Caspase-3 and gasdermin E mediate macrophage pyroptosis in periodontitis.

BACKGROUND AND OBJECTIVES: Periodontitis is a chronic inflammatory disease linked to pyroptosis, an inflammatory cell death process. Macrophages are essential for maintaining microenvironment homeostasis, which is crucial for periodontal health. This study explores the mechanisms underlying the relationship between macrophage pyroptosis and periodontitis. METHODS: Expression of the pyroptosis marker gasdermin E (GSDME) and the macrophage surface marker CD68 was examined by immunofluorescence double staining in healthy and periodontitis gingival tissues. In an in vitro pyroptosis model, RAW264.7 cells were irritated using Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) after treatment with either a nuclear factor kappa-B (NF-κB) agonist or inhibitor. The mRNA and protein levels of NF-κB, caspase-3, GSDME, and interleukin-1ß (IL-1ß) were evaluated through qRT-PCR, western blotting, and ELISA techniques. RESULTS: GSDME and CD68 were heavily elevated in inflamed gingival tissues compared to healthy tissues and co-localized in the same region. Furthermore, exposure to P. gingivalis-LPS resulted in a significant upregulation of NF-κB, caspase-3, GSDME, and IL-1ß at both the mRNA and protein levels in RAW264.7 cells. NF-κB agonist or inhibitor pretreatment enhanced or inhibited these effects. CONCLUSIONS: GSDME-mediated macrophage pyroptosis is implicated in periodontitis. Based on in vitro experiments, P. gingivalis-LPS causes pyroptosis in RAW264.7 cells through the caspase-3/GSDME pathway. Furthermore, NF-κB regulates this pyroptotic pathway.

Carboxymethyl chitin or chitosan for osteoinduction effect on the human periodontal ligament stem cells.

Human periodontal ligament stem cells (hPDLSCs) are seeding cells for tissue-engineered treatment of alveolar bone regeneration. To elucidate carboxymethyl chitosan (CMCTS) and carboxymethyl chitin (CMCT) effect on osteogenic differentiation, hPDLSCs were isolated and treated with CMCTS or CMCT. Cell viability and multiplication capacity were measured. The expression of classic osteogenic related molecules, including Alkaline Phosphatase (ALP), Phosphoprotein 1 (OPN), RUNX family transcription factor 2 (Runx2) and Osteocalcin (OCN), were determined. Mineralization levels were detected by Alizarin Red staining. Results showed that both CMCTS and CMCT treatment had the maximal promoting ability for hPDLSCs viability below the concentration of 100 µg/mL, while CMCTS improved hPDLSCs mineralization significantly. CMCTS induced multiple-factor high expression, including ALP, Runx2, OPN and OCN, whereas slightly osteoinductive bioactivity of CMCT was mainly due to ALP. Therefore, CMCTS had a more significant advantage for osteoinductive differentiation of hPDLSCs than CMCT, which may be a promising material for periodontal regeneration.

Isoliquiritigenin alleviates P. gingivalis-LPS/ATP-induced pyroptosis by inhibiting NF-κB/ NLRP3/GSDMD signals in human gingival fibroblasts.

OBJECTIVE: To investigate whether pyroptosis is induced by Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS)/ adenosine triphosphate (ATP) through NF-κB/NLRP3/GSDMD signaling in human gingival fibroblasts (HGFs) and whether isoliquiritigenin (ISL) alleviates pyroptosis by inhibition of NF-κB/NLRP3/GSDMD signals. DESIGN: Periodontitis was optimally simulated using a combination of P. gingivalis-LPS and ATP. The expression levels of genes and proteins of NF-κB, NLRP3 inflammasome, GSDMD, and IL-1ß was characterized by qRT-PCR, western blotting and ELISA. The 2',7'­dichlorodihydrofluorescein diacetate fluorescence probe was used to determine the intracellular ROS level. Hoechst 33342 and PI double staining, cytotoxicity assay, and caspase-1 activity assay were used to confirm the influence of ISL on pyroptosis in P. gingivalis-LPS/ATP-treated HGFs. RESULTS: P. gingivalis-LPS/ATP stimulation significantly promoted expression of NF-κB, the NLRP3 inflammasome, GSDMD, and IL-1ß at gene and protein levels. The proportion of membrane-damaged cells, caspase-1 activity, and the release of lactate dehydrogenase (LDH) were also elevated. However, pretreatment with ISL observably suppressed these effects. CONCLUSIONS: P. gingivalis-LPS/ATP induced pyroptosis in HGFs by activating NF-κB/NLRP3/GSDMD signals and ISL attenuated P. gingivalis-LPS/ATP-induced pyroptosis by inhibiting these signals. This evidence may provide a new direction for the treatment of periodontitis.

