CYP27A1^(−/−) mice were bred from C57BL/6J background CYP27A1^(+/−) heterozygous mice (five females, eight males, 009106 Bar Harbor, ME) purchased from Jackson Laboratories in the United States. All animals were raised in the Experimental Animal Center of Chongqing Medical University in an SPF level barrier environment, free of specific pathogens, with room temperature ranging from 18°C to 28°C, relative humidity of 40% to 70%, 12 h of light/12 h of darkness, and sufficient food and water.

Mouse genotyping was performed by digesting toe tissue using a commercial genotyping kit (Beyotime, D7283S). Polymerase chain reaction amplifier (PCR) amplification was performed using specific primer sequences, reaction components, and thermal cycling conditions as listed in Table 1. Dissolve 0.5 g of agarose in 50 mL of TAE solution, microwave heating for dissolution, add 4 μL of ethidium bromide after slightly cooling, mix well and pour into the gel making rack. The PCR product was mixed with 1 × DNA Loading Buffer, and 8 μL samples were loaded on each well. After 30 min of 120 V electrophoresis, gel imaging analysis was performed.

In the experiment, the normal diet (ND, cholesterol free and bile salt), was used as a standard laboratory animal diet. The high-cholesterol diet (HCD, containing 1.25% cholesterol and 4.5% bile salts) was applied to simulate metabolic disorders induced by excessive cholesterol intake. This dietary formulation has been widely used to establish disease models such as non-alcoholic fatty liver disease (NAFLD), atherosclerosis, and cholesterol metabolism disorders, owing to its experimental reproducibility and pathological relevance (19). All feed was purchased from Research Diets in Germany. See Table 2 for the list of dietary ingredients. Male CYP27A1 KO mice and WT mice were divided into four groups: WTND group (WT mice fed ND), WTHCD group (WT mice fed HCD), KOND group (KO mice fed ND), and KOHCD group (KO mice fed HCD). All mice were 6–10 weeks old, with 10 mice in each group. After 3 weeks of adaptive feeding, the mice were given diet intervention for 12 weeks, and weighed at a fixed time every week (Figure 1A).

Lung tissue samples (50 mg) were collected from mice in four experimental groups: WTND group (n = 5), WTHCD group (n = 5), KOND group (n = 4), and KOHCD group (n = 4). Total RNA was extracted using Trizol reagent, and RNA concentration and purity were measured and assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States). Library construction was performed using the Illumina NovaSeq Reagent Kit, and sequencing was carried out on an Illumina NovaSeq Xplus (Illumina, United States). The transcriptomic sequencing generated 126.15 Gb of clean data, with each sample yielding more than 6.12 Gb of clean data. The Q₃₀ base quality exceeded 96.1%, meeting the requirements for comprehensive gene expression analysis. All samples were submitted to Shanghai Meiji Biomedical Technology Co., Ltd. for RNA sequencing (Project No. MJ20240228062). Data analysis is conducted on the Majorbio cloud platform.¹ The software used for gene differential expression analysis is DESeq2.² The software used for KEGG pathway enrichment analysis is KOBAS.³

Mice were fasting before sample collection. Before euthanasia, 1% Pentobarbital Sodium was injected intraperitoneally at the concentration of 0.1 mL/10 g body weight. Then, cardiac blood was collected using a 1 mL syringe. After 2–3 h of storage at room temperature, the blood was centrifuged at 400 × g for 15 min, and the serum was collected and stored in a −80°C refrigerator before use. The serum was sent to the Affiliated Hospital of Chongqing Medical University, and the blood lipid spectrum, including total cholesterol (TC), triglyceride (TG) and high/low density lipoprotein cholesterol (HDL-C and LDL-C), was determined by automatic biochemical analyzer.

