The Molecular Mechanisms And Functions Of Unfolded Protein Response In Periodontitis

The nature of periodontitis is a kind of chronic infectious diseases of the periodontalsupporting tissues. The destruction of periodontal soft and mineralized connective tissuesis the main clinical manifestations of periodontitis. Microorganisms can bring aboutperiodontistis, but the destruction of periodontistis is mainly caused by the host immuneresponse to the infection. Lipopolysaccharides (LPS), an important Gram-negativebacteria pathogen-associated molecular pattern (PAMP), is the major causative agent ofperiodontitis. At one hand, LPS is a cytotoxic agent to periodontal ligament fibroblasts,inhibiting their viability, attachment, proliferation and matrix protein synthesis. At theother hand, LPS play an important role in the innate immune response by interacting withpattern recognition receptors. Periodontal ligament fibroblasts infected by LPS couldproduce a variety of cytokines such as interleukins, tumor necrosis factor and interferons, which induce more damage on tissues than LPS itself．Endoplasmic reticulum (ER) stress is defined as accumulation of unfolded ormisfolded proteins in the endoplasmic reticulum, which triggers a protective programcalled the unfolded protein response (UPR). UPR could maintain the endoplasmicreticulum steady-state and protect cells from apoptosis. UPR was marked by threemolecular markers named GRP78, XBP-1and CHOP respectively. It is becomingincreasingly apparent that the UPR also plays a role in the periodontal diseases. Inprevious studies, it has been demonstrated that UPR was associated with the severeness ofthe periodontal diseases, as the expression levels of the UPR related molecules weresignificantly higher in periodontitis than that in normal periodontal tissues. However, themolecular mechanisms of how UPR was induced in periodontitis remain unclear.To determine whether the UPR in periodontitis tissues was induced by LPS, theHPDLF were stimulated by LPS for different time periods and the relative expressionlevels of GRP78, XBP-1, CHOP mRNA were detected by qPCR. In order to study theimpact of the UPR on LPS inducing cytokine prodution, the HPDLF during UPR werestimulated by LPS and the cytokine production levels were detected by the ELISA and thereal-time quantitative PCR. The following results we got:1. For the first time, our results showed that LPS could up-regulate the GRP78, XBP-1and CHOP expression levels at the mRNA levels in HPDLF cells, suggesting that theUPR could be induced in HPDLF by LPS in vitro. Meanwhile, LPS could not onlyincrease the XBP-1mRNA expression levels, but also promote the splicing and activationof XBP-1. Low doses of LPS (0.1μg/mL) could only increase the expression levels ofGRP78and XBP-1mRNA, but could not increase the CHOP mRNA expression levels,indicating that the cells can be protected by UPR to sustain endoplasmic reticulumhomeostasis at low concentration of LPS. However, higher concentrations of LPS(1μg/mL,10μg/mL) could significantly increase the CHOP mRNA expression levels,indicating that the endoplasmic reticulum homeostasis was imbalanced and may inducethe apoptosis of the cells gradually. This may promote the development of theperiodontitis. 2. The activated XBP-1mRNA and GRP78mRNA expression levels were significantlyup-regulated in HPDLF by TM and TG treatments for6h, which suggested that HPDLFwere in UPR state after TM and TG treatments for6h. It has been found that the IL-1β,IL-6and IL-8mRNA levels were significantly decreased in the HPDLF at UPR statecompared with that of cells at normal state. Meanwhile, the IL-1β, IL-6and IL-8expression levels were significantly decreased in the cell culture supernatant (p <0.05)compared with that of cells which were not pretreated by TM or TG. In human fibroblastcell line1506, the LPS inducing expression levels of IL-1β, IL-6and IL-8mRNA at UPRstate and normal state were also detected to confirm the results.In summary, for the first time we found that LPS could induce the UPR of HPDLF invitro, suggesting that LPS may trigger the up-regulation of UPR related moleculesexpression levels in periodontitis tissues. Furthermore, our studies demonstrated that thecells at the UPR state could blunt the sensitivities of cells to LPS stimulation and inhibitthe cytokine production mediated by LPS. Our results not only found the new mechanismsof LPS in periodontitis, but also provided novel insight on clinical treatment forperiodontitis.