Jianpi Huazhuo Tiaozhi granules reduce oxidative stress injury in macrophages by inhibiting the nicotinamide adenine dinucleotide phosphate oxidase/reactive oxygen species-nuclear transcription factor kappa B pathway

OBJECTIVE:To assess the effect of Jianpi Huazhuo Tiaozhi granules(JHTG)on oxidative stress damage to macrophages and explore the relationship between the levels of this damage and the nicotinamide adenine dinucleotide phosphate oxidase(NOX)/reactive oxygen species(ROS)-nuclear transcription factor kappa B(NF-κB)signaling pathway.METHODS:Macrophages cultured in vitro were divided into seven groups:control,model control,inhibitor,positive control,2.5%JHTG,5%JHTG,and10%JHTG.An oxidative stress injury model was established by stimulating macrophages with oxidized low-density lipoprotein.Cell survival and apoptosis were detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide and flow cytometry assays,respectively.Malondialdehyde and superoxide dismutase levels were detected by enzyme-linked immunosorbent assays,while ROS levels were detected using a fluorescence probe.Proteins and m RNAs associated with the NOX/ROS-NF-κB pathway,including NOX4,p22 phox,inhibitor of NF-κB kinase-α(IKK-α),inhibitor of NF-κB kinase-β(IKK-β),and NF-κB were detected by Western blot and PCR,respectively.RESULTS:After JHTG treatment,there were fewer damaged and apoptotic macrophages,while superoxide dismutase levels were elevated.The JHTG-treated groups also showed reduced ROS levels.The molecular changes following JHTG treatment included decreased expression of NOX4 and p22 phox at the protein level and decreased IKK-α,IKK-β,and NF-κB expression at the m RNA level.All of these effects were correlated with the JHTG concentration.CONCLUSION:These results demonstrated that the molecular mechanism underlying the antioxidant activity of JHTG in macrophages is through inhibiting the NOX/ROS-NF-κB pathway.