Liver and lung tissues were fixed in 4% paraformaldehyde. The samples were embedded in paraffin and sectioned at a thickness of 4 μm for hematoxylin–eosin staining (HE). Sections were baked overnight at 60°C, then dewaxed in xylene and rehydrated through a graded ethanol series (100%-95%-80%-70%). After rinsing in distilled water three times, the sections were immersed in hematoxylin solution for 30 s and washed with running tap water. Nuclear staining intensity was assessed under a microscope and adjusted using 1% acid alcohol if necessary. The sections were then counterstained with eosin solution for 10 s at room temperature. Finally, the sections were dehydrated and mounted with neutral resin. Digital imaging was performed using a Pannoramic DESK digital slide scanner (3DHISTECH, Hungary).

Frozen liver and lung tissue sections stored at −20°C were rewarmed for 10 min. The sections were fixed in 10% formalin for 20 min, then rinsed on a shaker in distilled water. Afterward, the sections were differentiated in 60% isopropanol for 1 min and stained with freshly prepared Oil Red O stain (ORO) working solution at 37°C in the dark for 30 min. Following staining, the sections were immersed in 60% isopropanol for 1–2 min, and differentiation was assessed microscopically. After differentiation, the sections were washed in distilled water for 10 min. Hematoxylin counterstaining was performed for 20 s, followed by thorough rinsing with running water. Finally, the sections were sealed with glycerol gelatin.

Paraffin-embedded tissue sections were baked at 60°C for 2–4 h, dewaxed in xylene I and II for 15 min each, and rehydrated through a graded ethanol series (100%-95%-80%-70%) for 5 min at each step. Antigen retrieval was performed in citrate buffer. Immunohistochemical staining was conducted according to the instructions of the commercial kit (Fuzhou Maixin, KIT-9710). Diaminobenzidine (DAB) was used for color development, followed by hematoxylin counterstaining for 20 s. The sections were dehydrated through graded ethanol (70%-80%- 95%-100%), cleared in xylene I and II for 30 min each, and mounted with neutral resin.

Total RNA was extracted from tissue cells using Trizol reagent (DP424, Tiangen), followed by reverse transcription using 1,000 ng of total RNA (RR047A, Takara). The system was divided into a gDNA removal system and a reverse transcription system, with both systems having a volume of 10 μL. Perform quantitative real-time PCR (qRT-PCR) using SYBR Green qPCR premix (HY-K053, MCE). Calculate the relative mRNA expression fold changes based on endogenous β-actin. The specific primer sequences are shown in Table 3.

Total protein was extracted from cells or tissues using a lysis buffer containing protease inhibitors, phosphatase inhibitors, and PMSF. Protein concentration was determined using the bicinchoninic acid (BCA) method. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk at 37°C for 1 h, then incubated overnight at 4°C with primary antibodies. After washing, the membranes were incubated with secondary antibodies at 37°C for 1 h. Signals were detected using ECL substrate (BG0015, Bioground Biotech Co., Ltd.) and visualized with a chemiluminescence imaging system. The primary antibodies used in this study included: ABCA1 (1:1,000, Abclonal), LDLR (1:1,000, Abclonal), HMGCR (1:1,000, Abclonal), CYP27A1 (1:1,000, Abcam), sterol regulatory element-binding Protein 2 (SREBP2) (1:1,000, Proteintech), ATP-binding cassette subfamily G1 (ABCG1), (1:1,000, Proteintech), p-NF-κB/NF-κB (1:1,000, CST), ACTIN (1:1,000, Proteintech).

Paraffin-embedded tissue sections were baked at 60°C for 2–4 h, then dewaxed in xylene I and II for 20 min each. The sections were rehydrated through a graded ethanol series (100%-95%-80%-70%) for 5 min at each step, followed by antigen retrieval in citrate buffer for 30 min. Multiplex fluorescence staining was performed according to the manufacturer’s instructions of the staining kit (AFIHC024, AiFang Biotech). Finally, DAPI was applied in the dark for 5 min, and the sections were sealed with an anti-fade mounting medium CD68 (1:100, boster), SFTPC (1:100, Proteintech), ABCA1(1:100, Proteintech).

