Animals use approval and reagents
Sprague-Dawley rats weighing 100–150 g were purchased from the Experimental Animal Center of the Southern Medical University (Guangzhou, China). All animals were allowed food and tap water ad libitum and exposed to a 12 h light/12 h dark cycle in accordance with the Principles of Laboratory Animal Care approved by Southern Medical University. LPS (O111:B4) was purchased from Sigma Chemical Company (St. Louis, MO., USA) and dissolved in 0.9% saline before use. SYBR Premix Taq™ kit was purchased from TaKaRa Biotechnology Co., LTD (Shiga, Japan). Tissue protein extraction reagent was purchased from TaKaRa Biotechnology Co., LTD (Shiga, Japan). Antibody specific for total NF-κB p65 subunit was purchased from Abcam (Cambridge, MA., US). Enzyme-linked immunosorbent assay (ELISA) kit of TNF-α was purchased from Thermo Scientific Pierce Protein Research Products (Rockford, IL., USA).
RNA interference
Three siRNAs targeting NF-κB were synthesized by Shanghai GenePharma Co., LTD (Shanghai, China). All siRNAs (sense: 5′-GGA GUA CCC UGA AGC UAU AUU-3′; antisense: 5′- UAU AGC UUC AGG GUA CUC CUU -3′) were tested on lung tissue cells to choose the one with the highest gene silencing efficacy for further use. The scrambled siRNA (sense: 5′-UUC UCC GAA CGU GUC ACG UUU-3′; antisense: 5′-ACG UGA CAC GUU CGG AGA AUU-3′) was used as control. All siRNAs were dissolved by DEPC-treated water to a final concentration of 40 μg/ml.
Construction of LPS-induced ALI models
According to individual weights, SD rats were intraperitoneally injected with 5 mg/kg LPS dissolved in 2 ml sterile saline with a 20 gauge-needle syringe to establish ALI models. Since LPS administration can cause neutrophil infiltration, interstitial edema, hemorrhage and proinflammatory cytokine production, which effectually simulates the natural development of ALI, it provides us with an optimal platform to test siRNA efficacy.
Experimental protocol
All SD rats were randomly assigned to one of the following four groups (n = 12, each group). Saline + DEPC group received an intratracheal instillation of 1.5 ml DEPC-treated water immediately after an intraperitoneal injection of 2 ml saline. LPS + DEPC group received an intratracheal instillation of 1.5 ml DEPC-treated water immediately after LPS administration. LPS + scramble group received an intratracheal instillation of 1.5 ml scramble RNA (40 μg/ml) immediately after LPS administration. LPS + siRNA group received an intratracheal instillation of 1.5 ml siRNA (40 μg/ml) immediately after LPS administration. Six rats in each group were randomly sacrificed at 1 h and 8 h after the instillation respectively. Rectal temperatures were measured before both LPS injection and then sacrifice. Rectal temperature changes were calculated by taking the rectal temperature at time of sacrifice and subtracting the rectal temperature before LPS injection. Right lung was lavaged to collect bronchoalveolar lavage fluid (BALF). Then the lower lobe of right lung was rapidly excised and preserved in the liquid nitrogen for subsequent tissue RNA and protein extraction. The superior lobe of left lung was used for histopathological examination. The lower lobe of left lung was excised for analysis of lung wet/dry weight ratios. Blood samples were collected from iliac artery. The rats were anesthetized by chloral hydrate (3.5 ml/kg) throughout the surgical procedures.
Bronchotracho alveolar lavage (BAL)
Animals were anesthetized by intraperitoneal injection of chloral hydrate (3.5 ml/kg) and fixed on a board in a supine position. After sterilization, a median sternotomy was performed to expose both of lungs and trachea. The hilum of the left lung was ligated, and then the right lung was lavaged twice with 5 ml ice-cold phosphate buffered saline (PBS). Each time, 4.5 ml of the injected PBS was recovered. The collected BALF was immediately centrifuged at 500 × g for 10 min at 4 °C, and the cell-free supernatant were stored at -80 °C until TNF-α concentration assay. The concentration of TNF-α in BALF was quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacture’s recommendations.
