Time-dependent C5a and C5aR expression in dental pulp cells following stimulation with LTA and LPS.

Abstract

Clinically, deep decay can lead to inflammation in the dental pulp. Apart from the use of various materials to sooth the inflamed pulp, there is currently no adequate treatment, and the gold standard, calcium hydroxide, that is used to cover the dentin/pulp, has limited effect. Sometimes the pulp will remain infected and cause pulpitis, and ultimately, the pulp will need to be removed. The first principle of oral treatment is to protect the pulp. Therefore, it is necessary to study the immune response and regeneration of pulp cells in conditions of deep decay. Of the terminal complement system proteins, complement 5a (C5a) has the most potent effect compared to complement 3a (C3a) and complement 4a (C4a). C5a is 20- to 2,500-fold stronger than C3a and C4a. The purpose of this study was to elucidate the association between C5a, secreted by complement activation, and the duration of inflammation. Another key goal was to detect the expression of C5a and its receptor, complement 5a receptor (C5aR). To this end, the cells were divided into 4 groups as per stimulation with lipoteichoic acid (LTA) or lipopolysaccharide (LPS) as follows: i) The 1 µg/ml LTA group; ii) the 1 µg/ml LPS group; iii) the 1 µg/ml LTA and 1 µg/ml LPS group; and iv) the PBS-only group, which served as a control. There were 5 time points for all 4 groups: 1, 2, 3, 5 and 7 days. Reverse transcription-quantitative polymerase chain reaction was used to detect the gene expression levels of C5a, C5aR and interleukin (IL)-6 at different time points. Western blot analyses was carried out to detect the expression of C5aR. Transmission electron microscopy was also conducted to assess the ultra-structural features of dental pulp cells. The gene expression trends of C5a and C5aR mRNA were identical. C5a and C5aR mRNA was highly expressed on the second day of LTA or LPS stimulation. However, in the LTA and LPS co-stimulation group, C5a and C5aR mRNA were highly expressed on both the first and second day, with higher levels on the second day. IL-6 expression decreased as time progressed in the LTA only and in the LTA + LPS co-stimulation groups. However, a peak in its expression was observed on the second day in the LPS group. On the whole, this study demonstrates that a 1 µg/ml concentration of LTA and LPS stimulates human dental pulp cells to activate the expression of C5a.