Analysis of risk factors for perioperative death in patients undergoing aortic valve replacement using biological valves.

BACKGROUND: Aortic valve disease has become one of the important factors affecting human health. Aortic valve disease is a progressive disease, if not actively treated, the prognosis is poor. Aortic valve replacement (AVR) surgery is an important treatment for aortic valve disease. At present, the AVR surgery using biological valve accounts for about 40% of the total number of AVR surgery. There are still more perioperative deaths in China due to the large number of AVR patients using biological valves. The objective of this study is to explore measures to reduce perioperative mortality of patients after AVR surgery with biological valves. METHODS: The clinical data of patients undergoing AVR surgery with biological valves in Affiliated Hospital of Qingdao University from November 15, 2020 to December 31, 2022 were reviewed and analyzed. Patients were divided into death group and survival group according to their perioperative survival. Risk factors that may influence perioperative mortality were analyzed and compared between the 2 groups. DISCUSSION: This study was a retrospective analysis of risk factors that may influence perioperative mortality in patients undergoing AVR surgery using biological valves. The conclusions of this study can be used to guide clinical decisions-making and relevant guidelines-developing for perioperative treatment of patients undergoing AVR surgery using biological valves.

Role of gingival mesenchymal stem cell exosomes in macrophage polarization under inflammatory conditions.

OBJECTIVE: Exosomes have been shown to play a strong role in intercellular communication. While GMSCs have been extensively studied, less research exists on exosomes derived from GMSCs, especially on how exosomes affect macrophages. This study aimed to investigate the impact of GMSC-derived exosomes on macrophage polarization and phenotype under inflammatory conditions. METHODS: Exosomes were isolated from GMSCs-conditioned media by ultracentrifugation (UC) and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot (WB). In vitro, GMSC-derived exosomes were co-incubated with macrophages for 24 h in the absence or presence of M1 polarizing conditions in the six-well plate. The protein and mRNA expression levels of M1 and M2 macrophage markers were detected and the supernatants were collected for an enzyme-linked immunosorbent assay (ELISA). RESULTS: Exosomes were successfully isolated from GMSCs. Macrophages co-cultured with exosomes showed significantly decreased levels of the M1 markers Tumor Necrosis Factor-α (TNF-α), Interleukin-12 (IL-12), CD86 and Interleukin-1ß (IL-1ß). By contrast, M2 marker Interleukin-10 (IL-10) levels moderately increased. Meanwhile, similar results were acquired in the cell culture supernatants. CONCLUSION: GMSC-derived exosomes may promote M1 macrophage transformation into M2 macrophages, reducing the pro-inflammatory factors produced by M1 macrophages.

Doxycycline inhibits NAcht Leucine-rich repeat Protein 3 inflammasome activation and interleukin-1ß production induced by Porphyromonas gingivalis-lipopolysaccharide and adenosine triphosphate in human gingival fibroblasts.

OBJECTIVE: To investigate the effect of adenosine triphosphate (ATP) on inflammasome activation by Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) stimulation and the anti-inflammatory eff ;ect of doxycycline (Dox) in human gingival fibroblasts (HGFs). DESIGN: The optimal concentration of P. gingivalis-LPS (1.0 µg/mL) for cellular viability was determined by observing cell morphology and measuring the amount of formazan and the expression of pro-caspase-1. The expression of genes and proteins related to the NAcht Leucine-rich repeat Protein 3 (NLRP3) inflammasome, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), caspase-1 and its activated forms, and the inflammatory factor interleukin-1ß (IL-1ß) and its activated forms were measured. RESULTS: The NLRP3 inflammasome (i.e., NLRP3, ASC, caspase-1) was not affected by stimulation with P. gingivalis-LPS or ATP. However, a combination of P. gingivalis-LPS and ATP significantly enhanced inflammasome activation and IL-1ß production at the gene and protein levels as measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Furthermore, doxycycline addition markedly inhibited inflammasome activation and IL-1ß production induced by a combination of P. gingivalis-LPS and ATP. CONCLUSIONS: LPS, ATP, and doxycycline play critical roles in regulating host immune responses. This evidence provides guidance for the application of tetracycline drugs for the clinical treatment of periodontal disease.

