The Mechanism Of The Cigarette Smoke Extract Effects On Pulmonary Fibrosis And The Intervention Effect Of The Reduced Glutathione

Objective: Cigarette smoke contains many types of harmful chemicals. Itis one of the major causes of various diseases. It is also recognized as animportant risk factor of pulmonary interstitial fibrosis. Antioxidants and freeradical in cigarette smoke can generate oxidative stress through variouschannels. A lot of evidence shows that oxidative stress in the pathogenesis ofpulmonary fibrosis plays an important role. Whenever in the acute phase or inthe remission phase,all the patients had an oxidation/antioxidant imbalance.However, the molecular mechanism is still unclear. There is no effectiveclinical treatment in pulmonary fibrosis. Recently the antioxidant treatmentarouses a strong concern of many scholars. Studies have shown that thereduced glutathione (GSH) is an important reducing agent in human body,which has tioxidant properties and could eliminate free radicals and otherreactive oxygen species, protecting the lung tissue from oxidative damage.Therefore, this experiment is based on a model of A549cells which derivefrom alveolar type Ⅱcells. We stimulate A549cells in vitro with differentconcentrations of cigarette smoke extract (CSE), observing the morphologicalchanges in the growth of cells and the inhibitions. By detecting the oxidationindexes and the degree of fibrosis index, we discuss the possible mechanismof smoking on pulmonary fibrosis and the reverse effect of reducedglutathione on pulmonary fibrosis caused by smoking.Methods: The A549cells derived from alveolar type Ⅱcells werecultured in vitro. And the cells in logarithmic growth phase were tested.Following the method of Carp and Janoff[1]，we prepared the cigarette extract(CSE) from a special device. By pumping two filter cigarettes which removethe holder, we integrated the smoke in medium that excluding1640serums. The pH value was adjusted and the bacteria and large particles were removed.Then it was diluted into solutions with different concentrations, which wereused to test in30minutes. In this study, we attempted to apply the nutrientsolution of cigarette extract with the concentration of0%,2.5%,5%,7.5%,10%and12.5%onto the cells. The cell viability was measured by MTTmethod. And the effect of CSE on cell proliferation forms was detected underphase contrast microscopy. Based on the above results, the appropriateconcentration (0%,2.5%,5%,7.5%) were selected and divided reasonablyinto groups for further study on the related factors. The experiments weredivided into blank control group,2.5%CSE group,5%CSE group,7.5%CSEgroup and7.5%CSE+GSH group (GSH1mmol/L). We collected the superna-tants of the blank control group and the intervention groups24h hours after thecell culture. The superoxide dismutase (SOD) activity and malondialdehyde(MDA) content of supernatants were measured by colorimetric assay method.The levels of transforming growth factor1(TGF-β1) we detected by ELISAmethod. By observing the variation of the above factors, we explored themechanism of the cigarette smoke extract effects on pulmonary fibrosis andthe intervention effect of the reduced glutathione.Results:1Morphological changes observed on A549cells in solutions of differentconcentrations under microscopyThe result under the inverted microscope showed that the growth densityof A549cell was reduced under CSE. Also the gap between the cells and thenumber of floating cells was increased. The adherent cells also showed aretraction into a rounded posture with shrinking. There exists an increase inthe miscellaneous of the shrinking cells, which shows an obvious morpho-logical difference with good growth condition.2The growth inhibition effect of CSE solutions with different concentrationson the growth of A549cellsBy using MTT assay method, we found that the CSE has a stronginhibitory effect on the growth of A549cells in vitro. The effect is in a time dependent and dose dependent manner. We treated the cigarette smoke extractwith the same concentration under different stimulation time (12h,24h and36h). The result showed that the inhibition rate increases with the reaction time.And there was a significant difference (P <0.01). Also we treated the cigarettesmoke extract with the different concentration（0%、2.5%、5%、7.5%、10%、12.5%）under the same time. The result showed that the inhibition rateincreases with the concentration. And there was also a significant difference (P<0.01). For example, the inhibition rate was reached70.30±2.21%in10%CSE group when the reaction time is24hours. And the inhibition rate wasreached790.10±1.01%in12.5%CSE group when the reaction time is24hours.3The effect of different concentrations of CSE on TGF-β1, MDA, SOD levelsDifferent concentrations of CSE group TGF-β1, MDA was significantlyhigher than the control group. And the difference was significant (P<0.05).Wefound that higher concentrations of CSE caused a higher factor, which is in aconcentration-dependent manner. And the difference was also significant(P<0.05). Different concentrations of CSE SOD activity were lower than thecontrol group. The difference was significant (P<0.05). We found that higherconcentrations of CSE caused a lower activity. And the difference was alsosignificant (P<0.05).4Changes in TGF-β1, MDA, SOD levels after the effects of GSHThe TGF-β1, MDA levels of7.5%CSE+GSH group were significantlylower than the7.5%CSE group.The difference was significant(P<0.05).However, the SOD activity was higher than7.5%CSE group. And thedifference was also significant (P <0.05).Conclusion:1The cigarette smoke extract can inhibit the growth of alveolar epithelialcells. And the effect is in a time dependent and dose dependent manner.2The cigarette smoke can induce alveolar epithelial cells secretingTGF-β1, MDA, which would make the activity of SOD decrease. It caused anoxidative damage and a change of cell repairing after the damage. 3The glutathione can make CSE-stimulated A549cells generate lessMDA and TGF-β1, and more SOD respectively. There could be a reverseeffect of the oxidative damage caused by smoking.