Ursolic acid alleviates airway-vessel remodeling and muscle consumption in cigarette smoke-induced emphysema rats

Antibodies against TGF-β1, 8-OHdG, α-SMA, S100A4, and IGF1 were obtained from Abcam Biotechnology Company (Cambridge, UK). Antibodies against Smad2, p-Smad2, Smad3, p-Smad3, and cleaved-caspase3 were purchased from Cell Signaling Technology (Denver, CO). UA was purchased from Wanxiang Hengyuan Biotechnology (Tianjin, China). Masson and Alcian blue-periodic acid Schiff (AB-PAS) kits were obtained from Nanjing Jiancheng biological engineering research institute (Nanjing, China). BCA kit was obtained from Pierce (Thermo-Scientific, Rockford, IL, USA), ECL chemiluminescence kit from Applygen (Beijing, China).

Six-week-old male Wistar rats, weighing between 150 and 250 g, were bought from Chansheng Biotechnology Company (Liaoning, China). After two weeks of adaptation time, rats were randomized into one of five treatment groups (N = 10 each): Sham, CS, UA10, UA20, and UA40. UA rats were administrated 10 mg/kg, 20 mg/kg or 40 mg/kg body weight UA via gavage thirty minutes before the first CS exposure every day. Sham and CS rats were given vehicle instead. CS and UA rats were exposed to smoke of 16 filters removed 1R3F cigarettes for 30 min, two times a day, 6 days a week, for 3 months. Rats from the same group were placed five at a time into a glass chamber measuring 0.8 m × 0.6 m × 0.6 m, with a 2 cm × 2 cm spiracle on the top of the box. The time interval between the two exposures each day was 4 to 6 h. Rats in the Sham group were exposed to normal air. [46] The Animal Care and Use Ethics Committee of China Medical University approved this study.

Human bronchial epithelial cells (HBEs) and human umbilical vein endothelial cells (HUVECs) were purchased from Peking University Cancer Institute (Beijing, China). Cells were cultured in RPMI-1640 culture medium (Hyclone, UT, USA) containing 10% fetal bovine serum (Hyclone, UT) in a 5% CO2 humidified cell incubator (Thermo Fisher Scientific, Inc., USA) at 37 °C. We treated 1 × 10⁶ cells with 10 μM/L UA 2 h prior to 1% cigarette smoke extract (CSE) [49] intervention. The concentrations we used were according to the CCK8 cytotoxicity testing (Dojido, Japan).

The left lungs were inflated and fixed using 4% phosphate- buffered formaldehyde (pH 7.40) at 25cmH2O pressure for 24 h. The musculi soleus muscles were fixed using 10% formaldehyde for 24 h. Then lungs and musculi soleus muscles embedded with paraffin. The paraffin-embedded sections were used for histopathological examination. Right lung tissues were frozen in liquid nitrogen for 5 min before storing at − 80 °C.

We used hematoxylin and eosin (HE) staining to observe pathological changes to pulmonary airways and vessels. We measured and compared mean thickness of the airways and associated vessels in lung tissues. We observed and compared the pathological changes of musculi soleus using HE staining. To evaluate bronchial and vascular wall thickness, four sections that did not include cartilage but did include intact bronchial tracheal transections and concomitant vessels were randomly selected. For all bronchial sections, the ratio of minimum diameter to maximum diameter was> 0.5. Image Pro Plus 5.0 image analysis software (Media Cybernetics company, Maryland, American) was used to measure airway and vascular basement membrane perimeter (Pbm), airway wall area (total wall area [Wat]) and vascular wall area (total vascular area [Vat]). Wat/Pbm and Vat/Pbm values were calculated for each trachea and associated vessel, and the average value was used to compared airway and vascular wall thicknesses among groups.

