2.1 Preclinical study
2.1.1. Mouse model
C57BL/6J mice (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were obtained (male, 8 ~ 9 weeks old, average weight 23 ~ 25 g). They were hosted and bred in a specific pathogen-free environment with access to food and water ad libitum.
2.1.2. Lung fibrosis model and drug treatment
The mice were divided into 5 groups of 8 animals each: control group, bleomycin group (BLM + NaCl), bleomycin + DM group (BLM + DM), bleomycin + pirfenidone group (BLM + PFD), and bleomycin + pirfenidone + DM group (BLM + PFD + DM). The bleomycin 1.5U (1.5 mg/kg) (Nippon Kayaku Co., Tokyo, Japan) was intratracheally administered to the mice except for the control group at day 0. Mice in the different groups were pretreated with vehicle, pirfenidone alone, DM alone, or pirfenidone plus DM for one day and continuously administered until day 20. Pirfenidone (Beijing Kangdini Pharmaceutical Co., Ltd., China) was intragastrically administered once a day (100 mg/kg body weight). DM (Selleck Biotechnology Co., Ltd., USA) was subcutaneously injected three times a day (10 ng/kg body weight). The control group and the bleomycin group were given the same volume of saline every day. The mice were sacrificed on D21, and the lungs were removed to prepare for histological staining and hydroxyproline assays.
2.1.3. Histologic analysis
The mouse left lungs were dehydrated, paraffin-embedded, cut into 5-µm sections, and then stained with H&E and Masson’s trichrome. The degree of inflammation and fibrosis of the lung tissue was observed under an optical microscope. The pulmonary fibrosis area was quantified[15]. The slides were sampled with a random-start systematic sampling scheme using a 6-mm grid randomly superimposed on the slide to indicate the areas to evaluate. Digitized images of the slide were obtained using a ×2 objective on an Olympus BH-2 microscope (Olympus America, Melville, New York, USA) with a Sony DXC970 MD camera (Sony America, New York, New York, USA) and an IxTV capture card (IxMicro, San Jose, California, USA) on an Apple Macintosh G3 computer (Apple Computer Inc., Cupertino, California, USA). The images were then opened in Photoshop Elements (Adobe Systems Inc., San Jose, California, USA). The overall area of the lung was obtained by manual outlining. The area of the lung with fibrosis was then outlined, and the area was obtained. The pixels of the total versus fibrotic tissue were then summed over each lung, and a percentage was obtained.
2.1.4 Hydroxyproline assay
The collagen contents were evaluated with the conventional hydroxyproline method in the mouse right lungs after they had been cleared of blood [16]. Samples containing known amounts of purified collagen confirmed the capability of the assay to fully hydrolyse and recover hydroxyproline from collagen.
2.2 Human study
2.2.1. Patients
This study was a multicentre, open-label, randomized study conducted by the Tianjin Medical University General Hospital, Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital, Tianjin People’s Hospital, and Tianjin Beichen Hospital. This study was approved by the Ethics Committee of Tianjin Medical University General Hospital, China (No. IRB2020-YX-031-01). Informed consent was obtained from all study participants. All consecutive patients who had started pirfenidone treatment between May 2019 and December 2020 were candidates for our study. Patients were selected based on the following criteria: (1) The diagnosis of IPF was made in accordance with the 2018 ATS/ERS/JRS/ALAT guidelines [17], and the patient’s chest HRCT conformed or possibly conformed to the UIP pattern. (2) Glucocorticoids or immunosuppressants were not administered within 1 month. All patients took pirfenidone capsules (Beijing Kangdini Pharmaceutical Co., Ltd., 100 mg/capsule, H20133376). An initial dose of 200 mg of pirfenidone was administered three times daily with meals (600 mg/day). Thereafter, the dose was gradually increased by 200 mg every 2 weeks to a maximum of 600 mg per dose (1800 mg/day). Dextromethorphan (DM) hydrobromide tablets (Shijiazhuang Yiling Pharmaceutical Co., Ltd., 15 mg/tablet, H20066348) were added at 7.5–15 mg once a day for the pirfenidone-DM group. A total of 16 consecutive eligible patients were enrolled, with 8 in the pirfenidone group and 8 in the pirfenidone-DM group.
2.2.2. Laboratory tests and pulmonary function testing
Routine blood tests, C-reactive protein (CRP), liver and kidney function tests, and pulmonary function tests (PFTs) of the patients were collected before treatment, and at 6 months and 1 year after starting treatment. Pulmonary function tests (PFTs) were performed by standard techniques using CHESTAC-33 (Chest MI Co., Tokyo, Japan) and Fudac-77 (Fukuda Denshi, Tokyo). Pulmonary function test indexes included forced vital capacity (FVC), forced vital capacity percent (FVC%) predicted, forced expiratory volume in one second (FEV1), forced expiratory volume in one second percent (FEV1%) predicted and the diffusing capacity of the lung for carbon monoxide percent (DLCO%) predicted.
2.2.3. CT images
A high-resolution CT scan (Lightspeed-64, GE, America) was used, and CT images with 0.5 mm (or 1 mm) slice thickness were obtained (140 kVp and 200 mA). Each extent of normal lung and each lesion (ground glass opacities (GGOs), consolidation, reticular abnormality, honeycombing and emphysema) was recorded at 5% steps in three zones in each lung based on the method described by Best [18]. The upper zone, middle zone, and lower zone were defined as at or above the aortic arch, between the aortic arch and the pulmonary veins, and at or below the pulmonary veins, respectively. The mean extent of the three zones was calculated. The HRCT scans were scored by two expert thoracic radiologists using the above method, which assesses the degree of ground glass attenuation (HRCT alveolar score) and fibrotic change (HRCT interstitial score and honeycomb score) [19]. Radiologists were blinded to the patient’s diagnosis and grouping. The agreement between the radiologists was very good.
2.3. Statistical analysis
Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, La Jolla, California, USA). Multiple comparisons were performed using ANOVA tests with post hoc analysis. To compare the demographic data and baseline clinical characteristics between the pirfenidone group and the pirfenidone-DM group, a chi-square test and Fisher’s exact test for categorical variables and a Mann-Whitney U test for continuous variables were used as appropriate. The level of interobserver agreement between the two radiologists was evaluated by kappa statistic measurements. P values < 0.05 were considered to indicate statistical significance.