Objective To observe the effects of mechanical stretch on cytokines release from alveolar macrophages( AMs) and the expression of macrophage inflammatory protein-2( MIP-2) induced by lipopolysaccharide( LPS) . Methods AMs were divided into the following groups: ①AMs were subjected to 20% elongation by Flexercell 4000T cell stress system for 24 hours and the supernatant was collected to detect the levels of TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, macrophage inflammatory protein-1α( MIP-1α) , MIP-2, monocyte chemoattractant protein-1( MCP-1) , granulocyte /macrophage colony stimulating factors( GM-CSF) , interferon inducible protein-10( IP-10) , regulated on activation in normal T-cell expressed and secreted( Rantes) and keratinocyte chemoattractant( KC) , by using LiquiChip system. ② AMs were subjected to 5% , 10% , 15% and 20% elongation for 24 hours and the supernatant was collected to detect the levels of MIP-2. ③AMs were subjected to 20% elongation and MIP-2 in supernatant was detected 1, 3,6, 12, and 24 hours later. ④ AMs were subjected to 20% elongation and/ or LPS at a concentration of 10 ng/mL, and MIP-2 in supernatant was detected 24 hours later. Unstretched AMs were used as control in all kind of test. Results ①The levels of IL-1β, IL-6,MIP-2, MCP-1, IFN-γand IP-10 secreted by stretched AMs were 8. 7, 4. 3, 38. 6, 4. 8, 14. 2 and 5. 0 times those of the control group( all P lt; 0. 001) . ② The levels of MIP-2 secreted by AMs subjected to 10% , 15% and 20% elongation were ( 480. 5 ±93. 1) pg /mL,( 806. 3 ±225. 9) pg/mL and ( 1335. 7 ±18. 5) pg/mL respectively, all significantly higher than those oft he control group [ ( 34. 6 ±11. 4) pg/mL, all P lt;0. 001] . ③ Three hours after the stimulation of stretch the level of MIP-2 began to increase gradually. And 6, 12, and 24 hours after the stimulation the levels of MIP-2 secreted by the AMs were ( 819. 4 ±147. 5) pg/mL, ( 1287. 6 ±380 ±3 ) pg/mL and ( 1455. 9 ±436. 7) pg/mLrespectively, all significantly higher than those of the control group[ ( 33. 4 ±10. 2) pg/mL, all P lt; 0. 001] . ④When the AMs were stimulated individually by LPS( 10 ng /mL) or mechanical stretch ( 20% ) , the levels of MIP-2 increased to ( 1026. 3 ±339. 5 ) pg/mL and ( 1335. 7 ±318. 5 ) pg/mL respectively( both P lt; 0. 001) . When the AMs were costimulated by LPS and mechanical stretch, the level of MIP-2 increased to ( 2275. 3 ±492. 1) pg/mL, implicating a synergistic effect between mechanical stretch and LPS ( F = 121. 983, P lt; 0. 001) . Conclusions Mechanical stretch activates AMs to produce multiple inflammatory cytokines and induce AMs to secret MIP-2 in a strength- and time-dependent manner.Mechanical stretch also has synergistic effect with LPS in inducing MIP-2 release, which might play an important role in the development of ventilator-induced lung injury.
Objective To explore the effects of mechanical stretch with variant frequencies on the alignment and differentiation of the multilayer myotubes cultured in vitro, and to select the optimized cultural condition of regenerative skeletal muscle tissue with stress loading cultured in vitro. Methods C2C12 myoblasts cultured in vitro in the groove casts of Sylgard 184 were induced into the multilayer myotubes. Meanwhile the myoblasts were treated with various mechanical stretch withcells tensile instrument, at the amplitude of 10% and the frequency of 0 (group A), 0.25 (group B), 0.50 (group C), and 1.00 Hz (group D) for 1 hour, 3 times a day. The myotubes morphology was observed by inverted phase contrast microscope at 5, 7, and 10 days after continuous mechanical stretch. And the expressions of mRNA for myogenic differentiation antigen (MyoD), Myogenin, Desmin, and myosin heavy chain (MyHC) were detected by RT-PCR and real-time fluorescent quantitative PCR (QRT-PCR), respectively. Results The mechanical stretch could promote the al igned fusion and increase the number of myotubes. Indeed the multilayer myotubes arranged more closely in group B at 7 days. At the same group, as the time went on, the mRNA expressions of MyoD gradually decl ined in each group. There were significant differences in mRNA expressions of MyoD between 5 days and 7, 10 days (P lt; 0.05). The mRNA expressions of Myogenin, Desmin, and MyHC were highest at 7 days. There were significant differences between different time points (P lt; 0.05), except the mRNA expression of Desmin of group B between 7 and 10 days (P gt; 0.05). At the same time, with the increase of frequency, the highest mRNA expressions of MyoD, Myogenin, Desmin, and MyHC were in group B. There were significant differences at the same time between group B and the other groups (P lt; 0.05), except mRNA expression of Desmin at 5 days between groups B and C, and mRNA expression of MyHC at 10 days between groups A and B (P gt; 0.05). Conclusion Low frequency (0.25 Hz) and suitable time (7 days) periodic mechanical stretch is beneficial to the differentiation of the multilayer myotubes cultured in the groove casts of Sylgard 184, but as the stretch time goes on the aging of myotubes will be accelerated.