We used Masson staining to measure collagen deposition around the airways and vessels. Paraffin sections were dewaxed to water, then stained according to the Masson staining kit instructions (Nanjing Jiancheng Biotechnology, Nanjing, China). Areas of collagen deposition around airways and vessels were compared using the index wall area of collagen deposition (Wac)/Wat and vascular area of collagen deposition (Vac)/Vat measured using Image Pro Plus 5.0 image analysis software.

We used AB-PAS staining to count mucus-producing cells surrounding the airway. Paraffin sections were dewaxed to water, then stained according to the AB-PAS staining kit instructions (Nanjing Jiancheng Biotechnology, China).

Briefly, paraffin embedded tissues cut into 4-μm thick sections, and dewaxed, rehydrated. The slices were treated with H2O2 in methanol to inhibit endogenous peroxidase activity. Then antigen retrieval was performed using a microwave and 10-mM citrate buffer, pH 6. Slices were incubated with anti-8-OHdG, anti-cleaved caspase-3, anti-α-SMA, anti-TGF-β1, anti-p-Smad2, and anti-S100A4 antibodies overnight with the concentration of 1:500 at 4 °C. After washing, secondary antibodies were incubated room temperature for 1 h using the concentration of 1:1000. Then incubation with 3,3′-diaminobenzidine (DAB) and DAB Enhancer. One horizon in each quadrant of each section was assessed. Relative expression was compared using relative integrated optical density (IOD) surrounding the airway (IOD/Wat) and vessel (IOD/Vat), as measured using IPP 5.0 software. [46]

Lung tissues, HBE cells and HUVEC cells lysates were prepared. Briefly, tissues and cells were lysed in an ice-cold lysis buffer (Roche Applied Science, Indianapolis, IN). Samples were then homogenized for 15 s at 4 °C, 4–5 times. Cell lysates were centrifuged at 12,000×g for 30 min at 4 °C to remove cellular debris. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein (20–60 μg) were separated on 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany), blocked and incubated with diluted primary antibodies overnight at 4 °C refrigerator. Blots were stripped and re-probed with anti-GAPDH antibody to demonstrate equal loading. Incubated with secondary antibody, the chemiluminescent signal was detected using the Super Enhanced Chemiluminescence Kit (Bio-Rad Laboratories, Shanghai, China). Band density was quantified using Quantity One software (Bio-Rad Laboratories, Shanghai, China). [46]

We had 10 rats in each group at the beginning of the experiment, but 2 rats in the CS group died during CS exposure. The remaining 48 rats survived and were used in subsequent experiments. Musculi soleus weights were significantly reduced in the CS group, relative to the Sham group, and 40 mg/kg body weight UA administration significantly attenuated this muscle mass loss. The same trend was not observed for extensor digitorum longus (Fig. 1A). Examination of HE stained sections revealed marked muscle atrophy with characteristic muscle cell crinkle, cell vacuolation, and structural disorder in the musculi soleus of CS-induced emphysema model rats, relative to the muscles of Sham rats. In UA groups, musculi soleus atrophy related changes such as muscle cell crinkle, vacuolation, muscle fiber disorder, and gap dilation were all improved in a concentration dependent manner. Myoarchitecture in UA20 and UA40 groups were similar to that of the Sham group (Fig. 1B).

HE stained sections revealed that the airways and associated vessels of the emphysema rats underwent airway-vessel remodeling, UA administration alleviated CS induced airway and vessel remodeling. The pathology changes observed in CS rats included proliferation and exfoliation of airway epithelial cells, airway basement membrane thickening, collagen deposition, airway contracture, infiltration of inflammation cells around airways, and vessel thickening. UA administration at all three concentrations alleviated these changes (Fig. 2A). We used airway and vessel thickness to quantify changes associated with airway-vessel remodeling. Compared with Sham group, the thickness of airways and accompanying vessels were remarkably increased with CS, and UA administration alleviated these changes at all three concentrations tested (Fig. 2B).

