口腔疾病防治 (Sep 2024)
Analysis of differentially expressed mRNA in gingival tissue of hypertensive rats with or without periodontitis based on next-generation sequencing
Abstract
Objective To employ next-generation sequencing (NGS) to analyze differentially expressed mRNAs in the gingival tissue of hypertensive rats with or without periodontitis to provide a theoretical basis for the prevention and treatment of hypertension with periodontitis. Methods After obtaining approval from the Animal Experiment Ethics Committee, a hypertensive rat model was established by administering high-salt feed containing 8%（w/w） NaCl, and a periodontitis rat model was established by ligating the first molar of the mandibular region using 3-0 sterile silk thread. Rat models of the normal control (N), hypertension (H), and hypertension with periodontitis (PH) groups were established. The blood pressure, heart rate, alveolar bone resorption, and number of osteoclasts in the alveolar bone were measured, before harvesting the gingival tissues from the three groups for NGS to analyze the expression of significantly different genes. Gene ontology (GO) enrichment analysis was performed for all significantly differentially expressed genes between the H and PH groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed. Key genes were screened by protein-protein interaction (PPI) networks, and the key gene expression in each group was verified using immunohistochemistry (IHC). The expression of key genes in the systemic circulation of each group was analyzed using enzyme-linked immunosorbent assay (ELISA). Results At the end of the experiment (11th week), the blood pressure was higher in both the H and PH groups than that in the N group (P<0.001), but there was no statistically significant difference in blood pressure between the H and PH groups. There was no statistical difference in heart rate among the 3 groups. Micro-CT showed that the distance from the cemento-enamel junction to the alveolar bone crest (CEJ-ABC) of the mandibular first molar in the PH group was significantly higher than that in the N and H groups (P<0.016 7). The number of osteoclasts in the alveolar bone of the PH group was significantly higher than that of the N and H groups (P<0.0167). No common differentially expressed genes were found among the 3 groups. There were 235 significantly differentially expressed genes in the gingival tissue between the H and PH groups, and 137 upregulated genes (e.g., P-selectin, keratin 16, and S100 calcium binding protein A) and 98 downregulated genes (e.g., FK506 binding protein 5, mediator complex subunit 22, zinc finger and BTB domain containing 16) in the PH group compared to the H group. GO analysis showed that the major enriched biological processes (BP) were leukocyte migration, the major cellular component (CC) was complex of collagen trimers, and the significant molecular function (MF) was extracellular matrix structural constituent in the H and PH groups. KEGG pathway analysis showed that signaling pathways such as cytokine-cytokine receptor interaction, IL-17 signaling pathway, and TNF-α signaling pathway were significantly enriched in the H and PH groups. PPI analysis identified four key genes affecting periodontitis in hypertensive conditions, including interleukin-1β (IL-1β), matrix metalloproteinase-9 (MMP-9), collagen type I alpha1 (COL1α1), and chemokine ligand 1 (CXCL1). Compared to the N and H groups, the expressions of IL-1β and TNF-α were all upregulated in the gingival tissue and systemic serum in the PH group (P<0.016 7). Conclusion The differentially expressed mRNAs in hypertension with or without periodontitis included IL-1β and MMP-9, while the differentially expressed signaling pathways were IL-17 and TNF-α. These results provide a theoretical reference for further investigation of the molecular regulatory mechanism of hypertension with periodontitis in the future.
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