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.

scholarly journals TRIM16 Positively Regulates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Modulating the Ubiquitination and Degradation of RUNX2

2020 ◽  
Author(s):  
Yi Zhao ◽  
Qiaoli Zhai ◽  
Hong Liu ◽  
Xun Xi ◽  
Shuai Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Bone Regeneration ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Osteogenic Potential ◽  
Common Disease ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Periodontal Tissues

Abstract BackgroundPeriodontal disease is a common disease that compromises the integrity of tooth-supporting tissues. Bone regeneration is the ultimate goal of periodontal therapies, in which osteogenic differentiation of human periodontal ligament stem cells plays a critical role. The tripartite motif (TRIM)16 is downregulated in periodontal tissues of patients with periodontitis and involved in osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs).However, the role of TRIM16 in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is largely unknown.MethodshPDLSCs were isolated and identified by immunophenotype assays using flow cytometry. Overexpression plasmids and specific short-hairpin RNAs (shRNAs) were constructed to manipulate the expression of target molecules. Alkaline phosphatase (ALP) staining, alizarin red staining (ARS) and enzyme‐linked immunosorbent assays (ELISA) were used to evaluate osteogenic potential capacity. Reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis were performed to determine the expression of osteogenic-related markers and activation of relevant signaling pathways. Co-immunoprecipitation assays were performed to confirm the interactions between proteins and the ubiquitination of RUNX2. A LC-MS/MS analysis was performed to explore the different expression proteins in present of TRIM16.ResultsTRIM16 significantly promoted alkaline phosphatase activity and mineralized nodule formation, and positively regulated the osteogenic differentiation of hPDLSCs by enhancing protein expression of RUNX2, COL1A1 and OCN. Mechanistically, TRIM16 serves as a pivotal factor that stabilizes RUNX2 protein levels by decreasing CHIP-mediated K48-linked ubiquitination degradation of the RUNX2 protein. Besides, TRIM16 significantly increased expression of COL1A1 via activation of p38MAPK/RUNX2.ConclusionThis study identified a novel mechanism of TRIM16 in regulating stability of the RUNX2 protein, which may promote the osteogenic differentiation of hPDLSCs. TRIM16 may be a potential target of stem cell based-bone regeneration for periodontal therapies.

scholarly journals Effect of puerarin on osteogenic differentiation of human periodontal ligament stem cells

2019 ◽  
Vol 48 (4) ◽  
pp. 030006051985164
Author(s):  
Jun Li ◽  
Youjian Peng
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Calcium Deposition ◽  
Pcr Analysis ◽  
Cell Surface Markers ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Alp Activity ◽  
Experimental Group

Objective To investigate the effects of the flavonoid, puerarin, on osteogenic differentiation of human periodontal ligament stem cells (PDLSCs). Methods Human PDLSCs were isolated from patients undergoing orthodontic treatment, and the cell surface markers CD146, CD34, CD45, and STRO-1 were identified by immunofluorescence. Cell proliferation was detected by MTT assay; alkaline phosphatase (ALP) activity was measured, and calcium deposition was detected by alizarin red staining. PCR was then used to detect the distributions of COL-I, OPN, Runx2, and OCN, genes related to osteogenic differentiation. Results Staining was positive for cytokines CD146, CD34, CD45, and STRO-1 in the experimental group; staining was also positive for silk protein, but negative for keratin. After 7 days of culture, exposure to puerarin significantly promoted the level of intracellular ALP; increased puerarin concentration led to increased intracellular ALP. Red mineralized nodules appeared upon exposure to puerarin and the number of nodules was concentration-dependent. PCR analysis revealed that COL-I, OPN, Runx2, and OCN expression levels increased as puerarin concentration increased. Conclusions Exposure to puerarin can promote proliferation and ALP activity in human PDLSCs, thus promoting both molecular and osteogenic differentiation; these findings may provide a theoretical basis for the clinical treatment of periodontal disease with puerarin.

scholarly journals MicroRNA Hsa-Let-7b Regulates the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting CTHRC1

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Lin Fu ◽  
Na Li ◽  
Yu Ye ◽  
Xiaying Ye ◽  
Tong Xiao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Proliferation Ability ◽  
Polymerase Chain ◽  
Dual Luciferase Reporter Assay

