Research On DangGui BuXue Decoction's Effect On Ox-LDL Activating NF-κB Signal Transduction Pathway

Objectives:That the macrophage phagocytose lots of Ox-LDL and become foam cells, is the mainpathological change in early As. NF-κB transduction pathway plays an important role ofregulating and controlling in As process. Our research studied DangGui BuXueDecoction's effect on NF-κB activity induced by Ox-LDL in RAW264.7 cells, in orderto discuss the cell signal transduction mechanism of DangGui BuXue Decoction againstAs.Methods:1. Prepared rabbit serum containing DangGui BuXue Decoction herbs or not andOx-LDL, detected cell activity by trypanblue staining method, detected cell survival rateaffected by rabbit serum through MTT method.2. Detected NF-κBp65's expression by IHC method.3. Detected NF-κBp65's protein activity change by WB method.4. Detected NF-κBp65's mRNA expression by RFQ-PCR.Results:1. cells grew adherence to the well after 3 to 4 hours' culture, 2 to 3 days later, cells grewfully monolayer. cell activity detected by trypan blue staining was more than 95%. rabbitserum had nutrient action to facilitate cells' growth, but too highly concentration was badto cells. 10%concentration had small effect on cells.2. IHC results: positive stained materials were yellow brown granule inside the cytoplasmand nucleus. Ox-LDL group cells had great of positive stained materials, much more thancontrol group and PAR group. these results declared that, Ox-LDL induced RAW264.7 cells to activate more NF-κBp65 expression and PAR decreased its expression. Amongthe three serum groups containing herbs,the high concentration group cells had leastpositive staining materials,also least NF-κBp65 expression.so we chose the highconcentration serum to continue the later experiments.3. WB results: Ox-LDL group protein density area ratio is1.2883±0.0139, comparedwith control group, the difference had great meaning(p＜0.01),implied after Ox-LDLstimulate RAW264.7 cells,NF-κBp65's protein activity increased obviously.PARgroup(0.7713±0.0211) compared with Ox-LDL group,difference was obvious(p＜0.01), implied PAR could inhibit NF-κBp65's protein activity obviously.serum groupcontaining herbs (0.9189±0.0153) compared with Ox-LDL group, there's difference(p＜0.05), implied DangGui BuXue Decoctin could downregulate NF-κBp65's proteinactivity.4. RFQ-PCR results: Ox-LDL group mRNA relative expression ratio(0.007870±0.0024918) compared with control group(0.002637±0.0014726),difference wasgreat,implied NF-κBp65's mRNA increased after that Ox-LDL stimulate cells.and bothPAR group(0.004706±0.0010521) and serum group containing herbs(0.005335±0.0009869) compared with Ox-LDL group,all had obvious difference(p＜0.05).impliedthat PAR inhibited NF-κBp65's mRNA expression,and DangGui BuXue Decoctioncould downregulated it.Conclusions:100μg/mlOx-LDL stimulated RAW264.7 cells for 4 hours,NF-κB signal transductionpathway was activated obviously,NF-κBp65 protein activity and mRNA increasedgreatly.PAR inhibited NF-κBp65's activity obviously,DangGui BuXue Decoction coulddecreased NF-κBp65 protein activity and downregulated its mRNA expression.wededuced that during the process of lipid infiltration and foam cell formation in earlyAs,NF-κB pathway maybe play an important role,and DangGui BuXue Decoctionmaybe regulate and control relate inflammatory factors' expression through this pathwayto effect on As developing,so to block NF-κB pathway may be one of mechanisms ofDangGui BuXue Decoction's action against As injury.