Patients

We conducted this trial, enrolling 50 patients with EPLD from our hospitals between January 2022 and June 2023. These patients form the experimental group (N = 50), while 50 individuals with normal lung function during the same period serve as the control group (N = 50). All study participants were informed about the experiment and provided written consent, as approved by the Ethics Committee of our hospital. We will reference the key guidelines and literature supporting the use of the ASA physical status classification system in studies involving elderly patients with lung dysfunction [14].

Inclusion criteria:

① Age ≥ 65 years; ② ASA classification I ~ II, cardiac function I ~ II; ③ Emergency surgery with duration > 2 h; ④ Alert consciousness, no difficulty swallowing medications, and able to cooperate with medication treatment; ⑤ Acceptable control of basic diseases; ⑥ Not diagnosed with novel coronavirus pneumonia; and⑦ Signed informed consent form.

Exclusion criteria:

① Patients with incomplete clinical data; ② Patients with conditions such as pulmonary bullae or increased intracranial pressure, which preclude positive end-expiratory pressure (PEEP) ventilation; ③ Patients with severe complications in other organs temporarily unsuitable for surgery; ④ Patients with coagulation or platelet abnormalities; ⑤ Patients who have received corticosteroids or immunosuppressive therapy within 30 days before admission or during the course of the disease; and ⑥ Patients who refuse to cooperate and voluntarily withdraw.

Collection for clinical material from patients

Upon admission to the hospital, patients in the experimental and control groups were assessed for age, gender, body mass index (BMI), forced vital capacity (FVC), total lung capacity (TLC), superoxide dismutase (SOD), concurrent diseases such as diabetes mellitus and high blood pressure. Both the control and experimental groups underwent routine blood testing. Subsequently, blood samples were collected from both groups for further experimental analysis.

Animal experimentation

All animal experiments were approved by the Animal Research Ethics Committee of the Gongli Hospital of Shanghai Pudong New Area. We obtained 4-week-old BALB/c mice weighing 20–22 g from our hospital’s animal center. The mice were housed under standard conditions (23 ± 1 °C, 50 ± 5% relative humidity, 12-h light/dark cycle) with ad libitum access to food and water throughout the study. Following a one-week acclimation period, the mice were randomly assigned to one of three groups (n = 10 each): control, model, and MILPM groups. The lung dysfunction model was established by anesthetizing BALB/c mice with bleomycin sulfate (1–3 units/kg) for 3 weeks. After establishing the lung dysfunction model, the MILPM group received MILPM, including precise lung protective ventilation modes, drug therapy, standardized analgesia and sedation, and restricted fluid management strategies. The control and model groups were treated with conventional therapy. The mice were euthanized under anesthesia, and blood and lung tissue samples were collected from all groups for further testing.

HE staining

Before embedding in paraffin, lung tissues from each group of mice were fixed in formaldehyde. Subsequently, these specimens were sectioned into thin slices, each 5 µm thick, and stained using hematoxylin and eosin. A pathology specialist examined the stained tissue sections under a light microscope to assess the results.

Immunofluorescence assay

Initiate immunofluorescence assay by subjecting the lung tissues to stabilization and infiltration methods. Continue by placing lung tissues in a bath of primary antibodies. Following the designated time for incubation with the primary antibodies, wash the specimens immediately to remove any antibodies that have not bonded. Subsequently, let the cerebrospinal fluid undergo a second incubation with fluorescent secondary antibodies, and then, carry out an additional wash to dispel any lingering, non-bound antibodies. Finalize the process by examining the treated specimens with a fluorescence microscope to perform the analysis.

Measurement of reactive oxygen species (ROS)

The probe 2,7-dichlorofluorescin diacetate (DCFH-DA) was used to assess ROS production. According to the manufacturer’s instructions, lung tissues were first washed and then incubated with diluted DCFH-DA for 20 min in the dark. Subsequently, HNECs were washed with PBS and imaged using a confocal laser scanning microscope (Olympus FluoView FV1000, NY, USA). Finally, lung cells were added to 96-well plates, and their fluorescence was measured using a SpectraMax M5 microplate reader (Molecular Devices, CA, USA).

ELISA

Thaw the gradient-frozen serum samples at −20 °C and then at 4 °C. Equilibrate the ELISA kit to room temperature. Add the samples according to the kit instructions, incubate, wash the plate, and measure the levels of TNF-α (R&D Systems, Catalog No. DTA00 C), IL-6 (BioLegend, Catalog No. 430501), and IL-1β (Thermo Fisher Scientific, Catalog No. 88–7013-22) in the serum. Measure the absorbance at 450 nm using a microplate reader (Thermo Fisher Scientific, Catalog No. 51119000).

Flow cytometry assay

The cells were analyzed via flow cytometry in accordance with the manufacturer’s guidelines. Once collected, lung tissue cells were stained with Annexin V-FITC and propidium iodide (PI) in the dark, after which the levels of apoptosis in all groups were measured using a flow cytometer.

Western blotting

Protein extracts from the lung tissue were isolated using 10% SDS-PAGE, and the protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, Catalog No. 23225), and subsequently transferred to PVDF membranes. These membranes were then rinsed with TBST to remove nonspecific bindings. Specific primary antibodies NF-κB (Bioworld Technology, Catalog No.PMB-P44221) and TGF-β (Bioworld Technology, Catalog No. GRO-BJU100) against the target protein and actin were applied, and the membranes were incubated at 4 °C overnight. Following incubation, the membranes were washed with TBST to eliminate any nonspecifically bound primary antibodies. Secondary antibodies from the same supplier were then added, and the membranes were incubated at room temperature for two hours. Afterward, the membranes were washed with TBST to remove excess antibodies. Protein visualization was achieved using an ECL chemiluminescence reagent (Thermo Fisher Scientific, Catalog No. 34080), and the resulting bands were analyzed.

qRT-PCR

Total RNA was extracted from lung tissues using the TRIzol Reagent (Beyotime, Shanghai) according to the manufacturer’s protocol. Subsequently, mRNA was reverse transcribed into cDNA using the mRNA Reverse-Transcription Kit (Beyotime, Shanghai). Quantitative PCR assays were performed using the SYBR Green PCR Mix (Vazyme Biotech, Shanghai) on a Real-Time PCR System to analyze mRNA expression levels. Relative expression levels were calculated using the 2 − ΔΔCt method and normalized against β-actin. This procedure was repeated three times. The primers used were as follows: for NF-κB, Forward: 5’-GGAGATCCTTGCCGAGGAC-3’ and Reverse: 5’-GCTCGTAGGGCTCCTGTG-3’; for TGF-β, Forward: 5’-TGAACCGGCCTTTCCTGCTTCTCATG-3’ and Reverse: 5’-GCGGAAGTCAATGTACAGCTGCCGC-3’; and for β-actin, Forward: 5’-CGGTCAGGTCATCACTATC-3’ and Reverse: 5’-CAGGGCAGTAATCTCCTTC-3’.

Statistical analysis

Statistical evaluations were conducted using Prism 8 software. Data from measurements were depicted as the average value plus or minus the standard deviation and were consistently replicated a minimum of three times. To discern differences between pairs of groups, the statistical method of choice was the t test. Conversely, when making comparisons across three or more, a one-way ANOVA was applied. A p value less than 0.05 was regarded as the threshold for deeming results statistically meaningful.