Animals and laboratory protocols
Male c57bl/6 mice (8 weeks old and weighing 20± 2 g) were purchased from the Experimental Animal Center of Guangxi Medical University (animal certificate number: SYXK9 (GUI) 2014-0003). All animal experiments, including disposal of experimental animals, were approved by the animal ethics review of Guangxi Medical University. All animal surgeries were approved by the Ethics Committee of Experimental Animal Center of Guangxi Medical University (Nanning, China; Approval No. 202008010). Animals were assigned to one of four groups and treated as follows: LPS: 5 mg/kg intravenous LPS; LPS+UTI: 5 mg/kg intravenous LPS immediately followed by 50000 U/kg intraperitoneal UTI; Control group; UTI: 50000 U/kg intraperitoneal UTI. Lung tissue and bronchial alveolar lavage fluid (BALF) were collected on days 1, 3, 5 and 7 after LPS-modeling.
Reagents and antibodies
UTI was purchased from Guangdong Tempu Biochemical Pharmaceutical Co., LTD. (Guangzhou, China). LPS (Escherichia coli 055: B5) and PKH26 Red were purchased from Sigma Biochemical Corporation (St. Louis, USA). CellTrackerTM green B0DIPYTM was purchased from Invitrogen (California, USA). The ELISA kit was purchased from Wuhan Huamei Biological Engineering Co., LTD. (Wuhan, China). BCA test kit and Diff-Quick dye solution were purchased from Beijing Soleibao Technology Co., LTD. (Beijing, China). BIX02189 was purchased from MedChemExpress (New Jersey, USA). DMEM, RPMI1640 and FBS were purchased from Gibco BRL (Gaithersburg, Maryland, USA). Antibodies raised against the following proteins were used: ERK5 (Abcamab40809); Mertk (AbclonalA5443); Goat anti-rabbit immunoglobulin horseradish peroxidase (igg-hrp) (Ray antibody: RM3003); Fluorophile conjugated goat anti-rabbit secondary antibody (Alexa Fluor 488; Invitrogen: MA700184A488);
Cell culture
Mouse peritoneal macrophage cell-line, RAW264.7, and human acute myeloid leukemia cell-line, HL60, were purchased from Wuhan Procell Life Science and Technology Co., LTD. RAW264.7 cells were cultured in high glucose DMEM supplemented with 10 % (v/v) FBS and HL60 cells were cultured in RPMI 1640 medium supplemented with 12 % (v/v) FBS, 100 U/ mL penicillin and 100 μg/ mL streptomycin and in humidified air with 5 % CO2 at 37 ℃. UTI was dissolved in PBS and further diluted with cell culture media.
Specimen collection
The left lung was isolated immediately after anesthesia and fixed with 4 % paraformaldehyde. The upper lobe of the right lung was isolated, weighed and baked at 60 ℃ for 72 hours. Bronchoalveolar lavage fluid (BALF) was collected into 2 ml pre-cooled phosphate buffered saline (PBS) and the total number of cells were determined immediately. BALF was centrifuged and the supernatant was stored at -80 ℃ for determination of total protein and inflammatory factor levels. The cell pellet was immediately re-suspended in PBS and numbers of neutrophils were determined by Diff-Quick staining solution.
Lung histopathological analysis
The left lung was dehydrated, embedded, sectioned, stained with hematoxylin-eosin (HE) and histopathological changes were observed under light microscopy, as previously described[22,23]. Lung tissue was assigned an injury score, as follows: 0: normal; 1: mild injury affecting 25 % of lung tissue; 2: moderate injury affecting 25%-50 % of lung tissue; 3: severe injury affecting 50 %-75 % of lung tissue; 4: very severe injury affecting more than 75 % of lung tissue.
Inflammatory response
A wet-dry ratio was calculated for the lung tissue to indicate lung exudation during LPS-induced injury. The wet weight was determined immediately after collection and tissue dried in an oven for 72 hours before determination of the dry weight. BALF cells were counted by hemocytometer and concentrations of protein plus inflammatory factors were measured with a BCA test kit. The BALF cell pellet was re-suspended in PBS and cells were evenly distributed on a glass slide using a cell slicer before estimation of neutrophil number by Diff-Quick dye staining, as previously described[24].
Phagocytosis by alveolar macrophages in vivo
BALF was centrifuged and cell pellets resuspended in PBS with 30 % FBS to a density of 5×105 cells/ mL, as described previously[25]. 200 μL cell suspension was added to each sling hole and evenly distributed on the slide with a cytoprep (MOTIC, Xiamen, China). Diff-Quick dye was added, according to the manufacturer’s instructions. After staining, 300 macrophages were randomly selected under the microscope and numbers of apoptotic bodies within the macrophages were divided by 300 to obtain the phagocytic rate.
Preparation of apoptotic cells
HL60 cells were suspended in 10 ml RPMI1640 medium and seeded into a Petri dish, 9 cm from the ultra-clean cell for 30min UV irradiation. After incubation at 37°C for 3 h, apoptotic HL60 cells were stained with PKH26 Red.
Macrophage phagocytosis in vitro
In vitro phagocytosis was assessed as described previously[21]. Briefly, RAW264.7 cells were seeded into 24-well plates at 105 cells/ml. After adhesion, cells were pretreated with either 1000 U/mL or 5000U/mL UTI for 3 h[26]. Cells were stained with CellTrackerTM Green B0DIPYTM dye to a final concentration of 5 μM for 30 min. Cells were washed 3 times with PBS and 106 cells/well apoptotic HL60 cells were added with incubation in a cell incubator overnight. Wells were washed 3 times with PBS and anti-fluorescence quench agent added. Phagocytosis was observed under a fluorescence microscope.
Immunofluorescence
The cell experiment was first divided into 0 U/ mL, 1000 U/ mL and 5000 U/ mL groups. Mouse peritoneal macrophages RAW264.7 cells were seeded into 24 well plates at 105/ mL. After cell adherence, the cells were pretreated with ulinastatin at different concentrations for 3 h. ERK5 inhibitor BIX02189 was used to divide the cells into blank control group, BIX02189 group, 5000 U/ mL group and 5000 U/ mL +BIX02189 group. The cells were pretreated with ERK5 inhibitor BIX02189 for 1 h and then 5000 U/mlUTI for 3 h. Cells were washed 3 times with PBS and 4 % paraformaldehyde added to fix the cells at 4 ℃ overnight. After washing 3 times with PBS, primary antibodies raised against Mer (1:100) were added for overnight incubation at 4 ℃. Fluorescently conjugated secondary antibodies (AlexaFluor488) were used for staining and cells were incubated at room temperature in the dark for 1h. After 3 washes with PBS, DAPI was used for nuclear staining, slides observed by fluorescence microscope equipped with Nikon DS-U3 imaging system and fluorescence intensity quantified with Image-J software.
Western blotting
The cells were grouped as before. Total protein was extracted and protein loading buffer added. After boiling and denaturation, SDS-PAGE electrophoresis was carried out, proteins were transferred to a PVDF membrane and incubated with ERK5 primary antibody (1:1000) at 4 ℃ overnight. Goat anti-rabbit fluorescent secondary antibody (1:30,000) was added, incubated at room temperature for 1 h and bands developed by fluorescence scanner. Image-J software was used to determine the gray value of each band and relative expression levels of ERK5 protein represented by the ratio of the ERK5 gray band to the GAPPH band.
Statistical analysis
SPSS 23.0 software was used for statistical analysis. Student’s t test was used to compare means ± standard deviations (x±s), for two groups with normal distribution and ANOVA and SNK test to compare multiple groups. A value of p< 0.05 was considered statistically significant.