| BackgroundPulmonary fibrosis (PF) has always been the end of diverse interstitial lung diseases,and the pathogenesis of PF is complex. Smoking,drug, infection,environment and so on can induce the disease,but the mechanism of PF is still unknown.PF has a high fatality rate and poor prognosis, therefore an effectived therapeutic is needed imperiously.It was confirmed that mRNA expression of EGFR was increased significantly in BLM induced PF in rat. Madtes’s research demonstrated that the activity of EGFR was coherent with BLM induced PF in mice, because it’s not easy happen to PF in mice with EGFR gene mutation. It was supposed that EGFR played a key role in the development of PF. Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), which principally acts on the tyrosine kinase active area that inhibits the EGFR downstream signal. Gefitinib is the representative drug which is generally used for the targeted therapy of non-small cell lung cancer. Related research found that gefitinib could increase risk of PF, but recent II phase gefitinib clinical trials in Japan announced that there were no differences for incidence of PF between patients who use gefitinib and other chemotherapeutics, suggesting that gefitinib might not be the causation of PF.Our privious research find that large dose of gefitinib would not induce PF in mice.Meanwhile William’ research displayed that gefitinib could inhibit the activation of EGFR in TNF-α-transgenes mice to alleviate PF. Rho’research found that alveolar epithelial cell which was not insensitive to EGFR-TKI readily happen EMT.So we presume that EGFR-TKI could inhibit the development of PF.The pathomechanism of BLM inducing PF was local oxidative damage in lung tissue, therefore we suspected that EGFR-TKI might decrease the oxidative stress to alleviate the PF.The unbalance of oxidation and anti-oxidant palys an important role in the development of PF. The activation of EGFR could increase the expression of NADPH Oxidase (Nox) by downstream Erkl/2and ATF-1signal transduction to aggravating the oxidative damage. On one hand, excessive oxidative stress induces oxidative injuries immediately, on the other hand, it increases the activation of EGFR and downstream Akt/Erk signal transduction, increasing the expression of Nox.Oxidative stress and excessive expression of EGFR are interactive, and they aggravate the fibrosis collectively. The safety and efficacy of N-acetylcysteine (NAC) is accepted universally in PF, but the effect of NAC alone is limited, and a large dose or combination with glucocorticoids and cytotoxic drug is always needed,therefore the toxiferous adverse effect is hard to avoid. Seeking a more appropriate way to apply anti-oxidant therapeutics is necessary.Basing on that oxidative damage and excessive expression of EGFR are two ker points in lung fibrosis, we suppose that EGFR-TKI would inhibit the oxidative stress and abnormal regeneration, to alleviate the PF. If associating EGFR-TKI with antioxidant NAC to block the two key points simultaneously, would further inhibit the oxidative stress, then inhibit the development of PF. Our experiments adopt the method of intratracheal aerosol inhaled by MicroSprayer atomization devices to optimize the PF mice model. The purpose of this study is to examine the role and mechanism on oxidative stress of EGFR-TKI gefitinib in PF mice, then further evaluates the feasibility and effect when associating gefitinib with NAC deeply.Method1Optimizing a BLM induced mice model of PF by adopting MicroSprayer atomization devices.Mice were send to three groups,including intratracheal aerosol inhaled group(ITA group),intratracheally injected group(ITI group) and control group.ITA group mice received intratracheal aerosol inhaled of BLM(3mg/kg,100μL),while ITI group received intratracheally injected of BLM.Control group mice underwent no BLM challenge.Mice were sacrificed on14d after BLM treatment.Mice’s lung tissue was harvested. The concentration of hydroxyproline (HYP) was detected by the method of colorimetry.The lung was dissected and fixed in10%buffered formalin.After embedding in paraffin,the sections were evaluated by HE and Masson’s trichrome respectively.Lung injury and fibrosis grading were performed using Mikawar and Ashcroft scoring