Stretching magnitude-dependent inactivation of AKT by ROS led to enhanced p53 mitochondrial translocation and myoblast apoptosis.

Previously, we had shown that high magnitude stretch (HMS), rather than low magnitude stretch (LMS), induced significant apoptosis of skeletal muscle C2C12 myoblasts. However, the molecular mechanism remains obscure. In this study, we found that p53 protein accumulated in the nucleus of LMS-loaded cells, whereas it translocated into mitochondria of HMS-loaded cells. Knocking down endogenous p53 by shRNA abrogated HMS-induced apoptosis. Furthermore, we demonstrated that overaccumulation of reactive oxygen species (ROS) during HMS-inactivated AKT that was activated in LMS-treated cells, which accounted for the distinct p53 subcellular localizations under HMS and LMS. Blocking ROS generation by N-acetylcysteine (NAC) or overexpressing constitutively active AKT vector (CA-AKT) inhibited HMS-incurred p53 mitochondrial translocation and promoted its nuclear targeting. Moreover, both NAC and CA-AKT significantly attenuated HMS-induced C2C12 apoptosis. Finally, we found that Ser389 phosphorylation of p53 was a downstream event of ROS-inactivated AKT pathway, which was critical to p53 mitochondrial trafficking during HMS stimuli. Transfecting p53-shRNA C2C12s with the mutant p53 (S389A) that was unable to target p53 to mitochondria underwent significantly lower apoptosis than transfection with wild-type p53. Altogether, our study uncovered that mitochondrial localization of p53, resulting from p53 Ser389 phosphorylation through ROS-inactivated AKT pathway, prompted C2C12 myoblast apoptosis during HMS stimulation.

Characteristics of chitosan-modified glass ionomer cement and their effects on the adhesion and proliferation of human gingival fibroblasts: an in vitro study.

This study explores the possibility of adhering gingival tissue to a root surface that was restored with chitosan (CS)-modified glass ionomer cement (GIC) in the case of gingival recessions associated with root caries, which provides a theoretical basis for clinical application at the cellular level. The specimens were mixed after integrating 1, 2, and 4 wt% CS into the GIC fluid. The characteristics and cytocompatibility were then examined. As more CS was incorporated into the GIC fluid, the mechanical properties and cytocompatibility of chitosan-modified glass ionomer cement (CS-GIC) first improved but then reduced. Under scanning electron microscopy, microcracks were observed on the surface of all materials, but the fewest microcracks were observed on the surface of 2 wt% CS-GIC. The compressive strength of 2 wt% CS-GIC was significantly higher than that of the other groups at 5 days (P < 0.05) and the addition of chitosan didn't change the basic fracture mode of materials. Additionally, the integration 2 wt% CS into GIC can obviously reduce acidity of the original GIC (P < 0.01) when using extracts with concentrations of 100 and 50%. The Cell Counting Kit-8 assay and adhesion and proliferation of human gingival fibroblasts (HGFs) on the surface of the materials indicated that 2 wt% CS-GIC presented better cytocompatibility and was more suitable for the growth of HGFs. In summary, 2 wt% CS-GIC could be considered as a potential root filling material to allow the adhesion and growth of gingival tissue.

JAK2/STAT3 is associated with the inflammatory process in periapical granuloma.