Let-7 miRNA family has been proved as a key regulator of mesenchymal stem cells’ (MSCs’) biological features. However, whether let-7b could affect the differentiation or proliferation of periodontal ligament stem cells (PDLSCs) is still unknown. Here, we found that the expression of hsa-let-7b was visibly downregulated after mineralization induction of PDLSCs. After transfected with hsa-let-7b mimics or inhibitor reagent, the proliferation ability of PDLSCs was detected by cell counting kit-8 (CCK-8), flow cytometry, and 5-ethynyl-2-deoxyuridine (EdU) assay. On the other hand, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining and activity, alizarin red staining, Western blot, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). We verified that hsa-let-7b did not significantly impact the proliferation ability of PDLSCs, but it could curb the osteogenic differentiation of PDLSCs. Besides, we predicted CTHRC1 acts as the downstream gene of hsa-let-7b to affect this process. Moreover, the combination of CTHRC1 and hsa-let-7b was verified by dual luciferase reporter assay. Our results demonstrated that the osteogenic differentiation of PDLSCs was enhanced after inhibiting hsa-let-7b, while was weakened after cotransfection with Si-CTHRC1. Collectively, hsa-let-7b can repress the osteogenic differentiation of PDLSCs by regulating CTHRC1.

scholarly journals Downregulation of lncRNA DANCR promotes osteogenic differentiation of periodontal ligament stem cells

2019 ◽  
Author(s):  
Zhuo Wang ◽  
Yuanliang Huang ◽  
Luanjun Tan
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Marker Genes ◽  
Appreciable Effect ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Gene Markers ◽  
Related Transcription Factor

Abstract Backgrounds: Long non-coding RNAs (lncRNAs) have been widely known to have an appreciable effect in physiology and pathology. In tooth regeneration, periodontal ligament stem cells (PDLSCs) are regarded as a key effector, whereas, how lncRNA acts in the osteogenic differentiation of PDLSCs have not been completely understood. This study aims to find out the relationship between lncRNA DANCR and the proliferation and osteogenic differentiation of PDLSCs. Method: Microarray was used to observe the different expression of lncRNAs in differentiated and undifferentiated PDLSCs. And then osteogenic-related lncRNA, DNACR was screened out. To explore its effects on proliferation and osteogenic differentiation by constructing an overexpression and inhibition model. qRT-PCR was used to detect the mRNA expression of osteogenesis related genes. MTT assay was performed to assess the effects of DNACR on cell growth curve. To quantify the effects of DNACR on osteogenic differentiation of PDLCs, ALP staining and alizarin red was performed in basic culture medium and osteogenic medium. Data were statistically processed. Results: Compared with the undifferentiated PDLSCs, the alizarin red staining level was higher in differentiated PDLSCs. And the expressions of osteogenic differentiation marker genes Runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and bone morphogenetic protein (BMP-2) were significantly increased in the differentiated PDLSCs. Furthermore, we noticed that comparing with control groups, the expression of LncRNA DANCR decreases markedly in osteogenically induced PDLSCs. DANCR promoted proliferation of PDLSCs, as evidenced by cell viability. Further investigation has proven that the downregulation of DANCR shows in the calcium sediment forming, alkaline phosphatase (ALP) activation and some osteogenic-related gene markers’ upregulation including Runx2, OCN and BMP-2, which finally results in the osteogenic differentiation of PDLSCs following the transfection and induction. Conversely, DANCR upregulation was shown to repress the osteogenic differentiation potential of PDLSCs. Conclusions: The osteogenic differentiation of PDLSCs has proven to related to the down regulation of lncRNA DANCR. And this paper throws light on the effects of DANCR in the process of PDLSCs’ osteogenic differentiation.

scholarly journals CircRNA FAT1 Regulates Osteoblastic Differentiation of Periodontal Ligament Stem Cells via miR-4781-3p/SMAD5 Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Yu Ye ◽  
Yue Ke ◽  
Liu Liu ◽  
Tong Xiao ◽  
Jinhua Yu
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Periodontal Regeneration ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Mirna Sponge ◽  
Proliferation Capacity

