| Objective Idiopathic pulmonary fibrosis (IPF) is a chronic lung diseaseof unknown cause characterized by alveolitis in the early stage and fibrosis inthe advanced stage, the understanding of it’s pathogenesis remains unkown.Statins are3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductaseinhibitors, which are used to lower serum cholesterol synthesis through theinhibition of HMG-CoA reductase. Besides lipid-lowering, statins also has theeffects of anti-inflammation, antioxidant, anti-platelet aggregation andimproving endothelial function. Previous research has shown that theanti-inflammatory effects of statins can attenuate the fibrosis in the organs likelung, liver, kidney and so on, but its mechanism is still not clear. Krüppel-likefactor4is a transcription factor, with dual function, it is expressed in manytissues, and plays an important role in cell proliferation, differentiation andapoptosis in many physiological activities. Recent studies found thatcardiovascular disease, cancer and inflammatory diseases are closedly relatedto KLF4, and the role of KLF4in the inflammation is mainly manifested bythe regulation of monocyte and macrophage phenotype shifting. To investigatethe effect of KLF4on the inhibition of pulmomary fibrosis through thetreatment of atorvastatin, this study aimed to establish a mouse model ofpulmonary fibrosis by intratracheal instillation of bleomycin, to observe theexpression of KLF4gene and protein in the lung tissues of mice; to give theintervation of atorvastatin in mice with pulmonary fibrosis, to observe thechange of KLF4expression in the lung tissues of mice; and to acquire the cellline with stable KLF4gene silencing through RNA interference (RNAi) inmurine RAW264.7macrophages, to investigate the influence of KLF4inproliferation, apoptosis and the phnotype in this cell line, and then furtherexplore the mechanism of KLF4in inhibition of atorvastatin in experimental pulmonary fibrosis.Part one Dynamic Expression of Krüppel-like factor4in the LungTissues of Mice with Pulmonary FibrosisMethods:1C57BL/6mice were randomly divided into Control group and BLMgroup. Mice in the BLM group were given a single intratracheal injection ofbleomycin (2.5mg/kg), while those in the Control group were injected withisodose physiological saline. Groups were sacrificed on the hour12and theday1,2,3,7,14and28;2Hematoxylin and eosin stain （HE stain） and Masson’s TrichromeStain were used to detect the pathology of alveolar and the deposition ofcellularity and collagen;3Real time-polymerase chain reaction (RT-PCR) was performed toinvestigate the expression of KLF4gene in the lung tissues of mice;4Immunohistochemical technology was performed to investigate theexpression of KLF4protein in the lung tissues of mice.Results:1In the bleomycin-induced pulmonary fibrosis, acute inflammation wasperformed on the day1,2and3, and the inflammation was exacerbated andthe collagen deposition began to be observed on the day7, the architecture ofalveolar was destroyed and the collagen deposition was more obvious on theday14, while the alveolar structure was nearly recovered to normal and theinflammation and collagen deposition were attenuated on the day28;2The expression of KLF4mRNA increased from the day1, thendecreased, arrived at the minimum on the day3, and then gradually increaseduntil the day28;3The trend of KLF4protein showed roughly the same as the KLF4mRNA level, it started to increase on the day1, then decreased, arrived at theminimum on the day3, then gradually increased until the day14and thendecreased again.Part two The Effect of Atorvastatin on the Expression of KLF4in the Lung Tissues of Mice with Pulmonary FibrosisMethods:1C57BL/6were randomly divided into3groups: Control group,bleomycin group (BLM) and atorvastatin group (ATO). Group BLM and ATOwere given a single intratracheal injection of bleomycin (2.5mg/kg), whilegroup Control was injected with isodose physiological saline. Group ATO wastreated with atorvastatin10mg/(kg d) by intragastric administration the dayafter bleomycin instillation. All groups were sacrificed on the day3,14and28;2Hematoxylin and eosin stain （HE stain） and Masson’s TrichromeStain were used to detect the pathology of alveolar and the deposition ofcellularity and collagen;3Real time-polymerase chain reaction (RT-PCR) was performed toinvestigate the expression of KLF4gene in the lung tissues of mice;4Immunohistochemical technology was performed to investigate theexpression of KLF4protein in the lung tissues of mice;5Zymography was used to investigate the activation of matrixmetalloproteinase-2（MMP-2）.Results:1Pathological results showed that compared with the BLM group, theinflammation and the collagen deposition were significantly reduced in theATO group;2Compared with BLM group（0.336±0.195）, the expression of KLF4mRNA significantly（P<0.05） increased in the ATO group（2.050±1.564）on the day3, and the expression of KLF4mRNA significantly（P<0.05）decreased in the ATO group（0.464±0.110）compared with BLM group（2.000±0.435） on the day14;3Compared with BLM group, the expression of KLF4proteinsignificantly （P<0.05）decreased in the ATO group on each time point;4The activation of MMP-2significantly（P<0.05）increased in the groupBLM compared with the Control group, and significantly （P<0.05） decreased after the treatment of atorvastatin.Part three Effects of KLF4on Cell proliferation, Apoptosis andPhenotype shifting in macrophagesMethods:1KLF4deficient stable cell line was generated by selection in G418afterthe transfection of short hairpin (shRNA) plasmid with LipofectamineTM2000.The obtained cell line KLF4deficiency was named shKLF4, the controlplasmid expression cell line was named NC, wild type RAW264.7cells wasnamed WT;2RNAi efficiency was qualified by real-time quantitative polymerasechain reaction (RT-PCR) and Western-blot;3Macrophage phenotype shifting was detected by RT-PCR:After KLF4deficient stable cell line generated, LPS was used to induce the macrophageporlarization, and RT-PCR was used to detect the relative expression of M1macrophage biomarkers;4Proliferation ability was measured by CCK-8;5Cell cycle and apoptosis were analyzed by flow cytometry;Results:1Stable KLF4-deficient cell line was established and the expression ofKLF4was reduced by70%;2After LPS induced, the expression of M1macrophages biomarkerTNF-α，MCP-1and CCL5decreased in KLF4deficient stable cell line;3Decreased proliferation was observed in KLF4deficient stable cell lineby CCK-8(P<0.05);4Lower expression of KLF4in macrophages promote cell apoptosisthrough flow cytometry (P<0.05).Conclusion:1The expression of KLF4is dynamically changed in the process ofexperimental pulmonary fibrosis;2Ater the intervention of atorvastatin, the expression of KLF4isobviously decreased in the experimental pulmonary fibrosis; 3The cell cycle in the RAW264.7macrophages with KLF4lower-expression is not obviously different from the wide type macrophages.However, KLF4lower-expression could suppress the proliferation andpromote apoptosis, and inhibit macrophage M1polarization;4KLF4may be the mediator in the effects of anti-inflammation andanti-fibrosis of atorvastatin in the expremental pulmonary fibrosis. |
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