Targeting of G-protein coupled receptor 40 alleviates airway hyperresponsiveness through RhoA/ROCK1 signaling pathway in obese asthmatic mice

Reagents

DC260126 (MCE, New Jersey, USA) was employed to antagonize GPR40 expression. Y-27632, the inhibitor of ROCK, was obtained from Tocris Bioscience (Bristol, UK). High-fat diet (NO. MD12032) was purchased from Medicience Ltd (Jiangsu, China). Methacholine (Mch), Ovalbumin (OVA) and DMSO were bought from Sigma-Aldrich (St. Louis, MO), TRIzol reagents were used to extract total RNA (Takara, Otsu, Shiga, Japan). Oleic acid (OA) was purchased from Sigma-Aldrich Company, USA. The following antibodies: RhoA (1:5000, #AB187027, rabbit polyclonal, Abcam), ROCK1 (1:2000, #ab45171, rabbit polyclonal, Abcam), GPR40 (1:1000, #DF2745, rabbit polyclonal, Affinity Biosciences), GAPDH (1:5000, #AF7021, rabbit polyclonal, Affinity Biosciences) were applied to the western blot analysis. Mouse FFAs, IL-4, IL-5, IL-13, IL-1β, TNF-α, and IFN-γ ELISA kits were obtained from eBioscience (San Diego, CA).

Cell culture

Human airway smooth muscle (HASM) cells were sourced from the ScienCell Research Laboratories (Cat. No. 3410, Carlsbad, CA, USA). According to the manufacturer’s instructions, the cell type was confirmed by immunofluorescence with antibodies specific to α-smooth muscle actin. Cells between passages 3 and 10 were used for the subsequent experiments. HASM cells were grown in Smooth Muscle Cells Medium (SMCM) supplemented with 2% FBS, 1% smooth muscle cell growth supplement (5 ng/ml rh FGF, 5 µg/ml insulin, 50 µg/ml ascorbic acid, 5 µg/ml Transferrin, 10 mM L-glutamine, and 5 ng/ml rh EGF ng/ml), 10 mM HEPES buffer and 1% penicillin/ streptomycin, and were incubated at 37 °C in a humidified atmosphere containing 5% CO2.

Animals

Male C57BL/6 mice (3–4 weeks old, weighing 20 ± 2 g), were purchased from Zhejiang Experimental Animal Center (No. SCXK 2019-0002) and housed in Plexiglas cages under 12/12 h light/dark cycle, and given food and water ad libitum in the Laboratory Animal Center of Wenzhou Medical University. The Committee of the Ethics of Animal Experiments of the Wenzhou Medical University (license number: wydw2019-0223) approved this study. All the animals in our study received humane care.

Animals were randomly selected and allocated to two groups: the lean group, and the obesity group. The obesity model was established by feeding mice with high fat diet (HFD) (MD12032, Medicience Ltd, China) that containing 45% kcal from fat for 16 weeks, the lean mice were fed a normal chow diet (MD12031, Medicine Ltd, China) that containing 10% kcal from fat as the control. In order to establish an asthma model in lean or obese mice, mice were sensitized by i.p. injection of 10 μg OVA (Sigma, United States) emulsified in 20 mg Al (OH)3 gel in 0.1 mL normal saline on day 1 and 13 starting from 12th week, and then challenged with 10 mg/ml aerosolized OVA for 30 min a day for 7 consecutive days. The control mice were sensitized and challenged with 0.9% NS.

Treatment with DC260126 or Y-27632 in obese asthmatic mice

The lean and HFD-induced obese mice were randomly divided into 6 groups (n = 6): the control group (Normal chow diet), the OVA group (Normal chow diet + OVA), the HFD group (High fat diet), the HFD-OVA group (High fat diet + OVA), the DC260126 3 mg/kg group (High fat diet + OVA + DC260126 3 mg/kg) and DC260126 10 mg/kg group (High fat diet + OVA + DC260126 10 mg/kg). Herein, we selected DC260126 at 3 and 10 mg/kg as the intervention dose was based on our pretest that presented in Additional file 1: Fig. S1, for we found DC260126 at high dose (30 mg/kg) failed to exert a stronger lowering effect as expected, but probably aggravated the neutrophilic airway inflammation in asthma. DC260126 (DC), a GPR40 antagonist, was dissolved in DMSO (20 mg/ml) and stored at − 20 °C. Schedule of study design was showed in Fig. 1A, briefly, DC260126 was administered intraperitoneally for 7 consecutive days, 30 min before every OVA challenge in obese mice. 0.1% DMSO treatments were performed to the mice in the control and OVA model group to eliminate the interference errors that caused by the vehicle. Y-27632, a ROCK antagonist, was dissolved in 0.9%NS and stored at − 20 °C. Y-27632 at 3 mg/kg or 10 mg/kg was administered intraperitoneally to model mice refer to the schedule as described above.

