The human embryonic lung fibroblast cell line, MRC-5 (Cell Bank of Type Culture Collection, Chinese Academy of Sciences), was maintained in minimal essential medium (Invitrogen; Thermo Fisher Scientific, Inc.) which contained 2.0 mmol/l glutamine. The medium was supplemented with 10% fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc.) and cells were cultured at 37˚C in a humidified atmosphere containing 5% carbon dioxide and 95% air. The MRC-5 cells were inoculated on a flexible substrate culture plate (FX-5000, Flexcell International Corp.). MRC-5 cells in the logarithmic growth phase were cultured for 48 h with different concentrations of LPS (0, 5, 20 or 50 µg/ml). Subsequently, different amplitudes of mechanical stretch stimulation were applied to the cells via the Flexcell loading system (Flexcell Corp., USA) and culture was continued for 48 h (loading parameters: Frequency, 0.1 Hz; sine wave, stretching amplitude of 5, 10, 15 and 20%).

MRC-5 cells were collected after LPS stimulation and/or mechanical stretch treatment. The cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and cell proliferative activity was assessed by flow cytometry (BD FACSCalibur; BD Biosciences). CFSE is a non-toxic dye for cells and its chemical properties are stable. Once CFSE enters the cell, it cannot be released from the cell and will not be metabolized. The only way to reduce the CFSE content in cells is through cell proliferation and division. The CFSE contained in the cells enters the progeny cells as the cells proliferate. The cells were labeled with CFSE before inoculation on a flexible substrate culture plate where the initial fluorescence intensity was measured. Subsequently, the fluorescence intensity after proliferation was measured after LPS stimulation and/or mechanical stretch treatment by flow cytometry. To determine the proliferative activity of chondrocytes, the proliferation index was calculated as follows: Proliferation index=initial fluorescence intensity/fluorescence intensity after proliferation.

MRC-5 cells were treated by LPS stimulation and/or mechanical stretch for 48 h. The supernatant was collected after centrifugation (503.1 x g for 5 min at 25˚C) to remove impurities and refrigerated at -70˚C. An ELISA kit (1R443; Rapidbio) was used according to the manufacturer's protocol. The absorbance at 450 nm was detected with a microplate reader (iMark; Bio-Rad Laboratories, Inc.) and the content of TGF-β1 in the sample was extrapolated by using a standard curve.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from from the control and treated MRC-5 cells and reverse transcribe RNA into cDNA. The expression of TGF-β1 mRNA and collagen type I α1 (Col1α1) mRNA in human embryonic lung fibroblasts were detected by RT-qPCR (7). The PCR detection was determined using an Exicycler™ 96 real-time system (Bioneer) with SYBR Premix ExTaqII (Takara Bio, Inc.). The PCR conditions were as follows: Enzyme activation at 94˚C for 5 min, amplification at 94˚C for 10 sec, annealing at 60˚C for 20 sec and extension at 72˚C for 30 sec, 40 cycles, final extension at 72˚C for 6 min. The expression level of the target genes, TGF-β1 and Col1α1, were quantified using the 2^-ΔΔCq method (7). β-actin was used as a reference gene. The primers were as follows: β-actin forward, 5'-GACAGGATGCAGAAGGAGATTACT-3' and reverse, 5'-TGATCCACATCTGCTGGAAGGT-3', TGF-β1 forward, 5'-GCCCTGGACACCAACTATTGC-3' and reverse, 5'-AGGCTCCAAATGTAGGGGCAG-3'; Col1α1 forward, 5'-GCCTAGCAACATGCCAATC-3' and reverse, 5'-GCAAAGTTCCCACCGAGA-3'.

The deformation of control and treated MRC-5 cells under the negative pressure (range from 0.245 to 0.392 kPa) were examined using a micropipette aspiration system, which produced the association between aspirated lengths and time (8-10). The cellular viscoelastic parameters [the instantaneous modulus (E₀), the equilibrium modulus associated with long-term equilibrium (E_∞) and the apparent viscosity (µ)] were calculated using the Kelvin standard linear viscoelastic solid model as previously described, and the cellular viscoelastic parameters (E₀, E_∞ and µ) were calculated according to the association between aspirated lengths and time (8-10). The viscoelastic model and a cell sucked in by the microtubules are shown in Fig. 1.

