Background
Hypoxia refers to the pathological process of abnormal metabolic function and morphological structure of tissues caused by insufficient oxygen supply or oxygen use disorder, which has serious adverse effects on the body's metabolism and other normal life activities [
1,
2]. The lungs are extremely susceptible to the effects of a hypoxic environment, and symptoms such as dyspnea, chest tightness, palpitations, and insufficient oxygen supply to the body occur [
3]. In addition, a hypoxic environment can cause severe lung dysfunction and injuries such as high-altitude pulmonary edema (HAPE) and pulmonary hypertension [
4,
5]. In addition, if the body experiences severe trauma, infection, shock, acute lung injury, or acute respiratory distress syndrome can be rapidly induced, which can be life-threatening [
6]. It is considered that the occurrence of HAPE is a complex process that involves multiple factors and many genes [
7]. The happening and development of HAPE are related to obvious individual and racial diversity, and it is affected by environmental and genetic factors [
8]. However, drugs that prevent hypoxia-induced lung injury remain scarce [
9].
HAPE is a result of hypoxic pulmonary vasoconstriction and alveolar interstitial edema, yet its genetic mechanism remains elusive. The mechanisms of hypoxia-induced lung injury mainly include pulmonary arterial hypertension and acute non-bacterial inflammation, which are activated by increased activity of sodium ion channels and genetic changes [
10‐
12]. Genetic changes are primarily associated with hypoxia and individual susceptibility. It may be regarded as an important molecular marker for evaluating conditions [
13,
14].
Blood electrolyte balance means that the concentration of various ions in the blood of the human body is maintained within a certain range and maintains a stable state [
15,
16]. These ions include sodium and potassium, among others, which play an important regulatory role between the inside and outside of the cells [
17]. Blood electrolyte homeostasis is crucial for maintaining human health [
18]. In a normal physiological state, the disparity in concentration between extracellular and intracellular compartments ensures proper cellular function [
19]. However, certain conditions, such as hypoxia following carbon dioxide inhalation, can disrupt the delicate balance of electrolytes [
20]. Another study showed that long-term monitoring of blood sodium levels was associated with the progression of HAPE patients and persistent abnormal blood sodium was related to higher mortality [
11].
As the most active form of VD3, 1α, 25-dihydroxyvitamin D3 (1,25(OH)
2D
3) exerts various physiological activities, such as calcium and phosphorus regulation, immunoregulation, anti-cancer, and cardiovascular regulation [
21,
22]. Ten 1,25(OH)
2D
3 drugs are used for the treatment of osteoporosis, psoriasis, and hyperparathyroidism. Studies have shown that VD3 plays a protective role against lung injury [
23,
24]. Treatment with VD3 ameliorated seawater aspiration-induced inflammation and pulmonary edema by inhibiting NF-κB and RhoA/Rho kinase pathway activation [
25]. In the lungs of hamsters with acute lung injury, VD3 treatment alleviated lipopolysaccharide (LPS) instillation [
26]. High dietary VD3 intake results in elevated serum 25D3 levels and significant inhibition of lung tumor growth [
27]. Furthermore, VD3 significantly reduced the expression of TLR4, NF-κB, and the inflammatory cytokines TNF-α, IL-1β, and IL-6 [
24]. However, the modulatory mechanism of VD3 in protecting alveolar epithelial cells from hypoxia-induced lung injury has not yet been investigated.
In this study, we aimed to explore the mechanism underlying the protective effect of VD3 on hypoxia-induced lung injury using transcriptomic profiling. We found that the complement and coagulation cascades are downstream signaling pathways that affect the protective effect of VD3 against plateau lung injury. Thus, VD3 may play a cytoprotective role by inhibiting mitophagy.
Methods
Animals
Specific pathogen-free (SPF) healthy male SD rats (6–8 weeks old and weighing 180–220 g) were purchased from Chengdu Dossy Experimental Animals Co., Ltd. (Chengdu, Sichuan). The feeding environment was 25 °C ± 1 °C, relative humidity 50%-60%, and light/darkness for 12 h circulation. The rats were allowed free access to food and water. Animals and experimental protocols were conducted according to the ARRIVE guidelines.
