Protective Effect Of AEP On Acute Lung Injury In Mice And Its Anti-Inflammatory Mechanism Through TAK1/NF-κB Pathway

Objective:Acute lung injury(ALI)is a disease characterized by reduced lung volume,reduced lung compliance,and severe ventilation/blood flow ratio imbalance as pathophysiological features,clinically manifested by progressive hypoxemia and respiratory distress.An uncontrolled inflammatory response in the lungs or the whole body is the main pathogenesis of ALI.The efficacy of clinical treatment of ALI with glucocorticoids and cyclooxygenase inhibitors is still unclear.Therefore,searching for effective drugs for the prevention and treatment of ALI is an urgent issue at present.Andrographis paniculata(Burm.F.)Nees is a medicinal plant of Acanthaceae family.Andrographolide,the major active component from the aerial part of this plant,and its analogs were traditionally used for the treatment of pediatric colds and fevers,respiratory bacterial infections,and other diseases in clinical.It has also been proven that they can prevent ALI by inhibiting the inflammatory response.Andrographis diterpene lactone effective part(AEP)contains more than 50% diterpene lactones.Previous study showed that AEP significantly inhibited inflammation in the lung and alleviated lung injury in mice.In recent years,protein ubiquitination modification in regulating inflammation has become a hot spot.It is closely related to the NF-κB activation,and TRAF6-mediated TAK1 ubiquitination is involved in this process.Previous studies on the antioxidant aspects of Andrographolide in Nrf2 ubiquitination have been reported in the literature,but studies revealing the anti-inflammatory mechanisms of Andrographis paniculata lactones from the perspective of ubiquitination have not been reported.Therefore,an in-depth study of the anti-inflammatory mechanism of AEP against ALI lung inflammation based on ubiquitination could provide a solid experimental basis and theoretical foundation to promote its clinical application.1.Protective effect of AEP on the LPS-induced ALI in miceMice were randomly divided into the normal group,LPS group,dexamethasone group(5 mg/kg,Dex),andrographolide group(400 mg/kg,A),and AEP group(200,400 mg/kg,AEP-L,AEP-H).After a 7-day treatment,An ALI model was constructed with LPS to evaluate the effect of AEP on ALI in mice.Results:(1)AEP alleviated the LPS-induced ALI in mice.Compared with the normal group,lung index and lung wet-dry weight ratio were significantly larger in the LPS group.AEP-H and Dex significantly decreased the lung index.AEP-L,AEP-H,A,and Dex all significantly diminished the lung wet-dry weight ratio.Effects of AEP-H and A were comparable.The semiquantitative results of HE staining of lung tissue showed a significant increase in the lung injury score after being stimulated with LPS for 24 h,and this was significantly inhibited by AEP-L,AEP-H,A,and Dex.AEPH was superior to the equivalent dose of A.Myeloperoxidase(MPO)reflects the degree of neutrophil infiltration in tissues.The level of MPO significantly increased in the LPS group.AEP-H,A,and Dex groups significantly decreased the MPO content.The inhibitory effect of AEP-H on MPO was comparable to that of A and Dex.However,AEP-L had no significant effect on MPO.(2)AEP reduced the number of inflammatory cells and the level of inflammatory factors in the BALF of LPS-induced ALI mice.LPS stimulation significantly increased the total number of lymphocytes,macrophages,neutrophils and leukocytes in the mouse BALF compared with those in the normal group.AEP-H and Dex reduced the number of the above cells.The concentration of TNF-α,IL-1β,MCP-1,and IL-6 in the LPS group was significantly increased.Inhibitory effects of AEP-H and Dex on the above four cytokines,and that of A on TNF-α and IL-1β were observed.Moreover,the inhibitory efficacy of AEP-H on the inflammation response was better than A.2.Effects of AEP on the LPS-induced inflammatory response in RAW264.7 cellsThe expression of cytokines at the m RNA and protein levels was detected with Q-PCR and ELISA,respectively.Griess assay was carried out to detect the NO level in the medium.Results:The expression levels of TNF-α,IL-6,IL-1β,and i NOS m RNA were significantly increased after the LPS stimulation.The increase was hindered by AEP(3.5,7 μg/m L)with significance,but not A.Levels of NO,TNF-α,IL-6,and PGE2 were significantly increased in LPS-induced macrophage supernatants.Both A and AEP blocked the increase.AEP(3.5,7 μg/m L)significantly reduced NO levels,and AEP was much better than A.3.Effect of AEP on the TAK1/NF-κB pathwayWestern Blot and immunofluorescence techniques were employed to detect the effects of AEP on the phosphorylation and nuclear translocation of NF-κB.The effects of AEP on TRAF6 and TAK1 interaction and TAK1 ubiquitination were investigated by using immunoprecipitation techniques.Results:(1)The ratio of nuclear p65 and cytoplasmic p65 were significantly increased in the LPS group,and AEP significantly reduced this ratio and was superior to A.Immunofluorescence experiments further verified the effects of AEP on NF-κB nuclear translocation.IκBα phosphorylation levels were significantly upregulated after 6 h of LPS induction,A and AEP significantly reduced IκBα phosphorylation levels.AEP(3.5,7 μg/m L)significantly reduced TAK1 phosphorylation levels after LPS induction,and A had no significant effect on phosphorylated TAK1.Those indicated that AEP can inhibit the enhancement of NF-κB phosphorylation and nuclear translocation by LPS.(2)The total ubiquitination level of TAK1 as well as the K63-linked polyubiquitination level was significantly higher in the LPS group.Those were significantly lowered by AEP,rather than A.Furthermore, AEP inhibited the enhanced interaction of TRAF6 and TAK1 induced by LPS,and A showed no effects.Conclusion:AEP alleviated LPS-induced acute lung injury in mice by inhibiting the pulmonary inflammatory response,and its effect may be achieved by inhibiting TRAF6-mediated ubiquitination of TAK1 and decreasing phosphorylation of TAK1,thereby blocking the activation of NF-κB.The same dose of AEP was more effective than A,and A had no significant effect on the ubiquitination and phosphorylation of TAK1.