Estrogen deficiency aggravates apical periodontitis by regulating NLRP3/caspase-1/IL-1β axis

Abstract

Estrogen plays critical roles in apical periodontitis and subsequent bone loss, however the mechanism is not clear yet. In this study, we aimed to study the underlying mechanism of estrogen in apical periodontitis using both clinic samples and animal model. Clinically, as estrogen physiologically declines in elder female patients (premenopausal verses postmenopausal patients), we found that the expression level of NLRP3/Caspase-1/IL-1β signaling pathway was elevated in the infected apical tissues of postmenopausal patients as compared to the premenopausal patients, suggesting that this pathway is involved in the estrogen-mediated apical periodontitis. Furthermore, by analyzing the well-established OVX (estrogen deficiency model) animal model, we confirmed that the expression level of NLRP3/Caspase-1/IL-1β signaling pathway was also elevated in the infection areas of apical periodontitis in OVX animals. Importantly, as the periodontitis progressed, the subsequent bone loss was aggravated significantly. Thus, taken all these data together, our results demonstrated that the NLRP3/Caspase-1/IL-1β signaling pathway is involved in the estrogen-mediated apical periodontitis and the consequent bone loss in both human being and animal model. This study may provide a potential target for female apical periodontitis therapy.

Keywords: Estrogen; NLRP3/Caspase-1/IL-1β signaling pathway; apical periodontitis; bone loss; inflammation.

Figure 1

The expression levels of NLRP3, proCaspase-1, active Caspase-1 and IL-1β are increased in periapical tissues of periapical periodontitis in postmenopausal patients. The tissue lysates from the patients were separated on SDS-PAGE, and then immunoblotted with different specific antibodies. A. Representative immunoblotting images of different antibodies as indicated. B. Quantification analysis of the expression levels of the indicated proteins based on the immunoblotting results. The expression levels of NLRP3, Caspase-1, IL-1β were markedly up-regulated in periapical tissues of periapical periodontitis in postmenopausal patients. Data are presented as mean ± SEM (n=15 per group). ***P<0.001, *P<0.05. pre, premenopausal patients; post, postmenopausal patients.

Figure 2

Immunohistochemic analysis of the cellular expression of NLRP3, Caspase-1 and IL-1β in periapical tissues of periapical periodontitis in premenopausal and postmenopausal patients. A. Representative images of immunohistochemical staining using anti-NLRP3, Caspase-1 and IL-1β antibodies, respectively. The red arrows in the insert point to the immunoreactive positive cells. Scale bar, 100 µm. B. Quantification of the NLRP3, Caspase-1 and IL-1β positive cells in the periapical lesions of periapical periodontitis in postmenopausal patients vs premenopausal patients. The number of NLRP3, ASC, Caspase-1 and IL-1β immunostaining positive cells were significantly increased in the postmenopausal patients (grey bars) as compared to the premenopausal patients (black bars). Data are presented as mean ± SEM (n=15 per group). ***P<0.001, *P<0.05. Original magnification, 200×; Scale bar, 100 µm.

Figure 3

Measurement of serum levels of estrogen (E2) in Sham and OVX rats. Serum samples were prepared at different time points as indicated from both Sham and OVX groups, respectively. The serum level of estrogen was measured using immunochemiluminescent assay. At the start point right after ovariectomy (week 0), the serum levels of E2 in Sham and OVX rats are alike (P>0.05). However, during the first week after ovariectomy, the serum level of E2 was gradually reduced in the OVX group as compared to that in Sham group. After 1 week up to 5 weeks of ovariectomy, the serum levels of E2 were significantly reduced in the OVX groups as compared to that in Sham group, but no significant changes found within each group itself. Data are presented as mean ± SEM (n=5), ***P<0.001.

Figure 4

Estrogen deficiency promotes the proliferation of osteoclasts. A. Representative images of immunohistochemical staining using TRAP from normal, Sham, and OVX sections. Red arrows in the inserts indicate the immunoreactive positive osteoclasts, which have multinuclear. Original magnification, 200×; Scale bar, 50 µm. B. Quantitative analysis of osteoclast numbers in each group. On the 0^th and 28^th day after pulp exposure, the number of osteoclasts in Sham and OVX group are comparable. On the 7^th, 14^th and 21^st day after pulp exposure, there were significant more osteoclasts in OVX group as compared to that in the Sham group (P<0.01). Data is represented as mean ± SEM (n=5), ***P<0.001.

Figure 5

Estrogen deficiency upregulates the expression levels of NLRP3, capase-1 and IL-1β in periapical tissues of periapical periodontitis in OVX rats. A. Representative images of Immunohistochemical staining using anti-NLRP3 antibody at different time points as indicated (right panel), and the quantification analysis of the NLRP3 immunoactive positive cells in periapical tissues of periapical periodontitis of sham and OVX groups (left panel). B. Representative images of Immunohistochemical staining using anti-caspase-1 antibody at different time points as indicated (right panel), and the quantification analysis of the caspase-1 immunoactive positive cells in periapical tissues of periapical periodontitis of sham and OVX groups (left panel). C. Representative images of Immunohistochemical staining using anti-IL-1β antibody at different time points as indicated (right panel), and the quantification analysis of the IL-1β immunoactive positive cells in periapical tissues of periapical periodontitis of sham and OVX groups (left panel). The red arrows in the inserts point to the immunoreactive positive cells. Original magnification, 200×; Scale bar, 100 µm. Data are presented as mean ± SEM. ***P<0.001.

Figure 6

Estrogen deficiency aggravates the alveolar bone loss induced by apical periodontitis. A. Representative images of the radiologic photographs (upper panel) and the histological staining (lower panel) after 28 days of OVX. Original magnification, 400×; Scale bar, 50 µm. The periapical lesion areas were marked with the dot lines. B. Quantitative analysis of the bone loss area in periapical lesions of each group. Compared to the sham group, based on both radiologic and histological data, the bone loss area is significantly greater in the OVX group. Data is represented as mean ± SEM. ***P<0.001.