Establishment and grouping of animal models
Thirty SD rats were randomly divided into normoxia, hypoxia, and VD3 (1,25(OH)
2D
3) groups after 3 days of acclimatization in the animal experiment department, with 10 rats in each group. Rats in the VD3 group were administered 0.03 µg/kg VD3 (C9756, Sigma‐Aldrich, Missouri, USA) dissolved in peanut oil, as previously described [
28]. Rats in the normoxic and hypoxic groups were administered the same amount of peanut oil. The drug was administered continuously for seven days. From the 4th day, except for the normoxia group, the other groups were placed in the simulation chamber of a 6500 m altitude environment for 72 h in a hypobaric chamber (DYC-9070; Avic Guizhou Fenglei Aviation Armament Co., Ltd., Anshun, China) as described previously [
29,
30]. After entering the experimental chamber at 8:30 a.m. every day, the chamber ascended to an altitude of 4000 m at a constant speed of 2 m/s, while the simulated chamber descended to an altitude of 4000 m at a constant speed of 10 m/s. When the two chambers were stabilized, the experimenter entered the simulated chamber, administered the drugs by gavage, and changed the food and bedding. After each drug administration, the simulated chamber was returned to an altitude of 6500 m, and the experimental chamber was returned to the local altitude. During the experiment, the animals were fed and drank freely, and their survival status was monitored.
Arterial blood gas analysis
Arterial blood gas analysis was performed seven days after administration. The rats were anesthetized via intraperitoneal injection of pentobarbital sodium (45 mg/kg). Blood was drawn from the femoral artery via femoral artery catheterization under anesthesia. Arterial blood was measured within 15 min using an ABL800 blood gas analyzer (ABL 80 Flex Basic, Radiometer, Denmark). The indices included arterial partial pressure of carbon dioxide (PaCO2), arterial partial pressure of oxygen (PaO2), arterial oxygen saturation (SaO2), sodium (Na+), potassium (K+), and calcium (Ca2+) levels.
Measurement of pulmonary artery pressure
The right external jugular vein was separated, the distal end ligated, the proximal end clamped with an arterial clamp, and the external jugular vein removed. After endotracheal intubation and mechanical ventilation, changes in the pressure waveform were observed using the PowerLab system (PowerLab 7.8, AD Instruments, Colorado Springs, CO). The catheter was gradually inserted into the superior vena cava, progressing into the right atrium where a minor pressure waveform was observed. Subsequently, the catheter was further advanced into the right ventricle, enabling recording of the pressure curve in the right ventricle. The catheter is then sent to the pulmonary artery under the action of the right ventricular blood flow, in addition, changes in pulmonary arterial pressure waveforms were observed using PowerLab physiological loggers (ADI, Australia), and pulmonary arterial pressure was collected through a pressure sensor.
Lung water content
The water content of the lung tissue was detected using the dry–wet weight technique, as described previously [
31]. The isolated tissues of the left upper lobe of the lung were placed in a 55 °C electric heating constant temperature air drying oven until the dry weight error was within 0.0002 g [
32]. Water content = wet weight—dry weight.
Total RNA was extracted from lung tissues using the Trizol-centrifuge column method (Invitrogen, San Diego, CA, USA) according to the manufacturer's instructions. DNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer ( Thermo Scientific). RNA integrity number (RIN) was evaluated using an Agilent 2100 Bioanalyzer system (Santa Clara, CA, USA).
Library construction and sequencing
After total RNA extraction, the mRNA was enriched. Complementary DNA (cDNA) was synthesized from the fragmented RNA, followed by end repair. The ligated products were amplified by bridge PCR, using specific primers to construct cDNA libraries for sequencing. After the library was successfully constructed, the BGISEQ-500 platform of the BGI Genomics Institute (BGI-Shenzhen, China) was used for high-throughput sequencing.
