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. 2023 Apr 28;15(9):2115.
doi: 10.3390/nu15092115.

Resveratrol Alleviates Diabetic Periodontitis-Induced Alveolar Osteocyte Ferroptosis Possibly via Regulation of SLC7A11/GPX4

Yue Li  1 , Zhijun Huang  1 , Shuaifei Pan  1 , Yuhui Feng  1 , Haokun He  1 , Shuguang Cheng  1 , Lijing Wang  1 , Liping Wang  1 , Janak Lal Pathak  1
Affiliations

Resveratrol Alleviates Diabetic Periodontitis-Induced Alveolar Osteocyte Ferroptosis Possibly via Regulation of SLC7A11/GPX4

Yue Li et al. Nutrients. .

Abstract

The mode and mechanism of diabetic periodontitis-induced alveolar-osteocyte death are still unclear. This study aimed to investigate the occurrence of ferroptosis in alveolar osteocytes during diabetic periodontitis and the therapeutic potential of resveratrol to alleviate osteocyte ferroptosis. Diabetic periodontitis was induced in C57/BL6-male mice and treated with or without resveratrol. Periodontitis pathogenicity was analyzed by micro-CT and histology, and alveolar-osteocyte ferroptosis was analyzed by immunohistochemistry. MLOY4 osteocytes were treated with P. gingivalis-derived lipopolysaccharide (LPS)+advanced glycosylated end products (AGEs) mimicking diabetic periodontitis condition in vitro, with or without resveratrol or ferrostatin-1 (ferroptosis inhibitor). Osteocyte ferroptosis and expression of inflammatory mediators were analyzed. Diabetic periodontitis aggravated periodontitis pathogenicity and inhibited the expression of GPX4 and SLC7A11 in alveolar osteocytes and resveratrol alleviated these effects. LPS+AGEs triggered osteocyte ferroptosis in vitro as indicated by the downregulated GPX4 and SLC7A11, upregulated malondialdehyde, disrupted mitochondrial morphology, and overexpressed pro-inflammatory mediators IL-1β, TNF-α, SOST, RANKL, and IL-6, and ferrostatin-1 or resveratrol treatment reversed these effects. LPS+AGEs upregulated pIKBα and pNF-κB p65 expression in osteocytes, and resveratrol or ferrostatin-1 reversed this effect. In conclusion, diabetic periodontitis triggers alveolar osteocyte ferroptosis possibly via disruption of the SLC7A11/GPX4 axis, and resveratrol has therapeutic potential to correct this biological event.

