| Objectives:PM2.5 is one of the main risk factors for global diseases.It is reported that polycyclic aromatic hydrocarbons(PAHs),the main component of man-made PM2.5,are related to the morbidity and mortality of respiratory diseases.Airway epithelium is the first barrier of host defense between air and lung tissue,and it is extremely vulnerable to harmful effects such as cigarette smoke or PM2.5.Autophagy is a highly conservative "self-eating" process,and excessive self-degradation is harmful.Under various pathophysiological conditions,the abnormal level of autophagy may be related to programmed cell death(apoptosis)activated by the process of cell selfdigestion.There are evidences that inhibiting dysregulated autophagy can prevent the damage caused by fine particles.Hyperoside,also known as quercetin-3-O-β-Dgalactoside,is a flavonoid glycoside compound extracted from hypericum or hawthorn,which plays a protective role by regulating autophagy.However,at present,whether Hyperoside has a protective effect on PM2.5-induced lung injury and what mechanism it does need to be further studied.Methods:This experiment intends to study the protective effect and mechanism of Hyperoside in Beas-2b cells and PM2.5-induced lung injury in mice.In vitro,we prepared and compared the cytotoxicity of the water solvent-extractable fraction of SRM1649b(W-PMs)and the organic solvent-extractable fraction of SRM1649b(OPMs)to Beas-2b cells,and then used O-PMs as a stimulus in Beas-2b cells.Observe the molecular mechanism of cell damage and the mechanism of Hyperoside playing a protective role in regulating autophagy and apoptosis.In this process,molecular biology methods such as CCK-8 and Western Blot methods are mainly used.On the basis of in vitro experiments,we further studied the protective effects of Hyperoside on autophagy deregulation and inflammatory damage in the PM2.5-induced lung injury model.AMPK activator was been used for in-depth study of its molecular mechanisms that regulate autophagy and apoptosis.In this process,molecular biology methods such as enzyme-linked immunosorbent assay(ELISA)and western blotting(Western Blot)are mainly used.Results:Part 1:1.In vitro,O-PMs have stronger cytotoxicity,O-PMs can over-activate autophagy and apoptosis.It is shown that the levels of autophagy-related proteins(beclin-1,p62,atg3 and LC3Ⅱ)and apoptotic proteins(parp,bax,caspase 3)are upregulated in a dose-dependent manner.The activation of AMPK/mTOR pathway by O-PMs may be the upstream of its overactivation of autophagy.Hyperoside can inhibit O-PMs-induced cytotoxicity,autophagic protein overexpression,autophagosome accumulation,apoptosis,and P-AMPK expression,and up-regulate pmTOR expression.It is worth noting that 3-MA and Compond C can enhance the therapeutic effect of Hyperoside,while AICAR has a weakened effect.2.In vivo,Hyperoside significantly improved the pathological changes of lung tissue induced by PMs in mice,and inhibited the exudation of inflammatory cells(neutrophils)and cytokines(TNF-α and IL-6)secretion in BALF,reduced the expression of autophagy-related proteins(beclin-1,p62,atg3 and LC3Ⅱ)and apoptotic proteins(parp,bax and caspase-3)in lung tissue,and increased the expression of bcl-2.This effect may be closely related to its inhibition of AMPK/mTOR pathway.After the application of AICAR,the effects of Hyperoside on reducing PMs-induced pathological changes in lung tissue,inflammatory cells in BALF,and inhibiting the secretion of cytokines(TNF-α and IL-6)were significantly weakened,so as to the expression of LC3Ⅱ and caspase-3.It indicates that Hyperoside modulated autophagy and apoptosis through AMPK/ mTOR mediated.Part 2:1.Hyperoside significantly reduced the inflammatory response in the PM2.5-induced COPD exacerbated lung injury model.Compared with the COPD + PM2.5group,Hyperoside reduced the pathological score and the inflammatory cell count in BALF,TNF-α and IL-6 levels.At the same time,Hyperoside reduced the secretion of Muc5 ac and p-creb in the exacerbation of COPD lung injury model induced by PM2.5.2.Hyperoside significantly inhibited the activation of the NF-κB pathway and the NLRP3 inflammasomes,and reduced expressions of the autophagy-related proteins(beclin-1,atg3 and LC3Ⅱ)and apoptotic proteins(bax and caspase 3)in the lung tissue of the COPD exacerbated lung injury model induced by PM2.5.Conclusion:In summary,this study proved the protective effect of Hyperoside on PM2.5-induced lung injury,the exacerbation of COPD lung injury and the molecular mechanism of Hyperoside that inhibited the overexpression of autophagy-related proteins and apoptotic proteins.Provide strategies for the treatment of PM2.5-induced and aggravated respiratory diseases. |
