Computational analysis of curcumin-mediated alleviation of inflammation in periodontitis patients with experimental validation in mice.

AIM: Using network pharmacology and experimental validation to explore the therapeutic efficacy and mechanism of curcumin (Cur) in periodontitis treatment. MATERIALS AND METHODS: Network pharmacology was utilized to predict target gene interactions of Cur-Periodontitis. Molecular docking was used to investigate the binding affinity of Cur for the predicted targets. A mouse model with ligature-induced periodontitis (LIP) was used to verify the therapeutic effect of Cur. Microcomputed tomography (micro-CT) was used to evaluate alveolar bone resorption, while western blotting, haematoxylin-eosin staining and immunohistochemistry were used to analyse the change in immunopathology. SYTOX Green staining was used to assess the in vitro effect of Cur in a mouse bone marrow-isolated neutrophil model exposed to lipopolysaccharide. RESULTS: Network pharmacology identified 114 potential target genes. Enrichment analysis showed that Cur can modulate the production of neutrophil extracellular traps (NETs). Molecular docking experiments suggested that Cur effectively binds to neutrophil elastase (ELANE), peptidylarginine deiminase 4 (PAD4) and cathepsin G, three enzymes involved in NETs. In LIP mice, Cur alleviated alveolar bone resorption and reduced the expression of ELANE and PAD4 in a time-dependent but dose-independent manner. Cur can directly inhibit NET formation in the cell model. CONCLUSIONS: Our research suggested that Cur may alleviate experimental periodontitis by inhibiting NET formation.

Quercetin promotes autophagy to alleviate cigarette smoke-related periodontitis.

BACKGROUND AND OBJECTIVES: Cigarette smoking has been reported as an independent risk factor for periodontitis. Tobacco toxins affect periodontal tissue not only locally but also systemically, leading to the deterioration and recurrence of periodontitis. However, the mechanism of cigarette smoke-related periodontitis (CSRP) is unclear and thus lacks targeted treatment strategies. Quercetin, a plant-derived polyphenolic flavonoid, has been reported to have therapeutic effects on periodontitis due to its documented antioxidant activity. This study aimed to evaluate the effects of quercetin on CSRP and elucidated the underlying mechanism. METHODS: The cigarette smoke-related ligature-induced periodontitis mouse model was established by intraperitoneal injection of cigarette smoke extract (CSE) and silk ligation of bilateral maxillary second molars. Quercetin was adopted by gavage as a therapeutic strategy. Micro-computed tomography was used to evaluate the alveolar bone resorption. Immunohistochemistry detected the oxidative stress and autophagy markers in vivo. Cell viability was determined by Cell Counting Kit-8, and oxidative stress levels were tested by 2,7-dichlorodihydrofluorescein diacetate probe and lipid peroxidation malondialdehyde assay kit. Alkaline phosphatase and alizarin red staining were used to determine osteogenic differentiation. Network pharmacology analysis, molecular docking, and western blot were utilized to elucidate the underlying molecular mechanism. RESULTS: Alveolar bone resorption was exacerbated and oxidative stress products were accumulated during CSE exposure in vivo. Oxidative stress damage induced by CSE caused inhibition of osteogenic differentiation in vitro. Quercetin effectively protected the osteogenic differentiation of human periodontal ligament cells (hPDLCs) and periodontal tissue by upregulating the expression of Beclin-1 thus to promote autophagy and reduce oxidative stress damage. CONCLUSION: Our results established a role of oxidative stress damage and autophagy dysfunction in the mechanism of CSE-induced destruction of periodontal tissue and hPDLCs, and provided a potential application value of quercetin to ameliorate CSRP.

Three-Dimensional Matrix Stiffness Activates the Piezo1-AMPK-Autophagy Axis to Regulate the Cellular Osteogenic Differentiation.

Extracellular matrix (ECM) stiffness is a key stimulus affecting cellular differentiation, and osteoblasts are also in a three-dimensional (3D) stiff environment during the formation of bone tissues. However, it remains unclear how cells perceive matrix mechanical stiffness stimuli and translate them into intracellular signals to affect differentiation. Here, for the first time, we constructed a 3D culture environment by GelMA hydrogels with different amino substitution degrees and found that Piezo1 expression was significantly stimulated by the stiff matrix with high substitution; meanwhile, the expressions of osteogenic markers OSX, RUNX2, and ALP were also observably improved. Moreover, knockdown of Piezo1 in the stiff matrix revealed significant reduction of the abovementioned osteogenic markers. In addition, in this 3D biomimetic ECM, we also observed that Piezo1 can be activated by the static mechanical conditions of the stiff matrix, leading to the increase of the intracellular calcium content and accompanied with a continuous change in cellular energy levels as ATP was consumed during cellular differentiation. More surprisingly, we found that in the 3D stiff matrix, intracellular calcium as a second messenger promoted the activation of the AMP-activated protein kinase (AMPK) and unc-51-like autophagy-activated kinase 1 (ULK1) axis and modestly modulated the level of autophagy, bringing it more similar to differentiated osteoblasts, with increased ATP energy metabolism consumption. Our study innovatively clarifies the regulatory role of the mechanosensitive ion channel Piezo1 in a static mechanical environment on cellular differentiation and verifies the activation of the AMPK-ULK1 axis in the cellular ATP energy metabolism and autophagy level. Collectively, our research develops the understanding of the interaction mechanisms of biomimetic extracellular matrix biomaterials and cells from a novel perspective and provides a theoretical basis for bone regeneration biomaterials design and application.