Inflammatory Responses Of Leptomeningeal Cells And Microglia Induced By Porphyromonas Gingivalis And Porphyromonas Gingivalis LPS

Background and objective:Chronic periodontitis(CP)is defined as a chronic oral infectious disease caused by the bacteria plaque biofilm.This sustained low-toxicity stimulation may lead to the trigger of local innate immune system and systemic inflammatory response.Alzheimer s disease(AD),showing slow progressive cognitive decline,is considered to be the most common form of dementia.Chronic inflammation induced by microglial activation has emerged as a core to the pathogenesis of AD.Studies found that CP has a significant correlation with the development of AD.The various microorganisms of dental plaque biofilm can stimulate the host to secrete pro-inflammatory factors,which can spread into the brain therefore indirectly cause neuronal damage.In addition,microorganisms can also be transported to the brain directly through the systemic circulation or peripheral nerve pathways,inducing the inflammation of the central nervous system and the eventual cognitive impairment.Porphyromonas gingivalis(P.gingivalis)is currently considered to be the main pathogen involved in CP.Lipopolysaccharide(LPS),an outer cell-wall component,is served as the major virulence factor of P.gingivalis inducing host infections.The correlation between P.gingivalis and AD has been successively confirmed and explained in recent studies,but the detailed inflammatory transduction and nerve damage mechanisms have not yet been elucidated clearly.Leptomeningeal cells,covering the surface of the brain parenchyma,have been found to transducing inflammatory signals by secreting related factors.The purpose of this experiment is to investigate the inflammatory responses of leptomeningeal cells and microglia induced by P.gingivalis and its toxic component LPS,and to explore the relevant transduction pathway of P.gingivalis-induced neuroinflammation.Methods:1.Leptomeningeal cells were extracted from the brain of three-day-old C57BL/6N suckling mice,and were cultured and identified through immunofluorescence staining.BV2 microglia were purchased and cultured.2.Leptomeningeal cells were stimulated by P.gingivalis and P.gingivalis LPS for24 hours respectively.The cell viability was evaluated by CCK-8 method.The expressions of inflammatory factors including TNF-α,IL-1β,and IL-6 were estimated utilizing Quantitative Real-time PCR and Elisa.3.Microglia were stimulated by the culture medium which was collected and filtrated after the co-culture of leptomeningeal cells and P.gingivalis,P.gingivalis LPS.The morphological changes of microglia were observed by light microscope.The cell viability was evaluated by CCK-8 method.The expressions of TNF-α,IL-1β,and IL-6 were determined through Quantitative Real-time PCR and Elisa.The inflammatory responses of microglia transduced by leptomeningeal cells were assessed.4.Microglia were directly stimulated by P.gingivalis and P.gingivalis LPS,respectively.The morphological changes of microglia were observed through light microscope.The cell viability was evaluated by CCK-8 method.The expressions of TNF-α,IL-1β,and IL-6 were determined through Quantitative Real-time PCR and Elisa.The inflammatory reactions of microglia in response to P.gingivalis and P.gingivalis LPS were analyzed.5.The expressions of p-p38 MAPK protein in leptomeningeal cells stimulated by P.gingivalis and P.gingivalis LPS were estimated by cellular immunofluorescence staining.Results:1.Immunofluorescence analysis indicated a high purity of the extracted leptomeningeal cells.Most of the cells were oval,spindle or triangle in shape,with clear protrusions and large,clear nuclei.2.Leptomeningeal cells were activated directly by the infection of P.gingivalis and P.gingivalis LPS.The m RNA expression of TNF-α,IL-1β,IL-6,and the protein secretion of TNF-α,IL-6 were significantly increased in leptomeningeal cells.IL-1β protein couldn’t be detected and analyzed because the secretion volume didn’t reach the maximum sensitivity of the detection method.3.Microglia could be significantly activated by the culture medium of leptomeningeal cells,which was collected and filtered after the stimulation with P.gingivalis and P.gingivalis LPS.The m RNA expression of TNF-α,IL-1β,IL-6,and the secretion of TNF-α and IL-6 protein of microglia were significantly up-regulated.4.Microglia could be directly activated by the infection of P.gingivalis and P.gingivalis LPS.The m RNA expression of TNF-α,IL-1β,IL-6,and the secretion of TNF-α and IL-6 protein of microglia were promoted.Compared with this direct infection approach,the inflammatory response of microglia revealed much stronger to the inflammatory signals transduced by leptomeningeal cells.5.The protein expressions of p-p38 MAPK in leptomeningeal cells were significantly up-regulated when stimulated with P.gingivalis and P.gingivalis LPS.Conclusions:Microglia could be activated by inflammatory signals transduced from leptomeningeal cells indirectly,or by P.gingivalis and its toxic component LPS transported to the brain directly.The former approach triggered a stronger inflammatory response of microglia.Microglia activation could induce microglia activation-dependent brain inflammation and promote the progression of AD.Leptomeningeal cells up-regulate their own pro-inflammatory factors might through p38 MAPK pathway.The results of this study laid a foundation for further exploring the mechanism of brain inflammation caused by peripheral pathogens,leading to eventual neurocognitive impairment.