LPS-Induced AM Cell Modeling
Rattus norvegicus (NR) 8383 AM cells were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). NR8383 AM cells were cultured in Ham’s F-12K medium (Boster, China) supplemented with 15% (v/v) fetal bovine serum (FBS, Seo, China). AM cells were seeded into six-well plates at a density of 5 × 105 cells per well before being treated with 1 µg/mL lipopolysaccharide (LPS; Escherichia coli 055:B5; Sigma) for 6, 12 and 24 h. The negative control group was treated with an equal volume of phosphate-buffered saline (Biochem, Shenzhen, China). In subsequent experiments, the cells were randomly assigned to five groups: LPS + SC (PTP1B inhibitor, SC-222227; Santa Cruz Biotechnology, CA, USA), LPS + SC + tunicamycin (ER stress inducer, M4798; AbMoleBioScience, Houston, TX, USA), and LPS + SC + PP2 (Src inhibitor, HY-13805; MedChemExpress, USA). According to previous literature [15–17], the AM cells were pretreated with 2 µM SC for 30 min, 1.5 µg/mL tunicamycin for 24 h before SC, and 15 µM PP2 for 1 h before SC. Next, the AM cells in the LPS group were treated with LPS (1 µg/mL) for 6 h, and then collected for RNA or protein isolation. Inflammatory factors in the collected supernatant were measured using enzyme-linked immunosorbent assay (ELISA). AM cells were collected for western blotting analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
Animals
C57BL/6 mice (8 weeks old; body weight: 18–22 g) were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., China (certificate of conformity: SCXK2019-0001). According to institutional regulations, the mice were fed pathogen-free food and water under standard conditions (12-h light/dark cycle, 25–27°C, humidity of ~ 40%).We performed the invasive procedures with pentobarbital anaesthesia to minimize suffering and killed all the animals by inhaling excessive amounts of isoflurane anaesthetic.
Establishment of the Cecal Ligation and Puncture Model
Cecal ligation and puncture (CLP) was performed in mice as previously described [18]. First, after anesthesia and disinfection with betadine solution, laparotomy was performed on the abdomen to expose the cecum and adjoining intestine. Next, a 3.0 thread suture was used to tightly ligate the intestinal canal under the ileocecal section, after which a 20-gauge needle was used to puncture the canal wall, and a small amount of intestinal content was gently squeezed into the abdominal cavity to establish a sepsis mouse model. Finally, the mice received a subcutaneous injection of 1mL of saline for postoperative resuscitation. Mice in the sham group underwent laparotomy techniques without ligation and puncture. As previously described [10, 19–20], SC (10 mg/kg) and tunicamycin (1 mg/kg) were intraperitoneally injected into C57BL/6 mice 2 h before CLP surgery, and PP2 (1 mg/kg) injected 1 h before surgery.
Lentivirus Gene Delivery
Lentivirus gene delivery was conducted as previously described [21]. Lentiviruses silently expressing PTP1B were constructed by GeneChem Group (Gene Co., Ltd, Shanghai, China). AM cells were cultured in a medium with Ham’s F-12K containing 15% (v/v) FBS. The RNA and protein extraction groups were transfected with the indicated lentiviruses for 72 h in the culture medium.
Real-Time Quantitative PCR
TransZol Up (O41220; TRANS, China) was used to extract RNA from the samples, and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) used to assess the extracted RNA concentration and purity. EasyScript One-Step gDNA Removal and cDNA Synthesis Supermix (AE311; TransGen Biotech) were used to prepare cDNA using a StepOnePlus analyzer Real-Time PCR system (Applied Biosystems, USA). All qRT-PCR analyses were performed using the PerfectStart Green SuperMix kit (AQ601; TransGen Biotech) and an Applied LightCycler96 Real-Time PCR instrument. The qRT-PCR mixtures contained 5.0 µL of PerfectStart Green qPCR SuperMix, 3.6 µL of nuclease-free water, 0.4 µL of primers (10 µM), and 2.0 µL of the reverse transcription product. Optimization of the qRT-PCR amplification conditions was as follows: 94°C for 30 s, 40 cycles of each at 94°C for 5 s, 60°C for 30 s, 95°C for 10 s, 65°C for 60 s, 97°C for 1 s, and 37°C for 30 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The PCR primer sequences are presented in Tables 1 and 2.
