The Effect And Mechanism Of Irisin On The Migration,Proliferation,and Invasion Of Lung Cancer Cells And Inflammatory Response Of Acute Lung Injury In Mice

BackgroundLung cancer is one of the most common malignancies,incidence of the cases increased gradually and has become one of the top 2 leading causes of death.The different pathological types of lung cancer have the survival of different treatment methods and clinical characteristics.Numerous treatment options of lung cancer are available,such as new chemotherapy drugs,microwave ablation technique,improvement on surgical methods andradiation technique,but the poor 5-year overall survival rate for lung cancer patients is due to the obscure understanding of cancer pathogenesis,despite moderate improvements over the past 4 decades as a result of using many therapeutic modalities.Therefore,we are attempting to discover novel pathomechanisms that will be useful in drug development for tumor therapy.The pathogenic mechanism of cancer is very complicated.Normal tissuse and cells could regulating the produce and release of the signaling pathways involved in cell proliferation and apoptosis to control the cell growth and division cycle,to insure the cells are in normal homeostasis and quantity,to maintain organization has normal structure and function.However,the tumor cells escape the normal growth inhibition and apoptosis by removing the control of these signals.The expression of E-cadherin expression is an iconic event in the course of EMT and is positively correlated with the development of metastatic cancer.Epithelial-mesenchymal transition(EMT)occurs during the critical phases embryonic development of many animal species.During EMT,mesenchymal cells acquire a morphology that is appropriate for migration and set in organ formation,which involves interactions between epithelial and mesenchymal cells.EMT also contributes to repair the injured tissues,and it can cause organ fibrosis and carcinoma progression through a variety of mechanisms.EMT is the one of the major and early approaches from which cancers grow and metastasize.EMT made primary epithelial tumor cells change to ectomesenchymal cells capable of migration,and maintain certain characteristics of the epithelial cells at the same time,then the invasive tumor cells with the bloodstream or the lymphatic system transferred to different organs and set down to form micrometastasis fountain.Many signal pathway changes can cause the decrease of E-cadherin expression and promote the transformation of cells from epithelial to mesenchymal.It is accepted that the more physical activity you do the lower your risk of these cancers.Exercise is beneficial to our organ systems via mediating the transcriptional co-activator PPAR-γ co-activator-1 a(PGC1-α)which increases the expression of FNDC5 in muscle,a type I transmembrane protein of skeletal muscle,and then FNDC5 is proteolysed at amino acid position 30 and 140 to give rise to irisin,which is composed of 110 amino acids.As a biopharmaceutical protein,irisin is naturally produced in the muscles when a person exercises,and it has been proven to have protective effects on many systemic diseases,including malignant tumors.Thus far,irisin’s function has been mainly associated with energy homeostasis and metabolism(including cancer).Recent studies have revealed that irisin immunoreactivity was detected in both ovarian endometriosis and mucinous carcinomas compared to benign tumors.Higher baseline serum irisin concentrations might be secondary to insulin resistant conditions in women with polycystic ovary syndrome.The expression of serum irisin decreases in patients with chronic kidney disease and shows a consistent correlation with glomerular filtration rate and plasma bicarbonate levels.Furthermore,serum irisin levels were lower have been detected in patients with breast cancer.Irisin is involved in promoting metabolism,immune regulation,and affects chronic inflammation in many systemic diseases,including gastric cancer.However,the role of irisin in lung cancer is not well characterized.ObjectivesThe aim of this study was to investigate the effect of irisin on the migration,proliferation,and invasion of lung cancer cells and the the molecular mechanisms.Materials and methods1.Human NSCLC cell line A549 cells and SCLC cell line NCI-H446 cells culture.2.Proliferation detection of cells by MTT.3.Migration was detected by scratch-induced migration assay.4.Migration and invasion was detected by trans-well assay.5.Western blot analysis:Protein expression was detected by