Methods: BeWo were treated with A.a. LPS, and mRNA levels were detected using an Affymetrix GeneChip (Human Genome U133 plus 2.0 array, ca. 47,000 genes). GeneChip data was imported into Ingenuity Pathways Analysis (IPA) database. Altered mRNA levels in GeneChip results were confirmed by RT-PCR and real-time PCR. Moreover, Annexin V, calcein staining was carried out using Agilent 2100 Bioanalyzer (Agilent Technologies) to detect apoptotic cells after LPS treatment. Furthermore, carboxyfluorescein FLICA Apoptosis Detection Kit Caspase Assay (Immunochemistry Technologies, LLC) was used to detect the activation of caspases in BeWo.
Results: GeneChip and IPA analysis revealed increased mRNA levels of Cytochrome C, caspase 2 and caspase 3 and decreased mRNA levels of Bcl-xl, CAT and GNAS. Real-time PCR analyses successfully confirmed these mRNA level changes from the GeneChip data. All of these genes we tested are related to mitochondrial apoptotic signaling pathway. Annexin V, calcein staining showed increased percentage of apoptotic cells after treated with A.a. LPS. Fluorecent poly-caspases staining also detected increased activity of caspases in BeWo treated with LPS compared to control.
Conclusion: Taken these findings together, A.a LPS may induce apoptosis through increased expression of apoptosis-inducing genes and decreased expression of apoptosis-inhibiting genes involved in the mitochondrial apoptotic signaling pathway. Increased number of apoptotic cells and activation of cysteine proteases caspases were also observed in BeWo challenged with LPS. This might be one of the molecular mechanisms of pregnant ladies with periodontal diseases have a higher risk of bearing low birth weight babies.