Data processing and analysis
Low-quality bases, N-bases, or low-quality reads were filtered out, and high-quality clean reads were obtained. The fragments per kilobase million (FKPM) method was used to calculate the genes. Express quantity to |log2 (fold change)|> 1 and a false discovery rate (FDR) < 0.001 were used as the criteria to screen differentially expressed genes (DEGs). A Pearson Correlation test was performed for statistical analysis, and the results are presented in a volcano plot. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to determine the functions of DEGs. KEGG pathway enrichment statistical analysis was performed using KOBAS software.
Hematoxylin and eosin (H&E) stain
The lung tissues of the rats were collected and fixed in 4% paraformaldehyde overnight, processed, and embedded in paraffin. The tissue sections were stained with H&E to observe the degree of lesion and inflammatory cell infiltration under a 400 × magnification optical microscope (Olympus BH2, Tokyo, Japan).
Enzyme-linked immunosorbent assay (ELISA)
ELISA was used to detect the expression levels of Fga, Fgb, PAR1, PAR3, PAR4, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin-1beta (IL-1β) in the lung tissue according to the manufacturer’s instructions. In addition, the expression levels of tissue factor (TF), coagulation factor VII (FVII), coagulation factor II (FII), coagulation factor V (FV), coagulation factor X (FX), and thrombomodulin (TM), in liver tissue were detected by ELISA method according to the kit manufacturer’s instructions. The levels of lactate dehydrogenase (LDH), TNF-α, IL-6, and IL-1β in the cell supernatant were measured by ELISA according to the manufacturer’s instructions.
Western blot analysis
Lung tissue lysis solution or cells were prepared using RIPA buffer (Signaling Technology, Inc.). Protein concentration was determined using a BCA kit (Sigma-Aldrich; Merck KGaA). Total protein (30 µg/sample) was separated using 10% SDS-PAGE and nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk. Subsequently, the membranes were probed with primary antibodies. The membranes were washed with Tris-buffered saline/0.1% Tween (TBST) and incubated for 1.5 h with HRP Goat anti-rabbit IgG (Abcam, ab6721). Band visualization was carried out using the ECL system (Affinity Biosciences, Cincinnati, Ohio, USA) and β-actin was used as an internal control.
Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR)
Total RNAs were isolated using TRIzol® reagent (Thermo Fisher, Massachusetts, USA). cDNA was obtained using a reverse transcription kit (Invitrogen). The relative levels of target gene RNA transcriptomes were determined by qRT-PCR using the SYBR Premix Ex Taq kit (Bao Biological Engineering, Dalian, China). The reverse transcription reaction conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. The relative gene expression level was determined by applying the 2−△△Ct method to ABI software (Foster City, CA, USA).
Immunohistochemistry (IHC) stain
IHC staining was performed to detect protein expression in rat lung tissue. Complement (C) C3, C3a, C5, Fga, and Fgb protein expression levels were assessed according to the IHC protocol.
Immunofluorescence (IF) staining
After the experiments, lung tissues were dissected and fixed with 4% paraformaldehyde. Paraffin sections of the lung tissues were dewaxed and hydrated. The sections were incubated in QuickBlock Blocking Buffer (Beyotime, Shanghai, China) for 30 min at room temperature. Then, The sections were incubated with the anti-ZO1 tight junction (Abcam, ab221547; 1/100) at 4 °C overnight and washed three times with phosphate-buffered saline (PBS). Staining of the lung tissues was observed under a fluorescence microscope BX53 (Olympus, Tokyo, Japan) at 400 × magnification.
Cell culture
Rat type II alveolar epithelial cells (CP-R003) and hepatocytes (CL-0038) were purchased from Wuhan Procell Life Technology (Wuhan, China). The cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco).