Fig. 1figure 1

GPR40 expression is elevated in the lung tissues of obese asthmatic mice. A The mice were fed with normal chow diet or HFD for 12 weeks, followed by sensitized with OVA that emulsified in Al (OH)3 gel on day 1 and 13 from the 12th week, and then challenged with aerosolized OVA for 0.5 h a day from day 25 to 31. DC260126 was given to HFD-OVA model mice daily by i.p. injection at 0.5 h before each challenge, vehicle (0.1% DMSO) was given to the mice in control, OVA, HFD, and HFD-OVA instead. B The GPR40 expression in the pulmonary tissues of mice was assessed by immunohistochemical staining. The data were represented as the mean ± S.E.M. *P < 0.05, and **P < 0.01 compared with the control group. C FFAs level in serum was assessed by ELISA. D The body weight in lean mice (Control, OVA group) and obese mice (HFD, HFD-OVA group) was recorded every three weeks, the results were expressed as the percentage of body weight gain. E Mice body weight was measured with (HFD + OVA + DC260126 3 mg/kg, HFD + OVA + DC260126 10 mg/kg group) or without (Control, OVA, HFD, HFD-OVA group) DC260126 administration during 7 consecutive days. The data were represented as the mean ± S.E.M. (n = 6 per group). **P < 0.01 and ***P < 0.001 compared with the control group. #P < 0.05 compared with the HFD-OVA group

Determination of airway hyperresponsiveness (AHR)

Airway hyperresponsiveness (AHR) was determined following our previous research [12] by measuring changes in the airway resistance (Rrs, cmH2O/ml/s) after challenged with aerosolized methacholine (Mch) in a concentration gradient (3.125, 6.25, 12.5, 25 and 50 mg/mL) using the FlexiVent system (SCIREQ, Quebec, Canada). Anesthetized (1% pentobarbital sodium 50 mg/kg i.p.), tracheostomized (stainless-steel cannula, 12 G) mice were mechanically ventilated (at 150 breaths/min, with a tidal volume of 10 ml/kg, positive end-expiratory pressure of 3 cmH2O). Rrs was measured at every dose to calculate the percentage of each concentration to baseline. The baseline values of Rrs were measured at the stage of aerosolized saline nebulizing.

Bronchoalveolar lavage fluids (BALFs) preparation and cell counts

After the left upper lobe lung was ligated, BALFs were obtained by flushing the right lung three times with 0.5 ml of phosphate buffer solution (PBS) containing 1% BSA and 5000 IU/L heparin. The sample was centrifuged and its supernatant was analyzed by ELISA. The pelleted BALF cells were resuspended in PBS, and the total number of leucocytes was counted using a Neubauer chamber according to our previous study [27]. An aliquot of 200 BALF cells were stained with Wright-Giemsa and differentially identified under a light microscope according to classical cell morphology. The total number of each cell type was determined by multiplying the percentage by the total number of cells. The results were expressed as the numbers of each type of cell population in BALFs (1 ml).

Histopathological examination

Each mouse’s left middle lobe lung was collected and fixed in 4% paraformaldehyde. It was then embedded with paraffin, sliced into 3–4 μm sections, and examined with hematoxylin and eosin (H&E) staining. The severity of lung inflammation was determined under light microscopy and scored based on the following histologic grading system (scored 0–4) according to our previous study [12]. Collagen deposition around the bronchial airway was observed by Masson's trichrome staining. The severity of collagen deposition was evaluated using the Image Pro 6.1 software system, the results were expressed as collagen volume fraction (%) = area of collagen fiber/area of total view. Goblet cell hyperplasia was observed with PAS staining. The percentage of PAS staining-positive cells in the airway epithelium was quantified [28]. To perform the immunohistochemistry analysis, the experimental protocol in Streptavidin–Biotin Complex kit that bought from Boster Bio-engineering Ltd. Co., (Wuhan, China) was employed. The diluted primary anti-GPR40 (1:100) were incubated the slices overnight at 4 °C. Afterwards, the immunolabeling were visualized using 3,3′-diaminobenzidine (DAB) and analyzed by DP2-BSW software (Olympus, Tokyo, Japan). Semiquantitative evaluations of histopathological images in the present study were performed by a blinded observer.

