Anti-inflammatory Activity Of 20-Hydroxy-3-oxolupan-28-oic Acid And Its Mechanism

The incidence of inflammation-related diseases in people's daily live is increasing,which is seriously plaguing people's life and health.However,non-steroidal anti-inflammatory drugs(NSAIDs)and steroidal anti-inflammatory drugs(SAIDs)can cause adverse reactions such as gastric mucosa,cardiovascular damage,adrenal hyperfunction,and safety issues that cannot be ignored.Therefore,researchers have turned their attention to Chinese herbal medicines with anti-inflammatory effects.The diversity of structure and biological activity makes drug design have unique chemical structure and pharmacological activity.The Chinese Pharmacopoeia describes the Mahonia bealei(Fort.)Carr for the treatment of inflammation and related diseases.Pharmacological studies have shown that alkaloids in their polar parts have anti-inflammatory,anti-bacterial,anti-cancer,blood pressure-lowering and anti-tumor effects.Up to now,there are few studies were carried out on non-alkaloids in this genus.20-Hydroxy-3-oxolupan-28-oic acid(HOA)is a lupane-type triterpene compound extracted from the Mahonia bealei(Fort.)Carr.leaves.Studies have shown that the lupane-type triterpene have good anti-inflammatory effects.The aim of this study was to investigate the anti-inflammatory activity and anti-inflammatory mechanism of HOA on lipopolysaccharide(LPS)-induced RAW 264.7 macrophages.(1)By measuring cell viability and the release of nitric oxide(NO),tumor necrosis factor-?(TNF-?)and interleukin-6(IL-6),it was found that HOA suppressed the release of NO,TNF-?,and IL-6 after macrophage activation.Under inverted fluorescence microscope and laser confocal microscope,it was found that HOA could effectively improve the activation of RAW 264.7 cells stimulated by LPS.(2)Transcriptome sequencing analysis showed that after LPS stimulated RAW 264.7macrophages,the differential genes in C/LPS group were mainly enriched in inflammatory process,immune process and cellular response to interferon beta.After HOA pretreated cells,the differential genes in the LPS/HOA group were significantly enriched in the chemotaxis process,the response to laminar fluid shear stress and the positive regulation ofGTPase activity,and the KEGG results showed that the differential genes were mainly enriched in inflammatory-related NF-?B signaling pathway,cytokine-cytokine interaction,Rap1 signaling pathway,chemokine signaling pathway and Jak-STAT pathway.The RT-qPCR verification results of 15 differential genes associated with inflammatory responses were consistent with the trend of gene expression results in transcriptome sequencing.(3)HOA was able to inhibit the expression of inductible Nitric Oxide Synthase(iNOS),IL-6 and TNF-? mRNA in LPS-stimulated RAW 264.7 cells in a dose-dependent manner by semi-quantitative and real-time quantitative PCR.In addition,immunofluorescence and western blotting assay revealed that HOA inhibited the phosphorylation of upstream signalling molecules including p85,PDK1,Akt,ERK,and JNK,as well as the nuclear translocation of nuclear factor ?B(NF-?B)/p65.Interestingly,HOA had no effect on the LPS-induced nuclear translocation of activator protein 1(AP-1).Taken together,HOA has basic anti-inflammatory activity,and the RNA-Seq sequencing technology to reveal the metabolic pathway in the process of HOA treatment of RAW 264.7 cells.The results of western blot indicate that HOA inhibits the production of cytokine by downregulating iNOS,TNF-?,and IL-6 gene expression via the downregulation of phosphatidylinositol 3-kinase(PI3K)/Akt and mitogen-activated protein kinases(MAPKs),and the inhibition of NF-?B activation.Our findings indicated that HOA could potentially be used as anti-inflammatory agent for medical use.