Masson staining showed that blue-dyed collagen deposition areas were significantly increased in airway and accompanying vessel walls of CS rats, compared with those in the Sham rats. Blue-dyed areas around airways and accompanying vessels were reduced significantly by all doses of UA administered (Fig. 2C). We used blue-stained areas in airway or vessels divided by the total areas of airway or vessels to compare the relative extent of collagen deposition. Proportion of collagen deposition areas to total airway or vessel areas are increased significantly in CS group compared with Sham. UA administration reduced the proportion of collagen deposition around airways and accompanying vessels (Fig. 2D).

We used AB-PAS staining to assess mucus secreting cell expression surrounding the airways. Many large blue-dyed goblet cells were present in the epithelial layer in the airways of CS rats compared with Shams. UA decreased the size and number of goblet cells in epithelial layer of CS rats. We also noted metaplasia of large blue-dyed mucous glands in the large airway in the CS group (Fig. 2E).

To investigate smooth muscle cell proliferation, fibroblast to myofibroblast differentiation, EMT, and EndMT, we examined α-SMA expression in the area surrounding the airways and accompanying vessels using IHC. α-SMA expression around airways and vessels were significantly increased in the CS group compared with the Sham group, especially in the epithelial and endothelial layers. UA decreased α-SMA expression in the area surrounding airways and vessels, particularly in the epithelial and endothelial layers, at each dose of UA (Fig. 3A). The percentage of α-SMA staining areas per airway or vessel areas were significantly increased in CS rats compared with Sham rats, but UA administration decreased the percent of α-SMA staining area at all doses (Fig. 3B).

We used S100A4 expression to assess EMT and EndMT around airways and vessels. IHC analyses showed that S100A4 expression surrounding airways and accompanying vessels was significantly increased in the CS group compared with the Sham group. This increase was suppressed by UA, especially in the epithelial and endothelial layers (Fig. 3C). The percentage of S100A4 staining areas per airway or vessel areas were significantly increased in CS rats compared with Sham rats, and UA administration reversed this effect at all doses (Fig. 3D).

We used 8-OHdG and cleaved-caspase3 expressions to assess cell apoptosis around airways and vessels. IHC analyses showed that 8-OHdG and cleaved-caspase3 expression surrounding airways and vessels was significantly increased in the CS group compared with the Sham group. This increase was suppressed by UA, particularly in the epithelial and endothelial layers (Fig. 3E, G). The IOD/area values are consistent with the results above (Fig. 3F, H).

IHC analysis further showed markedly upregulated TGF-β1 expression in airways and accompanying vessels, while in UA groups, TGF-β1 expression was downregulated in all of the UA groups, especially in the epithelial and endothelial layers (Fig. 4A). These IOD/area values are consistent with the results above (Fig. 4B). Western blot analysis of TGF-β1, p-Smad2, p-Smad3, Smad2, Smad3, α-SMA, and IGF1 expression showed that the activated forms of TGF-β1, p-Smad3, and α-SMA were upregulated in rat lung in the CS group compared with the Sham group. In the UA groups, active forms expression of TGF-β1, p-Smad3, and α-SMA was down-regulated. IGF1 expression was down-regulated in the CS group compared with the Sham group. UA administration upregulated IGF1 expression in rat lung (Fig. 4C and D). Contrary to expectations, the p-Smad2 expression seemed to be down-regulated in the CS group but upregulated in the UA groups. Thus, we used IHC to assess p-smad2 expression around airways and accompanying vessels. It revealed markedly upregulated p-Smad2 expression in airways and accompanying vessels, but not pulmonary parenchyma regions, downregulated p-Smad2 expression in airways and accompanying vessels after UA treatment, which is inconsistent with the IOD results (Fig. 4E and F).

Western blot analysis showed upregulated expression of activated forms of TGF-β1, p-Smad2, p-Smad3, and S100A4 in HBEs and HUVECs after CSE administration compared with vehicle control cells, that could be attenuated by UA treatment. CSE administration downregulated IGF1 expression in HBE cells, UA alleviated the IGF1 down-regulation (Fig. 5A-D).