Objective To explore the molecular mechanism of CAFs promoting energy metabolism of EC9706 cells by collecting conditioned media of cancer-associated fibroblasts(CAFs).Methods Cell viability was detected by MTT,and the CAFs conditioned medium(CAFM)most suitable for indirect co-culture was selected with EC9706 cultured in DMEM high glucose medium as control.The contents of lactic acid and glucose in the supernatant of EC9706 cells were determined by colorimetry.The energy metabolism of EC9706 cells in DMEM and CAFM was detected by seahorse system energy metabolism analysis system.Real time quantitative polymerase chain reaction(RT-qPCR)and western blotting were used to detect the mRNA and protein expression of energy metabolism related molecules.Results Compared with normal esophageal fibroblast conditioned medium(NFM),CAFs conditioned medium of 50%-60%and 70%-80%cell fusion degree promoted the proliferation of EC9706 cells(P<0.01),and CAFM groups with 70%-80%cell fusion degree promoted the proliferation of EC9706 cells compared with the control group.When the CAFM content was 60%,the proliferation of EC9706 cells was significantly increased(P<0.01).Compared with DMEM,CAFM could increase glucose uptake,superlactate content,basal respiratory value,basal glycolysis,compensatory glycolysis(P<0.05),non-mitochondrial oxygen consumption,maximum respiratory value,oxygen consumption for ATP synthesis,and reserve respiratory capacity(P<0.01)of EC9706 cells.RT-qPCR results showed that CAFM could also up-regulate the Hypoxic-inducible factor-1α(HIF-1α),Hexokinase-2(HK2)and Monocarboxylic acid transporter 1(MCT1)of EC9706 cells(P<0.05).Expression of Glucose transporter 1(GLUT1),Pyruvate kinase 2(PKM2)mRNA(P<0.01).Western blot showed that compared with DMEM,the protein expression of HK2,PKM2,MCT1 and GLUT1 was significantly increased(P<0.01).Conclusion CAFM can promote energy metabolism of EC9706 cells by promoting mRNA and protein expressions of HK2,PKM2,HK2,GLUT1,MCT1 and MCT4 under in vitro culture conditions.

Objective:To observe the stimulation of cigarette smoke extract(CSE)at different times influencing the alveolar macrophage efferocytosis and phagocytosis function,and to explore the effect of CSE on inflammatory response in lung diseases and molecular mechanism.Methods:Rat alveolar macrophages NR8383 were intervened with 10%CSE for 6 h,12 h,24 h,48 h,divided into 6 h blank control group,6 h-10%CSE group,12 h blank control group,12 h-10%CSE group,24 h blank control group,24 h-10%CSE group,48 h blank control group,48 h-10%CSE group.10%CSE intervention 12 h in combination with PPAR inhibitor/ago-nist,divided into PPAR inhibitor group and PPAR agonist group.Alamar Blue colorimetry was used to detect the proliferation and toxi-city of PPAR agonist on NR8383 cells.Flow cytometry was used to detect the efferocytosis and phagocytosis of NR8383 cells,and M1 and M2 polarizations.The contents of TNF-α,TGF-β1 and MFG-E8 were detected by enzyme-linked immunosorbent assay.Western blot was used to detect the expressions of PPARγ and CD36 protein.mRNA expressions of PPARγ and CD36 were detected by qPCR.Results:After 6 hours of 10%CSE stimulation,the phagocytosis and efferocytosis of NR8383 cells were increased,the expression of PPARγ was down-regulated,the expression of CD36 mRNA was increased,and the expressions of TNF-α,TGF-β1 and MFG-E8 were increased,but there was no obvious polarization direction.After 12 hours CSE stimulation,the efferocytosis and phagocytosis functione of NR8383 cells were significantly decreased,the expressions of PPARγ and CD36 were significantly down-regulated,the expression of TNF-α was increased,the expressions of TGF-β1 and MFG-E8 were decreased,and the cells were polarized to M1-type macrophages.After 24 hours of intervention,the efferocytosis rate of NR8383 cells were decreased,but