The Effect Of Aα-LPS On The Expression Of Membrane Receptors，ROS And NO In Tissue-specific Monocytes/Macrophages

Objective To study the heterogeneity in Phagocytosis of Actinobacillusactinomycetemcomitans(Aa) by monocytes/macrophages from four different tissuesof New Zealand Rabbits, to compare the expression of membrane receptor CD14,TLR2, TLR4, SR-A and MHC-Ⅱ in healthy and hyperlipidemic rabbit tissue-specificmonocytes/macrophages activated by Aa-lipopolysaccharide（LPS）with the effectof additional of exogenous IL-10, and examin the amount of ROS and NO production.This study is to provide a theoretical basis for understanding the relationship betweenthe periodontal diseases and the systemic disorders.Methods Establishing the research model of healthy and hyperlipidemic NewZealand rabbits and isolation, identification, purification and culture of tissue-specificmonocytes/macrophages from different tissues (blood, lungs, abdominal cavity, liver)1. Mo, AM, PM and KC from3healthy New Zealand rabbits were detected by flowcytometry at different time in the phagocytic index (PI) of FITC-labeled Aa.2.Monocytes/macrophages from blood, lungs, abdominal cavity, liver cells in6healthyand6hyperlipidemia New Zealand rabbits were divided into5groups: RPMI1640+monocytes/macrophages,1μg/ml Escherichia.coli-LPS+monocytes/macrophages,1μg/ml Aa-LPS+monocyte/macrophage cells,100ng/ml IL-10+monocyte/macrophage,100ng/ml IL-10+1μg/ml Aa-LPS+monocyte/macrophage cells, RT-PCR was usedto detect the expression of CD14, TLR2, TLR4, SRA and MHC-Ⅱ.3. Nitratereductase assay and DCFH-DA fluorescence detection were used to assess the amountof ROS and NO production. Results1. The phagocytosis of Monocytes/macrophages from rabbits’ four partswere different, within1.5h, the AM, PM phagocytic index rise as the time increase;the phagocytosis ability of Mo and KC reached the peak at1h; the obvious differenceof phagocytosis between Mo, AM, PM and KC has been observed. At30min, thephagocytic index is: AM and PM＞Mo, KC; at1h, AM＞PM＞Mo and KC; at1.5h,AM＞PM＞KC＞Mo.2. Realtime PCR detection results:①After stimulation by1μg/ml Aa-LPS for24h, Mo, AM, PM and KC from healthy New Zealand rabbits, thegene expression of TLR2, SRA and MHC-Ⅱ were raised compared with the controlgroup (P<0.05), addition to the down-regulation of CD14in Mo and inhibition ofTLR4in KC (P<0.05), and the remaining gene expression were raised (P<0.05).②the CD14, SRA and MHC-Ⅱ gene expression was increased (P<0.05) after the1μg/ml Aa-LPS stimulation for24h of hyperlipidemia New Zealand rabbits.③healthy and hyperlipidemia groups, the100ng/ml exogenous IL-10affect Aa-LPSactivated monocytes/macrophages, the membrane receptor CD14, TLR2, TLR4,MHC-Ⅱ gene expression was inhibited (P<0.05), SRA was increase (P<0.05).3.①healthy and hyperlipidemia groups, after the1μg/ml Aa-LPS stimulation the Mo,AM, PM, KC release of a large amount of ROS (P<0.05) compared with the controlgroup, the NO emission of AM, PM and KC groups were increased (P<0.05).Conclusions1.Monocytes/macrophages from different parts of rabbit showed thefunction and phenotypic heterogeneity, the different phagocytosis of Aa bymonocytes/macrophages of various tissues may be the important reason why there aredifferent effects on different parts in the periodontitis.2. KC of hyperlipidemic rabbitsin a defensive immune response, AM and PM enter into the inflammatory stage. IL-10can reached anti-inflammatory effects by increasing SR-A and decreasing CD14,MHC-Ⅱ, TLR4.3. IL-10can inhibit the inflammatory response and maintain the stable internal environment, protect the host from inflammatory mediators injury viathe suppress in the ROS, NO production of LPS-activated macrophages.