OBJECTIVE: It has been shown that JAK2/STAT3 is involved in the occurrence of various inflammatory diseases. The purpose of this study was to associate the expression of Janus kinase 2 (JAK2) and its receptor signal transducer and activator of transcription 3 (STAT3) and suppressors of cytokine signaling 3 (SOCS3), to periapical granuloma. METHODS: Samples were collected from 40 patients who were divided into two groups, namely, healthy control (N=20) and periapical granuloma (N=20) groups in accordance with classified standards. The samples were prepared for histological analysis, immunohistochemistry, and double immunofluorescence staining. RESULTS: Only slight inflammatory cell infiltration was observed in the tissues from the healthy control group. Extensive infiltration of inflammatory cells was observed in patients with chronic periapical disease. The periapical granuloma group had higher levels of JAK2, STAT3, p-JAK2, p-STAT3 and SOCS3 (all P<0.05) than the control group. Double immunofluorescence staining results showed the presence of JAK2-positive and STAT3-positive cells in the periapical lesion areas. CONCLUSIONS: This study demonstrated that JAK2, STAT3, and SOCS3 can be observed and may be associated with the inflammatory process in periapical lesions. The results of this study will provide new insights into the pathological mechanisms of human periapical granuloma.

Refinement and Evaluation of Modified Minimally Invasive Harvest Technique for Subepithelial Connective Tissue.

INTRODUCTION: The aim of this paper was to refine the modified minimally invasive single-incision technique (MSIT) into 6 steps that are easy to execute. The advantage of this modification was evaluated and compared with the traditional trap-door incision technique (TDIT). Several other harvesting techniques, suturing techniques, indications, contraindications, and limitations were also summarized. MATERIALS AND METHODS: A total of 40 patients presenting with multiple areas of gingival recession were recruited for this study. All patients were randomly assigned to either the MSIT or TDIT group. Standard periodontal instruments and crossed horizontal suspension sutures were used for both procedures. Harvesting and suturing time, verbal rating scale (VRS), and an early wound-healing index (EHI) were recorded. RESULTS: The total operating time, and particularly the suturing time, was shorter in the MSIT group (267.70 ±â€Š20.24 seconds) than the TDIT group (298.20 ±â€Š21.07 seconds), and the difference was statistically significant (P < 0.05). However, there was no significant difference in pain level between the 2 groups according to the VRS evaluation (P = 0.3658). One week postsurgery, the EHI of the MSIT group (2.00 ±â€Š0.95) was significantly lower than the TDIT group (2.85 ±â€Š1.15) (P < 0.05). DISCUSSION: The 6-step MSIT is more predictable and easy to execute, which decreases the challenge for both dentists and patients. Favorable outcomes occurred because of the streamlined minimally invasive procedure and favorable postoperative recovery.

An experimental hyperlipidemia model with periodontitis in mice.

PURPOSE: In many animal models and clinical trials, the relationship between periodontitis and hyperlipidemia is bidirectional and interlinked. In this study, an experimental hyperlipidemia model with periodontitis in mice is introduced. METHODS: C57BL/6J mice were assigned into group A and B and Apolipoprotein E-deficient (ApoE-/-) mice into group C. After 4 weeks of a high fat diet (HFD), group B and C were ligated on the maxillary second molar tooth, and mice were sacrificed after 8 weeks of the HFD. Levels of lipids, interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α in serum after 0, 4, and 8 weeks were determined. Alveolar bone loss (ABL) was assessed under stereomicroscope. Maxillary bones and atherosclerotic lesion area in the aorta were collected for hematoxylin and eosin (HE) staining. RESULTS: After feeding with a HFD for 4 weeks, group C demonstrated dramatic increases in serum lipid levels. The ABL and levels of IL-6, IL-10, and TNF-α in group C was significantly higher than those of group A and B (P<0.05). Atherosclerotic lesions were observed in group C. CONCLUSIONS: These data demonstrate that an experimental hyperlipidemia model with periodontitis in mice is successfully established by ligation in ApoE-/- mice. This method is economical and time saving, and worthy of more general application.

Glucose-regulated protein 78 contributes to the proliferation and tumorigenesis of human colorectal carcinoma via AKT and ERK pathways.