Table 1
The rat primers for qRT-PCR.
| Primer (5’-3’) |
GAPDH | Forward :GTCATCAACGGGAAACCCAT Reverse :ACGCCAGTAGACTCCACGACAT |
PTP1B | Forward:TGGGCGGCTATTTACCAGGA Reverse:CACCATCTCCCAGAAGTGCC |
eif2α | Forward:TTCGCCATGTTGCTGAGGTA Reverse :TGACAGCTTGTGGGGTCAAA |
CHOP | Forward:AACCTGAGGAGAGAGAAACCG Reverse :TGCAGATCCTCATACCAGGC |
GSDMD | Forward:AGCATCCTTGCATTCCGAGT Reverse :TAAAGTCATGCCGCCTCTGG |
Caspase-1 | Forward: TTTCCTGGACCGAGTGGTTC Reverse: AGGTCAACATCAGCTCCGAC |
NLRP3 | Forward: GCATGCCGTATCTGGTTGTG Reverse: AGGGTACCCCATAGACTGGC |
IL-1β | Forward: AGCTTCAGGAAGGCAGTGTC Reverse: TCAGACAGCACGAGGCATTT |
IL-18 | Forward: TCAGCTCTTCTACCAGCAAACA Reverse: TTCCAACTGAGAGGCTGTGC |
TNF-α | Forward: GGCTTTCGGAACTCACTGGA Reverse: GGGAACAGTCTGGGAAGCTC |
Table 2
The mice primers for qRT-PCR.
| Primer (5’-3’) |
GAPDH | Forward :TTTTGTCTACGGGACGAGGC Reverse :TACGGGTCTAGGGATGCTGG |
PTP1B | Forward:GGCTATTTACCAGGACATTCGAC Reverse:TCCATGATGCGGTTGAGCAT |
eif2α | Forward:TCCTCGTTGCCACTAAGCAG Reverse :AACAAGCTGACATAGGCCCC |
CHOP | Forward:TCTTGAGCCTAACACGTCGATT Reverse :ACGTGGACCAGGTTCTCTCT |
GSDMD | Forward:GATCAAGGAGGTAAGCGGCA Reverse :CACTCCGGTTCTGGTTCTGG |
Caspase-1 | Forward: CACTCCGGTTCTGGTTCTGG Reverse: TGATCACATAGGTCCCGTGC |
NLRP3 | Forward: GGCTGCTATCTGGAGGAACTT Reverse: GGGATACAGCCTTTCTCGGG |
IL-1β | Forward: GCCACCTTTTGACAGTGATGAG Reverse: AGCTTCTCCACAGCCACAAT |
IL-18 | Forward: TGAGGCATCCAGGACAAATCA Reverse: GAACCACAGAGAACCCCCAC |
TNF-α | Forward: AGGCACTCCCCCAAAAGATG Reverse: CCACTTGGTGGTTTGTGAGTG |
Western Blot
Total protein was extracted from the cells using lysis buffer containing a protease inhibitor, and protein levels were quantified using a protein quantification kit (Beijing Pulilai Gene Technology Co., Ltd., China). Proteins were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk for 1 h at room temperature (RT) and then incubated with primary antibodies for 24 h at 4°C. Subsequently, the membranes were incubated with secondary antibodies at RT for 2 h. GAPDH was used as the internal reference. Blots were visualized using an enhanced chemiluminescence assay kit (Millipore), developed with a Bio-Rad Gel Doc EZ imager (Bio-Rad, USA), and band intensities were analyzed using ImageJ software (NIH Image analysis). Primary antibodies used included anti-PTP1B (1:1000, ab244207; Abcam, UK), anti-p-eif2α (1:1000, 3398; Cell Signaling Technology, USA), anti-eif2α (1:1000, 5324T; Cell Signaling Technology), anti-CHOP (1:1000, T56694; Abmart, China), anti-GSDMD (1:1000, P30823; Abmart), anti-NLRP3 (1:1000, ab263899; Abcam, UK), anti-caspase-1 (1:1000, 22915-1-Ap; Proteintech, China), anti-p-src (1:200, sc-166860; Santa Cruz), anti-Src (1:1000, 2109; Cell Signaling Technology), and anti-GAPDH (1:20000, 60004-1-Ig; Proteintech).
Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay (ELISA) was performed to assess the concentration of inflammatory cytokines according to the manufacturer’s instructions. Specific ELISA kits were purchased from Fine Test (Wuhan Fine Biotech Co., Ltd, China). Levels of IL-1β and TNFα in mouse plasma and bronchoalveolar lavage fluid (BALF) harvested from each group were detected using commercial mouse IL-1β and TNFα kits. IL-18, IL-1β, and TNF-α release levels in the supernatants were measured using specific rat ELISA kits.
Hematoxylin and Eosin Staining and Histopathological Analysis
Mice were euthanized after anesthesia, and fresh lung tissues collected, fixed in 4% paraformaldehyde (PFA) buffer, embedded, and sliced into 5-µm-thick sections. The slices were stained with hematoxylin and eosin to assess lung injury. Hematoxylin and eosin-stained images were captured using a microscope (Zeiss) and evaluated by two pathologists to assess the degree of lung injury, as previously reported [22]. The sections were analyzed to assess inflammatory cell infiltration, epithelial desquamation, edema, and hemorrhage. Each characteristic was scored as 3 (prominent), 2 (moderate), 1 (mild), or 0 (absent).
Lung Wet/Dry Ratio
Lung edema was evaluated by calculating the lung wet/dry (W/D) ratio. Immediately after euthanasia, the wet weight of the right lung from each mouse was calculated and then dried in an incubator at 65°C for 24 h. The dry weight was measured, and the W/D ratios were calculated.
Immunofluorescence Staining
Fresh lung tissue was fixed with 4% PFA, dehydrated in a sucrose gradient solution, wrapped in Tissue-Tek OCT, and sectioned into 5-µm-thick slices. The tissue slices were fixed in 4% PFA, permeabilized, blocked with 5% goat serum to minimize nonspecific staining, and then incubated with primary antibodies overnight at 4°C. The primary antibodies used were anti-PTP1B (1:50, ab244207; Abcam, USA), anti-NLRP3 (1:200, WL02635; Wanleibio, China), and anti-caspase-1 (1:100, 22915-1-Ap; Proteintech). After incubation with the secondary antibodies Alexa Fluor 488 (Thermo Fisher Scientific, USA) and Alexa Fluor 594 (Thermo Fisher Scientific) for 1 h at RT, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Boster) for 5 min without exposure to light. The samples were visualized using a confocal laser scanning microscope (Olympus) or a fluorescence microscope (Zeiss).
Co-immunoprecipitation Assay
Cells were lysed in equal proportions of immunoprecipitation (IP) buffer (Beyotime, China) with a complete protease inhibitor cocktail (MedChemExpress) on ice for 30 min. After centrifugation, protein samples were collected and prepared for IP assays. Protein A/G magnetic beads (MedChemExpress) were coupled to anti-p-Src antibodies (Santa Cruz) at 1–2 µg per 100–500 µg of total protein or anti-PTP1B (1:30; Abcam, UK) and rotated for 2 h at 4°C. After washing three times, 500 µg of total protein was added and co-rotated with the antibody-magnetic bead complex overnight at 4°C. Equivalent amounts of cognate mouse or rabbit IgG antibodies were used as IP controls. Finally, immunoprecipitated proteins were dissolved in 25 µL 1× loading buffer (Solarbio, China) by boiling for 10 min for western blot analysis.
Statistical Analysis
Three biological replicates were used for all the experiments. Data are expressed as mean ± standard deviation. All data were analyzed using SPSS (version 19.0, IBM, USA) and statistical charts were generated using GraphPad Prism 8 software. Student’s t-test was used to compare differences between two groups and analysis of variance (ANOVA) was used to compare differences between multiple groups. Dunn’s multiple comparison test was used for one-way ANOVA, and Fisher’s least significant difference test was used for two-way ANOVA. The survival of the sham, CLP, and CLP + SC groups was analyzed using Kaplan–Meier curves. Statistical significance was set at P < 0.05. significant.