The Effects Of Cigarettes Extract On The Secretion Of MMP-9、TIMP-1in Human Alveolar TypeⅡ Epithelial Cells

Objective：According to research，cigerette smoking extracts including asmany as thousands of harmful toxic substances，are common risk factors of avariety of pulmonary diseases. Also some studies put forward that smokingmay be closely related to the pathogenesis of idiopathic pulmonary fibrosis(IPF), but the specific mechanism is not fully clear. Cigarette smoke contains avariety of inflammatory mediators，including antioxidants (coxidant) andproteolytic enzymes (such as proteases). These inflammatory mediators canultimately lead to protease/resistance protease imbalances that involved in thepathogenesis of many lung disease. In recent years, in the formation ofpulmonary fibrosis mechanism protease/resistance protease imbalancegradually become the research hotspot and focus. Among them, MMP-9as akind of important matrix metalloproteinases plays an important role in manyphysiological and pathological process such as the extracellular matrixdegradation and the lung tissue structure reshape and participate in a variety oflung disease, its active regulated by the specific inhibitors (TIMP-1). Underthe action of various factors stimulation, the proportion and the interactionhappens disorder, can lead to the extracellular matrix degradation andextracellular protein deposition, the lung tissue reconstruction and otherpathological changes,which involved in the pathogenesis of pulmonaryfibrosis.This experiment aims to study the cigarette extract on in vitro culturedhuman A549cells secrete matrix metalloproteinases9and its tissue inhibitorsand the effect of explore cigarette extract as a risk factor for possiblemechanisms involved in the process of pulmonary fibrosis.Methods：The human A549cells are cultivated in vitro, the exponentialphase cells are used in the experiment.Two cigarettes are continuously and evenly burned by vacuum extractor，comes from50ml syringe filters throughmodification,.The smoke is inhaled into1640medium20ml and made intosuspension. Two cigarettes are burnt out within15minutes.The suspension isfully shaked and dissolved, the pH is adjusted about7.4by NAOH.Thesolution is used in experiments in30minutes after filtered by0.22u mdiameter,then removed bacteria and impurity.Human A549cells are intervenedwith different concentrations for24hours, and experiments can be dividedinto four groups according to the different concentration:: The control group,group A(2.5％CSE interfered group), group B(5％CSE interfered group),group C(10%CSE interfered group). And collect group C intervened A549cells respectively0h,3h,6h, h,12h,48h of culture supernatant. Themorphology change of four groups cells is observed by Optical inversionelectron microscope after intervened24hours; Enzyme linked immunosorbentassay (ELISA) method is used to detect the concentration of MMP-9andTIMP-1in supernatant fluid; Collected separately intervention group after24h of culture cell, extract the protein in the cell, check the protein concentrationwith BCA method, detect the cells the expression of MMP-9and TIMP-1protein with Western Blot method.Results:1The changes of cell morphologyThe electronic microscope observes the cell morphology after traing24hours. The control performance for stick wall, flat spindle cell growth, cellmorphological rules and cells thrive. After different concentration ofCSE intervention, A, B, C three group’s cell morphology are diffrent formthe control group. Presents the higher the concentration of intervention, theworser the characteristics of cell growth situation. Main show is the decreasein the number of cells, shrink volume, cell adherent ability to drop, more spacebetween cells.As for group C’s(10%CSE interfered group) cell morphologicalchanges more significant than the control group. Most cells have retractionand cell density reduced obviously, it become intracellular granular materialincreased obviously and cell adherent ability greatly reduced. 2The changes of A549cells secrete MMP-9after CSE interventionAccording to t he ELISA results，the control group’s content of MMP-9was(1.70±0.12ng/ml). The A, B, C three group’s concentration showed a trendof rising, three groups were statistically significant compared with controlgroup (P <0.05), and the comparison between A, B, C three groups are alsostatistically significant (P <0.05). Group C at the same time, to observe theeffect of different time the expression of MMP-9we can find that, as the roleof the extension of time its expression showed a trend of rising. In0h when itsconcentration is(1.76±0.31ng/ml),at the same time the48h’ concentration isup to (9.29±0.76ng/ml).After statistical analysis can be concluded that the3h-48h group compared with0h were statistically significant (P <0.05).3The changes of A549cells secrete TIMP-1after CSE interventionAccording to the ELISA results，the control group’s content of TIMP-1was (2.20±0.31ng/ml). The A, B, C three groups concentration showed atrend of rising, three groups were statistically significant compared withcontrol group (P <0.05), and the comparison between A, B, C three groupsare also statistically significant (P <0.05). Group C at the same time, toobserve the effect of different time the expression of TIMP-1we can find that,as the role of the extension of time, its expression showed a trend of rising. In0h when its concentration is (2.52±0.41ng/ml), at the same time the48h’concentration is up to (16.31±0.96ng/ml).After statistical analysis can beconcluded that the3h-48h group compared with0h were statisticallysignificant (P <0.05).4The changes of A549cells produce TIMP-1after CSE interventionELISA results showed in A549cells are cultured after24hours thecontrol of MMP-9/TIMP-1value was (0.77±0.05). After the CSEintervention, the A, B, C three group’s values are lower than the controlgroup.The three groups are compared with the control group had statisticalsignificance (P <0.05), but both two comparison between groups have nostatistical significance (P>0.05). Observes the changes of MMP–9/TIMP-1in group C in different intervention time we find that,12h,24h and48h compared with0h with statistical significance (P <0.05), and3h,6h and9hcompared with0h has no obvious statistical significance. Then3h comparedwith0h,6h have no obvious statistical significance,but it did with9h,12h,24h,48h (P <0.05).5The expression of MMP-9and TIMP-1protein in each groupWestern Blot showed that the expressions of MMP-9and TIMP-1proteinin CSE-interfering cells were higher than those without CSE-interfering cells.Conclusions:1. CSE can cause the human alveolar type Ⅱ epithelial cells secreteMMP-9and TIMP-1increases.2. CSE can induce the human alveolar type Ⅱ epithelial cells whichexpress MMP-9in a time-dependent and concentration-dependent manner.3. CSE can cause the imbalance of MMP-9/TIMP-1in the humanalveolar type Ⅱ epithelial cells, and showed the trend of the rising afterdownward trend.4. CSE can cause the unbalance of MMP-9/TIMP-1in the human alveolartype Ⅱ epithelial cells,and its as a risk factor may involved in the formationprocess of cigaretting related pulmonary interstitial fibrosis.