Cell co-culture
To investigate the regulatory role of hypoxic or/and VD3-induced alveolar epithelial cells in the ability of hepatocytes to secrete coagulation factors, we developed a co-culture system. Logarithmic growth phase rat type II alveolar epithelial cells (1.0 × 10
5/mL) were inoculated on the base of a 6-well plate (2 mL per well). For experiment 1, cells were divided into four groups: normoxia, hypoxia, hypoxia + low-concentration VD3, and hypoxia + high-concentration VD3. For experiment 2, the cells were divided into five groups: normoxia, hypoxia, hypoxia + VD3, hypoxia + VD3 + mitochondrial autophagy inhibitor (5 μM Mdivi-1), and hypoxia + VD3 + mitochondrial autophagy agonist (50 μM CCCP). The Rat type II alveolar epithelial cells were incubated under normoxic (95% O
2, 5% CO
2, 37 °C) conditions for 1 h and then in hypoxia (1% O
2, 94% N
2, 5% CO
2, 37 °C) for 24 h, as the previously described [
33]. For VD3 treatment, rat type II alveolar epithelial cells were treated with low VD3 (20 nM) or high VD3 (40 nM) for 24 h. For the mitochondrial fission inhibitor Mdivi-1 (Beyotime, Shanghai, China), rat type II alveolar epithelial cells were treated with 5 μM Mdivi-1. For treatment with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma-Aldrich, Missouri, USA), rat type II alveolar epithelial cells were treated with 50 μM CCCP. Logarithmic growth phase rat hepatocytes (1.0 × 10
5/mL) were inoculated into the upper chamber of Transwell ventricles (2 mL per well). Rat hepatocytes were cultured at 37 °C and 5% CO
2, and Traswell was placed in a 6-well plate for co-culture after the rat hepatocytes were attached to the wall overnight.
CCK-8 assay
The viability of type II alveolar epithelial cells was measured using the Cell Counting Kit 8 (CCK-8, Thermo Fisher Scientific) according to the manufacturer’s instructions. The absorbance was recorded at 450 nm.
JC-1 mitochondrial membrane potential detection assay
The JC-1 assay was performed to measure mitochondrial membrane potential (ΔΨm) using the JC-1 mitochondrial membrane potential assay kit (KeyGen, China). Briefly, cells were seeded in a 6-well plate and ΔΨm was detected according to the guidelines of the JC-1 kit. All samples were analyzed using an Accuri or LSRII flow cytometer (BD Biosciences).
Transmission electron microscopy (TEM)
Transmission electron microscopy (TEM) was performed to observe autophagosomes. Briefly, prefixed with 3% glutaraldehyde, then the cells were postfixed in 1% osmium tetroxide, dehydrated in a series of acetone, infiltrated in Epox 812 for a longer period, and embedded. The semi-thin sections were stained with methylene blue, and ultrathin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate. The sections were examined using a JEM-1400-FLASH Transmission Electron Microscope (JEOL JEM-1400, JEOL, Ltd., Tokyo, Japan).
Statistical analysis
Means and standard deviations (SD) were used to represent the data. All data were analyzed using the SPSS software (version 22.0; IBM Corp., Armonk, NY, USA). Kolmogorov–Smirnov tests revealed that the data were normally distributed, that is, variables were parametric. Statistical analyses were performed using the independent sample Student’s t-test for comparisons between two groups. One-way ANOVA with Tukey’s post hoc test of means was used for multiple group comparisons. Statistical significance was set at P < 0.05.
Discussion
In our study, we observed increases in blood K
+ and Na
+ concentrations under hypoxic conditions. VD3 treatment significantly reduced K
+ and Na
+ concentrations and ultimately maintained electrolyte homeostasis. Patients with lung injury caused by hyperbaric hypoxia may also present with generalized edema and pleural-pericardial effusion, and the resulting heart failure may be the cause of elevated K
+ and Na
+ levels [
35,
36]. Similarly, a previous study showed that Compared with controls, patients with lung injury caused by hyperbaric hypoxia had significantly higher serum Na + and K
+ [
37]. In addition, when the altitude increased to 3200 m, the subjects' blood potassium levels increased significantly [
38]. Mechanistically, the hypoxia-induced closure of K
+ conductance in alveolar epithelium results in fluid clearance, thereby promoting fluid retention and contributing to the development of pulmonary edema [
39]. A further investigation into alveolar epithelial cells has confirmed that hypoxia leads to a down-regulation of the expression and epithelial Na
+ channels (ENaC) and Na, K-ATPase, thereby diminishing salt and water clearance [
40]. Moreover, in response to alveolar hypoxia, a mitochondrial sensor dynamically changes reactive oxygen species and redox couples in pulmonary artery smooth muscle cells (PASMC), inhibiting potassium channels, and ultimately inducing depolarization of PASMC [
41]. Hypoxia inhibited several potassium channels (voltage-gated and TASK), leading to membrane depolarization. The inhibition of potassium channels results in high cytosolic levels of potassium. These lead to the inhibition of apoptosis and an increase in cellular proliferation [
42].
