Gan-Lu-Yin (Kanroin), Traditional Chinese Herbal Extracts, Reduces Osteoclast Differentiation In Vitro and Prevents Alveolar Bone Resorption in Rat Experimental Periodontitis

Gan-Lu-Yin (GLY), a traditional Chinese herbal medicine, shows therapeutic effects on periodontitis, but that mechanism is not well known. This study aims to clarify the precise mechanism by investigating the inhibitory effects of GLY extracts on osteoclastogenesis in vitro and on bone resorption in periodontitis in vivo. RAW264.7 cells are cultured with soluble receptor activator of nuclear factor-kappa B (sRANKL) and GLY extracts (0.01-1.0 mg/mL), and stained for tartrate-resistant acid phosphatase (TRAP) to evaluate osteoclast differentiation. Experimental periodontitis is induced by placing a nylon ligature around the second maxillary molar in rats, and rats are administered GLY extracts (60 mg/kg) daily for 20 days. Their maxillae are collected on day 4 and 20, and the levels of alveolar bone resorption and osteoclast differentiation are estimated using micro-computed tomography (CT) and histological analysis, respectively. In RAW264.7 cells, GLY extracts significantly inhibit sRANKL-induced osteoclast differentiation at a concentration of more than 0.05 mg/mL. In experimental periodontitis, administering GLY extracts significantly decreases the number of TRAP-positive osteoclasts in the alveolar bone on day 4, and significantly inhibits the ligature-induced bone resorption on day 20. These results show that GLY extracts suppress bone resorption by inhibiting osteoclast differentiation in experimental periodontitis, suggesting that GLY extracts are potentially useful for oral care in periodontitis.

Local administration of calcitonin inhibits alveolar bone loss in an experimental periodontitis in rats.

Calcitonin (CTN), a calcium regulatory hormone, promotes calcium diuresis from the kidney and suppresses bone resorption. The objective of this study was to evaluate whether the topical and intermittent application of CTN inhibits alveolar bone resorption using ligature-induced experimental periodontitis in rats. Experimental periodontitis was induced by placing a nylon ligature around maxillary molars of 8-week-old male Wistar rats for 20 days. Thirty-two rats were divided into four groups: basal sham control group, periodontitis group, periodontitis plus 0.2 U CTN (low dose), and periodontitis plus 1.0 U CTN (high dose) group. To investigate the effects of CTN on alveolar bone resorption, CTN was topically injected into the palatal gingivae every 2 days after ligature removal (day 0). Micro-computed tomography (CT) analysis was performed for linear parameter assessment of alveolar bone on day 5 and day 14. Periodontal tissues were examined histo-pathologically to assess the differences among the study groups. Micro-CT images showed that alveolar bone resorption was induced statistically around the molar of ligatured rats on day 5 and day 14. The amount of bone resorption was more severe on day 14 than that on day 5. On day 5, only high-dose CTN treatment significantly suppressed bone resorption. In addition, both doses of CTN significantly suppressed bone resorption on day 14. Histological examination clarified that there were fewer TRAP-positive cells in the CTN treatment groups than in the periodontitis group on day 5. Local administration of CTN decreased alveolar bone resorption by regulating osteoclast activation in rats with periodontitis.

Effect of Hangeshashinto on calprotectin expression in human oral epithelial cells.

Oral epithelial cells produce antimicrobial peptides (AMPs) to prevent microbial infection. Calprotectin (S100A8/S100A9) is one of these AMPs in oral epithelial cells, the expression of which is up-regulated by interleukin-1α (IL-1α). Hangeshashinto (HST) is a traditional Japanese herbal medicine that has anti-inflammatory effects. The purpose of this study was to investigate the effect of HST on the expression of calprotectin through the regulation of IL-1α in oral epithelial cells. Human oral epithelial cells (TR146) were cultured with HST in the presence or absence of anti-IL-1α antibody or IL-1 receptor antagonist, or with six major components of HST (3,4-dihydroxybenzaldehyde, baicalin, ginsenoside Rb1, glycyrrhizin, oleanolic acid and berberine). The expression of S100A8, S100A9, other AMPs and cytokine mRNAs was examined by RT-PCR and quantitative real-time PCR. Calprotectin expression and IL-1α secretion were investigated by ELISA. HST (6 µg/ml) increased the expression of S100A8/S100A9 mRNAs and calprotectin protein, and also up-regulated ß-defensin 2 (DEFB4) and S100A7 expression. The expression of IL-1α mRNA and its protein was slightly but significantly increased by HST. A neutralizing antibody against IL-1α and IL-1 receptor antagonist inhibited HST-up-regulated S100A8/S100A9 mRNA expression. Although 3,4-dihydroxybenzaldehyde, baicalin and ginsenoside Rb1 as HST components increased S100A8/S100A9 expression, oleanolic acid and berberine decreased their expression. These results suggest that HST increases the expression of calprotectin, DEFB4 and S100A7 in oral epithelial cells. In response to HST, up-regulation of calprotectin expression may be partially induced via IL-1α.