systems. At the same time, Evans Blue instead of Bleomycin was intratracheal aerosol inhaled or intratracheally injected to observe liquid distribution in lung over all.2Gefitinib attenuated lung oxidative damage and fibrosis in PF miceOptimize the PF mice model by adopting MicroSprayer atomization devices. Mice were divided into three groups,such as Control group,BLM group,BLM+gefitinib group.After BLM treatment,mice was given gefitinib orally. Control group mice received noamal saline intratracheal aerosol inhaled. Mice were sacrificed on14d after BLM treatment. The lung tissue was harvested to measure the concentration of tissue HYP.Lung injury and fibrosis grading were performed using Mikawar and Ashcroft scoring systems.The concentration of MDA and T-AOC were detected by colorimetric method in serum.Total protein was extracted from lung of mice to detecte the phosphorylation EGFR by western blot.3Associating gefitinib with NAC attenuated lung oxidative damage and fibrosis in PF miceOptimize the PF mice model adopting MicroSprayer atomization devices. Mice were divided into five groups,such as Control group,BLM group,BLM+gefitinib group, BLM+NAC group and BLM+NAC+gefitinib group.After BLM treatment,mice was given gefitinib or/and NAC orally. Control group mice received noamal saline intratracheal aerosol inhaled. Mice was sacrificed on14d after BLM treatment. The concentration of MDA and T-AOC were detected by colorimetric method in serum.Total RNA and protein were extracted from lung of mice.The gene expression of Nox1/2/4were detected by PCR. The protein expression of Nox1/2/4and phosphorylation EGFR were detected by western blot respectively.4Associating gefitinib with NAC decreased the activation of EGFR and downstream Akt/MAPKs signal transductionMice were treated with BLM following the procedure mentioned in "Associating gefitinib with NAC attenuated lung oxidative damage and fibrosis in PF mice".Total protein was extracted from lung of mice.The phosphorylation and total protein expression of EGFR, Akt,Erk1/2,p38and JNKwere detected by Western blot.Results1Optimizing a BLM induced mice model of PF by adopting MicroSprayer atomization devices.1.1The concentration of HYP in lung tissue in ITA group and ITI group were significantly higher than that in control group.The concentration of HYP in ITA group increased after BLM treament than that in ITI group 1.2HE trichrome for mice lung revealed that all lobe of lung had a average degree of lung injury in ITA group.but lung local injury was serious and the lung architecture was destroyed apparently in ITI group.The lung injury score in ITA group and ITI group were higher than that in control group.The lung injury score in ITA group was similar to that in ITI group. Uneven degree of lung injury was seen in every different lobe of lung in ITI group, but not in ITA group.1.3Masson’s trichrome for mice lung revealed that all lobe of lung had a average degree of lung fibrosis in ITA group, but in ITI group airway collagen deposition was serious in local lung tissue which the lung architecture could not be identified.The lung fibrosis scores in ITA group and ITI group were higher than that in control group. The lung fibrosis scores in ITA group was similar to that in ITI group. Uneven degree of lung injury was seen in every different lobe of lung in ITI group, but not in ITA group.1.4After5minutes of treatment with Evans Blue in mice, Evans Blue was dispersed distribution in ITA group, but was regional and plaquelike in ITI group.2Gefitinib attenuated lung oxidative damage and fibrosis in PF mice2.1The concentration of HYP in BLM group was apparently higher than that in control group.After gefitinib treatment the concentration of HYP was markedly decreased.2.2The scores of lung injury and fibrosis in BLM group were apparently higher than that in control group. After gefitinib treatment the scores of lung injury and fibrosis were markedly decreased.2.3The phosphorylation EGFR expression in BLM group was apparently higher than that in control group.After gefitinib treatment the phosphorylation EGFR was markedly decreased.2.4The concentration of MDA in BLM group was apparently higher than that in control group,the ability of total anti-oxidant was lower than that in control