The Adjustment Role Of Macrophage TLR4 Signaling By High Glucose

ABSTRACTObjective: Diabetic nephropathy is diabetes cause serious hazardness and larger a chronic complications.Its pathogenesis is associated with chronic inflammation, animal experiment and clinical study of patients with DN were found in kidney tissue of infiltration of inflammatory cells is given priority to with macrophages.Its infiltration degree and degree of kidney damage were positively correlated. Therefore, macrophage mediated inflammatory reaction is one of the important reasons to accelerate the development of DN.TLR4 is pattern recognition receptors on the surface of the macrophages, the role of TLR4 signaling pathways in the pathogenesis of DN gradually by the attention of people, many clinical and experimental showed that TLR4 in the condition of high glucose increased expression.LPS under physiological conditions in combination with surface of macrophages and dendritic cells to induce proinflammatory TLR4 of signal and the signal transduction of anti-inflammatory, respectively to produce proinflammatory factor TNF-α and IL-β, anti-inflammatory factor IL-10, to maintain the balance of inflammatory cytokines.So, in the condition of diabetes macrophage mediated activation of TLR4 whether to change the state of equilibrium, cause the DN? Now also did not see the detailed report.Therefore, this topic on the basis of preliminary results and literature material, with high glicose cultivation of macrophages in vitro, with different ligand stimulation of TLR4, observation of TLR4 mediated proinflammatory and anti-inflammatory the expression of signal transduction molecules, discusses high glucose influence on macrophage TLR4 signaling, clear macrophages in DM and the inflammation mechanism in the DN.Methods:1 By PMA activation of THP-1 cells at the same time with normal culture medium and high glucose medium after 24 hours, then use exogenous ligands LPS stimulation, at 0, 2, 4, 8, 16, 24 h cells. With Real- time PCR detection of inflammatory factors IL-10,IL-1β,TNF-α m RNA expression level.2 By PMA activation of THP-1 cells at the same time with normal culture medium and high glucose medium after 24 hours, then use exogenous ligands LPS stimulation, at 0, 2, 4, 8, 16, 24 h collecting cells supernatant; With endogenous ligand HSP60 stimulates cells for 24 h to collect supernatant with ELISA detection of inflammatory factors on IL-10,IL-1β,TNF-α m RNA expression level.3 By PMA activation of THP-1 cells at the same time with normal culture medium and high glucose medium after 24 hours, then use exogenous ligands LPS stimulation, at 0, 15, 30, 60, 120, 180 min collecting cells. Another group of exogenous ligands LPS and endogenous ligand HSP60 stimulation, at 0, 30, 60, 180 min collecting cells, using Western blot detection p110δ, P-NF-κBp65, NF-κB p65,P-AKT, AKT expression level.Results:1 Compared with the blank control group, IL- 10 m RNA in normal group and high glucose in 2 h, 4 h expression increased obviously, IL-1β m RNA in normal group and high glucose in 2 h, 4 h, 8 h significantly higher, the expression of m RNA in TNF-α normal group and high glucose in 2 h, 4 h, 16 h, 24 h expression increased obviously. Control the amount of IL- 10 m RNA expression peak value appeared in 2 hours after LPS stimulation, high glucose group in 4 hours, appear delay phenomenon. High glucose group in 4 hour amount of IL-1β m RNA expression is significantly higher than normal group. High glucose group in 2 hours of TNF-α m RNA expression is significantly higher than normal group.2 Supernatant ELISA result, IL- 10 has no obvious change, LPS stimulation, IL-1β and the production of TNF-α extended over time are increased, and the high glucose group were higher than normal at any point. LPS and HSP60 stimulus for 24 h at the same time, under the LPS stimulation IL-1β, changes s TNF-α ignificantly, and the high glucose group was higher than normal group, HSP60 does not change significantly under the stimulus.3 Western blot results showed that LPS stimulation cell 0, 15, 30, 60, 120, 120 min, P-NF-κBp65 expression increase 15 minutes, normal group of 60 minutes, 15 minutes every peak, high glucose group increased expression of P- AKT60 minutes, at the same time high glucose group were higher than normal group; LPS and HSP60 stimulate cell 0, 30, 60, 180 min, p110 δ high glucose condition in LPS and HSP60 stimuli expression level decreased, P-NF-κBp65, P- AKT in under the action of LPS change obviously.Conclusion:1 Under different ligand stimulation, proinflammator factor IL-1β,TNF-α expression at ELISA level and P-NF-κBp65, P- AKT protein levels, LPS was significantly more than HSP60.2 p110 δ in the condition of high glucose LPS or HSP60 stimulation expression levels are reduced.3 TLR4 mediated pro-inflammatory signaling pathways high gucose condition is significantly higher than normal group. Anti-inflammatory signaling molecules change is not obvious.