Isolation and extraction of hAMSCs. Human fetal placentas were obtained from the Department of Obstetrics and Gynecology at the Affiliated Hospital of Zunyi Medical University (Zunyi, China). The present study was approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University, and all volunteers signed informed consent form before participating. The human amniotic membrane is a thin film composed of a single layer of interconnected epithelial cells that contains human amnion epithelial stem cells (hAESCs) and hAMSCs (14). After amniotic membrane separation, the tissue samples were washed with PBS three times and the tissue was shredded by mechanical methods. Next, 0.25% trypsin-EDTA was added to a final concentration of 0.05 mg/ml, and the tissues were digested by shaking at 37˚C for 30 min before being filtered with a 300-mesh cell strainer. Then, the tissue in the strainer was transferred to a 50 ml centrifuge tube and digested with collagenase IV at 37˚C for 2 h until tissue lysis was complete. The samples were then rinsed with PBS and filtered, and the filtrate was collected and centrifuged at 790 x g (2500 rpm) for 10 min at 4℃. The supernatant was discarded, and original hAMSC medium was added. The cells were mixed and seeded in 25 cm2 cell culture flasks at a density of 1×106 cells/ml for later use after cell counting.
Culture of cell lines and collection of hAMSC-CM. A549 human alveolar type II-like epithelial cells from Beina Chuanglian Biotechnology Co., Ltd (Beijing, China) and hAMSCs (between passages 3 and 5) were seeded at a density of 2x106 cells/ml (determined with a fully automatic multifunction microplate reader; Thermo Fisher Scientific, Inc.) in an incubator (37˚C, 5% CO2) and cultured in DMEM/low-glucose medium (HyClone; GE Healthcare Life Sciences) containing 10% fetal bovine serum (FBS; Gemini Bio Products). The medium was changed once every two days. When the cells reached 80% confluence, they were subjected to 1:2 passaging. When the hAMSCs covered 80–90% of the culture flask, they were washed with PBS twice, and the medium was replaced with 5 ml of DMEM/low- glucose medium containing 10% FBS. After additional culture for 24 h at 37℃, the culture supernatant was aspirated, filtered through a 0.22 µm filter, dispensed into an EP tube and stored at -80˚C.
Experimental protocol. A549 cells were cultured in 96-well plates, treated with PQ to establish the model (Sigma-Aldrich; Merck KGaA) and immediately treated with hAMSC-CM for 24 h before Cell Counting Kit-8 (CCK-8) detection. A549 cells with appropriate PQ and hAMSC-CM concentrations were selected for intervention experiments and were divided into the following groups: i) control group; ii) PQ group (exposed to PQ only); iii) PQ + CM group (treated first with PQ and then with hAMSC-CM); and iv) CM group (the hAMSC-CM control group). A549 cells were also cultured in 6-well plates, and apoptosis was measured by flow cytometry 24 h after treatment. Other A549 cells were cultured in a 25 cm2 cell culture flask. At 24 h after intervention, the morphology of the cells was observed, and the levels of ROS were detected. The expression levels of related proteins in the cells were detected by western blot analysis. The cytokine content in the supernatant was detected by ELISA.
CCK-8 experiment. A549 cells were cultured and then exposed to different concentrations of PQ solution (0, 250, 500, 750 and 1,000 µM) for 24 h at 37℃ (6 replicate wells per group). Then, 10 µl of CCK-8 reagent (Beijing Soleil Technology Co., Ltd.) was added to each well, and the cells were incubated at 37℃ for 2 h. The absorbance value at a wavelength of 450 nm was measured, and cell proliferation activity was calculated according to the following formula: cell proliferation activity (%) = (experimental group OD value - zero well OD value) / (control group OD value - zero well OD value). Analyses were conducted in the same manner after hAMSC-CM was administered at concentrations of 25, 50 and 75% volume fraction (ie, medium volume/total volume). The order of addition was medium, PQ solution and hAMSC-CM, and the cells were analyzed after 24 h of culture at 37℃.
Qualitative observation of intracellular ROS. 2,7-Dichlorofluorescein diacetate (DCFH-DA) itself has no fluorescence and is hydrolyzed into DCFH by esterase after entering cells. In the presence of active oxygen, DCFH is oxidized to form a substance with strong green fluorescence, DCF, that cannot penetrate the cell membrane (15). The fluorescence of DCF has an excitation wavelength of 502 nm, the emission peaks near 530 nm and the intensity is proportional to the level of intracellular ROS. Reactive oxygen species Assay Kit was obtained from Nanjing Jiancheng Bioengineering Institute. (catalog number: E004-1-1and was used according to the manufacturer’s protocol. After 24 h of culture at 37℃, the cells in each group were washed once with PBS. Next, 40 µM DCFH-DA dye was added to each group, and the mixture was incubated for 30 min at 37℃. The development of fluorescence in the cells of each group was observed under a green light source with a fluorescence microscope (Olympus Corporation),100×.
