Objective:To screen the time points of high survival rate and efferocytosis dysfunction of rat alveolar macrophages stimulated by cigarette smoke extract (CSE), establish an in vitro model of alveolar macrophage efferocytosis function, and study chronic respiratory diseases with chronic inflammatory reaction as the main pathological changes. Methods:① Time point screening experiment: rat alveolar macrophages (NR8383 cells) were cultured in vitro, and the cells in logarithmic growth phase were divided into blank control group (100 μL complete medium) and 5% CSE group (90 μL complete medium + 10 μL 100% CSE). Alma blue method was used to detect the effect of 5% CSE on the activity of NR8383 cells at 6, 12, 24 and 48 hours. ② Apoptosis induction experiment: rat type Ⅱ alveolar epithelial cells (RLE-6TN cells) were cultured in vitro as phagocytic target cells of NR8383 cells, and the cells in logarithmic growth phase were divided into blank control group and 10, 30 and 60 minutes groups after ultraviolet exposure (apoptosis was induced by 30 000 μJ/cm 2 ultraviolet irradiation for 15 minutes). Flow cytometry was used to detect the apoptosis rate of RLE-6TN cells cultured for 10, 30 and 60 minutes after ultraviolet exposure. ③ Cell efferocytosis experiment: NR8383 cells in logarithmic phase were divided into blank control group and 5% CSE group. Two hours before NR8383 cells were stimulated by CSE for 6, 12 and 24 hours, RLE-6TN cells were exposed to ultraviolet to induce apoptosis, and the RLE-6TN cell suspension was added to NR8383 cells (the ratio of RLE-6TN cells to NR8383 cells was 5∶1). Flow cytometry was used to detect the efferocytosis rate of NR8383 cells to RLE-6TN cells at different time points treated with 5% CSE. Results:① Compared with the blank control group, the activity of NR8383 cells significantly decreased after treatment with 5% CSE for 48 hours [cell reduction rate: (68.5±4.1)% vs. (73.6±2.3)%, P < 0.05]. However, there were no significant differences when the activities of NR8383 cells treated with 5% CSE for 6, 12 and 24 hours were compared with the blank control group, so these three time points were selected for the subsequent establishment of alveolar macrophage cell efferocytosis dysfunction in vitro model experiment. ② Compared with the blank control group, the apoptosis rate of RLE-6TN cells significantly increased at 10, 30 and 60 minutes after ultraviolet exposure [(66.87±8.63)%, (85.51±2.39)%, (96.13±2.74)% vs. (9.13±3.17)%, all P < 0.01] in a time-dependent manner. Considering that it taked about 50 minutes for RLE-6TN cells to be labeled with PKH26 membrane labeling probe, 10 minutes after ultraviolet exposure was selected to label RLE-6TN cells. ③ Compared with the blank control group, the efferocytosis function of NR8383 cells was significantly decreased after treatment with 5% CSE for 12 hours [cell efferocytosis rate: (33.64±1.30)% vs. (44.02±2.71)%, P < 0.01], but there was no significant effect on the efferocytosis function of NR8383 cells at 6 hours and 24 hours. Conclusions:CSE can induce alveolar macrophage cell efferocytosis dysfunction. Based on the test results of the effect of 5% CSE on NR8383 cell activity and cell efferocytosis function, 12 hours with high survival rate and weak efferocytosis effect of NR8383 cells can be selected as the in vitro model condition of alveolar macrophage cell efferocytosis dysfunction.

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.

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.