Cell proliferation assay

Cell proliferation assay was determined by Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology, Shanghai, China) assay. A total of 100 µL of cell suspension (5000 cells/well) was pre-incubated for 24 h (at 37 °C, 5% CO2). After adding 10 µL of various concentrations of oleic acid with or without DC260126 pretreatment for 30 min, we incubated each well of the plates with 10 µL of CCK-8 solution for 3 h. The absorbance was measured at 450 nm by a microplate reader (Bio-Tek, Winooski, VT, USA).

Real-time PCR

TRIzol reagent bought from Takara company was applied to extract total RNA from lung homogenates. The process of PCR was carried out referred to our previous study [29]. Briefly, the first-strand cDNA was generated from 4 μg of total RNA using oligo-dT to prime the reverse transcription reaction. The PCR mixture consisted of 10.4 μl of 2 × SYBR Green 1 Master Mix, 0.4 μl of both sense and antisense primers, 2.0 μl of sample cDNA solution, and distilled water to a final volume of 20 μl. Mouse β-actin was used as an internal control. The mRNA levels were normalized to the β-actin expression level and expressed using the comparative parameter threshold cycle (Ct). The PCR primers were bought from Shanghai Bioengineering Ltd (Shanghai, China). Table 1 shows all primers’ sequences that were used in our study.

Table 1 Sequence for primers in the present studyScratch wound healing assay

Human airway smooth muscle (HASM) cells were plated at 2 × 105 cells/well into 6-well plates. The cells monolayer was scratched manually with a yellow plastic pipette tip, and washed with PBS to remove cell debris. Then, the cells were pretreated with or without DC260126 for 30 min, followed by incubation with oleic acid (OA) for another 24 h. The photographs of the scratch wound were recorded at 0 and 24 h to investigate and analyze the scratch wound assay using different samples. Digital photographs were obtained using an inverted microscope (Olympus, Japan), and the scratch area was measured using the Image-J software. The results were expressed as Migration aera (%) = (initial wound area at hour zero—the remaining wounded area) / initial wound area at hour zero.

Cell cycle analysis

HASM cells were seeded in a 6-well tissue culture plate (2 × 105 cells/well), after treatment, the cells were collected and washed with PBS, and fixed in 1.5 ml 95% ethanol at 4 °C overnight, followed by incubation with RNase and staining with propidium iodide (Multi Sciences Biotech Co., Ltd) for 30 min. The DNA content was detected using a Cytomics FC500 Flow Cytometer (Beckman Coulter, USA). The percentage of cells in the G1 phase, the S phase, and the G2 phase was analyze.

ELISA assay

Mice serum and BALFs were collected at 24 h after the last OVA challenge. The indicators (IL-4, IL-5, IL-13 in serum, and IL-1β, TNF-α, IFN-γ in BALFs) were separately detected by ELISA kits following the manufacturer's instruction. A Bio-Rad microplate reader was used to measure the absorbance of the tested sample at 450 nm.

RhoA pull-down activation assay

GTP-bound RhoA protein measurement was carried out using RhoA activation assay kit (Cytoskeleton, Inc., USA). Briefly, lung tissues were fragmented and lysed in 1 × ice-cold assay/lysis buffer, and then centrifuged. Each sample's supernatant was fixed with 40 μl of either rhotekin RBD or PAK PBD agarose bead slurry, and the mixture was incubated at 4 °C for 1 h with slow agitation. Afterwards, the agarose beads were centrifuged, pelleted, and then resuspended after washed for 3 times. The precipitated GTP-RhoA expression was detected by western blot using anti-RhoA antibody.

Western blot analysis

Briefly, the lung samples were fragmented and lysed in RIPA buffer (Beyotime, Biotechnology, Shanghai, China) containing 1% PMSF (Haoxin Biotechnology, Hangzhou, China). Sample protein (40 µg) were separated on 8–10% Tris/Glycine SDS-PAGE gel and subsequently transferred to PVDF membrane. Followed by 5% fat-free milk blocking for 1 h at room temperature, the blots were then incubated with primary Abs at 4℃ overnight. After rinsing in TBST, goat anti-rabbit 800 antibodies (1:5000) was used to probed the PVDF membranes. The immunoreactive bands were detected by a two-color infrared imaging system (Odyssey; LI-COR, Lincoln, NE, USA).

Statistical analysis

The results are expressed as the mean ± S.E.M. One-way ANOVA followed by the Student–Newman–Keuls test was employed to determine multiple comparisons. All statistical calculations were performed using 18.0 SPSS software (Chicago, IL). P < 0.05 was considered statistically significance.

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