Western blot.The cells were collected and lysed.The extracted protein concentration was measured using the BCA protein assay kit.SDS-PAGE was used to examine the expression of the target protein.6.Statistical analysis.Results1.Irisin inhibits the proliferation of lung cancer cells.Irisin can significantly inhibit A549 cells proliferation over a range of concen-trations(20-50 nM)as detected by MTT assay.20nM irisin significantly inhibited A549 cells proliferation after 24 h stimulation.2.Irisin inhibits the migration and invasion of lung cancer cells.A549 cells were treated with irisin at the concentration of 20nM for 24 h,the number of invading cells migrating from the upper to the lower surface was also significantly reduced after treatment with irisin,and healing over the scratch was significantly reduced after treatment with irisin.3.Irisin affects the expression of EMT markers via inhibiting the PI3K/AKT pathway in lung cancer cells.(1)Various concentrations of irisin(0,5,10,and 20nM)were respectively added to the cells,and the cancer cells were cultured for another 24 h.We choiced two cell lines:human NSCLC cell line A549 and human SCLC cell line NCI-H446.(2)We observed irisn treatment significantly decreased the expression of mesenchymal markers N-cadherin and vimentin and increased the expression of epithelial marker E-cadherin in two cell lines.(3)20nM irisin significantly decreased phosphorylation of PI3K/AKT proteins and increased the total PI3K/AKT protein expression in A549 and NCI-H44 cells respectively.The results above indicate that irisin inhibit the PI3K/AKT signal pathway,and LY294002 significantly reversed irisin-induced E-cadherin expression,and irisin-induced N-cadherin and vimentin suppression4.Irisin induces Snail down-regulation via inhibiting activation of PI3K/AKT signaling pathway.(1)Snail is the main regulator of E-cadherin,irisin clearly suppresses the expression of Snail,in particular at the 20nM concentration.(2)After treating A549 and NCI-H446 cells with lOuM LY29400 for 2 h,the expression of Snail increased compared to untreated samples.The results indicate that irisin inhibits the expression of Snail through blocking the PI3K/AKT pathway.Conclusions1.This report is the first to examine the relationship between irisin and lung cancer.2.In this study,we discoverd irisin was a new class of anticancer agents with a novel mode of action,suppressed the migration,proliferation,and invasion of lung cancer cells.3.Irisin inhibited EMT and reduced the invasion of lung cancer cells via the PI3K/AKT/Snail pathway.BackgroundAcute lung injury and ARDS are severe clinical conditions that are associated with excessive inflammatory response,sepsis,impaired gas exchange,alveolar-capillary barrier disruption and pulmonary edema.The most severe form of ALI is defined as acute respiratory distress syndrome(ARDS).Patients with ALI/ARDS present with acute onset,decreased oxygenation capacity,bilateral lung ground-glassedge on chest radiographs,increase in breathing rate,edema formation and non-cardiogenic respiratory failure.ALI and ARDS carry a high mortality morbidity and mortality in the clinic.Meanwhile,previous studies have revealed some important mechanism leading to ALI/ARDS:the accumulation of ROS linked with excessive oxidative stress,inflammatory responses.The molecular mechanism of ALI/ARDS might be related to the activation of inflammatory signaling,including the p-38MAPK,AKT and the NF-κB signal pathways.Irisin,a metabolism associated factor,induces the browning of adipose tissue,has potentially protective effect on the development of obesity-related states,such as arteriosclerosis,insulin resistance and type 2 diabetes.Recent studies demonstrated that irisin has an effect on promoting fatty acid oxidation and glucose utilization by regulating the AMPK signaling pathway,and inhibiting hepatic gluconeogenesis by the PI3K/AKT signaling pathway in type 2 diabetes.Irisin decreases blood pressure by augmenting acetylcholine-mediated vasodilation via AMPK-Akt-eNOS-NO signal pathway.Some studies revealed AMPK pathway was involved in the regulation of irisin on depressive-like behaviors in chronic unpredictable stress rats.Irisin could be therapeutic for atherosclerotic vascular diseases in diabetes through activation of AMPK-PI3K-Akt-eNOS signaling pathway.Irisin restored ox-LDL-induced human umbilical vein endothelial cell dysfunction via inhibiting the reactive oxygen species(ROS)/p38 MAPK/NF-κB signaling pathway activation.Irisin has been demonstrated the suppression of inflammation by