Ethics Statement.
Adult male wild-type (WT) mice of genetic background C57BL/6N and ApoE-/- mice [25,26] (20-25g; 8-10 weeks old) were used in this study. All animals were kept at the Key Laboratory of State Administration of Traditional Chinese Medicine (Cardio-Cerebral Vessel Collateral Disease) (Shijiazhuang, China). All procedures with animals were conducted under the guidelines on the Care and Use of Laboratory Animals for biomedical research published by National Institutes of Health (No.85-23, revised 1996) and according to the ethical guidelines of the Ethics Committee of Hebei Yiling Pharmaceutical Research Institute (No. N2020051).
Animals and treatments
All mice were housed in an animal center under a 12-h light-dark cycle for 7 days to acclimatize to the environment prior to experimentation. The animals had free access to standard rodent diet and water at all times. Wild type C57BL/6N mice were divided into control group, and ApoE-/- mice were divided random into five groups (n=10/group): 1) AS; 2) COPD+AS; 3) TXL; 4) Ato; 5) TXL+Ato. According to the preliminary experiments, the dose of TXL was determined to be 1.5 g/kg/day (about 2 times of human dose), and atorvastatin was 10 mg/kg/d[27]. As previously described [28,29], the smoke was pumped into a plastic box, measuring 42 cm (length) × 28 cm (width) × 27 cm (height), containing the animals that passively inhaled the cigarette smoke. The AS was induced in ApoE-/- mice fed a high fat diet (SCXK2019-0003, Beijing Keao Xieli Feed Co., Ltd. Beijing, China) for 20 weeks. Briefly, mice were exposed to smoke twice daily, burning 20 cigarettes each time for an hour, 5 days a week for up to 20 weeks. The control group mice were fed a normal chow diet and exposed to an airy room. The AS group mice were fed a high-fat diet for 20 weeks. The COPD+AS group, TXL group, Ato group, and TXL+Ato group mice were exposed to cigarette smoke combined with a high-fat diet for 20 weeks. TXL group, Ato group, and TXL+Ato group mice were given TXL (1.5 g/kg/day), Ato (10 mg/kg/day), and TXL+Ato once a day.
Mouse pulmonary functions
Mice were anesthetized with 50 mg/kg sodium pentobarbital (Sigama–Aldrich, USA) via intraperitoneal injection, and then tracheostomized and placed in a forced pulmonary maneuver system (Buxco Research Systems). To acquire the lung function of mice, the Boyle’s low functional residual capacity (FRC), quasistatic PV, and fast flow volume maneuver were performed using the Buxco system [30]. The FRC was determined using the Boyle’s law FRC maneuver; the dynamic compliance (cdyn) was acquired by the quasistatic PV maneuver; the forced vital capacity (FVC), forced expiratory volume (FEV) at 50 ms (FEV50), and inspiratory resistances (RI) were recorded with the fast flow volume maneuver. Each maneuver was repeated at least three times.
Lung microvascular permeability
Lung microvascular permeability was determined by the Evans Blue dye method [31]. According to, 20 mg/kg body weight, 1% of Evans blue dye (Sigma-Aldrich, USA) was solubilized in 250 µL phosphate-buffered saline (PBS) and injected into the tail vein of the mice. After 2 h, the mice were anesthetized with pentobarbital sodium and perfused with 10 mL of 0.9% sodium chloride solution to remove the blood. Then, the lung tissue was weighed, incubated, and homogenized with formamide (Sigma–Aldrich, USA) for 48 h at 37 °C. The concentration of the dye (µg/mg tissue) was determined in the supernatant using a spectrophotometer at 620 nm against a standard curve.
Tissue collection and morphological analysis
Mouse left lung without lavage and the total length of aortic arch were fixed in 4% phosphate-buffered paraformaldehyde (pH 7.4), embedded in paraffin, and sliced into 8-µm-thick sections that were stained with hematoxylin and eosin (HE) solution. The alveolar enlargement was determined by the average linear intercept and the ratio of the total length of alveoli to the number of alveoli per field.