the phagocytosis function of E.coli was increased,the expression of PPARγ was down-regulated,the expressions of CD36 protein was decreased,the expression of TNF-α was decreased,while there was no statistical difference,the expressions of TGF-β1 and MFG-E8 were still decreased,and there was obvious tendency of M1 polarization.After 48 h,the efferocytosis rate of NR8383 cells were still decreased,but the phagocy-tosis ability was significantly increased,the expression of PPARγ was significantly decreased,the expression of CD36 was significantly increased,the expression of TNF-α was decreased,the expressions of TGF-β1 and MFG-E8 were increased,and the polarization of macrophages towards M1 and M2 were increased.After 12 h stimulation with 10%CSE combined with PPAR agonist and inhibitor,it was found that PPAR agonist enhanced the efferocytosis and phagocytosis of NR8383 cells,up-regulated the expression of PPARγ and CD36,inhibited the expression of inflammatory factor TNF-α,and promoted the expressions of anti-inflammatory factor TGF-β1 and cytokinesis cofactor MFG-E8.Conclusion:With the stimulation time of CSE,alveolar macrophages gradually change from the activated state of early inflammatory response to chronic inflammatory response,which leads to alveolar macrophage efferocytosis and phagocyto-sis dysfunction,and the mechanism is related to the inhibition of PPARγ pathway.

ObjectiveTo explore the mechanism of Qigesan （QGS） in intervening in the migration and invasion of esophageal carcinoma TE-1 cells. MethodMicroarray technology was used to screen differentially expressed genes （DEGs） in the normal group and the QGS group， and the ontological functions and signaling pathways of DEGs were analyzed. The thiazolyl tetrazolium （MTT） assay was used to detect the effect of QGS on the viability of TE-1 cells. In the subsequent experiments for verification， a blank group， a transforming growth factor-β₁ （TGF-β₁） group， a TGF-β₁ + QGS group， and a TGF-β₁ + SB431542 group were set up. The cell morphology in each experimental group was observed by microscopy. The migration and invasion abilities of cells were detected by wound healing assay， and the mRNA expression levels of E-Cadherin， vimentin， Smad2， and Smad7 were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression of E-Cadherin， vimentin， p-Smad2/3， Smad2/3， and Smad7 was detected by Western blot. ResultThere were 1 487 DEGs between the QGS group and the blank group， including 1 080 down-regulated ones （accounting for 72.63%） and 407 up-regulated ones. The down-regulated genes were mainly involved in biological processes such as cytoskeletal protein binding， ATP binding， adenylate nucleotide binding， and adenylate ribonucleotide binding， and the involved Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways included TGF-β signaling pathway， cell cycle， extracellular matrix-receptor interaction protein， tumor pathways， and oocyte meiosis. The up-regulated genes were mainly involved in RNA binding， DNA binding， transcriptional regulator activity， transcriptional activator activity， and nucleotide binding， and the KEGG pathways involved mainly included mitogen-activated protein kinase （MAPK） signaling pathway， bladder cancer， renal cell carcinoma， cancer pathways， and p53 signaling pathway. Compared with the blank group， the inhibition rate of cell viability of TE-1 cells increased after QGS （20， 30， 40， 60， 80 mg·L^-1） intervention for 12， 24， 36， 48， 60 h （P<0.05）， and the inhibition rate was time- and dose-dependent. Compared with the blank group， the TGF-β₁ group showed lengthened cells with fibroblast phenotype. Compared with the TGF-β₁ group， the TGF-β₁ + QGS group showed shortened cells with normal morphology and epithelial phenotype. The cell morphology in the TGF-β₁ + SB431542 group was similar to that of the TGF-β₁ + QGS