Histopathological examination
The superior lobe of left lung of each rat was harvested when rats were sacrificed, fixed in 10% neutral buffered formalin for 24 h, and embedded in paraffin. Tissues were cut into a series of microsections (4 μm), and then stained with hematoxylin-eosin using standard protocols.
Lung wet/dry weight ratio
As an indication of lung edema, the lung wet/dry weight ratio was calculated by dividing the wet weight by the dry weight. The lower lobe of the left lung was excised and weighed to obtain the wet weight. After drying in an oven at 80 °C for 24 h, the lobe was weighed again to obtain the dry weight.
Western blot analysis
The lower lobe of the right lung was harvested and stored in −80 °C liquid nitrogen. After homogenization of tissue samples, total proteins were extracted and their concentrations were measured by BCA protein assay. Cell lysates (20 μg protein/lane) were separated on 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, which were subsequently blocked with 5% skim milk in a PBST solution (100 mM NaCl,50 mM Tris,0.1% Tween-20,PH 7.5)for 1 h at room temperature and probed overnight at 4 °C with appropriate primary antibodies. Horseradish peroxidase (HRP) conjugated anti-rabbit IgG and anti-rat IgG were used as secondary antibodies according to the primary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence.
Total RNA extraction and qRT-PCR
The lower lobe of the right lung was harvested and stored in −80 °C liquid nitrogen. Total RNA was extracted from tissue samples using Trizol reagent. 2 μl of total RNA from each sample in a final reaction volume of 10ul was converted into single-stranded cDNA using SuperScript II Reverse Transcriptase (Invitrogen) following the manufacturer’s recommendations. The RT PCR reaction was carried out with 37 °C for 15 min and 85 °C for 50 s. qRT-PCR was performed using SYBR Premix Taq™ kit with the 95 °C activation step for 30 s; 95 °C denaturation step for 5 s, 62 °C annealing step for 30 s (NF-κB) or 58 °C annealing step for 30 s (TNF-α), 72 °C extension step for 34 s for 40 cycles; and a final extension step of 72 °C for 4 min. The following primers were used: NF-κB forward: 5′-GGG ACT ATG ACT TGA ATG CGG TCC -3′, reverse: 5′-CAG GTC CCG TGA AAT ACA CCT CAA-3′; TNF-α forward: 5′-CTT CTG TCT ACT GAA CTT CGG-3′, reverse: 5′-GTG CTT GAT CTG TTG TTT CC-3′; GAPDH forward: 5′-ACC ACA GTC CAT GCC ATC AC-3′, reverse: 5′-TCC ACC ACC CTG TTG CTG TA-3′. Results in triplicate were expressed as relative transcriptions of the NF-κB mRNA and TNF-α mRNA using the following formula: \( \mathrm{Folds} = {2^{\hbox{-}}}^{\varDelta \varDelta \mathrm{C}\mathrm{t}};\kern0.5em \varDelta \varDelta \mathrm{C}\mathrm{t} = {\left(\mathrm{C}{\mathrm{t}}_{{}_{\left(\mathrm{target}\ \mathrm{genes}\right)}}-\mathrm{C}{\mathrm{t}}_{{}_{\kern0.5em \left(\mathrm{GAPDH}\right)}}\right)}_{\mathrm{experimental}\ \mathrm{group}}-{\left(\mathrm{C}{\mathrm{t}}_{{}_{\left(\mathrm{target}\ \mathrm{genes}\right)}}-\mathrm{C}{\mathrm{t}}_{\left(\mathrm{GAPDH}\right)}\right)}_{\mathrm{control}} \).
Statistical analysis
Statistical analysis was conducted using SPSS 13.0 software package. All data were expressed as mean ± S.D. and compared using the student’s t-test for two-group analysis and one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls method for multiple-group analysis. Differences in values were considered significant at P < 0.05.