This study used SPSS 25.0 for data analysis, GraphPad Prism 8.0 for plotting, Student’s t-test or Wilcoxon rank sum test for comparison between two groups, and one-way ANOVA or Kruskal–Wallis H test for comparison between three or more groups. All statistical analyses were two-tailed with a significance level set at 0.05. Data were presented as mean ± SEM. Differences were considered statistically significant at p < 0.05. Statistical significance was indicated as follows: ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

ORO staining revealed distinct cholesterol metabolic responses in the liver and lung tissues of WT mice fed HCD.

In the liver, compared with the ND group, the HCD group exhibited markedly enlarged lipid droplets and significant lipid accumulation (Figure 1B). In contrast, no obvious lipid droplets were observed in lung tissue, and the difference was not statistically significant. Western blot (WB) analysis further showed that in the liver, HCD feeding led to a feedback downregulation of the low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), key regulators of cholesterol uptake and synthesis. Concurrently, the expression of CYP27A1 and ABCA1, both involved in cholesterol clearance, was also significantly downregulated (Figure 1C).

In contrast, the lung tissue exhibited a distinct pattern. Similar to the liver, LDLR and HMGCR were downregulated; however, the expression of CYP27A1 and ABCA1 was significantly upregulated in the HCD group (Figure 1E). This tissue-specific pattern was further confirmed by qRT-PCR (Figures 1D,F): CYP27A1 and ABCA1 mRNA levels were downregulated in the liver, while significantly upregulated in the lung under HCD. These findings suggest that CYP27A1 and ABCA1 may participate in tissue-specific regulation of cholesterol metabolism in WT mice. Given that CYP27A1 acts upstream of ABCA1, we next employed whole-body knockout of CYP27A1 to further investigate its role in tissue-specific metabolic regulation.

Body weight was monitored weekly throughout the 12 weeks feeding period. At the end of the experiment, major organs including the liver, lung, kidney, and spleen were collected and weighed. Compared with the WTND group, mice in the WTHCD group exhibited a significant reduction in body weight (Figure 2A), while liver body weight ratio were significantly increased. In contrast, no significant changes were observed in the weights of other organs such as the lung or kidney (Figure 2B). Serum lipid analysis revealed that HCD feeding elevated TC and LDL-C levels, while TG and HDL-C levels in WT mice (Figure 2C).

In CYP27A1 KO mice, compared with the KOND group, the KOHCD group resulted in an increased liver body weight ratio with lipid level changes similar to those observed in WT mice.

Under ND conditions, KO mice exhibited lower body weight than WT mice, along with an increased liver body weight ratio. No significant differences were observed in the weights of other organs such as the lung and kidney. Serum TG levels were markedly reduced in KO mice, while other lipid parameters remained unchanged.

In conclusion, HCD had a significant impact on body weight, liver index, and lipid profiles in both WT and KO mice. However, the global metabolic phenotype of CYP27A1 KO was relatively mild, it may still influence tissue-specific lipid regulation under both physiological and dietary stress conditions.

After 12 weeks of HCD feeding, measurements of TC and TG in liver and lung tissues were performed (Figure 3A). compared with the ND group, HCD significantly increased hepatic TC levels in WT mice. Consistently, HE staining revealed increased lipid droplets in the liver (Figure 3C), and ORO staining showed abundant lipid droplets (Figure 3E), indicating notable hepatic lipid accumulation. In contrast, the lung tissue exhibited metabolic tolerance to HCD (Figures 3B,D,F), although TC levels in the lung showed an increasing trend, the difference was not statistically significant, and ORO staining revealed no obvious lipid accumulation.

In CYP27A1 KO mice, compared with the KOND group, the KOHCD group further increased the TC level in liver tissue, while the TG level decreased. HE staining revealed a marked increase in hepatic lipid droplets, and ORO staining confirmed extensive lipid droplet accumulation, indicating exacerbated hepatic steatosis. However, the lung tissues of KO mice showed no significant lipid accumulation, either in TC levels or ORO staining, suggesting metabolic tolerance to both HCD and CYP27A1 deficiency.

Under ND conditions, KO mice displayed elevated hepatic TC and TG levels compared with WT mice. Mild lipid droplet was observed in the liver, and ORO staining revealed an increased number of lipid droplets, indicating enhanced hepatic lipid accumulation. In contrast, TC levels and ORO staining in the lungs of KO mice did not show evidence of lipid accumulation.

