Male C57BL/6 mice and PICK1 KO mice (aged 8–10 weeks) were used in this study. The original breeding pairs of PICK1−/− mice were obtained from Ying Shen (Zhejiang University School of Medicine, Hangzhou) and maintained at the Experimental Animal Center of Wenzhou Medical University. Animals were approved by the Animal Experimentation Ethics Committee of Wenzhou Medical University (Approval No. SYXK 2010 − 0150).
Sepsis-associated acute lung injury model
C57BL/6J mice and PICK1 KO mice were intraperitoneally injected with LPS (10 mg/kg) or saline(7).After all treatment for 12h, the mice were euthanized and the lung tissues were collected and used for subsequent staining and tests.
The mouse monocyte/macrophage cell line RAW264.7 was incubated in DMEM with 10% FBS. PDZ domain inhibitor FSC-231 was purchased from sigma (Ref: 529531-10mg) .CaMKII inhibitor KN93 and its inactive analog KN92 was purchased from GLPBIO (Ref:GC12501-10mM, GC15988-10mM )
Cell apoptosis detection
1 × 106 cells were seeded in 6-well plates and treated with LPS for 18h, and then cells were suspended for further study. Apoptosis was determined using an Annexin-V fluorescein isothiocyanate and propidium iodide (FITC/PI) apoptosis detection kit (Life Technologies, Waltham, MA) with flow cytometry in accordance with the manufacturer’s instruction. Annexin-V (+)/PI (−) and Annexin-V (+)/PI (−) represented the RAW264.7 cells in early apoptosis and late apoptosis/necrosis, respectively. Flow cytometry data was collected on a FACScan (Becton Dickinson) using CellQuest software, then analyzed using FlowJo software (version 9). Each sample was assayed in triplicate to ensure the authenticity of the experiments.
Cell Viability Assay
Cell viability was determined by MTT assay. RAW264.7 cells were cultured in 96-well plates at a density of 10^5 cells/mL and exposed to different concentrations of LPS (500 ng/mL) or 0.1% DMSO as control for 48 h and 10 µL of MTT (5 mg/mL) was added to each well and incubated for 4 h. The supernatant was discarded and the formation was resolved with 150 µL/well of DMSO. The absorbance at 570nm was measured on a microplate reader (BioTek Epoch, Winooski, VT, USA). Concentrations were determined for three wells of each sample, and each experiment was performed in triplicate.
The protein concentration of the supernatant was determined by the BCA protein assay. Then, equal amounts (20µg) of proteins were denatured by heating at 100°C for 5min in 1×nuPAGE loading buffer (Life Technologies, Carlsbad, NM, USA), fractionated by 10% SDS-polyacrylamide gel electrophoresis (Life Technologies), and blotted to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.05% Tween-20 (TBST) and cut into strips, which were incubated with PICK1 (Neurolab, L20/8, 1:1000), PKCα (Abcam, Ab32376, 1:1000), GAPDH (Bio-world, AP0063, 1:1000), Bcl-2 (CST, 3498s, 1:1000), BAX (Abcam, Ab32503, 1:1000), cleaved-Caspase 3(CST, 9663s, 1:1000), respectively, at 4°C overnight. After three washes with TBST for 10min, the strips were incubated with HRP-linked anti-mouse (DC-02L) or anti-rabbit IgG (DC-03L) (Calbiochem), and then followed by ECL (Amersham, RPN2106). The strips were detected using an ECL detector (Image Lab Software, Bio-Rad). The signals were scanned and quantified using Image Lab software (Bio-Rad).
Quantitative polymerase chain reaction
Total RNA was extracted from lung tissues of mice with TRIzol reagent (Invitrogen, Carlsbad, CA).The RNA concentration was measured with absorbance at a wavelength of 260 nm.Then, cDNA was synthesized by reverse transcriptase reaction with cDNA Synthesis kit (TaKaRa, Dalian, China). The resulting cDNA was subsequently amplified with the following IL-6 primers: forward 5’-ACAGGGAGAGGGAGCGATA-3’ and reverse 5’- CCAGTCCTCTTTGTTGGGGAT-3’.