group. Compared with the blank group， the TGF-β₁ group showed potentiated ability of cell migration and invasion （P<0.05）. Compared with the TGF-β₁ group， the TGF-β₁ + QGS group and the TGF-β₁ + SB431542 group showed inhibited and weakened migration and invasion abilities of cells （P<0.05）. However， there was no significant difference in migration and invasion abilities between the TGF-β₁ + QGS group and the TGF-β₁ + SB431542 group. The mRNA expression levels of vimentin and Smad2 in the TGF-β₁ group were higher （P<0.05）， and the mRNA expression levels of E-Cadherin and Smad7 were lower （P<0.05） than those in the blank group. Compared with the TGF-β₁ group， the TGF-β₁ + QGS group and the TGF-β₁+ SB431542 group exhibited decreased expression levels of vimentin and Smad2 mRNA （P<0.05）， and elevated expression levels of E-Cadherin and Smad7 mRNA （P<0.05）. Compared with the blank group， the TGF-β₁ group showed up-regulated protein expression levels of vimentin， p-Smad2/3， and Smad2/3 （P<0.05）， and reduced protein expression levels of E-Cadherin and Smad7 （P<0.05）. Compared with the TGF-β₁ group， the TGF-β₁ + QGS group and the TGF-β₁ + SB431542 group displayed decreased protein expression levels of vimentin， p-Smad2/3， and Smad2/3 （P<0.05）， and increased protein expression levels of E-Cadherin and Smad7 （P<0.05）. ConclusionThe ethyl acetate extract of QGS inhibits the epithelial-mesenchymal transition （EMT） of TE-1 cells through the TGF-β₁ pathway to reduce the migration and invasion of TE-1 cells.

ObjectiveTo investigate the mechanism of icariin in ameliorating efferocytosis dysfunction and inflammatory response of alveolar macrophages induced by cigarette smoke extract via the peroxisome proliferator-activated receptor gamma （PPARγ） signaling pathway. MethodThe untreated rat alveolar macrophages （NR8383） were taken as the blank group. The NR8383 cells treated with 10% cigarette smoke extract were divided into model， low-， medium-， and high-dose （10， 20， 40 μmol·L^-1） icariin， PPARγ inhibitor， and PPARγ inhibitor + low-， medium-， and high-dose icariin groups. Alamar blue colorimetry was employed to examine the proliferation and toxicity of icariin on NR8383 cells. The efferocytosis rate of NR8383 cells was detected by flow cytometry. Enzyme-linked immunosorbent assay was employed to measure the levels of tumor necrosis factor-alpha （TNF-α）， transforming growth factor-β₁ （TGF-β₁）， and milk fat globule-epidermal growth factor 8 （MFG-E8）. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were employed to determine the protein and mRNA levels， respectively， of PPARγ， CD36， and RAS-related C3 botulinum toxin substrate 1 （Rac1）. ResultThe efferocytosis dysfunction model of NR8383 was established with the cigarette smoke extract. Compared with the blank control group， the model group showed decreased efferocytosis rate （P<0.05）， elevated TNF-α level （P<0.05）， lowered TGF-β₁ and MFG-E8 levels （P<0.01）， and down-regulated mRNA and protein levels of PPARγ， CD36， and Rac1 （P<0.05， P<0.01）. Compared with the model group， the treatment with icariin increased the efferocytosis rate （P<0.05， P<0.01）， lowered the TNF-α level （P<0.01）， elevated TGF-β₁ and MFG-E8 levels （P<0.05）， and up-regulated the protein and mRNA levels of PPARγ， CD36， and Rac1 （P<0.05， P<0.01）. Compared with icariin alone， PPARγ inhibitor + icariin decreased the efferocytosis rate （P<0.05） and down-regulated the protein and mRNA levels of PPARγ （P<0.05， P<0.01）. In addition， PPARγ inhibitor + low-dose icariin down-regulated the protein level of CD36 （P<0.01） and PPARγ inhibitor + low-/medium-dose icariin up-regulated the protein level of Rac1 （P<0.05）. ConclusionIcariin ameliorates the cigarette smoke extract-induced efferocytosis dysfunction of alveolar macrophage by regulating the PPARγ signaling pathway and cytoskeletal structure rearrangement.