For the quantification of atherosclerotic lesions, Oil Red O-positive lesion surface areas on en face preparation of whole aorta were measured. Briefly, the aorta from root to the abdominal area was dissected and fixed with formalin, followed by removal of the connective tissues carefully, and longitudinal opening with the intima towards the outside. Then, the tissues were stained in prewarmed Oil Red O solution for 10 min and differentiated in 85% propylene glycol for 5 min at room temperature. The extent of atherosclerotic lesion development was defined as the percentage of total Oil Red O-positive lesion area over the total surface area.
For detection of VE-cadherin (anti-VE-cadherin, sc-9989, 1:50, Santa Cruz, Texas, USA), frozen sections were stained with 8 μM dihydroetidium (DHE; Wako) for 30 min, followed by counterstaining and sealing with mounting medium, antifading (with DAPI) (Solarbio, Beijing, China). The fluorescence images were captured under a ZEISS confocal microscope (Oberkochen, Germany).
Transmission electron microscopy (TEM)
The ERCs were centrifuged at 2,000 × g for 5 min and the pellets were fixed with 2.5% glutaraldehyde (Sigma-Aldrich) in PBS buffer (pH 7.2) at room temperature for 2 h. After several rinses with PBS, the cells were post-fixed with 1% osmium tetroxide (Sigma-Aldrich) for 1 h at 4°C, dehydrated in a graded series of acetone, and embedded in Epon 812. Ultrathin sections were cut (60–80 nm thick), and then double-stained with uranyl acetate and lead citrate (both from Amresco, Solon, OH, USA) prior to examination under a transmission electron microscope (H-7700; Hitachi, Tokyo, Japan).
Serum lipid analysis
Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were determined using enzymatic kits from Jiuqiang Biotechnology (Beijing, China) according to the manufacturer’s protocols.
Cell culture and treatment
Human pulmonary microvascular endothelial cells (HPMECs) were obtained fro m Yaji Biological (Shanghai, China). The cells were maintained in DMEM me dium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, New York, USA) and 1% antibiotic-antimycotic in a humidified incubator under 5% CO2 at 37 ℃. All cells between passages 3-4 were used for subsequent e xperiments.
The HPMECs were divided into five groups: (i) Control: HPMECs were incubated in regular cell culture conditions; (ii) cigarette smoke extract (CSE): HPMECs were incubated with diluted CSE for 24 h to establish CSE-induced pulmonary microvascular barrier dysfunction; (iii) CSE + 200 µg/mL TXL: HPMECs were pretreated with TXL (200 µg/mL) for 6 h and underincubated with diluted CSE for 24 h; (iv) CSE + 400 µg/mL TXL: HPMECs were pretreated with TXL (400 µg/mL) for 6 h and underincubated with diluted CSE for 24 h; (v) CSE + 800 µg/mL TXL: HPMECs were pretreated with TXL (800 µg/mL) for 6 h and underincubated with diluted CSE for 24 h. All experiments were performed in triplicate.
Preparation of CSE
CSE was generated as described previously [32]. A syringe-driven apparatus device was designed and operated to allow a stream of smoke to flow into a tube-shaped trap. The smoke then entered a flask submerged in liquid nitrogen. The amount of smoke obtained was calculated by the increase in the weight inside the flask. The smoke particulates were collected in dimethylsulfoxide (DMSO) at a concentration of 40 mg/mL, filtered through a 0.22-μm-pore filter, and stored at −80 °C for subsequent use.
Preparation of TXL
TXL ultrafine powder (Shijiazhuang Yiling Pharmaceutical Co., Shijiazhuang, China) was solubilized in TXL in serum-free DMEM and sonicated. The pellet was collected by centrifugation at 10000 ×g for 10 min. Subsequently, the precipitate was dried at 60 ℃, and the supernatant was filtered (0.22 µm pore size, Costar, Cambridge,
Massachusetts, USA) to calculate an accurate weight of the dissolved ingredients [33]. According to the previous reports[34,35] and MTS experiments, the doses of TXL were determined to be 200, 400 and 800 μg/ml in HPMECs.
HPMECs monolayer permeability assays
Test of the permeability to FITC-dextran
Cells were grown on Transwell compartments. Tracer experiments with fluorescent
dextran were performed using the 12-well Transwell system (0.4-μm-pore size,12-mm diameter, transparent, Costar, Cambridge, Massachusetts, USA). HPMECs (1×105 cells/well) were plated in the Transwell chambers that were supplemented with 500 and 1500 µL culture media in the upper and lower compartments, respectively. The cells were grown for 5 days and replaced with
serum-free medium for 1 h before the treatment. The permeability was measured by the addition of FITC-dextran (40 kDa; Sigma–Aldrich, New York, USA) for 1 h.