Previously published data have demonstrated in cases of high-altitude pulmonary edema, there is an increase in the infiltration of inflammatory cells, specifically macrophages and neutrophils, as well as elevated levels of cytokines such as IL-6, TNF-α, and IL-1β [
43]. Rats exposed to acute hypobaric hypoxia and exhibiting signs of high-altitude pulmonary edema displayed an upregulation of NF-κB levels in the nuclear fraction [
44]. This increase in NF-κB activity regulates the production of inflammatory molecules, including IL-1, IL-6, and TNF-α, within the lung tissue under hypoxic conditions [
44]. Furthermore, this study highlights an elevation in the levels of cell adhesion molecules ICAM-1 and VCAM-1 [
44]. Furthermore, a separate study conducted on rats experiencing high-altitude pulmonary edema (HAPE) as a result of acute hypobaric hypoxia (9142 m for 5 h) also demonstrated elevated levels of proinflammatory molecules, including TNF-α, monocyte chemoattractant protein-1 (MCP-1), INF-γ, IL-6, and TNF-β, within the bronchoalveolar lavage. Additionally, an increase in NF-κB levels was observed in lung nuclear extracts [
45]. Our data suggest that VD3 inhibited the protein levels of inflammatory biomarkers, such as IL-6, TNF-α, and IL-1β, caused by a hypoxic environment in vivo and in vitro.
The disruption of the integrity of the lung vascular endothelial barrier leads to an increase in permeability, resulting in pulmonary edema [
46]. VE-Cadherin is specifically expressed in endothelial cells and is considered to be a structurally and functionally critical part of adherence junctions [
47]. A previous study indicated that hypoxia selectively disrupts microvascular endothelial tight junction complexes in the lung through the permeability-inducing factors VEGF and VE-cadherin [
48]. In addition, increased capillary pressure was found to induce pulmonary permeability edema by disrupting endothelial adhesion junctions, including activation of the calcium-dependent protease calpain and degradation of adhesion junction proteins VE-Cadherin, β-catenin, and p120-Catenin [
49]. Tight junction (TJ) proteins occludin and ZO-1 are reported to be critical for regulating pulmonary vascular permeability. Ulinastatin ameliorates pulmonary edema by upregulating the expression of ZO-1 and occludin, thereby reducing pulmonary permeability and stimulating alveolar fluid clearance [
50]. The TJ protein (ZO-1, JAM-C, claudin-4, occludin) expression in the lungs of the HAPE model group has been restored to a normal high level upon quercetin pre-treatment [
51]. The results of the present study demonstrate that VD3 treatment effectively ameliorated hypoxia-aggravated pulmonary barrier injury, as evidenced by the significant increase in the expression of ZO-1, occludin, and VE-Cadherin.
