Identification of Genes Differentially Expressed in Simvastatin-Induced Alveolar Bone Formation

Abstract

Local delivery of simvastatin (SIM) has exhibited potential in preventing inflammation and limiting bone loss associated with experimental periodontitis. The primary aim of this study was to analyze transcriptome changes that may contribute to SIM's reduction of periodontal inflammation and bone loss. We evaluate the global genetic profile and signaling mechanisms induced by SIM on experimental periodontitis bone loss and inflammation. Twenty mature female Sprague Dawley rats were subjected to ligature-induced experimental periodontitis around maxillary second molars (M2) either unilaterally (one side untreated, n = 10) or bilaterally (n = 10). After the ligature removal at day 7, sites were injected with either carrier, pyrophosphate (PPi ×3), 1.5-mg SIM-dose equivalent SIM-pyrophosphate prodrug, or no injection. Three days after ligature removal, animals were euthanized; the M1-M2 interproximal was evaluated with μCT, histology, and protein expression. M2 palatal gingiva was harvested for RNA sequencing. Although ligature alone caused upregulation of proinflammatory and bone catabolic genes and proteins, seen in human periodontitis, SIM-PPi upregulated anti-inflammatory (IL-10, IL-1 receptor-like 1) and bone anabolic (insulin-like growth factor, osteocrin, fibroblast growth factor, and Wnt/ β-catenin) genes. The PPi carrier alone did not have these effects. Genetic profile and signaling mechanism data may help identify enhanced pharmacotherapeutic approaches to limit or regenerate periodontitis bone loss. © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Keywords: ANABOLICS; DENTAL BIOLOGY; GH/IGF‐1; MOLECULAR PATHWAYS–REMODELING; WNT/Β‐CATENIN/LRPS.

Figure 1

μCT and histology of experimental periodontitis. (A) Normal interproximal bone height between maxillary first and second molars (white bar, control). (B) Experimental periodontitis caused bone loss (ExP; red bar), whereas (C) ExP followed by local simvastatin injections has been shown to cause bone preservation/regeneration after 28 days9 (shorter red bar, ExP + simvastatin [SIM]). (D) Furthermore, histology of ExP showed increased lymphocytic infiltrate between the periodontal pocket (P) and alveolar bone (B), and apical migration of the epithelial attachment along the root surface (arrow). White box indicates the area of interest for the immunofluorescence evaluations in the current study. (E) Untreated control histology showed epithelial attachment at the cementoenamel junction (arrow where enamel has been decalcified away) and minimal inflammatory infiltrate.

Figure 2

Differentially expressed genes by RNA‐seq in ExP and ExP + SIM‐PPi models. (A) Statistically significantly differentially expressed (adjusted p value <0.05) genes between each pair of sample groups. (B) Unsupervised hierarchical clustering analysis of the three sample groups (control, ExP, ExP + SIM‐PPi) using all the transcripts (B). ExP = experimental periodontitis; SIM‐PPI = simvastatin‐pyrophosphate.

Figure 3

Overlap of genes that are dysregulated in ExP and ExP + SIM‐PPi models compared to the unmanipulated controls by RNA‐seq. Hierarchical clustering of and functional groups and pathways over‐represented by the 596 + 702 genes uniquely dysregulated in the ExP + SIM‐PPi model. ExP = experimental periodontitis; SIM‐PPI = simvastatin‐pyrophosphate.

Figure 4

Immunostaining analysis of protein expression of IGF‐1, MMP‐9, and TNF‐α in different sample groups. (A) IGF‐1 showed significantly elevated expression in response to simvastatin treatment(s) compared to experimental periodontitis (ExP and ExP + PPi) and unmanipulated controls. Unmanipulated controls have limited expression of MMP‐9 and TNF‐α, whereas in ExP groups, MMP‐9 and TNF‐α expression was significantly upregulated. However, SIM treatment remarkably reduced the expression of MMP‐9 and TNF‐α. Each bar value, as stated, represents the size and magnification of the image. (B) Quantification of staining intensity of IGF‐1, MMP‐9, and TNF‐α in different sample groups. Statistical analysis was performed by one‐way ANOVA. Error bars represent 95% CI. ExP = experimental periodontitis; SIM‐PPI = simvastatin‐pyrophosphate. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

mRNA expression by real‐time qPCR in primary human gingival fibroblasts. (A) Fold change (log2) expression of mRNA relative to reference control genes (18rSRNA, GAPDH, and β‐actin) in primary human gingival fibroblasts treated with LPS and simvastatin for 24 hours. Bar heights indicate mean expression of the genes in samples. Error bars indicate 95% CI estimates of the mean expressions. One asterisk indicates statistically significant difference between the means of a sample set compared to the mean of the control sample set to 5% (correspond to a p value < 0.05); two asterisks indicate statistically significant difference to 1% (correspond to a p value < 0.01). (B) Fold‐change (log2) expression of mRNA relative to reference control genes (18rSRNA, GAPDH, and β‐actin) in primary human gingival fibroblasts treated with LPS and simvastatin for 48 hours. Bar heights indicate mean expression of the genes in samples. Error bars indicate 95% CI estimates of the mean expressions.

Figure 6

Schematic diagram of simvastatin induced pathways and gene network during rat periodontitis model. Simvastatin directly regulates by IGF‐1, FGF7, Wnt/β‐catenin, and IL‐1 receptor‐like (1IL1rl1) that may play decisive roles in (1) activating periodontal ligament/fibroblast growth and homeostasis, (2) induction of alveolar bone repair and regeneration, and (3) immune response and repression of periodontitis.