2.1. Materials and Reagents
Fresh fish (Pneumatophorus japonicus) heads, were provided by Zhejiang Xingye Group Co., Ltd., China. Two proteases (trypsin, alcalase) were provided by Shanghai Yuanye Biological Technology Co., Ltd. Both 10 kDa and 3 kDa ultrafiltration membranes were purchased from Millipore, 1 kDa ultrafiltration membranes were purchased from PALL. The Gastric Mucin was purchased from Beijing Jinming Biotechnology Co., Ltd. MTT Cell Proliferation and Cytotoxicity Assay Kit were provided by Shanghai Yuanye Biological Technology Co., Ltd. Lactate dehydrogenase LDH activity quantitative determination kit was purchased from Beijing Applygen Technology Co., Ltd.
2.2. Preparation of Pneumatophorus japonicus heads peptides (PHPs)
An appropriate number of fish heads were thawed, cleaned, drained crushed, and placed them in a 500 mL conical flask, then, according to the best results of the laboratory's early single factor test, shaken in a water bath for hydrolysis. The solid-liquid ratio was 1:4 (W/V), the amount of enzyme added was 0.32% (Alkaline protease and trypsin enzyme activity ratio was 1:3), the enzymatic hydrolysis time was 4.03 h, and the enzymatic hydrolysis temperature was 50.95℃, after enzymatic hydrolysis was completed, the enzyme was inactivated at a constant temperature of 90℃ water bath 15 min. The enzymatic solution was cooled to room temperature, and then centrifuged in a centrifuge for 10 minutes at 10,000 rpm/min. The supernatant was collected and filtered to obtain PHPs. Finally, PHPs was separated step by step through ultrafiltration membranes with molecular weight cutoffs of 10 kDa, 3 kDa, and 1 kDa, and PHPs of different molecular weights were collected and freeze-dried for 48 h before use.
2.2.1. Cell culture
Human gastric mucosal epithelial cells (GES-1) were purchased from Feng Hui Biotechnology Limited Company (Hunan, China). The cells were maintained RPMI-1640 complete medium (Gibco Company Inc., USA) supplemented with 10% Fetal Bovine Serum (FBS) (TIANHANG Biotechnology Company Inc., China) and 100 U. mL-1 penicillin, 100 U. mL-1 streptomycin in a humidified incubator with 5% CO2 and 37℃. Cells were collected for use after entering the logarithmic growth phase.
2.2.2. Cell damage model exploration
GES-1 in logarithmic growth phase were digested and counted, then seeded in 96-well cell culture plate with 1 x 104 cells per well, and cultured in a humidified incubator at 5% CO2 and 37 ℃ for 24 h until cells were essentially attached. GES-1 were incubated in medium containing alcohol (0, 2%, 4%, 6%, 8% and 10%) for 2 h, 3 h, 4 h, 5 h and 6 h respectively, following which MTT was performed. The test parameters at a cell survival inhibition rate of approximately 40% could be used as the best GES-1 injury model [20]. Set up 5 replicate wells for each dilution, and set up a blank control group at the same time.
2.2.3. Cell grouping and treatment
GES-1 were divided into normal group, model group, positive drug gastric mucin group (0.05 mg/mL) and PHPs group (Table S1). Among them, the PHPs group was divided into six groups with three molecular weight ranges of high concentration group (0.3 mg/mL) and low concentration group (0.06 mg/mL). GES-1 in logarithmic growth phase were digested and counted, then inoculated in 96-well cell culture plates with 1x104 cells per well, 100 μL per well, placed in 37 ℃, 5% CO2 incubator until the cells were basically stick to the wall, draw out the culture medium in the well carefully with a syringe and discarded. The positive drug gastric mucin and PHPs medium with different molecular weight segments were added before damage (BD) and after damage (AD) induced by alcohol, 100 μL per well, and the normal group and model group were not added, and cultured for 24 h. Each group was set up with 5 replicate wells, the edge wells of the culture plate were filled with sterile PBS, and a blank control group (without cells) was set.
2.2.4. MTT assay
The cells were treated as above in Cell Grouping and Treatment, and then performed the MTT test. Removed the old culture medium, replaced with 100μL fresh culture medium, added 10 μL of MTT working solution (5 mg/mL) to each well, mixed at low speed and incubated in 37°C incubator for 4 h, then, aspirated the culture medium, added 100 μL per well formazan dissolving solution, mixed well, put into incubator and continued to incubate for 4 h. After incubation, placed it on the microplate reader and mixed well, and measured the absorbance at 570 nm [21]. Recorded the result, adjusted the colorimetric to zero with blank, and calculated the cell survival rate.
2.2.5. LDH cytotoxicity assay
Cell processing was the same as in Cell Grouping and Treatment above. Old culture medium was collected simultaneously during the MTT experiment. Determining the LDH activity of the medium according to the 2,4-dinitrophenylhydrazine method, the result was expressed as U [22].
2.2.6. Mitochondrial membrane potential determination
Analysis of mitochondrial membrane potential with fluorescent dye JC-1 purchased in Beyotime Biotechnology (Beijing China). When the mitochondrial membrane potential was high, JC-1 aggregated in the mitochondrial matrix to form J-aggregates, producing red fluorescence; when the mitochondrial membrane potential was low, JC-1 was incapable of aggregate in the mitochondria at this time, JC-1 was a monomer and produced green fluorescence. Consequently, it was extremely convenient to detect the change of mitochondrial membrane potential through the transition of fluorescence color. Preparation of the sample according to the requirements of the kit, and photographed with Laser Scanning Confocal Microscope.
2.2.7. Acquisition of infrared transmission imaging
Took 10 μL of single cell suspension sample and dropped on CaF2 sheet (13 mm x 2 mm) at room temperature (25°C, air humidity less than 40%) and dried for 2 h. After the sample was dried, the CaF2 sheet was placed on the transmission imaging accessory stage of the infrared microscope imaging system, and an infrared light absorption map (100 μm x 100 μm) was collected after finding a suitable area in the view of the optical microscope. Infrared microscope images of the cell samples were acquired by Spectrum Image software with a spectral resolution of 4 cm-1, an image pixel size of 6.25 μm, 16 scans per pixel, and a wave number range of 4000-900 cm-1 for the acquired spectra. Infrared microscope images of the cell samples were collected and analyzed by Spectrum Image software. After removing atmospheric noise and baseline correction, analysis was performed.
2.2.8. Acquisition of IR spectra
Fourier transform infrared spectrometer (PerkinElmer, Inc., USA), equipped with DTGS detector and ATR accessories.
Took 10 μL of GES-1 single cell suspension, dropped it on the center of CaF2 sheet (12 mm x 1 mm), and dried it in a sealed container at room temperature (25℃, air humidity less than 40%) for 2 h, and placed it in a sealed and dry container to prevent the interference of moisture in the air before measuring the spectra. The IR spectra of the samples were detected with air as the background to eliminate the interfering effect of H2O and CO2 on the IR spectra of the samples. Then the spectra were collected by 32 scans in reflection mode, in the range of 4000-650 cm-1 with a resolution of 4 cm-1.
2.2.9. Acquisition of second derivative IR spectra (SD-IR spectra)
SD-IR spectra were obtained after baseline correction and 13-point Savitsky Golay smoothing of average IR spectra using Spectrum10™ software (Version 10.6.0, PerkinElmer, Inc., USA).
2.3. Statistical Analysis
Experimental data were expressed as mean ± standard deviation, and analyzed by ANOVA using SPSS 22.0 with p-value less than 0.05. Spectrum fitting was done by PeakFit v4. The related pictures were completed by Origin 2018.