| Objective1To investigate the mechanism of macrophage receptor with collagenousstructure (MARCO) induced alveolar macrophage (AM) mitochondrial apoptosis bydetecting levels of reactive oxygen specises (ROS) and mitochondrial membranepotential (MTP) in AM, MARCO, cytochrome C (CytC), cysteinyl aspartate specificprotease-9(Caspase-9), Caspase-3, B cell lymphoma/lewkmia-2(Bcl-2), Bcl-2assioatedX protein (Bax) in rats’ lung tissue after inhibiting MARCO and mitochondrial apoptosisrelated targets.2To investigate the role of mitochondrial apoptosis in silicosis fibrosis bymeasuring mRNA expression levels of procollagen Ⅰ and Ⅲ and observingpathological changes of rats’ lung tissue.3To investigate the different effects betweenpreventive intervention and treated intervention.Methods176specific pathogen free adult male Sprague-Dawley (SD) rats weighing180-220g were randomly divided physiological saline control group (24rats), silicosismodel group (24rats), N-Acetylcysteine (NAC) group (32rats), cyclosporin A (CsA)group (32rats), Caspase-9inhibitor group (32rats) and polyguanylic acid (PolyG) group(32rats), each group was divided into preventive group and treated group according todifferent intervention time point, both of them were matched with28d prolonged group,rats in all groups but physiological saline control group were intratracheally given silica(50g/L)1ml to establish silicosis model, the physiological saline group was injected withsame dose physiological saline in parallel. Rats in different preventive groups wereadopted different interventions from the day of establishing silicosis model, and theywere sacrificed at28d and56d time point with8rats per time; rats in different treatedgroups were also adopted different interventions after28days from establishing silicosismodel, and they were sacrificed at56d and84d time point with8rats per time, the leftside lung lavage were collected, AMs were separated and purified. ROS contents andMTP changes of AMs were identified by flow cytometry. mRNA expression levels oftype I and type Ⅲ procollagens were measured by real-time PCR. The contents ofMARCO, Bcl-2, Bax, CytC, Caspase-9and Caspase-3were detected by Western blotting.The right side lung tissue was fixed with paraformaldehyde and stained withhematoxylin-eosin (HE). The pathological change and the severity of pulmonary fibrosis was observed under light microscope.Results1Pathological change of rats’ lung tissue in different groups. The lungstructure of physiological saline control group was clear, the alveolar wall was thin,normal alveolar cells can be seen in the alveolar corner, but there was also slightinterstitial inflammation. Focal interstitial inflammation with exudate which wascomposed by lymphocytes and macrophages was obviously found in the lung tissue ofsilicosis model group, what’s more, there were typical silicotic nodules formed byfibroblast cells, macrophages and collagen, different degrees of alveolar hyperplasiawere also observed, especially cells in alveolar wall near the nodular lesion were moresignificant, the number of silicotic nodule increased with time going by and somesilicotic nodules even mixed together. There were no cell nodules or silicotic nodulesformation in lung tissues in NAC group, CsA group, Caspase-9inhibitor preventivegroup or PolyG group, some sporadic cell nodules can be seen in Caspase-9inhibitorgroup. However, there were widely distributed inflammatory cells infiltration in allgroups, and it alleviated in different levels with time prolonged, especially in PolyGgroup.2Comparison of AM mitochondrial apoptosis related proteins in different groups.The contents of MARCO, CytC, Caspase-9, Caspase-3, Bax and Bcl-2were of nostatistical significance among28d,56d and84d time point after establishing silicosismodel (P>0.05), Bcl-2showed an decreasing trend with time extending in silicosis modelgroup, but other proteins increased (P>0.05). MARCO, CytC, Caspase-9, Caspase-3andBax in NAC group and CsA group (both preventive and treated) were all higher thanthose in the same time point physiological saline control group, but lower than the ones inthe same time point silicosis model group, however, the changes of Bcl-2showed acontrary (P<0.05). There was no statistical significant of CytC in the comparison betweenCaspase-9inhibitor group and the same period silicosis model group at28d and56d timepoint after establishing silicosis model, other variables showed a same trend as NACgroup (P<0.05). Comparing with the corresponding