The ability of human periodontal ligament stem cells (PDLSCs) to differentiate into osteoblasts is significant in periodontal regeneration tissue engineering. In this study, we explored the role and mechanism of circRNA FAT1 (circFAT1) in the osteogenic differentiation of human PDLSCs. The proliferation capacity of PDLSCs was evaluated by EdU and CCK-8 assay. The abilities of circFAT1 and miR-4781-3p in regulating PDLSC differentiation were analyzed by western blot, reverse transcription-polymerase chain reaction (RT-PCR), alkaline phosphatase (ALP), and Alizarin red staining (ARS). A nucleocytoplasmic separation experiment was utilized for circFAT1 localization. A dual-luciferase reporter assay confirmed the binding relationship between miR-4781-3p and circFAT1. It was showed that circFAT1 does not affect the proliferation of PDLSCs. The osteogenic differentiation of PDLSCs was benefited from circFAT1, which serves as a miRNA sponge for miR-4781-3p targeting SMAD5. Both knockdown of circFAT1 and overexpression of miR-4781-3p suppressed the osteogenic differentiation of PDLSCs. Thus, circFAT1 might be considered as a potential target of PDLSCs mediated periodontal bone regeneration.

scholarly journals Downregulation of lncRNA DANCR promotes osteogenic differentiation of periodontal ligament stem cells

2019 ◽  
Author(s):  
Zhuo Wang ◽  
Yuanliang Huang ◽  
Luanjun Tan
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Marker Genes ◽  
Appreciable Effect ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Gene Markers ◽  
Related Transcription Factor

Abstract Backgrounds: Long non-coding RNAs (lncRNAs) have been widely known to have an appreciable effect in physiology and pathology. In tooth regeneration, periodontal ligament stem cells (PDLSCs) are regarded as a key effector, whereas, how lncRNA acts in the osteogenic differentiation of PDLSCs haven’t been completely understood. This study aims to find out the relationship between lncRNA DANCR and the proliferation and osteogenic differentiation of PDLSCs. Results: Compared with the undifferentiated PDLSCs, the alizarin red staining level was higher in differentiated PDLSCs. And the expressions of osteogenic differentiation marker genes Runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and bone morphogenetic protein (BMP-2) were significantly increased in the differentiated PDLSCs. Furthermore, we noticed that comparing with control groups, the expression of LncRNA DANCR decreases markedly in osteogenically induced PDLSCs. DANCR promoted proliferation of PDLSCs, as evidenced by cell viability. Further investigation has proven that the downregulation of DANCR shows in the calcium sediment forming, alkaline phosphatase (ALP) activation and some osteogenic-related gene markers’ upregulation including Runx2, OCN and BMP-2, which finally results in the osteogenic differentiation of PDLSCs following the transfection and induction. Conversely, DANCR upregulation was shown to repress the osteogenic differentiation potential of PDLSCs. Conclusions: The osteogenic differentiation of PDLSCs has proven to related to the down regulation of lncRNA DANCR. And this paper throws light on the effects of DANCR in the process of PDLSCs’ osteogenic differentiation.

scholarly journals Dose-Dependent Effects of Zoledronic Acid on Human Periodontal Ligament Stem Cells: An In Vitro Pilot Study

2020 ◽  
Vol 29 ◽  
pp. 096368972094849 ◽  
Author(s):  
Anna Di Vito ◽  
E. Chiarella ◽  
F. Baudi ◽  
P. Scardamaglia ◽  
A. Antonelli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Zoledronic Acid ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Periodontal Ligament ◽  
Chronic Exposure ◽  
Periodontal Ligament Stem Cells

