Curcumin modulates the immune response associated with LPS-induced periodontal disease in rats

Abstract

Curcumin is a plant-derived dietary spice ascribed various biological activities. Curcumin therapeutic applications have been studied in a variety of conditions, but not on periodontal disease. Periodontal disease is a chronic inflammatory condition initiated by an immune response to micro-organisms of the dental biofilm. Experimental periodontal disease was induced in rats by injecting LPS in the gingival tissues on the palatal aspect of upper first molars (30 µg LPS, 3 times/week for 2 weeks). Curcumin was administered to rats daily via oral gavage at 30 and 100 mg/kg body weight. Reverse transcriptase-qPCR and ELISA were used to determine the expression of IL-6, TNF-α and prostaglandin E(2) synthase on the gingival tissues. The inflammatory status was evaluated by stereometric and descriptive analysis on hematoxylin/eosin-stained sections, whereas modulation of p38 MAPK and NK-κB signaling was assessed by Western blot. Curcumin effectively inhibited cytokine gene expression at mRNA and protein levels, but NF-κB was inhibited only with the lower dose of curcumin, whereas p38 MAPK activation was not affected. Curcumin produced a significant reduction on the inflammatory infiltrate and increased collagen content and fibroblastic cell numbers. Curcumin potently inhibits innate immune responses associated with periodontal disease, suggesting a therapeutic potential in this chronic inflammatory condition.

Figure 1

Curcumin inhibits cytokine gene expression induced by LPS in gingival tissues. Animals were treated with 30 or 100 mg/Kg of curcumin by oral gavage daily for 15 days. Control animals received the same volume of the vehicle by oral gavage. LPS or an equivalent volume of PBS vehicle were injected in palatal aspect of the gingival tissues around the upper first molars three times/week for 2 weeks. Total RNA was isolated from gingival biopsies and used for RT-qPCR. All samples were assayed in duplicate and the results were analyzed by the delta-Ct method and expression of target genes was normalized to GAPDH expression. (*) indicates significant reduction (p< 0.01) in comparison to vehicle control in LPS-injected tissues. Bars indicate means and vertical lines the standard error of mean of at least three animals in each experimental group.

Figure 2

Effect of curcumin administration on IL-6 and TNF-α protein production in gingival tissues with and without LPS injections. Total protein was extracted from gingival biopsies obtained 15 days after the start of LPS injections and used in ELISA tests. These tests were performed according to the manufacturer’s instructions and the results for each sample were normalized to the concentration of total proteins determined by a Lowry-based microassay (DC assay, Bio-Rad). For TNF-alpha production, bars indicate means and vertical lines standard error of mean of at least three animals in each experimental group. IL-6 concentration was determined in pooled samples from three animals in each experimental group due to the low level of expression, and the bar indicate the average of the triplicate measurement from these pooled samples.

Figure 3

Curcumin inhibits activation of NF-kB, but not of p38 MAPK, in the LPS model of periodontal disease. Total protein was isolated from gingival biopsies obtained 15 days after the start of LPS injections with a detergent-containing buffer (T-Per, Pierce) supplemented with protease and phosphatase inhibitor cocktails (Complete and Phos-Stop, Roche). Protein concentration was determined with a Lowry-based microassay (DC assay, Bio-Rad) and 60 µg of each sample were used for the Western blot. Activation of the signaling pathways was assessed by the detection of phosphorylated forms of p65 (NF-kB) and p38 MAPKinase. Expression levels of constitutive housekeeping GAPDH are shown to confirm equal protein loading (A). In (B) the bars represent means and vertical lines the standard error of mean of the densitometric quantification of three western blots performed with three different pools of protein samples. The densitometry of target proteins was normalized to that of GAPDH for the same samples. (*) indicates a significant (p<0.05) difference in comparison with vehicle-treated and PBS-injected control. The images in (A) are representative of samples from three different animals in each experimental group.

Figure 4

Stereometry analysis of gingival tissues injected with LPS, according to the experimental group: vehicle control, 30 or 100 mg/Kg of curcumin administered for 15 days by oral gavage. Negative control groups (PBS injections and vehicle administration by oral gavage) were analyzed for comparative purposes. A total of three images obtained from equally spaced slides were evaluated from each animal. Slides from at least three different animals in each experimental group were used. Both doses of curcumin markedly reduced the inflammatory infiltrate in LPS-injected gingival tissues. There was a significant increase in collagen content in curcumin-treated animals associated with a non-statistically significant trend of increasing numbers of fibroblastic cells. (*) indicates significant reduction (p< 0.01) in comparison to healthy controls and (!) indicates significant difference (p<0.01) in comparison to vehicle-treated and LPS-injected tissues. Bars indicate means and vertical lines standard error of mean of at least four animals in each experimental group.

Figure 5

Histological aspect of the gingival tissues according to the experimental group (PBS vs LPS injections) and administration of curcumin (vehicle, 30 mg/Kg and 100 mg/Kg). Semi-serial sections with 4 µm thickness were routinely processed and stained with hematoxylin and eosin. A total of three sections spaced 100 µm were evaluated for each first molar in a minimum of four animals in each experimental group. A thick keratinized epithelium layer corresponding to the palatal gingival with an underlying dense connective tissue with reduced number of cellular infiltrate and a smooth bone crest surface characterize the gingival tissues of control (PBS-injected) animals (A). LPS injections (B) produced a decrease on the thickness of the epithelium layer and an intense cellular infiltrate and irregular bone crest surface with the presence of multinucleated osteoclast-like cells; whereas in animals treated with 30 mg/Kg (C) and 100 mg/Kg (D) of curcumin, the cellular infiltrate was markedly reduced and the collagen content was increased in comparison with the LPS-injected sites (B). Images were obtained at 100 X magnification and the insert depicts the alveolar bone crest area at 200 X magnification. These images are representative of the histological aspect observed.