2.1 Grouping and establishment of experimental animals
Forty-eight healthy adult female SPF grade C57/BL6 mice aged 6-8 weeks with the body weight of 20-22 g were randomly divided into six groups: normal control group (NC group), bleomycin-induced SSc-ILD model group (BLM group), low-dose PESV intervention group (PESV-L group), medium-dose PESV intervention group (PESV-M group), high-dose PESV intervention group (PESV-H group), and dexamethasone intervention group (DXM group), with eight mice in each group. The study was carried out in accordance with the Declaration of Helsinki. This animal experimental program has been reviewed by the Experimental Animal Management and Ethics Committee of Zhejiang University of Traditional Chinese Medicine and conforms to the principles of animal protection, animal welfare and ethics, as well as the relevant provisions of China's National Experimental Animal Welfare Ethics. Animal Experiment Ethics Approval Number: IACUC-20210524-03.
SSc-ILD animal modelling in this study is based on reports from Aso Y16 and Yamamoto T17,18 et al. After removing the 2.0 × 2.0 cm hair from the center of the back in each group mice with 10% sodium sulfide solution, mice in the NC group were subcutaneously injected with 0.1 ml of normal saline. Except for the NC group, the rest groups were subcutaneously injected with 0.1 ml bleomycin solution (1 mg/ml, Hanhui Pharmaceutical Co., Ltd., 2006741) once a day for 4 weeks to establish a mouse SSc-ILD model. In addition, PESV-L group, PESV-M group, PESV-H group, and DXM group were intragastrically administered with 5 mg/kg PESV solution (Zhengzhou Licheng Biotechnology Co., Ltd.), 10 mg/kg PESV solution, 20 mg/kg PESV solution , and 0.15 mg/kg dexamethasone sodium phosphate injection (Suicheng Pharmaceutical Solution Co., Ltd.) for 4 weeks from the day of modeling, respectively.19 The activity status, mental status, respiration, fur change, food intake, water intake and other general conditions of mice in each group were observed during the experiment.
2.2 Bronchoalveolar lavage fluid (BALF), lung and serum collection
After 4 weeks of administration, the mice in each group were sacrificed, and blood samples were collected from the mice in each group (eyeball removal method) and placed in sterilized EP tubes overnight (4 °C). Blood was centrifuged after clotting (4 °C, 10 min, 2000 rpm). The collected supernatant was stored at -80 °C.
The skin of the anterior cervical region of the mice was cut open. A red intravenous catheter was placed at the distal end of the trachea and ligated and fixed. One milliliter of normal saline was then infused into the lung tissue. After the lung was significantly filled, it remained for 10 seconds for pumping back, which was repeated three times. Finally, the collected BALF was centrifuged (4 °C, 800 rpm, 5 min) and stored at -80 °C until use. After that, lung tissues form the each group mice were dissected, a portion was fixed in 10% neutral formaldehyde, another portion was stored at -80℃.
2.3 HE staining
Lung tissue samples (10% neutral formaldehyde (Fuzhou Feijing Biotechnology Co., Ltd.)) from mice in each group were fixed and cut into 4-μm-thick paraffin sections. Deparaffinize to water. Hematoxylin staining solution was added, followed by differentiation (0.5% hydrochloric acid in alcohol, 10 s) and reversion to blue (1% diluted ammonia, 90 s). Eosin staining solution (60 s) was then added for staining, and the results were observed under a microscope after fixation with neutral resin.
2.4 Masson's staining
The lung tissues of mice in each group were deparaffinized to water after sectioning, stained with iron hematoxylin staining solution (10 min), refluxed with blue solution (5 min), and finally stained with red staining solution (15 min). After differentiation (1% acetic acid, 10 s) they were fixed with neutral gum. Observations were performed and assessed microscopically.
2.5 Immunohistochemical staining
Lung tissue sections from mice in each group were deparaffinized to water and subjected to antigen heat retrieval. Primary TGF-β1 antibody (ImmunoWay Biotechnology Company, USA) was added followed by overnight incubation at 4 °C. Secondary antibodies were added the following day and incubated for half an hour at 37 °C. Then DAB chromogenic solution was added, and the staining results were observed microscopically at 200 × field after counterstaining with hematoxylin.
2.6 Western blot
The lung tissue samples of mice in each group were crushed and lysed, centrifuged and the total protein concentration was measured with a BCA kit, followed by SDS-PAGE electrophoresis, and then transferred to a PVDF membrane. At the end of the transfer membrane, blocking was performed at room temperature for 1 h (5% non-fat dry milk), and then the corresponding primary antibodies (E-cadherin Antibody (Affinity, AF0131), Collagen I Antibody (Affinity, AF7001), Vimentin Antibody (Affinity, AF7013), N-cadherin Antibody (Affinity, AF4039), α-SMA Antibody (Affinity, BF9212), TGF-β1 Antibody (Affinity, AF1027), TβRⅠ Antibody (Affinity, DF7309), TβRⅡ Antibody (Affinity, DF13307), Phospho-Smad2 (Ser250) Antibody (Affinity, AF3450), Smad2 Antibody (Affinity, AF6449), Phospho-Smad3 (Ser423+Ser425) Antibody (Affinity, AF8315), Smad3 Antibody (Affinity, AF6362), Smad7 Antibody (Affinity, AF5147)) were incubated at 4 °C overnight. After incubation of HRP-labeled secondary antibody at room temperature for 1 h, the developing solution was added for luminescence and development and fixation. After scanning the film, the gray values of each band were determined with image analysis software, and the ratio with the internal reference β-actin was used as the basis for semi-quantitative analysis.
2.7 ELISA test
Take standby BALF and serum, place them at room temperature for 20 min, add distilled water to dilute 20 times concentrated washing solution into original washing solution. The corresponding inflammatory factor concentrations in BALF were measured according to the manufacturer's instructions of the ELISA kit. The tested ELISA kits include Mouse IL6 (Interleukin 6) ELISA Kit (ELK1157, ELK Biotechnology), Mouse TGFβ1 (Transforming Growth Factor Beta 1) ELISA Kit (ELK1186, ELK Biotechnology), and Mouse TNFα (Tumor Necrosis Factor Alpha) ELISA Kit (ELK1387, ELK Biotechnology).
2.8 Statistical analysis
All data from this experiment were analyzed and processed using SPSS 16.0 as well as GraphpadPrism7 software. All data were expressed as mean ± standard deviation (x̄ ± s), P < 0.05 was considered statistically significant. One-way-ANOAY analysis of variance was used for multiple groups of measurement data, and SNK test was used for comparison. Kruskal-WallisH test was used for unequal or non-normal variance.