Study On Anti-inflammatory And Antioxidant Mechanism Of Anti-asthmatic Catecholic Alkaloids BTQ And ITQ From Portulaca Oleracea Based On NF-κB/MAPK Signaling Pathways

Portulacra oleracea L.is an annual succulent herb belonging to the Portulaca genus of the Portulaceae family,which is a potherb and also a medicinal plant.As recorded in the Chinese Pharmacopoeia,P.oleracea can clear heat and detoxify,cool blood,stop bleeding,and stop dysentery.In addition to various pharmacological effects such as antibacterial,anti-inflammatory,and antioxidant,ancient Iranian medical books indicated that P.oleracea can also treat asthma.Clinical,animal and in vitro experiments have also proven that P.oleracea has anti-asthmatic effects,however,its anti-asthmatic pharmacodynamic substances are not yet clear.P.oleracea mainly contains alkaloids,organic acids,and polyphenols.Catecholic alkaloids are characteristic chemical constituents in P.oleracea,with a large number(60)and diverse structural types.In view of the fact that the activation of β2-AR to relieve bronchospasm and anti-inflammation are the two major strategies for the treatment of asthma,our research group carried out a preliminary in vitro bioactivity screening and found that a number of catecholic alkaloids in P.oleracea have different degrees of activation of β2-AR and anti-inflammatory dual functions,indicating these alkaloids might be the anti-asthmatic substances in P.oleracea.Among them,1-benzyl6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline(BTQ)and 6,7-dihydroxy-1-isobutyl1,2,3,4-tetrahydroisoquinoline(ITQ)are β2-AR strong agonist or selective agonist.Moreover,animal experiments have shown that BTQ and ITQ have the effects of relaxing the trachea of guinea pig and alleviating OVA or LPS-induced airway inflammation in mice models,but their anti-inflammatory molecular mechanisms are unknown.NF-κB/MAPK signaling pathways are key signal transduction pathways that regulate inflammation and oxidative stress,which are involved in airway inflammation and epithelial cell injury in asthma.In view of our previous discovery that tetrahydroisoquinoline alkaloids BTQ and ITQ in P.oleracea have tracheal diastolic and antiinflammatory dual functions,this paper further explored the anti-inflammatory and antioxidant effects of BTQ and ITQ and their molecular mechanisms at the cellular level,mainly based on the NF-κB/MAPK signaling pathways.The research content of this paper includes the following two parts:1.Effects and underlying mechanisms of BTQ and ITQ on LPS-induced inflammation in RAW 264.7 macrophages.Objective:To investigate the effects of BTQ and ITQ on LPS-induced inflammatory response in RAW 264.7 murine macrophages and their molecular mechanisms.Methods:The levels of inflammatory mediators NO,TNF-α and IL-1β were measured by colorimetric method or enzyme linked immunosorbent assay.The cell viability was measured by MTT assay.The mRNA levels of TNF-α,IL-1β,IL-6,iNOS and COX-2 were detected by RT-PCR.The expression levels of iNOS,COX-2,and NF-κB/MAPK signaling pathways-related proteins were determined by Western blot analysis.The cellular localization of NF-κB p65 was detected by immunofluorescence assay.Results:Compared with the LPS-induced inflammatory cellular model.BTQ and ITQ inhibited the NO production in a dose-dependent manner,with EC50 values of 67.91 and 80.40μM,respectively.BTQ and ITQ(50～100 μM)significantly reduced the mRNA and protein expression of TNF-α,IL-1β,IL-6,iNOS and COX-2.BTQ and ITQ(100 μM)significantly increased the level of IκBα,an inhibitor protein of NF-κB,and prevented IκBα degradation,and both of them significantly inhibited the phosphorylation of IκBαand p65,and reduced the nuclear translocation of NF-κB p65.Moreover,BTQ and ITQ(25～100 μM)could significantly inhibit the JNK phosphorylation in MAPK signaling pathway,and BTQ also significantly reduced the phosphorylation level of p38 MAPK,however,both of them had no effect on ERK activation.Conclusion:It was clarified for the first time that BTQ and ITQ can inhibit LPS-stimulated inflammation in RAW 264.7 murine macrophage cells,and their anti-inflammatory mechanisms are related with the inhibition of the activation of NF-κB and MAPK signaling pathways.2.Effects and underlying mechanisms of BTQ and ITQ on sodium arseniteinduced oxidative damage in Beas-2B human lung epithelial cells.Objective:To study the effects of BTQ and ITQ on sodium arsenite-induced oxidative damage in Beas-2B human lung epithelial cells and their molecular mechanisms.Methods:The in vitro DPPH·and ·OH scavenging activites of the compounds,GSH content and the activities of CAT and T-SOD were determined by colorimetric method.The cell viability was determined by MTT assay.The proteins expression of MAPK signaling pathway were determined by Western blot analysis.Results:The scavenging capacity of BTQ against DPPH·(EC50=34.20μM)was slightly stronger than ITQ(EC50=53.47μM),and both of them were slightly weaker than VC(EC50=26.94μM).The ability of BTQ to scavenge ·OH was also slightly higher than ITQ,and their EC50 values were 447.85 and 577.17 μM,respectively,weaker than VC(EC50=213.59 μM).BTQ(50～100 μM)significantly increased the cell viability of Beas-2B cells injured by sodium arsenite in a dose-dependent manner,while ITQ had no protective effect.BTQ and ITQ(100 μM)not only significantly increased the GSH content and T-SOD activity in normal Beas-2B cells,but also significantly enhanced the GSH content,CAT and T-SOD activity in Beas-2B cells damaged by sodium arsenite,thereby alleviating the intracellular oxidative stress.In the MAPK singaling pathway,BTQ at 100 μM significantly inhibited the phosphorylation of JNK and ERK in sodium arsenite-injured cells,but had no effect on p38 MAPK phosphorylation.ITQ at 100 μM only down-regulated the phosphorylation level of ERK,whereas it had no effect on JNK and p38 MAPK activation.Conclusion:It was discovered for the first time that BTQ plays a protective role in Beas-2B cells injured by sodium arsenite through inhibition of oxidative stress,as well as JNK and ERK phosphorylation in MAPK signaling pathway.In summary,BTQ and ITQ alleviated LPS-induced inflammation in RAW 264.7 macrophage cells and sodium arsenite-induced oxidative stress injury in Beas-2B lung epithelial cells by regulating NF-κB/MAPK signaling pathways.The abovementioned results provide a scientific basis for elucidating the anti-asthmatic substances of P.oleracea and their anti-inflammatory and antioxidant mechanisms.