Advanced glycation end products induce inflammaging in periodontal ligament fibroblasts through RAGE/AKT/mTOR/glycolysis pathway

Abstract

Background: Inflammaging plays a pivotal role in the pathogenesis of multiple age-related diseases, including periodontitis. Advanced glycation end products (AGEs) are known to induce inflammaging and exacerbate periodontitis. However, the mechanisms by which AGEs promote inflammaging remain unclear. This study aimed to investigate the mechanisms underlying AGE-induced inflammaging.

Methods and results: Human periodontal ligament fibroblasts (hPDLFs) were extracted and stimulated with lipopolysaccharide (LPS), with prior treatment using AGEs. The expression of pro-inflammatory cytokines was measured to explore the role of AGEs in LPS-induced inflammation. Subsequently, hPDLFs were treated with AGEs and pre-incubated with 2-deoxyglucose (2-DG, a glycolysis inhibitor), Ly294002 (an AKT/mTOR pathway inhibitor), and FPS-ZM1 (a receptor for advanced glycation end product [RAGE] antagonist) to assess the levels of inflammaging markers, glycolysis, AKT/mTOR pathway activation, and RAGE expression, along with the potential relationships among these factors. Our findings demonstrated that AGEs significantly increased the expression of pro-inflammatory cytokines in response to LPS stimulation. Additionally, AGEs alone elevated the levels of inflammaging factors, including cell senescence, senescence-associated secretory phenotype factors, SA-β-Gal expression, glycolysis markers, and AKT/mTOR pathway activation. Furthermore, inhibiting glycolysis reduced AGE-induced inflammaging, while blocking the AKT/mTOR pathway, suppressed both AGE-induced inflammaging and glycolysis. Antagonizing RAGE effectively blocked AGE-induced inflammaging, glycolysis, and AKT/mTOR pathway activation.

Conclusions: Our study indicated that AGE-induced inflammaging through binding to RAGE to activate the AKT/mTOR pathway and eventually enhancing glycolysis level, which may contribute to the increased inflammatory response triggered by LPS. These findings suggest that inflammaging is a critical mechanism through which AGEs exacerbate periodontitis.

Figure 1

Extraction and identification of hPDLFs. (A) The photo of hPDLFs in generation 1 (100x). (B) The photo of hPDLFs in generation 1 (100x). (C–F) Results of the expression of surface antigens CD90, CD105, CD14, and CD31 analyzed by flow cytometry, respectively. hPDLFs: human periodontal ligament fibroblasts.

Figure 2

AGE-induced inflammaging in hPDLFs. (A) Western blotting of senescence characteristic proteins of p16, p21, and p53 in the control (oh) group and AGE group (12 h, 24 h, and 48 h). (B) mRNA expression of SASP factors, including IL-1β, IL-6, TNF-α in the control (oh) group and AGE group (12 h, 24 h, and 48 h). (C) Protein expression of SASP factors, including IL-1β, IL-6, and TNF-α in the control (oh) group and AGE group (12 h, 24 h, and 48 h). (D) SA-β-Gal staining in the control and AGE group. AGE: advanced glycation end product; SASP: senescence-associated secretory phenotype; hPDLFs: human periodontal ligament fibroblasts. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus control group.

Figure 3

Suppression of glycolysis-alleviated AGE-induced inflammaging in hPDLFs. (A) Western blotting of key enzymes of glycolysis, including HKII, PKM2, GLUT1, LDHA in the control, AGEs, and AGEs+2-DG group. (B) Glucose uptake in the control, AGEs, and AGEs+2-DG group. (C) Lactate production in the control, AGEs, and AGEs+2-DG group. (D) Western blotting of senescence characteristic protein of p16, p21, and p53 in the control, AGEs, and AGEs+2-DG group. (E) mRNA expression of SASP factors including IL-1β, IL-6, and TNF-α in the control, AGEs, and AGEs+2-DG group. (F) Protein expression of SASP factors including IL-1β, IL-6, and TNF-α in the control, AGEs, and AGEs+2-DG group. (G) SA-β-Gal staining in the control, AGEs, and AGEs+2-DG group. AGEs: advanced glycation end products; SASP: senescence-associated secretory phenotype; hPDLFs: human periodontal ligament fibroblasts; 2-DG: 2-deoxyglucose. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

Inhibition of AKT/mTOR pathway activation downregulated AGE-induced glycolysis and inflammaging. (A) Western blotting of AKT, p-AKT, mTOR, p-mTOR in the control, AGEs, and AGEs+Ly294002 group. (B) Western blotting of senescence characteristic protein of p16, p21, and p53 in the control, AGEs, and AGEs+Ly294002 group. (C) mRNA expression of SASP factors including IL-1β, IL-6, and TNF-α in the control, AGEs, and AGEs+Ly294002 group. (D) Protein expression of SASP factors including IL-1β, IL-6, and TNF-α in the control, AGEs, and AGEs+Ly294002 group. (E) SA-β-Gal staining in the control, AGEs, and AGEs+Ly294002 group. (F) Relative OD value of SA-β-Gal staining in each group. (G) Western blotting of key enzymes of glycolysis including HKII, PKM2, GLUT1, and LDHA in the control, AGEs, and AGEs+Ly294002 group. (H) Glucose uptake in the control, AGEs, and AGEs+Ly294002 group. (I) Lactate production in the control, AGEs, and AGEs+Ly294002 group. AGEs: advanced glycation end products; SASP: senescence-associated secretory phenotype; OD: optical density. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

Block of RAGE-inhibited AGE-induced activation of AKT/mTOR pathway, glycolysis, and inflammaging. (A) RAGE expression in the control, AGEs, and AGEs+FPS-ZM1 group. (B) Western blotting of senescence characteristic protein of p16, p21, p53 in the control, AGEs, and AGEs+FPS-ZM1 group. (C) mRNA expression of SASP factors including IL-1β, IL-6, and TNF-α in the control, AGEs, and AGEs+FPS-ZM1 group. (D) Protein expression of SASP factors including IL-1β, IL-6, TNF-α in the control, AGEs, and AGEs+FPS-ZM1 group. (E) SA-β-Gal staining in the control, AGEs, and AGEs+FPS-ZM1 group. (F) Relative OD value of SA-β-Gal staining in each group. (G) Western blotting of key enzymes of glycolysis including HKII, PKM2, GLUT1, LDHA in the control, AGEs, and AGEs+FPS-ZM1 group. (H) Glucose uptake in the control, AGEs, and AGEs+FPS-ZM1 group. (I) Lactate production in the control, AGEs, and AGEs+FPS-ZM1 group. (J) Western blotting of AKT, p-AKT, mTOR, p-mTOR in the control, AGEs, and AGEs+FPS-ZM1 group. RAGE: receptor for advanced glycation end products; AGEs: advanced glycation end products; SASP: senescence-associated secretory phenotype; OD: optical density. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6

AGEs significantly promote LPS-induced secretion of inflammatory factors. (A) mRNA expression of inflammatory factors including IL-1β, IL-6, and TNF-α in the AGEs, LPS, AGEs+LPS, AGEs+LPS+FPS-ZM1, AGEs+LPS+2-DG, and AGEs+LPS+Ly294002 group. (B) Secretion of inflammatory factors including IL-1β, IL-6, TNF-α in the AGEs, LPS, AGEs+LPS, AGEs+LPS+FPS-ZM1, AGEs+LPS+2-DG, and AGEs+LPS+Ly294002 group. AGEs: advanced glycation end products; LPS: lipopolysaccharide; 2-DG: 2-deoxyglucose. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.