Bisphosphonates (BPs) are widely used to treat several metabolic and oncological diseases affecting the skeletal system. Despite BPs’ well-known therapeutic potential, they also displayed important side effects, among which is BPs-related osteonecrosis of the jaw, by targeting osteoclast activities, osteoblast, and osteocyte behavior. The aim of this study is to evaluate the biological effects of zoledronic acid (ZOL) in an in vitro model of periodontal ligament stem cells (PDLSCs) by using an experimental setting that resembles the in vivo conditions. PDLSCs were treated with different concentrations of ZOL ranging from 0.1 to 5 μM. The effects of ZOL exposure were evaluated on cell viability via 3-[4,5-Dimethylthiaoly]-2,5-diphenyltetrazolium bromide (MTT), cell cycle analysis, apoptosis detection, and immunofluorescence. Quantitative real-time polymerase chain reaction (PCR), colorimetric detection of alkaline phosphatase activity, and Alizarin Red S staining were performed to investigate the osteogenic potential of PDLSCs exposed to ZOL. MTT analysis showed that the viability of PDLSCs exposed to ZOL concentration ≥1.5 μM for 3 and 6 days was significantly lower ( P < 0.001) than that of untreated cells. The percentage of apoptotic cells was significantly higher in PDLSCs exposed for 4 days to ZOL at 2 μM ( P < 0.01) and 5 μM ( P < 0.001) when compared to the control. Moreover, ZOL treatment (3 days) accounted for alterations in cell cycle distribution, with an increase in the proportion of cells in G0/G1 phase and a reduction in the proportion of cells in S phase. Chronic exposure (longer than 7 days) of PDLSCs to ZOL accounted for the downregulation of ALP, RUNX2, and COL1 genes at all tested concentrations, which fit well with the reduced alkaline phosphatase activity reported after 7 and 14 days of treatment. Reduced Col1 deposition in the extracellular matrix was reported after 14 days of treatment. Increased calcium deposits were observed in treated cells when compared to the control cultures. In conclusion, chronic exposure to 1 μM ZOL induced significant reduction of osteogenic differentiation, while ZOL concentrations ≥1.5 μM are required to impair PDLSCs viability and induce apoptosis.

scholarly journals Evaluation of the Effects of Cuminum cyminum on Cellular Viability, Osteogenic Differentiation and Mineralization of Human Bone Marrow-Derived Stem Cells

Medicina ◽  
2021 ◽  
Vol 57 (1) ◽  
pp. 38
Author(s):  
Hyunjin Lee ◽  
Youngmin Song ◽  
Yoon-Hee Park ◽  
Md. Salah Uddin ◽  
Jun-Beom Park
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Alizarin Red S ◽  
Control Group ◽  
Cellular Viability ◽  
Alizarin Red

Background and Objectives: Cuminum cyminum L. has long been used in the treatment of various diseases in multiple geographical regions. This study was performed to determine the effects of C. cyminum methanolic extract (CCT) on the cellular viability, alkaline phosphatase activity and mineralization of human mesenchymal stem cells. Materials and Methods: Bone marrow-derived stem cells were cultured in the presence of CCT at concentrations of 0, 0.001, 0.01, 0.1 and 1 μg/mL. Evaluations of cell morphology were performed on days 1, 3, 7 and 14. Cellular viability was evaluated on days 1, 3, 5 and 7. On the 7th and 14th day, alkaline phosphatase activity measurements and Alizarin red S staining were conducted to assess the osteogenic differentiation of stem cells. A real-time polymerase chain reaction was used to determine the expression levels of RUNX2, BSP, OCN, COL2A1 and β-catenin mRNAs. Results: Stem cells in the control group showed fibroblast-like morphology and the addition of CCT at 0.001, 0.01, 0.1 and 1 μg/mL did not generate noticeable changes in morphology compared with the untreated control group. The application of CCT did not produce significant changes in cellular viability or alkaline phosphatase activity compared with controls. Alizarin Red S staining was significantly increased with the application of CCT. Treatment with CCT increased the expressions of RUNX2, BSP and OCN. Conclusions: These results indicate that CCT enhanced the osteogenic differentiation of stem cells derived from bone marrow by regulating the expressions of RUNX2, BSP and OCN. Thus, the use of CCT may be applied to achieve beneficial effects on the mineralization of stem cells.

Melatonin Promotes Osteogenic Differentiation in Lipopolysaccharide-Stimulated Human Periodontal Ligament Stem Cells Through Bone Morphogenic Proteins-2-Related Signaling

2019 ◽  
Vol 9 (5) ◽  
pp. 679-686
Author(s):  
Na Yu ◽  
Jinghui Zhang ◽  
Lijuan Han ◽  
Cunjirigala Na ◽  
Xiaoguang Yuan
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Wnt Signaling ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Biological Activities ◽  
Phase Cell ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Protein Expressions