The clotting factors, anticoagulation factors, and fibrinolytic system of the coagulation pathway are in a dynamic balance and work together to maintain [
52,
53]. Once this balance is disturbed, abnormal bleeding or thrombosis occurs. Fibrinogen (Fg) is an important factor in the coagulation pathway and consists of an alpha chain (Fga), beta chain (Fgb), and gamma chain (Fgg) [
54]. Fibrinolytic inhibition prevents an increase in lung vascular permeability after pulmonary thromboembolism [
55]. Furthermore, fibrinogen augmented mean arterial pressure and reduced histopathological injury and lung permeability by inhibiting MMP-9-mediated syndecan-1 cleavage in obese mice [
56]. Tissue factor (TF) is a crucial component of the coagulation pathway. Upon tissue injury, exposure of plasma to TF expressed on non-vascular cells or activated endothelial cells leads to the formation of the TF-factor VIIa (FVIIa) complex [
57,
58]. Subsequent catalysis of the initial activation of factor X (FX) to FXa is facilitated by the TF-FVIIa complex. Subsequently, the enzymatic conversion of prothrombin into thrombin (TM) is facilitated by the collaborative action of FXa and activated factor V (FVa) [
59]. Sustained coagulation is achieved through the catalytic action of thrombin, synthesized via the initial TF-FVIIa-FXa complex, which activates FXI, FIX, FVIII, and FX [
60,
61]. The study showed that uncontrolled activation of the coagulation cascade contributes to acute and chronic lung diseases [
58]. FX expression of FX was locally increased in human and murine fibrotic lung tissues [
62]. FX inhibitors attenuated bleomycin-induced pulmonary fibrosis in mice [
62]. Significantly increased levels of TF-enriched neutrophil extracellular traps (NETs) have been observed in patients with acute respiratory distress syndrome (ARDS) patients and mice. The blockade of NETs in ARDS mice alleviated disease progression, indicating a reduced lung wet/dry ratio and PaO
2 levels [
63]. In this study, we found that VD3 prevented the upregulation of Fga and Fgb expression in the lungs of rats with high-altitude pulmonary edema. Meanwhile, VD3 treatment markedly blunted the hypoxia-induced increase in the protein levels of TF, FVII, FII, FV, FX, and TM. Meanwhile, in the co-culture system of alveolar epithelial cells and hepatocytes, VD3 treatment blunted the hypoxia-induced increase in the protein levels of TF, FVII, FII, FV, FX, and TM.
Protease-activated receptors (PARs) are a superfamily of G protein-coupled receptors that mediate transmembrane signaling and regulate cellular functions, including four members: PAR1, PAR2, PAR3, and PAR4. They are mainly distributed in the airways, intestines, skin, and other tissues where inflammatory responses are likely to occur [
64,
65]. The biological effects of PARs include inducing a coagulation response, releasing inflammatory factors to regulate the local inflammatory response, increasing vascular exudation and neutrophil chemotaxis, regulating vascular tone, and promoting cell division and proliferation [
64,
66]. Current research indicates that PARs play important roles in embryonic development, atherosclerosis, vascular stenosis, and the physiological and pathological processes of tumors [
64,
67,
68]. Studies have shown that the activity of PARs after activation is mainly characterized by the high expression of inflammatory mediators such as IL-6, IL-8, cyclooxygenase, prostacyclin, angiogenesis, and regulation of vascular barrier function [
69‐
71]. PAR1-mediated enhancement of alpha (v) beta 6-dependent TGF-β activation could be one mechanism by which activation of the coagulation cascade contributes to the development of acute lung injury [
72]. In an ischemia/reperfusion-induced acute lung injury (ALI) model, the specific PAR-1 antagonist SCH530348 decreased lung edema and neutrophil infiltration, attenuated thrombin production, reduced inflammatory factors, including cytokine-induced neutrophil chemoattractant-1, IL-6, and TNF-α, mitigated lung cell apoptosis, and downregulated phosphoinositide 3-kinase (PI3K), nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) pathways [
73]. PAR1, PAR3, and PAR4-induced epithelial-mesenchymal transition (EMT) have been suggested to be a possible mechanism underlying the expanded (myo) fibroblast pool in lung fibrosis [
74]. γ-Tocotrienol (γ-TE) inhibits IL-13/STAT6-activated eotaxin secretion in human lung epithelial A549 cells via upregulation of PAR4 expression and enhancement of atypical protein kinase C (aPKC)-PAR4 complex formation [
75]. Our experiments demonstrated that hypoxia enhanced the expression of PAR1, PAR3, and PAR4, both in vivo and in vitro. VD3 treatment decreased PAR1, PAR3, and PAR4 levels in the lungs of rats with high-altitude pulmonary edema and hypoxia-induced alveolar epithelial cells. Additionally, in the co-culture system of alveolar epithelial cells and hepatocytes, VD3 treatment blunted the hypoxia-induced increase in the protein levels of TF, FVII, FII, FV, FX, and TM. Significant increases in PAR1, PAR3, and PAR4 levels were observed in the hypoxia-induced co-culture system of alveolar epithelial cells and hepatocytes, which were decreased by VD3.