group.After gefitinib treatment the concentration of MDA was markedly decreased and T-AOC was increased.3Associating gefitinib with NAC attenuated lung oxidative damage and fibrosis in PF mice3.1The concentration of HYP in BLM group was apparently higher than that in control group.After NAC,gefitinib and NAC+gefitinib treatments the concentration of HYP were markedly decreased.Its concentration was significantly lower in BLM+NAC+gefitinib group than that in BLM+NAC group and BLM+gefitinib group.3.2The scores of lung injury and fibrosis in BLM group were apparently higher than that in control group.After NAC,gefitinib and NAC+gefitinib treatments the scores of lung injury and fibrosis were all markedly decreased.The scores in BLM+NAC+gefitinib group were significantly lower than that in BLM+NAC group and BLM+gefitinib group.3.3The serum concentration of MDA in BLM group was apparently higher than that in control group, the ability of total anti-oxidant was lower than that in control group.After NAC, gefitinib and NAC+gefitinib treatments the serum concentration of MDA were markedly decreased and T-AOC were increased.There were no differences among the three treatment groups.3.4The tissue genes transcription of Nox1/2/4in BLM group were apparently higher than that in control group.After gefitinib and NAC+gefitinib treatments the genes transcription of Nox1/2/4were markedly decreased. Nox4gene transcription in BLM+NAC+gefitinib group was more lower than that in BLM+gefitinib group, and there were no differences for Nox1/2genes transcription between the two treatment groups. The Nox1/2/4genes transcription in BLM+NAC group were similar to that in BLM group. 3.5The tissue protein expression of Noxl/2/4in BLM group were apparently higher than that in control group.After gefitinib and NAC+gefitinib treatments the protein expression of Noxl/2/4were markedly decreased. Nox4protein expression in BLM+NAC+gefitinib group was more lower than that in BLM+gefitinib group, and there were no differences for Noxl/2protein expression between the two treatment groups. Nox4protein expression in BLM+NAC group was lower than that in BLM group, and Noxl/2protein expression were similar to that in BLM group.4Associating gefitinib with NAC decreased the activation of EGFR and downstream Akt/MAPKs signal transduction4.1The tissue protein expression of p-EGFR in BLM group was apparently higher than that in control group.After gefitinib and NAC+gefitinib treatments the expression of p-EGFR were markedly decreased, and with no difference between the two group.The expression of p-EGFR in BLM+NAC group was simlar to that in BLM group.4.2The tissue protein expression of p-Akt in BLM group was apparently higher than that in control group.After NAC, gefitinib and NAC+gefitinib treatments the expression of p-Akt were markedly decreased.It was lower in BLM+NAC group and BLM+NAC+gefitinib group than that in BLM+gefitinib group, and with no difference between BLM+NAC group and BLM+NAC+gefitinib group.4.3The phosphorylation protein expression of Erkl/2,p38and JNK in BLM group were apparently higher than that in control group.After gefitinib and NAC+gefitinib treatments the phosphorylation protein expression of Erkl/2,p38and JNK were markedly decreased. P-Erkl/2protein expression in BLM+NAC+gefitinib group was more lower than that in BLM+gefitinib group, and there were no differences for p-p38and p-JNK between the two treatment groups. The phosphorylation protein expression of Erkl/2in BLM+NAC group was lower than that in BLM group, and p-p38and p-JNK protein were similar to that in BLM group.ConclusionGefitinib and gefitinib associating with NAC could inhibit the EGFR activation and its downstream signal transduction with decreasing the related Nox gene expression, alleviating the oxidative stress, depressing the architecture cell injuries, abnormal regeneration and collagen deposition Subsequently, meanwhile associating gefitinib with NAC could further decrease the Nox4expression compared with gefitinib or NAC alone, alleviating the development of PF utmostly. Associating EGFR-TKI with antioxidant provided a new pathway with better target, less adverse effect and more safety for PF clinical therapy. |
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