Periodontitis is one of the most widespread infectious diseases that troubled the majority of adults. Human periodontal ligament stem cells (hPDLSCs) have been reported as a promising therapy for the treatment of periodontitis. Melatonin, an indoleamine hormone from pineal gland, has various biological activities such as anti-inflammation, anti-cancer and so on. However, whether it is functional in periodontitis is still unclear. The aim of this study was to investigate the effect of melatonin in periodontitis and elucidate the molecular mechanism. Lipopolysaccharide (LPS) was used to stimulate hPDLSCs, and viability of hPDLSCs that was treated with melatonin (0, 1, 10, 50 and 100 μmol/L) for 24 h or 48 h was determined by MTT assay. Flow cytometry analysis was carried out to detect the influence of melatonin on cell proliferation. Osteogenic differentiation ability of melatonin was determined by Alkaline phosphatase (ALP) assay kit and Alizarin Red Staining. Lastly, western blot was used for the determination of protein expressions related to proliferation, differentiation and ERK/Wnt signaling activity. The results showed that LPS significantly inhibited cell viability, which was reversed by melatonin, especially at 10 μM for 48 h and at 50 μM for 24 h. Melatonin (10 μM, 48 h) and melatonin (50 μM, 24 h) notably induced G0/G1 phase cell arrest, increased the expression of CDK2, cyclin E and decreased the expression of p27 in LPS-stimulated hPDLSCs. Besides, melatonin significantly promoted cell differentiation through increasing ALP activity, mineralization and protein expressions of Oct4, Sox-2, Runx2 and bone morphogenic protein-2 (BMP-2). Additionally, BMP-2 related ERK and Wnt signaling was activated with the treatment of melatonin in LPS-stimulated hPDLSCs. Collectively, melatonin could improve cell proliferation and osteogenic differentiation in LPS-stimulated hPDLSCs, partly through regulating BMP2-related ERK/Wnt pathway.

Attenuation of Porphyromonas Gingival Lipopolysaccharide-Induced Periodontal Ligament Stem Cells Injury and Inflammation by Blocking Cell Pyroptosis

2021 ◽  
Vol 11 (10) ◽  
pp. 1940-1946
Author(s):  
Shuangfeng Jiang ◽  
Shanjuan Huang ◽  
Jin Liu ◽  
Qi Zhou ◽  
Xiaosheng Liu
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Osteogenic Differentiation ◽  
Inflammatory Cytokines ◽  
Chronic Periodontitis ◽  
Cell Apoptosis ◽  
Periodontal Ligament ◽  
High Mobility ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red

Periodontitis is a chronic inflammation of periodontal tissue, and programmed cell death plays an important role in chronic periodontitis induced by P. gingivalis. Studies have shown that the increased expression of pyroptosis-related NLRP3 inflammasome and the pro-inflammatory cytokines IL-1β and IL-18 in gingivitis, invasive periodontitis, and chronic periodontitis patients. The present study aimed to investigate whether the inhibition of pyroptosis could protect porphyromonas gingival lipopolysaccharide (pg-LPS)-induced human periodontal ligament stem cells (hPDLSCs) injury and inflammation. The hPDLSCs were treated with pg-LPS and ATP in the presence of caspase1/4 inhibitor VX765. The cell proliferation and survival were assessed by CCK-8, the osteogenic differentiation capacity was evaluated by Alkaline Phosphatase (ALP) assay and alizarin red staining. Then, cell apoptosis, cleavage of gasdermin D (GSDMD) and generation of inflammatory cytokines were estimated. Lastly, western blotting was used to detect the expression of potential target proteins. Results showed that the treatment of pg-LPS plus ATP significantly inhibited the proliferation, survival and osteogenic differentiation of hPDLSCs, while inducing cell apoptosis, pyroptosis and inflammation. However, the presence of VX765 partially recovered the cell proliferation, survival and osteogenic differentiation. At the same time, VX765 inhibited cell apoptosis, cleavage of GSDMD and generation of inflammatory cytokines. Besides, the expression of related proteins including Bax, Bcl-2, cleaved (c)-caspase3, c-caspase4, c-caspase1, Toll Like Receptor 4, High Mobility Group Box 1 (HMGB1) and NLRP3 was all rescued by VX765. In conclusion, our results revealed that the blocking of cell pyroptosis could protect hPDLSCs from pg-LPS-induced injury. Therefore, the application of pyroptosis inhibitor may be a valuable therapeutic approach for treating periodontitis.

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