HDAC9-Mediated Pyroptosis Promotes Orthodontically Induced Inflammatory Root Resorption

Abstract

Introduction and aims: Orthodontically induced inflammatory root resorption (OIIRR) is a common iatrogenic outcome of orthodontic treatment. Both epigenetic modifications and pyroptosis have demonstrated a certain role in OIIRR. This study aims to investigate whether epigenetic modifications regulate pyroptosis to be involved in OIIRR.

Method: Rat model of OIIRR was established, and the periodontal tissues were utilized for H&E staining, TRAP staining, immunofluorescence, transcriptome sequencing, and RT-qPCR analysis. Human periodontal ligament fibroblasts (hPDLFs) were overexpressed with HDAC9, treated with pyroptosis inhibitor, incubated with osteoclast, and then subjected to CUT&Tag sequencing.

Results: Orthodontic force increased the distance of orthodontic tooth movement and the abundance of osteoclast. Transcriptome sequencing identified that Hdac9 was upregulated in the periodontal tissues of OIIRR rats compared to the control. Immunofluorescence revealed that HDAC9 was present in periodontal ligament fibroblasts, with reduced fluorescence of HDAC9 in OIIRR compared to the control. HDAC9 overexpression in hPDLFs induced pyroptosis and promoted osteoclast differentiation. These effects were reversed by pyroptosis inhibitor. CUT&Tag analysis showed that HDAC9 overexpression resulted in an enrichment of deacetylated genes on mitochondrial dysfunction-associated pathways. CUT&Tag-PCR analysis confirmed reduced H3K9ac enrichment on the mitochondrial dysfunction-associated genes VPS13D, AQP1, PEX2, CDK1, and PLEKHA1 after HDAC9 overexpression, and RT-qPCR analysis revealed a corresponding decrease in their respective expression levels. Accordingly, the ROS level was also increased by HDAC9 overexpression.

Conclusion: HDAC9-mediated histone deacetylation induces mitochondrial dysfunction and pyroptosis in hPDLFs, thereby promoting osteoclast differentiation and OIIRR progression.

Clinical relevance: This study reveals the regulatory mechanism of pyroptosis in OIIRR from the perspective of epigenetic modifications, providing new insights into the pathogenesis of OIIRR.

Keywords: HDAC9; Mitochondrial dysfunction; Orthodontically induced inflammatory root resorption; Periodontal ligament fibroblasts; Pyroptosis.

Fig. 1

Establishment of OIIRR model of rats. A, Photos of dental orthodontic treatment in rats (n = 5). All animals were housed for 7 days post-surgery. B, Statistic data of OTM distance in rats. C, H&E staining revealing the pathologic characteristics of maxilla tissues of rats. D, TRAP staining showing the osteoclasts in maxilla tissues of rats. The black arrow points to an osteoclast. **P < .01.

Fig. 2

RNA sequencing reveals DEGs between the OIIRR and sham groups. A, Volcano plot depicting the DEGs between the OIIRR and sham groups. B, Bubble plot illustrates the enriched GO terms of upregulated DEGs. C, Bubble plot indicates the KEGG enrichment pathways of downregulated DEGs. D, RT-qPCR analysis validated the mRNA expression levels of epigenetic modification-associated genes in the OIIRR and sham groups, including Krt1, Krt10, Igf2bp2, Ldhb, and Hdac9. *P < .05, ***P < .001.

Fig. 3

HDAC9 is present in periodontal ligament fibroblasts. A-C, Representative images of immunofluorescence revealing the localization of HDAC9 in fibroblasts (CD90, A), osteoblasts (OCN, B), and osteoclasts (TRAP, C) within periodontal tissues in the OIIRR and sham groups.

Fig. 4

HDAC9 overexpression in hPDLFs promotes osteoclast differentiation. A, RT-qPCR was used to assess the mRNA levels of HDAC9 in hPDLFs after transfection with plasmids that overexpress HDAC9. B, Western blotting was used to examine the protein levels of HDAC9 in hPDLFs after transfection with plasmids that overexpress HDAC9. C, Schematic diagram of cell co-culture. D, After co-culturing of HDAC9-overexpressed hPDLFs with osteoclast precursor cells, the expression of osteoclast marker genes was examined using RT-qPCR. E TRAP staining revealed that HDAC9 overexpression in hPDLFs promoted osteoclast differentiation. *P < .05, **P < .01, ***P < .001.

Fig. 5

HDAC9 regulates pyroptosis in hPDLFs. A, Representative images of immunofluorescence revealing the co-localization of HDAC9 with the inflammatory NLRP3 within periodontal tissues in the OIIRR and sham groups. B, Flow cytometry detecting apoptosis in HDAC9-overexpressed hPDLFs. C, LDH activity in HDAC9-overexpressed hPDLFs was assessed using an LDH activity assay kit. D CCK-8 assay indicated the proliferation of HDAC9-overexpressed hPDLFs. E Western blotting was used to evaluate the levels of NLRP3 in HDAC9-overexpressed hPDLFs. D, The expression levels of IL-18 and IL-1β in HDAC9-overexpressed hPDLFs were tested using RT-qPCR.

Fig. 6

HDAC9-induced pyroptosis involves osteoclast differentiation. A, Schematic diagram of pyroptosis inhibitor VX765 treatment. B, After treatment with pyroptosis inhibitor VX765, the expression levels of osteoclast marker genes were assessed in HDAC9-overexpressed hPDLFs using RT-qPCR. C, TRAP staining revealed the impact of inhibition of pyroptosis in HDAC9-overexpressed hPDLFs on osteoclast differentiation. *P < .05, **P < .01.

Fig. 7

HDAC9 mediates mitochondrial damage in hPDLFs. A, Pie chart showing the distribution of H3K9ac at annotated genomic regions in NC and HDAC9-overexpressed hPDLFs. B, The top 2 enriched motifs in the NC and HDAC9^OE groups. C, Volcano plot showing differential deacetylated and acetylated H3K9ac-marked genes between the NC and HDAC9^OE groups. D, Bubble chart showing the GO terms of differential genes with decreased H3K9ac modification. E, Genome browser tracks of genes modified by H3K9ac. F, CUT&Tag-qPCR analysis revealing the H3K9ac enrichment levels on VPS13D, AQP1, PEX2, CDK1, and PLEKHA1. G, Gene expression analysis by RT-qPCR. H, Mito-SOX assay depicted the production of ROS in mitochondria in HDAC9-overexpressed hPDLFs. ns = no significance, *P < .05, **P < .01.

Fig. 8

Proposed mechanism by which HDAC9 regulates pyroptosis in hPDLFs through inducing mitochondrial damage to promote OIIRR progression. HDAC9-mediated histone deacetylation induces mitochondrial dysfunction in hPDLFs. This consequence activates NLRP3-driven pyroptosis in hPDLFs, leading to the release of IL-1β and IL-18, osteoclast differentiation, and OIIRR progression.