Keywords: NF-κB signaling; diabetic periodontitis; ferroptosis; inflammatory mediators; resveratrol.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Diabetic periodontitis aggravated periodontitis pathogenicity and reduced the expression of GPX4 and SLC7A11 in alveolar osteocytes. (A) Micro-CT 3D reconstruction images of the maxilla of wildtype and diabetic periodontitis mice. (B) CEJ-ABC distance, BV/TV, BS/TV, Tb.Th, Tb.N, and Tb.Sp were analyzed from micro-CT images. (C) H&E stained histological images of periodontal tissues and the quantitative analysis of CEJ-ABC distance from histological images. (D) Representative immunohistochemistry images of GPX4 and SLC7A11 staining and quantification. Data are presented as mean ± SD (n = 5). Significant difference compared to the control group, * p < 0.05, ** p < 0.01, and *** p < 0.001. Con, control; DP, diabetes periodontitis; red line, CEJ; black dotted line, ABC; yellow arrow, immunostained osteocyte.
Figure 2
Figure 2
Diabetic periodontitis induced osteocyte death and osteocyte-mediated inflammation. (A) Osteocyte viability. Protein level expression of TNF-α (B) and IL-1β (C) in AGEs+LPS-induced osteocytes analyzed by ELISA. Relative mRNA expression of GPX4 (D), SLC7A11 (E). (F) Relative expression of pro-inflammatory markers SOST, IL-6, TNF-α, and RANKL, and anti-inflammatory markers IL-10 and IL-4. Data are presented as mean ± SD (n = 3). Significant difference compared to the control group, * p < 0.05 and ** p < 0.01. Con, Control; AGEs, advanced glycation end products; LPS, lipopolysaccharide.
Figure 3
Figure 3
Diabetic periodontitis condition promoted osteocyte ferroptosis and inflammation. (A) Osteocyte viability. (B,C) Relative mRNA expression of ferroptosis markers SLC7A11 and GPX4 in osteocytes. (D) Relative mRNA expression of pro-inflammatory markers and anti-inflammatory markers in osteocytes. Data are presented as mean ± SD (n = 3). Significant difference compared to control and ferrostatin-1 group, * p < 0.05, ** p < 0.01, and *** p < 0.001, and compared to AGEs+LPS group, # p < 0.05, ## p < 0.01, and ### p < 0.001. Con, control; AGEs, advanced glycation end products; LPS, lipopolysaccharide; Fer-1, ferrostatin-1.
Figure 4
Figure 4
Resveratrol reversed the diabetic periodontitis condition-induced osteocyte ferroptosis in vitro. (A) The half maximum inhibitory concentration (IC50) of resveratrol (RSV) in osteocytes. (B) Osteocyte viability. Protein level expression of TNF-α (C) and IL-1β (D) in osteocytes analyzed by ELISA. (E,F) Capillary-based immunoassay analysis and quantification of GPX4 and SLC7A11 in osteocytes. (G,H) Relative mRNA expression of GPX4 and SLC7A11 in osteocytes. (I) Relative mRNA expression of pro-inflammatory markers and anti-inflammatory markers in osteocytes. (J) TEM images of osteocytes showing mitochondrial damage (white arrow: normal mitochondria; yellow arrow: damaged mitochondria). (K) Analysis of MDA level in osteocyte cell lysate. Data are presented as mean ± SD (n = 3). Significant difference compared to the control or the first group, * p < 0.05, ** p < 0.01, and *** p < 0.001, and compared to the AGEs+LPS group, # p < 0.05, ## p < 0.01, and ### p < 0.001. Con, control; AGEs, advanced glycation end products; LPS, lipopolysaccharide; Fer-1,ferrostatin-1; RSV, resveratrol.
Figure 5
Figure 5
Resveratrol treatment mitigated diabetic periodontitis-induced osteocyte ferroptosis in mice. (A) Representative H&E staining images and CEJ-ABC distance analysis. (B) Representative immunohistochemistry images of GPX4 and SLC7A11 staining and quantification of GPX4 and SLC7A11 positive osteocytes in the alveolar bone. Data presented as mean ± SD (n = 5). The significant difference between the groups, * p < 0.05 and ** p < 0.01. Con, control; DP, diabetes periodontitis; RSV, resveratrol; red line, CEJ; black dotted line, ABC; yellow arrow, immunostained osteocyte.
Figure 6
Figure 6
(A) pathway enrichment analysis of differential genes in Con vs. DP. (B) pathway enrichment analysis of differential genes in DP vs. DP+RSV. (C,D) Capillary-based immunoassay analysis and quantification of p-IKBα/IKBα and NF-κB p-p65/NF-κB p65 proteins in MLOY4 cells treated under different conditions. Data are presented as mean ± SD (n = 3). Significant difference: compared to the control group, ** p < 0.01 and *** p < 0.001, and compared to the AGEs+LPS group, ## p < 0.01 and ### p < 0.001. Con, control; AGEs, advanced glycation end products; LPS, lipopolysaccharide; Fer-1, ferrostatin-1; RSV, resveratrol; blue box, cell death related pathway.
Figure 7
Figure 7
Scheme showing the protective effect of resveratrol against diabetic periodontitis-induced alveolar osteocyte ferroptosis.
See this image and copyright information in PMC

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