The complement system comprises a group of non-specific proteins found in human and vertebrate serum [
76]. It is an important component of the intrinsic immune response [
77]. The complement system can be activated in three ways: the classic, alternative, and lectin pathways. An activated complement system can eliminate invaders and protect the body. This system can also modify self-cells, such as apoptotic particles and cellular debris, but can also regulate the cell cycle through its ligands and receptors. C3 is a key protein in the complement cascade response and its multiple molecular binding sites are essential for its role [
78]. C3 binds to its corresponding receptor protein and plays an important role in the pathological mechanisms of immune defense and inflammation [
79]. The binding of C5a and its receptor (C5aR) stimulates the aggregation of neutrophils and macrophages and generates inflammatory mediators during inflammation [
80,
81]. In LPS-induced human lung type II pneumocytes (A549), C3 production was increased [
82]. C3 was found to play a dominant role in pathogen-specific T-cell and B-cell responses, contributing to the amelioration of
Chlamydia psittaci-induced pneumonia in mice [
83]. Inhibition of the C3a receptor mitigates sepsis-induced acute lung injury by suppressing pyroptosis in pulmonary vascular endothelial cells [
84]. Systemic activation of C5a leads to neutrophil (NEUT) activation, sequestration, and adhesion to the pulmonary capillary endothelium, resulting in damage and necrosis of vascular endothelial cells and ALI [
85]. In the present study, the expression of C3, C3a, and C5 was increased in the lungs of rats with high-altitude pulmonary edema and a hypoxia-induced co-culture system of alveolar epithelial cells and hepatocytes. The expression of C3, C3a, and C5 were inhibited by VD3.
Autophagy is a highly conserved self-eating process, in which cells degrade long-lived proteins and organelles for recycling. Autophagy is generally activated by conditions of nutrient deprivation but has also been associated with physiological and pathological processes. Autophagy appears to be involved in pulmonary diseases, either beneficially or adversely [
86,
87]. Pharmacological inhibition of autophagy with 3-methyladenine (3-MA) significantly improved pulmonary appearance, edema, microvascular dilatation, and arterial oxygenation in rats with hepatopulmonary syndrome (HPS) rats [
88]. Autophagy was activated in common bile duct ligation (CBDL) rats and cultured pulmonary microvascular endothelial cells induced by CBDL rat serum [
88]. Autophagy activation reduces the sepsis-induced release of inflammatory factors and pulmonary edema through autophagy activation [
86]. mTOR in the epithelium promotes LPS-induced ALI through downregulation of autophagy and subsequent activation of NF-κB [
89]. A previous study found that sodium tanshinone II sulfonate A (STS) alleviated hypoxia-induced lung edema by promoting apoptosis, inhibiting inflammatory responses, and upregulating autophagy [
90]. Additionally, in vivo experiments, LPS-induced severe pulmonary edema was further exacerbated by inhibiting autophagy [
91]. An increase in autophagy is associated with the promotion of inflammation, which leads to lung injury [
92]. SARS-CoV-2 spike pseudovirions (SCV-2-S) promote autophagic responses [
93]. SCV-2-S-induced autophagy triggered inflammatory responses and apoptosis in infected human bronchial epithelial and microvascular endothelial cells [
93]. The ethanol extract of the tuber of A. orientale Juzepzuk (EEAO) relieved the pathological features of chronic obstructive pulmonary disease (COPD) by suppressing lung emphysema and autophagy and inducing TNF-α, IL-6, and TGF-β in a mouse model [
94]. In this study, we confirmed that the activation of autophagy in a hypoxia-induced co-culture system of alveolar epithelial cells and hepatocytes and the lungs of rats with pulmonary edema was inhibited by VD3, which was further inhibited by the autophagy inhibitor Mdivi-1 and reversed by the autophagy activator CCCP.