Glucose-regulated protein 78 (GRP78), a molecular chaperon in the endoplasmic reticulum (ER), is overexpressed in a variety of tumor types and plays a critical role in cancer cell proliferation, migration, invasion and drug resistance. However, the mechanisms underlying the role of GRP78 in tumor carcinogenesis remain largely unknown. In the present study, we found that GRP78 knockdown in colorectal carcinoma (CRC) cells significantly inhibited cell proliferation, colony formation and tumorigenesis in vitro and in vivo. The proliferation inhibition of CRC cells by GRP78 knockdown was associated with an S phase arrest, a reduced G1/S transition, and a downregulation of phosphorylation of AKT and ERK1/2, key cell cycle regulatory proteins. In addition, GRP78 knockdown enhanced the apoptosis induced by 5-fluorouracil (5-FU) in CRC cells. Taken together, our results indicate that GRP78 plays an important role in the development and progression of CRC and may have therapeutic potential for CRC patients.

Systemically transplanted human gingiva-derived mesenchymal stem cells contributing to bone tissue regeneration.

As novel postnatal stem cells, gingiva-derived mesenchymal stem cells (GMSCs) have been considered as an ideal candidate cell resource for tissue engineering and cell-based therapies. GMSCs implanted into sites of injury have been confirmed to promote the injury repair. However, no studies have demonstrated whether systemically transplanted GMSCs can home to the bone injuries and contribute to the new bone formation in vivo. In this study, we transplanted human GMSCs into C57BL/6J mice with defects in mandibular bone via the tail vein to explore the capacity of transplanted GMSCs to promote bone regeneration. Results showed that the transplanted GMSCs were detected in the bone defects and employed in new bone formation. And the newly formed bone area in mice with GMSCs transplantation was significantly higher than that in control mice. Our findings indicate that systemically transplanted GMSCs can not only home to the mandibular defect but also promote bone regeneration.

Modulation of pro-inflammatory mediators in LPS-stimulated human periodontal ligament cells by chitosan and quaternized chitosan.

The aim of this study was to evaluate the effects of chitosan and quaternized chitosan (HTCC) modulate IL-1ß and TNF-α in LPS-stimulated human periodontal ligament cells (HPDLCs). MTT assay revealed that chitosan stimulated the proliferation of HPDLCs. However, HTCC inhibited the proliferation of HPDLCs at concentrations of 1000 and 100 µg/mL more than the control, especially after 5d (P<0.001). ELISA assay exhibited that chitosan inhibited the production of IL-1ß and TNF-α at 24, 48 and 72 h. IL-1ß and TNF-α secreted by HPDLCs with LPS and treated with 1000 µg/mL of HTCC significantly increased compared to both the control and the chitosan group (P<0.001). The bioactive role for bFGF in modulating the responses of HPDLCs cells to LPS via inhibiting IL-1ß and TNF-α production was demonstrated. All results were necessary to enhance our understanding of the biomedical properties of chitosan and HTCC for modulation of pro-inflammatory mediators.

[Cytocompatibility of chitosan-based thermosensitive hydrogel to human periodontal ligament cell].

PURPOSE: The aim of this investigation was to evaluate the cytocompatibility of an in situ chitosan-quaternized chitosan/α, ß-glycerophosphate (CS-HTCC/GP) thermosensitive hydrogel in vitro. METHODS: The primary cells were isolated from human periodontal ligament and cultured. The role of different concentrations of CS-HTCC/GP extract to HPDLCs was evaluated by MTT assay and alkaline phosphatase (ALP) activity. Also, the ultra-architecture of HPDLCs was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) respectively. SPSS13.0 software package was used for statistical analysis. RESULTS: By immunocytochemical method, the cells were stained positively to antibodies against vimentin, and negatively to antibodies against cytokeratin, which indicated that they were external embryo mesenchymal cell without epithelial cell mixure. CS-HTCC/GP thermosensitive hydrogel promoted proliferation of HPDLCs,especially at 3d and 5d, the results was significantly different (P<0.001). ALP activity was significantly greater in group 2 and 3 than in group 4 after 5d (P<0.001). Also, no negative influence to ultrastructure of HPDLCs was found through SEM and TEM. CONCLUSIONS: The results indicate that CS-HTCC/GP thermosensitive hydrogel exhibits excellent cytocompatibility and has potential to be used as an in situ injectable local periodontal drug delivery vehicle and a tissue-engineering scaffold for periodontal disease therapy.