Local Application of a New Chalconic Derivative (Chalcone T4) Reduces Inflammation and Oxidative Stress in a Periodontitis Model in Rats

Abstract

Chalcones are phenolic compounds with biological properties. This study had the aim to evaluate the effects of topical administration of a new synthetic chalcone, Chalcone T4, in an animal model of periodontitis induced by ligature. Forty rats were distributed in the following experimental groups: negative control (without periodontitis and topical application of distilled water), positive control (periodontitis and topical application of distilled water), chalcone I and II (periodontitis and topical application of 0.6 mg/mL and 1.8 mg/mL, respectively). Chalcone or distilled water was administered into the gingival sulcus of the first molars daily for 10 days, starting with the ligature installation. The following outcomes were evaluated: alveolar bone loss (µCT and methylene blue dye staining), quantification of osteoclasts (histomorphometry), cell infiltrate and collagen content (stereometry), gene expression of mediators (Nfact11, Tnf-α, Mmp-13, iNos, Sod and Nrf2) by (RT-qPCR); expression of BCL-2 and Caspase-1 (immunohistochemistry). Chalcone T4 inhibited bone resorption and prevented collagen matrix degradation. Reduction in the expression of inflammatory markers (Nfact11, Tnf-α, Mmp-13, and Caspase-1), attenuation of oxidative stress (iNOS reduction, and increase in Sod), and pro-apoptotic effect of the compound (BCL-2 reduction), were associated its effects on periodontal tissues. Topical application of Chalcone T4 prevented bone resorption and inflammation, demonstrating potential in the adjunctive treatment of periodontitis.

Keywords: antioxidant activity; chalcone; periodontitis; topical application.

Figure 1

Chalcone T4 (0.6 mg/mL and 1.8 mg/mL) reduced the exposed root surface area of the mandibular first molar, indicating a reduction of bone resorption (I). Chalcone T4 (0.6 mg/mL) decreased the distance between CEJ and ABC (II) and the volume of bone tissue in the ROI in the ligature-induced periodontitis model (III). Results are representative of 8 animals per experimental group. Representative images of each experimental group acquired in the sagittal, axial, coronal, and 3D planes (IV). The bar graph indicates the number of osteoclasts in a defined region (furcation of the first molar and interproximal region between the first and second molars) (V). Image of the ABC region between the first and second molars. Arrows indicate osteoclasts identified by morphology and location in histological sections stained with Masson’s trichrome (at a 40× magnification 40×) (VI). The bars illustrate the mean values, and the vertical lines the standard error of the mean (SEM). (*) (p < 0.05) compared to Ø− group. (†) (p < 0.05) compared to the Ø+ group.

Figure 2

Chalcone T4 inhibits collagen fiber degradation (I). A grid was overlaid on the digitized images of the interest area to conduct a quantification analysis of tissue components (cellular infiltrate and collagen fibers) relative to the total number of points counted. Chalcone T4 did not alter the cell infiltrate but prevented collagen content destruction (I). Histological appearance of the studied group’s experimental groups (200× magnification) (II). The bars represent the mean percentage value, and the vertical lines denote the standard error of the mean (SEM). (*) p < 0.05 compared with the Ø− group. (†) p < 0.05 compared to the Ø+ group.

Figure 3

Administration of Chalcone T4 inhibited the expression of inflammatory genes Nfact1, Mmp-13, and iNOS and increased the expression of an antioxidant gene Sod in the gingival tissues. Data from RNA extracted from gingival tissues from at least four hemi-mandibles of individual animals from each experimental group. The bars illustrated the mean values, and the vertical lines the standard error of the mean (SEM). (*) p < 0.05 compared with the Ø− group. (†) p < 0.05 compared to the Ø+ group.

Figure 4

Chalcone T4 (0.6 mg/mL and 1.8 mg/mL) reduced Caspase-1 expression in the region of interest (I,II). Chalcone T4 (0.6 mg/mL) reduced the expression of BCL-2 (III). Images (II,IV) show the immunohistochemical staining of the studied groups (200× magnification). The bars indicate the mean percentage value, and the vertical lines indicate the standard error of the mean (SEM). * p < 0.05 compared to the Ø− group. † p < 0.05 compared to the Ø+ group. Scale bar (100 µM).