Taken together, both HCD and CYP27A1 deficiency promoted lipid accumulation in the liver, while the lung tissue exhibited metabolic tolerance, with no significant lipid deposition.

Although pronounced lipid accumulation was observed in the liver under either CYP27A1 KO or HCD conditions, no similar changes were found in the lung tissue (Figures 3C–F). To investigate the underlying cause, we examined the expression of genes and proteins involved in cholesterol uptake, synthesis, and efflux. WB and qRT-PCR analyses revealed that ABCA1 expression exhibited a tissue-specific pattern (Figures 4A–D). Under both ND and HCD conditions, CYP27A1 KO downregulated ABCA1 expression in the liver, whereas ABCA1 expression in the lung was significantly upregulated. In addition, no significant differences were observed in the expression levels of ABCG1 and SREBP2 in either the liver or lung tissues among the four groups (Supplementary Figures 1A,B). Meanwhile, in the liver, compared with the KOND group, the expression levels of LDLR and HMGCR protein and mRNA in the KOHCD group were significantly decreased. In lung tissue, compared with the KOND group, the expression of LDLR in the KOHCD group was significantly decreased, while there were no significant differences in other groups. In addition, no significant differences in LDLR or HMGCR protein levels were found in either liver or lung tissues after CYP27A1 KO. Immunohistochemical results further supported the tissue specific regulation of ABCA1 (Figures 4E–H), showing a progressive decrease in the liver and a gradual increase in the lung.

Immunofluorescence analysis (Figures 5A,B) showed that ABCA1 was abundantly expressed in lung tissue and primarily localized to alveolar type II epithelial cells. In contrast, ABCA1 expression in macrophages did not show a clear increase. These findings suggest that alveolar type II epithelial cells may play a critical role in pulmonary cholesterol homeostasis, potentially by promoting cholesterol efflux to prevent excessive lipid accumulation in the lung.

In summary, ABCA1 expression in the liver and lung exhibited tissue specific differences under HCD or CYP27A1 KO conditions, which may be related to distinct physiological functions and regulatory mechanisms of ABCA1 in different cell types.

To explore the molecular mechanism of ABCA1 upregulation in lung tissue and its potential role in cholesterol clearance, RNA-seq analysis was conducted on lung tissues from WT and CYP27A1 KO mice fed with either ND or HCD. Differentially expressed genes were screened using the criteria of |log₂FC| >0 and p-value <0.05. The results showed distinct gene expression differences in lung tissues under different dietary conditions (Figure 6A). In the volcano plots, compared with the WTND group, the WTHCD group showed 1,015 upregulated genes and 1,232 downregulated genes. Compared with the KOND group, the KOHCD group showed 1,006 genes upregulated and 875 genes downregulated (Figures 6C,D). Venn diagram analysis between the DEGs of WTND vs. WTHCD and KOND vs. KOHCD revealed 288 Common genes (Figure 6B). Further clustering and KEGG enrichment analysis (Figure 6E) showed that the upregulated genes were significantly enriched in pathways associated with immune regulation, such as Fc gamma receptor (FcγR)-mediated phagocytosis, the NF-κB signaling pathway, and other signal transduction processes. In contrast, the downregulated genes were enriched in pathways related to immune signaling modulation, metabolic regulation, endocrine functions, and cancer-related biological processes (Figures 6F,G).

NF-κB, as a classical inflammation pathway, has been reported to regulate ABCA1 expression (20). Therefore, we selected the NF-κB pathway for subsequent validation. We examined the expression of the NF-κB signaling pathway and its related inflammatory genes using WB and qRT-PCR (Figures 6H,I). The results were consistent with the RNA-seq data: both HCD and CYP27A1 KO enhanced the expression of NF-κB pathway proteins, which paralleled the expression trend of ABCA1 observed (Figure 4B). Meanwhile, proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) showed an increasing trend in lung tissue following HCD treatment, while the expression level of the anti-inflammatory cytokine interleukin-10 (IL-10) was decreased.