Observation of cell morphology. After cells were cultured for 24 h, cell morphology was observed under an inverted phase-contrast microscope (OLYMPUS, 200×). The cells in each group were washed twice with PBS, digested and centrifuged (2,200 × g; 5 min) at 25℃. The supernatant was discarded. The cells in the pellets were fixed with 1% citric acid for 24h at 4℃, dehydrated with different concentrations of ethanol and acetone, soaked, embedded, trimmed, sectioned and stained (stains: Uranyl Acetate and Lead Citrate were purchased from HEAD (BeiJing) Biotechology Co. Ltd). A transmission electron microscope (Zeiss, Germany, bar = 2µm) was used to observe changes in tissue structure.
Detection of oxidation index values and inflammatory factor levels in cells and their supernatants. Samples were prepared according to the corresponding kit instructions. The activity of superoxide dismutase (SOD) in A549 cells was determined using the xanthine oxidase method (Superoxide Dismutase assay kit, Nanjing Jiancheng Bioengineering Institute). The MDA content in A549 cells was detected by the thiobarbituric acid method (Cell Malondialdehyde assay kit, Nanjing Jiancheng Bioengineering Institute). The levels of interleukin (IL)-1β, IL-6 and IL-10 in the supernatants of A549 cells were detected by ELISA (Wuhan Sanying Biotechnology). Blank control wells and standard wells were included in the experiment. The absorbance was measured at a wavelength of 450 nm to prepare a standard curve, and the concentrations of the samples to be tested were calculated.
Apoptosis detection with Annexin V-FITC/propidium iodide (PI). Annexin V-FITC (TransDtetce Annexin V-FITC/PI Cell Apoptosis Detection Kit) was purchased from TransGen Biotech (), and it can specifically bind to phosphatidylserine, which translocates to the membrane during apoptosis, and emit green fluorescence under excitation by blue light to enable apoptotic cells and normal cells to be distinguished. PI is a nucleic acid dye that cannot penetrate intact cell membranes; however, it can penetrate damaged cell membranes (including those of late apoptotic and dead cells) and stain the nuclei red. The signals of these two labels can be compared to distinguish between early and late apoptotic cells. After 24 h of culture, cells (density 2×106)were digested with 0.25% trypsin without EDTA for 1 min and then collected, washed twice with cold PBS, and resuspended in binding buffer. Annexin V-FITC and PI were added to the cell suspensions, and the solutions were mixed and protected from light for 5 min. Flow cytometry (BD, FACSCanto™ II Flow Cytometry System) was performed within 1 h. BD FACSDiva Software (v8.0.2) was used for analysis. Quantification of the respective flow plots. Q1, Q2, Q3 and Q4 represent necrotic cells, late apoptotic cells, early apoptotic cells and normal cells, respectively.
Western blot detection of related protein expression. A549 cells of each group were collected, appropriate amounts of lysis buffer were added, and the mixtures were centrifuged at 4˚C (39,588 × g, 10 min; Beckman Coulter, Inc.). The supernatants were collected, and the protein concentrations were determined by the BCA method. Loading buffer was then added, and the proteins were denatured at 100˚C for 5 min. A total of 20 µg of each protein sample was added to each lane for SDS-PAGE (separation gel concentration 12% and concentrated gel 5%). After electrophoresis, the proteins were transferred to a PVDF microporous membrane at 200 mA for 70 min and blocked with 3% BSA (Beijing Solarbio Science & Technology Co., Ltd)in TBST (5% of Tween 20) blocking solution for 2 h at room temperature. Then, the membrane was incubated with diluted primary antibodies, including a b-actin monoclonal antibody (cat. no. 66009-1-lg; dilution 1:20,000), a Bcl-2-related X protein (Bax) polyclonal antibody (cat. no. 50599-2-lg; dilution 1:7,000), a Bcl-2 polyclonal antibody (cat. no. 12789-1-AP, dilutions 1:3,000), and a CHOP polyclonal antibody (cat. no. 15204-1-AP, dilution 1:1,000) (Wuhan Sanying Biology Technology Co., Ltd.), overnight at 4˚C and washed three times with TBST (15 min each time). The membrane was then incubated with secondary antibodies, including HRP-conjugated Affinipure goat anti-rabbit IgG (H + L)(dilution 1:5,000; Wuhan Sanying Biology Technology Co., Ltd.) and HRP-conjugated Affinipure goat anti-mouse IgG (H + L) (dilution 1:5,000; Wuhan Sanying Biology Technology Co., Ltd.) for 2 h at room temperature before being washed three times with TBST. A mixture of luminescent liquids A and B was evenly dropped onto the membrane༈ECL ENhanced Kit, Beijing Dining Biotechnology Co., Ltd༉, which was then placed in a Bio-Rad gel imaging system for imaging. The Western blot results were analyzed by grayscale scanning (Bio-Rad Laboratories, Inc.) using ImageJ software (National Institutes of Health༌v5.2).
Statistical analysis. Experimental and measurement data are expressed as the mean ± standard deviation (x̅ ± s). SPSS 18.0 statistical software (SPSS, Inc.) was used for statistical analysis. One-way ANOVA was conducted among groups, the post hoc test used following one way ANOVA was Tukey’s HSD, and P < 0.05 was considered to indicate statistical significance.