression of miR-125b was down regulated during the process of ostoblast differentiation of PDLCs. When the expression of miR-125b was up regulated, the osteogenic differentiation of PDLCs was inhibited. During this process, the over-expressed miR-125b led to the activation of NF-κB. NF-κB inhibitor interacting RAS-like 2 (NKIRAS2) is one of target gene of miR-125b, and it is a regulator of NF-κB signaling that plays various roles in osteoblastic differentiation. We demonstrate thatmiR-125b is involved in osteogenic differentiation of PDLCs. Conclusion: Our data support the hypothesis that that miR-125b attenuates PDLCs osteoblastic differentiation by targeting NKIRAS2 and enhancing NF-κB signaling.

scholarly journals Downregulation of lncRNA ANRIL Inhibits Osteogenic Differentiation of Periodontal Ligament Cells via Sponging miR-7 through NF-κB Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Xinwei Liu ◽  
Yue Zhou
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Periodontal Ligament Cells ◽  
Reporter Assay ◽  
Protein Levels ◽  
Alp Activity ◽  
Dual Luciferase Reporter Assay

Background. Long noncoding RNAs (lncRNAs) are dysregulated in periodontitis development and involved in osteogenesis. The current study was aimed at investigating the function of lncRNA ANRIL in periodontal ligament cells (PDLCs) and potential molecular mechanisms. Methods. Firstly, the level of ANRIL was tested by qPCR. Then, PDLCs were treated with a mineralizing solution to induce osteogenic differentiation. ALP activity was measured, and protein levels of BMP2, Osterix, and OCN were measured by Western blot. A target of ANRIL was verified using dual-luciferase reporter assay. miR-7 level was measured by qPCR, and the signals of the NF-κB pathway were tested by Western blot. Results. ANRIL expression was downregulated in PDL tissues. Next, ALP activity and protein levels of BMP2, Osterix, and OCN were increased to show that PDLCs were differentiated. ANRIL level was increased in differential PDLCs, in which knockdown inhibited osteogenic differentiation. Then, miR-7 was found as a target of ANRIL. The miR-7 level was upregulated in PDL tissues and reduced in differential PDLCs. Inhibition of miR-7 suppressed ALP activity and BMP2, Osterix, and OCN expression. Moreover, inhibition of miR-7 reversed the effects on the osteogenic differentiation induced by knockdown of ANRIL. Besides, the levels of p-P65 and p-IκBα were elevated by ANRIL downregulation and were rescued by suppressing miR-7. Conclusions. Knockdown of ANRIL inhibited osteogenic differentiation via sponging miR-7 through the NF-κB pathway, suggesting that ANRIL might be a therapeutic target for periodontitis.

scholarly journals Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Minxia Bian ◽  
Yan Yu ◽  
Yuzhi Li ◽  
Zhou Zhou ◽  
Xiao Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Luciferase Reporter ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Non Coding Rna ◽  
Protein Expressions

BackgroundLong non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is a base length of about 3.8 kb lncRNA, which plays an important role in several biological functions including cell proliferation, migration, and senescence. This study ascertained the role of lncRNA ANRIL in the senescence and osteogenic differentiation of inflamed periodontal ligament stem cells (iPDLSCs).MethodsHealthy periodontal ligament stem cells (hPDLSCs) and iPDLSCs were isolated from healthy/inflamed periodontal ligament tissues, respectively. The proliferation abilities were determined by CCK-8, EdU assay, and flow cytometry (FCM). The methods of Western blot assay (WB), quantitative real-time polymerase chain reaction (qRT-PCR), alizarin red staining, alkaline phosphatase (ALP) staining, ALP activity detection, and immunofluorescence staining were described to determine the biological influences of lncRNA ANRIL on iPDLSCs. Senescence-associated (SA)-β-galactosidase (gal) staining, Western blot analysis, and qRT-PCR were performed to determine cell senescence. Dual-luciferase reporter assays were conducted to confirm the binding of lncRNA ANRIL and miR-7-5-p, as well as miR-7-5p and insulin-like growth factor receptor (IGF-1R).ResultsHPDLSCs and iPDLSCs were isolated and cultured successfully. LncRNA ANRIL and IGF-1R were declined, while miR-7-5p was upregulated in iPDLSCs compared with hPDLSCs. Overexpression of ANRIL enhanced the osteogenic protein expressions of OSX, RUNX2, ALP, and knocked down the aging protein expressions of p16, p21, p53. LncRNA ANRIL could promote the committed differentiation of iPDLSCs by sponging miR-7-5p. Upregulating miR-7-5p inhibited the osteogenic differentiation of iPDLSCs. Further analysis identified IGF-1R as a direct target of miR-7-5p. The direct binding of lncRNA ANRIL and miR-7-5p, miR-7-5p and the 3′-UTR of IGF-1R were verified by dual-luciferase reporter assay. Besides, rescue experiments showed that knockdown of miR-7-5p reversed the inhibitory effect of lncRNA ANRIL deficiency on osteogenesis of iPDLSCs.ConclusionThis study disclosed that lncRNA ANRIL promotes osteogenic differentiation of iPDLSCs by regulating the miR-7-5p/IGF-1R axis.