Measurement of transendothelial electrical resistance
The cellular barrier properties were assessed by measuring the resistance values across confluent HPMECs using Millicell-ERS (MERS00002, Millipore, Molsheim, France) [36]. The 12-well Transwell system was inserted into the 12-well plate.
HPMECs were plated in Transwell chambers. The electrode tips were inserted into the upper and lower compartments, respectively, and the resistance (R sample) was measured. The value for the blank well represented the blank resistance (R blank). The TER value was calculated based on the resistance per unit area as (R sample − R blank) × 1.33 cm2 (the area of a 12-well Transwell chamber is 1.33 cm2) [37].
Immunofluorescence staining
After pretreated HPMECs reached 70% confluency, the cells were washed with PBS, fixed in 4% paraformaldehyde for 15 min, and permeabilized for 5 min with the buffer containing 0.1% Triton X-100, followed by blocking with goat serum for 1 h at room temperature. The cells were further incubated with primary antibodies
(VE-cadherin, 1:200, SC9989, Santa Cruz, Texas, USA; β-catenin, 1:200, ab32572, San Francisco, USA) at 4 ℃ overnight. After washing, the cells were incubated with secondary antibodies (goat anti-mouse, SA00013-3, 1:200; goat anti-rabbit
SA00013-2, 1:200). Finally, the cell nuclei were stained using DAPI for an additional 15 min and visualized under a ZEISS confocal microscope (Oberkochen, Germany) [38].
ELISA for inflammatory cytokines
Lungs were homogenized in a saline solution (0.9% NaCl), the mice serum and the cell culture supernatants were collected via centrifugation (1200 rpm, 10 min, 4 °C) to remove cell debris and were stored at -80℃ until subsequent analyses. The levels of inflammatory cytokines were quantified using enzyme linked immunosorbent assays (ELISA). The kits were purchased from Proteintech (mouse IL-1β ELISA kit, cat.
No.KE10003, Rosemont, USA; Mouse IFN-γ ELISA kit, cat. No.KE10001; Mouse TNF-α ELISA kit, cat. No.KE10002; Human IL-6 ELISA kit, cat. No. KE00139; Human IL-1β ELISA kit, cat. No.KE20005) and Zcibio (Human hsCRP ELISA kit, cat. No. ZC-M6088, Shanghai, China). The operation steps were strictly carried out according to the instructions of manufacturer.
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from mouse pulmonary and aorta tissue and HPMECs by Eastep Super Total RNA extraction kit and measured by Nanodrop2000 (Thermo Scientific, Waltham, MA, USA). Subsequently, reverse-transcription was performed using a Prime-ScriptTM RT Reagent kit, according to the manufacturer’s instructions. Quantitative PCR was carried out on a 7900 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). GAPDH was used as an internal reference, and the primer sequences are shown in Table 1.
Western blotting analysis
Protein concentration of mice lung tissue and HPMECs was determined using the BCA protein assay kit before SDS-PAGE(Genscript, Nanjing, China). Then, the proteins were transferred to nitrocellulose blotting membrane (Life Sciences, Mexico).
After blocking with Odyssey® blocking Buffer (LI-COR, Lincoln, USA) for 1 h at 37 ℃, the membranes were incubated with primary antibodies (VE-cadherin, SC9989, Santa Cruz, Texas, USA; β-catenin, ab32572, San Francisco, USA; Claudin5, ab131259, San Francisco, USA; NF-қB, CST8242s, MA, USA; β-actin, GB11001, Servicebio, Wuhan, China) at 4 ℃ overnight, followed by incubation with secondary antibodies at 37 ℃ for 1 h. Finally, the membranes were rinsed with TBST before scanning by Odyssey two-color infrared imaging system (Li-Cor, Lincoln, NE, USA). The β-actin was used as an internal control.
Statistical analysis
Statistical significance of differences was assessed using one-way ANOVA or repeated measures analysis, followed by post-hoc analysis using Fisher’s LSD multiple comparison test. Differences at P <0.05 were regarded as statistically significant. Data are presented as means ± SEM.