IL-1β: forward 5’-TGATGGCTTATTACAGTGGCA-3’ and reverse 5’-CGGAGATTCGTAGCTGGATG-3’.
TNF-α: forward 5’-CACAGTGAAGTGCTGGCAAC-3’ and reverse 5’-GATCAAAGCTGTAGGCCCCA-3’
GAPDH : forward 5′-AAGAAGGTGGTGAAGCAG-3′ and reverse 5′-AGGTGGAAGAGTGGGAGT-3′. (8)
Lung wet/dry weight ratio
Fresh lung samples were dissected and weighed immediately to obtain the wet weight. Then, lungs were placed in a drying oven at 65°C for 72 h until a constant weight was obtained. The wet to dry weight ratio was calculated to represent the degree of lung edema.(6)
Homogenized tissues and cells were collected and lysed using lysis buffer (50mM Tris, 150mM NaCl, 0.5% Nonidet-40, 5mM EDTA, 1mM PMSF (pH 8.0)) and centrifuged. The decimus supernatant was used as the input, and the remainder was used for coimmunoprecipitation (co-IP). Add 10 µL Mouse anti-PICK1 antibody and 20 µL of resuspended volume of Protein A/G PLUS-Agarose incubate at 4°C on a rocker platform or rotating device for 1 hour to overnight. Collect immunoprecipitates by centrifugation and wash pellet 4 times with 1.0 mL RIPA buffer, each time repeating centrifugation step above. After final wash, aspirate and discard supernatant and resuspend pellet in 40 µL of 2×nuPAGE loading buffer (Life Technologies, Carlsbad, NM, USA) and boiled for 5 minutes at 100°C.The samples were then run on SDSPAGE gels and transferred to PVDF membrane for Western blotting with the indicated antibodies.
Flow cytometry assay for ROS
RAW264.7 cells were seeded at a density of 2 ×105/well in 12-well cell culture plates. Following FSC-231 (50 µM) pre-treatment at 37°C, the cells were stimulated with LPS (500 ng/mL) for 18 hours, and intracellular total ROS was detected using a Reactive Oxygen Species Assay kit (Shanghai Beyotime Bio-Tech Co., Ltd.) according to the manufacturer's protocol. Briefly, following treatment, the cell culture medium was removed, and dichloro-dihydro-fluorescein diacetate (DCFH-DA) was added to a final concentration of 10 µM. Then, the cells were incubated in a CO2 incubator for 20 min at 37°C and washed 3 times with PBS to completely remove the DCFH-DA from the cells. RAW264.7 cells were collected by centrifugation, aspirating and discarding supernatant and resuspending with PBS. DCF fluorescence intensities were detected by flow cytometry or a multi-detection reader at excitation and emission wavelengths of 485nm and 535 nm, respectively. Each sample was assayed in triplicate to ensure the authenticity of the experiments.
Lung superoxide measurement
Superoxide production was evaluated by lucigenin chemiluminescence. Briefly, lungs were homogenized in Krebs-Hepes buffer and centrifuged at 4500 g for 15 min. The supernatants were transferred to scintillation vials containing 5 mM lucigenin (Sigma) in the same buffer, and incubated in the dark for 10 min at 37°C. The chemiluminescence was measured by a luminometer (AB-2200 LuminescencerPSN, ATTO Co., Nagoya, Japan) at 1-min intervals over a 5-min period.Data are expressed as relative light units (RLU) per minute per milligram protein.(6)
All measured data in this experiment were expressed as Mean ± SEM.GraphPad Pro Prism 7.00 software was used for statistical analysis. The comparison between the two groups was performed by Student's t test, and the differences between the groups were compared by one-way ANOVA and corrected by Bonferroni.P < 0.05 was considered statistically significant.