scholarly journals Downregulation of lncRNA ANRIL Inhibits Osteogenic Differentiation of Periodontal Ligament Cells via Sponging miR-7 through NF-κB Pathway

2021 ◽  
Author(s):  
Xinwei Liu ◽  
Yue Zhou
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Periodontal Ligament Cells ◽  
Protein Levels ◽  
Alp Activity ◽  
Non Coding Rnas ◽  
Dual Luciferase Reporter Assay

Abstract Background Long non-coding RNAs (lncRNAs) are dysregulation in periodontitis development and involved in osteogenesis. The current study aimed was to investigate the function of lncRNA ANRIL in periodontal ligament cells (PDLCs) and potential molecular mechanisms.Methods Firstly, the level of ANRIL was tested by qPCR. Then PDLCs were treated with a mineralizing solution to induce the osteogenic differentiation. ALP activity was measured and protein levels of BMP2, Osterix, and OCN were measured by western blot. A target of ANRIL was verified using dual-luciferase reporter assay. MiR-7 level was measured by qPCR and the signalings of NF-κB pathway were tested by western blot.Results ANRIL expression was downregulated in PDL tissues. Next, ALP activity and protein levels of BMP2, Osterix, and OCN were reduced to show PDLCs were differentiated. ANRIL level was increased in differential PDLCs, and which knockdown inhibited osteogenic differentiation. Then, miR-7 was found as a target of ANRIL. The miR-7 level was upregulated in PDL tissues and reduced in differential PDLCs. Inhibition of miR-7 suppressed ALP activity and BMP2, Osterix, and OCN expression. Moreover, inhibition of miR-7 reversed the effects on the osteogenic differentiation induced by knockdown of ANRIL. Besides, the levels of p-P65 and p-IκBα were elevated by ANRIL downregulation and were rescued by suppressing miR-7.Conclusions Knockdown of ANRIL inhibited osteogenic differentiation via sponging miR-7 through the NF-κB pathway, suggesting that ANRIL might be a therapeutic target for periodontitis.

Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells

2014 ◽  
Vol 88 (2-3) ◽  
pp. 33-41 ◽  
Author(s):  
Tadashi Yamamoto ◽  
Yuki Ugawa ◽  
Keisuke Yamashiro ◽  
Masayuki Shimoe ◽  
Kazuya Tomikawa ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Rho Kinase ◽  
Periodontal Ligament Cells

scholarly journals Azithromycin Promotes the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells after Stimulation with TNF-α

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Tingting Meng ◽  
Ying Zhou ◽  
Jingkun Li ◽  
Meilin Hu ◽  
Xiaomeng Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Alkaline Phosphatase Activity ◽  
Periodontal Ligament ◽  
Caspase 3 ◽  
Caspase 8 ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Induced Apoptosis

Background and Objective. This study investigated the effects and underlying mechanisms of azithromycin (AZM) treatment on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) after their stimulation with TNF-α in vitro. Methods. PDLSCs were isolated from periodontal ligaments from extracted teeth, and MTS assay was used to evaluate whether AZM and TNF-α had toxic effects on PDLSCs viability and proliferation. After stimulating PDLSCs with TNF-α and AZM, we analyzed alkaline phosphatase staining, alkaline phosphatase activity, and alizarin red staining to detect osteogenic differentiation. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to detect the mRNA expression of osteogenic-related genes, including RUNX2, OCN, and BSP. Western blotting was used to measure the NF-κB signaling pathway proteins p65, phosphorylated p65, IκB-α, phosphorylated IκB-α, and β-catenin as well as the apoptosis-related proteins caspase-8 and caspase-3. Annexin V assay was used to detect PDLSCs apoptosis. Results. TNF-α stimulation of PDLSCs decreased alkaline phosphatase and alizarin red staining, alkaline phosphatase activity, and mRNA expression of RUNX2, OCN, and BSP in osteogenic-conditioned medium. AZM enhanced the osteogenic differentiation of PDLSCs that were stimulated with TNF-α. Western blot analysis showed that β-catenin, phosphorated p65, and phosphorylated IκB-α protein expression decreased in PDLSCs treated with AZM. In addition, pretreatment of PDLSCs with AZM (10 μg/ml, 20 μg/ml) prevented TNF-α-induced apoptosis by decreasing caspase-8 and caspase-3 expression. Conclusions. Our results showed that AZM promotes PDLSCs osteogenic differentiation in an inflammatory microenvironment by inhibiting the WNT and NF-κB signaling pathways and by suppressing TNF-α-induced apoptosis. This suggests that AZM has potential as a clinical therapeutic for periodontitis.

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