regulating cellular signaling pathways,and inflammation response was precisely the feature of ALI/ARDS.Therefore,we hypothesized that irisin could affect cell survival,cell apoptosis,expression of inflammatory mediators and fibrogenesis via mediating the signaling pathway which was closely relative with inflammation in ALI/ARDS.However,no evidence has been reported for the direct effect of irisin on ALI/ARDS.The present study was designed to investigate the effects of irisin on ALI.ObjectivesThe aim of this study was to investigate the role of irisin on on LPS-induced ALI,and alveolar epithelial cells proliferation and apoptosis,and further explore the possible signaling mechanisms by which irisin exerts its effects.Materials and methods1.Animal models of induction of acute lung injury.Working solution of LPS was prepared at a concentration of 2mg/ml.ALI was induced by intranasal instillation of LPS(5 μg/g in 100 μl PBS)as described previously.Male C57BL/6J mice(age:6-8 weeks)in the control and LPS groups were received intranasal instillation of saline(SAL)and LPS,respectively,while mice in LPS + irisin groups treated daily with purified irisin at a dose of 0.5 μg/g body weight by intraperitoneal(i.p.)2.The fibrosis tissue with Hematoxylin&eosin(H&E)-staining to reflect the degree of ALI objectively by image pattern analysis.3.Smith score analysis of the mice’s lung tissue injury degree.4.LPS-induced inflammationary factors expression in A549 cells were derected by Real-time PCR.5.Protein expression was detected by Western blot.6.A549 cells culture to do the following experiments.7.Alveolar epithelial cells proliferation was detected by cell MTT assay.8.Alveolar epithelial cells apoptosis was detected by TUNEL staining.9.Statistical analysis.All data in the present study were evaluated with predictive analytics software(PASW)statistics 18.0.The normally distributed data were analyzed by one-way ANOVA and the non-parametric variables were analyzed by the Mann-Whitney U test.Results1.Irisin inhibited the inflammation reactivity of cells and pathological changes of LPS-induced lung injury in mice.Mice were treated with LPS and irisin,and then lung tissue samples were collected.We observed significantly pathological alterations such as inflammatory cells infiltration,interstitial and intra-alveolar edema,hemorrhage,hyaline membrane formation and diffuse alveolar damage in LPS-induced group without irisin.Irisin markedly reduced the extent of tissue damage.The histology Smith score also showed that irisin time-and dose-dependently alleviated lung tissue damage.2.Irisin inhibited mRNA expression of inflammatory cytokines induced by LPS in A549 cells.A549 cells were treated with LPS(10 μg/ml),and with different concentrations of irisin(0,10,20 and 40nM)for 0,6,12,24 and 48 h,and 20 nM irisin significantly inhibited the cell viability as detected by MTT assay.As a result,we chose 20 nM of irisn for the subsequent experiments.The mRNA levels of IL-6,MCP-1,IL-1β and TNF-awere remarkably increased after LPS challenge.Irisin significantly inhibited the expression of pro-inflammatory cytokines induced by LPS in A549 cells.3.Irisin inhibited apoptosis induced by LPS in the injured lung.Apoptotic index(number of TUNEL-positive cells/number of DAPI-stained cells)significantly decreased in LPS-induced A549 cells treated with irisin compared to controls.Irisin significantly inhibited the expression of caspase-3 and increased the expression of Bcl-2 and decreased the expression of Bax.The data suggested that irisin inhibited the apoptosis in LPS-induced A549 cells4.Irisin reduced LPS-induced activation of APK and NF-κB signaling pathways.We analyzed the effects of irisin on LPS-induced MAPK and NF-κB activation using western blot.The results showed that the levels of p38 phosphorylation and NF-κBp65 phosphorylation dramatically increased in A549 cells exposed to LPS,whereas decreased following irisin pretreatment.Furthermore,NF-κB(p65)levels were evaluated of cytoplasmic and nuclear extracts from the A549 cells.The results suggested irisin that significantly reduced nuclear levels and promoted the expression in cytoplasm of NF-κB(p65).Conclusions1.Irisin inhibits the inflammation reactivity of cells of LPS-induced lung injury in mice.2.Irisin inhibits apoptosis induced by LPS in the injured lung.3.Irisin increases the expression of Bcl-2 and decreases the expression of Bax in LPS-induced A549 cells.4.Irisin mediates inflammatory cytokines and apoptosis induced by LPS in A549 cells through the MAPK and NF-κB signaling pathway.