physiological saline control group,the differences of the contents of MARCO and CytC in PolyG preventive group at28dtime point after establishing silicosis model were of no statistical significance, besides,the differences of MARCO, CytC, Caspase-3and Bax at56d time point after establishingsilicosis model were also of no statistical significance (P>0.05), other variables showed asame trend as NAC group (P<0.05).3AM ROS and mitochondrial transmembrane potential changes in different groups. The content of AM ROS and mitochondrialtransmembrane potential in physiological saline control group were all lower than theircorresponding silicosis model group at28d,56d and84d time point (P>0.05), and both ofthem in NAC or CsA group (preventive and treated) were higher than the same time pointphysiological saline control group but lower than the same time point silicosis modelgroup (P<0.05). The difference of AM ROS positive rate was no statistical significancebetween Caspase-9inhibitor preventive group than the corresponding silicosis modelgroup at28d time point after establishing silicosis model (P>0.05), other variablesshowed a same trend as NAC group (P<0.05); similarly, the difference of AM ROSpositive rate was no statistical significance between Caspase-9inhibitor preventive groupthan the corresponding silicosis model group at28d time point after establishing silicosismodel (P>0.05), other variables showed a same trend as NAC group (P<0.05).4Analysisof typeⅠand type Ⅲ procollagen mRNA expression levels in different groups. ThemRNA expression level of procollagenⅠincreased gradually in physiological salinecontrol group at28d,56d and84d time point, mRNA expression level of procollagen Ⅲwere of no statistical significance (P>0.05); typeⅠand type Ⅲ procollagen mRNAexpression levels in silicosis model group showed an increasing trend along with timeextending (P<0.05); the two variables in NAC, CsA and Caspase-9preventive groupwere all higher than the same time point physiological saline control group, lower thanthe corresponding silicosis model group (P<0.05); comparing with the correspondingphysiological saline control group, typeⅠand type Ⅲ procollagen mRNA expressionlevels in PolyG preventive group were of statistical significance (P>0.05), but lower thansilicosis model group (P<0.05); both of them in PolyG treated group showed a sametrend as NAC group (P<0.05).5The effects of different intervention time point on AMmitochondrial apoptosis related variables in different groups. The comparison between allpreventive groups at28d time point and all treated groups at56d time point showed thatthe content of MARCO, CytC, Caspase-9, Caspase-3, Bax and Bcl-2, type Ⅲprocollagen mRNA expression level, the positive rate of AM ROS and mitochondrialtransmembrane potential in physiological saline control group were of no statisticallsignificance (P>0.05); the positive rate of AM ROS and mitochondrial transmembranepotential in silicosis model group were of no statisticall significance (P>0.05), the contentof Bcl-2increased in former, other varibles decreased (P<0.05); as for NAC group, CsA group and PolyG group (preventive and treated), the content of Bcl-2all increased, othervaribles decreased (P<0.05); the mRNA expression level of typeⅠand type Ⅲprocollagen, AM mitochondrial depolarization rate of Caspase-9inhibitor group were ofno statisticall significance (P>0.05), the content of Bcl-2all increased, other variblesdecreased (P<0.05).Conlusions1Variables which include ROS, MTP, MARCO, CytC, Caspase-9,Caspase-3and Bax/Bcl-2showed an descending trend in different degrees after inhibitingMARCO and mitochondrial apoptosis related targets, suggesting that the combination ofMARCO and silica dust plays an important role in mitochondrial apoptosis pathwaystarting.2mRNA expression levels of procollagen Ⅰ and Ⅲ, pathological changes ofrats’ lung tissue in different groups alliaveted after inhibiting MARCO and mitochondrialapoptosis related targets, indicating that the starting of mitochondrial apoptosis pathwayplays an important role in pulmonary fibrosis.3The development of pulmonary fibrosiscan be control when preventive intervention were adopted which including the inbition ofthe combination of MARCO and silica dust by PolyG and mitochondrial apoptosissignaling inhibitor, what’s more, it can be reversed when treated intervention wereadopted, and the effect was more obvious when the uptream target was blocked in earlytime. |
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