The Effects Of Hypoxia Augments On The Lipopolysaccharide Induced Cytokine Expression In Periodontal Ligament Cells

Periodontitis is a chronic inflammatory disease characterized by the destruction oftooth supporting tissues. Hypoxia, the mainly changes of the plateau environment, caninduces severe periodontitis in animal experiments. Our previous study indicated that thedegree of periodontal lesion in rats was more severe under imitational altitude inducedhypoxic environment than that under normoxic conditions. However, there is very littleinformation on hypoxia and lipopolysaccharide (LPS) induced cytokine expression inperiodontal ligament (PDL) cells. In the present study, we characterized the effects ofhypoxia or P. gingivalis Lipopolysaccharide (Pg LPS) on the TNF-α, IL-1β and IL-6expression in human periodontal ligament (hPDL) cells. We found that hypoxia augmentedPg LPS increased the TNF-α, IL-1β and IL-6expression in hPDL cells. We alsodemonstrated that NF-κB pathway was involved in the hypoxia augmenting Pg LPSinduced cytokine expression in hPDL cells. Thus, our results suggest that the degree ofperiodontal lesion in rats was more severe under imitational altitude induced hypoxicenvironment than under normoxic conditions partly because the hypoxic environmentenhances the immune function of hPDL cells that is induced by Pg LPS.Methods and results1. The effects of hypoxia on LPS-stimulated TNF-α, IL-1β, and IL-6expression inhPDL Cells1). To identify the hPDL cells, we showed that the cytoplasm was positive for vimentinby IHC testing of the hPDL cells (fourth passage). However, keratin was not found in thecytoplasm. The results demonstrated that hPDL cells are mesenchymal cells derived fromthe embryonic mesoderm. MTT assays showed that hPDL growth curve was similar to an‘S’.2). To investigate whether hypoxic conditions could have an additional effect onLPS-stimulated inflammatory cytokine expression in hPDL cells, we measured the TNF-α, IL-1β, and IL-6expression at both mRNA and protein levels. A six-hour exposure to bothhypoxia and Pg LPS significantly increased the secretion of TNF-α, IL-1β, and IL-6inhPDL cells compared with normoxic conditions (18.5%O2)(P <0.05). What’s more,Addition of Pg LPS combination with hypoxia (ranging from10%to1%O2) caused afurther increase compared to Pg LPS alone (P <0.05).3). To study the time-course effects of hypoxia (1%O2) on LPS induced cytokineproduction in hPDL cells，the hPDL cells were stimulated by Pg LPS (10μg/ml) underhypoxic or with normoxic conditions for6h,12h,24h, or48h. We found that LPS orhypoxia (1%) independently increased the TNF-α, IL-1β and IL-6secretion, whereasco-treatment with hypoxia and LPS significantly increased inflammatory cytokine secretioncompared to cells challenged with LPS alone (P <0.05).4). Real-time PCR was also performed to confirm the effect of hypoxia and LPS oncytokine expression. Co-treatment of the cells with LPS and hypoxia also increased themRNA expression of TNF-α, IL-1β and IL-6compared to the LPS challenged group (P <0.05).5). To further explore the effect of LPS and hypoxiaon cytokine production, theintracellular levels of TNF-α, IL-1β and IL-6were also investigated. LPS or hypoxia (1%)also independently up-regulated the intracellular levels of TNF-α, IL-1β and IL-6comparedwith controls. Moreover, hypoxia also enhanced the stimulatory effects of LPS onintracellular inflammatory cytokine expression (P <0.05).2. The signaling pathway involved in hypoxia and LPS induced cytokine expression inhPDL cells.1). To investigate the individual pathway in hypoxia or LPS induced cytokineexpression in hPDL cells, we examined the effects of the NF-κB inhibitor BAY11-7082,ERK1/2inhibitor PD98059, p38inhibitor SB203580,JNK inhibitor SP600125, and HIF-αinhibitor YC-1on hypoxia and LPS induced TNF-α, IL-1β and IL-6expression in hPDLcells. Hypoxia and LPS combination induced TNF-α, IL-1β and IL-6expression wasblocked by BAY11-7082but not by PD98059, SB203580, SP600125or YC-1.2). To further study the NF-κB signaling pathway involved in both hypoxia and LPSinduced cytokine expression in hPDL cells, the Pretreated cells were stimulated withhypoxia (1%O2) and Pg LPS (10μg/ml) for6h respectively, and the expression of phosphorylated I-κB and phosphorylated NF-κB p65was analyzed by western blot.Weobserved that hypoxia or LPS elevated the expression of phosphorylated IkBa in the cytosoland phosphorylated NF-kB p65in the nucleus compared with the control group. Whereas,co-treatment hPDL cells with hypoxia and LPS greatly increased phosphorylated IkBa andphosphorylated NF-κB p65compared to LPS challenged hPDL cells. These resultsindicated that the NF-kB pathways are involved in hypoxia and LPS induced cytokineexpression in hPDL cells.3). Effects of hypoxia (1%O2) and LPS on OPG and RANKL expression in hPDLcells.To confirm that the effects of hypoxia on LPS-stimulated OPG and RANKLexpression, we examined the amounts of OPG and RANKL by real-time PCR and ELISA.Within72h of LPS or hypoxia treatment, no change of OPG expression was observed,whereas cells that were co-treated with LPS and hypoxia significantly increased theRANKL expression compared with LPS challenged group. These results indicated thathypoxia may，at least in part, regulate RANKL/OPG system to aggravate the boneresorption of periodontitis which is induced by LPS.In conclusion, the present study demonstrated that hypoxia augments Pg LPS inducedTNF-α, IL-1β and IL-6expression in hPDL cells. In addition, NF-κB pathway is involvedin hypoxia augmented Pg LPS induced cytokine expression in hPDL cells. Our resultssuggest that the degree of periodontal lesion in rats was more severe under imitationalaltitude induced hypoxic environment than under normoxic conditions partly because thehypoxic environment enhances the immune function of hPDL cells that is induced by PgLPS. However, further studies are necessary to investigate the exact mechanisms involvedin the hypoxic environment of periodontitis.