In the present study, all the independent experiments were repeated three times. Data processing and mapping were performed using SPSS 22.0 statistical software (IBM Corp.) and GraphPad Prism 6.0 (GraphPad Software, Inc.). The measurement data were expressed as the mean ± standard deviation. Differences between the groups were compared by one-way analysis of variance with the Student-Newman-Keuls post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

MRC-5 cells were treated with different concentrations of LPS and the cell proliferation activity was analyzed by flow cytometry. It was indicated that LPS induced cell damage and decreased the proliferative activity. As the concentration of LPS increased, the cell proliferation activity continued to significantly decrease (P<0.01; Fig. 2A). MRC-5 cells were cultured under different stretch amplitudes and the cell proliferation activity was analyzed by the same method. The results suggested that the proliferative activity of MRC-5 cells cultured under a low-amplitude stretch (5 and 10%) was significantly increased, but as the stretch amplitude increased (15 and 20%), the proliferative activity of MRC-5 cells exhibited a significant decrease (P<0.01; Fig. 2B). According to the experimental results obtained with different concentrations of LPS combined with clinical practice to simulate ARDS induced injury, cells treated with LPS at low concentrations (5 µg/ml) were selected to construct a cell injury model similar to the pathophysiological state of ARDS, and culture was then continued at different stretch widths to observe the effects on the proliferative activity. The results also indicated that the proliferation activity of MRC-5 cells stimulated by LPS cultured with a low-amplitude stretch (5 and 10%) was improved; however, under high traction (15 and 20%), the cell proliferation was significantly decreased (P<0.05; Fig. 2C).

As provided in Fig. 3, it was indicated that low concentrations of LPS upregulated TGF-β1 and collagen I expression after cell injury, while high concentrations of LPS affected the cell proliferation activity and reduced the expression of TGF-β1 and collagen I in the supernatant (Fig. 3A and C). The RT-qPCR results suggested that low concentrations of LPS led to upregulated expression of TGF-β1 and Col1α1 mRNA, while high concentrations of LPS decreased their expression (Fig. 3B and D).

MRC-5 cells were cultured under different mechanical stretches and the concentration of TGF-β1 and collagen in the cells or supernatant was detected (Fig. 4). The results indicated that mechanical stimulation with a stretch of 5% had no effect on the protein levels of TGF-β1 and collagen I, while a 10% stretch induced a significant increase in TGF-β1 and collagen I levels, and a much greater mechanical stimulation (15 and 20%) significantly increased the levels of TGF-β1 and collagen I than the 10% stretch (Fig. 4A and C). Total RNA was extracted from the control and mechanical stretch-treated MRC-5 cells, and the expression of TGF-β1 and Col1α1 mRNA was detected by RT-qPCR. Mechanical stimulation with a stretch of 10% induced upregulation of TGF-β1 and Col1α1 mRNA expression and a much larger mechanical stretch stimulation (15 and 20%) significantly increased the expression of TGF-β1 mRNA and Col1α1 mRNA in the cells (Fig. 4B and D).

MRC-5 cells were treated with LPS at 5 µg/ml to construct the cell injury model and those cells were cultured for a further 48 h under mechanical stimulation with different stretch amplitudes. The results indicated that mechanical stimulation with a stretch amplitude of up to 10% did not result in any significant increase in TGF-β1 or collagen I protein, or their gene expression levels in cells following culture with 5 µg/ml LPS (P>0.05). However, much larger mechanical stimulation (15 and 20%) caused further damage to the LPS cell injury model. At the same time, the levels of TGF-β1 and collagen I, and their gene expression levels were significantly increased (P<0.05; Fig. 5).

MRC-5 cells treated with 5 µg/ml of LPS were cultured under different mechanical stimulation conditions. A cell microtubule suction test indicated that MRC-5 cells exhibited typical viscoelastic solid creep characteristics under constant negative pressure. MRC-5 cells exhibited an instantaneous viscoelastic deformation. The trend of cell inhalation length changing with time at a constant negative pressure of 294 Pa in each group suggested that LPS stimulation and mechanical stretch treatment increased the inhalation length of cells and the length of inhalation was positively correlated with the stretch amplitude (Fig. 6). The viscoelastic parameters (E₀, E_∞ and µ) of treated and control MRC-5 cells are presented in Fig. 7. The results showed that the LPS treatment significantly reduced all viscoelastic parameters of MRC-5 cells (P<0.05). After LPS treatment, the biomechanical properties of MRC-5 cells were reduced and the cells were softened. A mechanical stretch of 5% had no effect on the biomechanical properties of MRC-5 cells treated with only 5 µg/ml LPS. As the mechanical stretch increased, the viscoelasticity of MRC-5 cells gradually decreased (P<0.05), but there was no significant difference in cell viscoelasticity between the two groups of MRC-5 cells cultured with a 15 and 20% mechanical stretch (P>0.05).