Objective To investigate the effects of different mechanical stretch conditions on the differentiation of rat tendon stem cells (TSCs), to find the best uniaxial cyclic stretching for TSCs tenogenic differentiation, osteogenic differentiation, and adipogenic differentiation. Methods TSCs were isolated from the Achilles tendons of 8-week-old male Sprague Dawley rats by enzymatic digestion method and cultured. The TSCs at passage 3 were randomly divided into 5 groups: group A (stretch strength of 4% and frequency of 1 Hz), group B (stretch strength of 4% and frequency of 2 Hz), group C (stretch strength of 8% and frequency of 1 Hz), group D (stretch strength of 8% and frequency of 2 Hz), and group E (static culture). At 12, 24, and 48 hours after mechanical stretch, the mRNA expressions of the tenogenic differentiation related genes [Scleraxis (SCX) and Tenascin C (TNC)], the osteogenic differentiation related genes [runt related transcription factor 2 (RUNX2) and distal-less homeobox 5 (DLX5)], and the adipogenic differentiation related genes [CCAAT-enhancer-binding protein-α (CEBPα) and lipoprteinlipase (LPL)] were detected by real-time fluorescent quantitative PCR and the protein expressions of TNC, CEBPα, and RUNX2 were detected by Western blot. Results The mRNA expressions of SCX and TNC in group B were significantly higher than those in groups A, C, D, and E at 24 hours after mechanical stretch (P<0.05). The mRNA expressions of CEBPα and LPL in group D were significantly higher than those in groups A, B, C, and E at 48 hours after mechanical stretch (P<0.05). The mRNA expressions of RUNX2 and DLX5 in group C were significantly higher than those in groups A, B, D, and E at 24 hours after mechanical stretch (P<0.05). Western blot detection showed that higher protein expression of TNC in group B than group E at each time point after mechanical stretch (P<0.05), and the protein expression of CEBPα was significantly inhibited when compared with group E at 24 hours after mechanical stretch (P<0.05). At 24 hours after mechanical stretch, the protein expression of RUNX2 in group C was significantly higher than that in group E (P<0.05); and the protein expression of TNC was significantly lower than that in group E at 24 and 48 hours after mechanical stretch (P<0.05). At 48 hours after mechanical stretch, the protein expression of CEBPα was significantly increased and the protein expression of TNC was significantly decreased in group D when compared with group E (P<0.05), but no significant difference was found in the protein expression of RUNX2 between groups D and E (P>0.05). Conclusion The mechanical strain could promote differentiation of TSCs, and different parameter of stretch will lead to different differentiation. The best stretch condition for tenogenic differentiation is 4% strength and 2 Hz frequency for 24 hours; the best stretch condition for osteogenic differentiation is 8% strength and 1 Hz frequency for 24 hours; and the best stretch condition for adipogenic differentiation is 8% strength and 2 Hz frequency for 48 hours.
Objective To study the effect of mechanical stretch on the microenvironment of BEAS-2B on macrophage polarization and the role of polarized macrophages in the epithelial-mesenchymal transition (EMT) of BEAS-2B. Methods Using enzyme linked immunosorbent assay to detect the changes in the levels of cytokines such as interferon-γ, granulocyte-macrophage colony stimulating factor, tumor necrosis factor-α, interleukin (IL)-4, IL-6, IL-10 in the supernatant of lung epithelial cells cultured statically and mechanically stretched. The M0 macrophages (derived from THP-1) were stimulated by stretch/static conditioned medium of BEAS-2B. The surface markers of M1 (CD197) /M2 (CD206) macrophages were detected by flow cytometer. Stretch/static conditioned medium were used to stimulate the co-culture system of macrophages and BEAS-2B in the presence or absence of platelet-derived growth factor receptor inhibitor (PDGFRi), then the protein expression level of EMT makers was examined by Western blot. Results Exposure of BEAS-2B to mechanical stretch resulted in significantly higher production of the pro-M1/M2 polarized factor. The EMT of the co-culture system of M0 and BEAS-2B could be induced by stretch conditioned medium, epithelial marker cytokeratin (CK)-8 and E-cadherin were decreased, while mesenchymal marker α-smooth muscle actin, N-cadherin and vimentin were increased in stretch conditioned medium group. The expression of platelet-derived growth factor (PDGF) was significantly higher in stretch conditioned medium group. The PDGFRi can block the EMT in stretch conditioned medium group. Conclusions The lung epithelial cell supernatant induced by mechanical stretch can promote the polarization of macrophages to M1 and M2. Polarized macrophages promote EMT in human lung epithelial cells via PDGF, and blocking PDGF might attenuate the VILI-associated lung fibrosis.
Idiopathic pulmonary fibrosis (IPF) is a progressive scar-forming disease with a high mortality rate that has received widespread attention. Epithelial mesenchymal transition (EMT) is an important part of the pulmonary fibrosis process, and changes in the biomechanical properties of lung tissue have an important impact on it. In this paper, we summarize the changes in the biomechanical microenvironment of lung tissue in IPF-EMT in recent years, and provide a systematic review on the effects of alterations in the mechanical microenvironment in pulmonary fibrosis on the process of EMT, the effects of mechanical factors on the behavior of alveolar epithelial cells in EMT and the biomechanical signaling in EMT, in order to provide new references for the research on the prevention and treatment of IPF.