The human lung adenocarcinoma cell lines A549, H1975 and H1299, as well as the human bronchial epithelial cell line BEAS-2B, were used in the present study. These cell lines were provided by The Cell of Bank of Type Culture Collection of the Chinese Academy of Sciences. The cell lines used in the presented study were tested for mycoplasma contamination, ensuring that the experiments were conducted with uncontaminated cell cultures. Cells were cultured in DMEM High Glucose (Hyclone; Cytiva) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). The cells were cultivated in a cell incubator at 37˚C in a humidified atmosphere with 5% CO₂.

Rabbit anti-human GAPDH antibody (1:6,000 dilution; cat. no. AP0063) was purchased from Bioworld Technology, Inc. Peroxisome proliferator-activated receptor (PPAR)-γ rabbit polyclonal antibody (1:1,000 dilution; cat. no. 16643-1-AP) was purchased from Proteintech Group, Inc. HRP-conjugated goat anti-rabbit IgG (1:6,000 dilution; cat. no. BS13278) was purchased from Bioworld Technology, Inc. and was used as a secondary antibody (26).

23-HBA (Shanghai Aladdin Biochemical Technology Co., Ltd.) was used in the present study. It is known by the chemical names is ‘(3β)-3,23-Dihydroxylup-20(29)-en-28-oic acid’ or ‘(1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aS,13bR)-9-hydroxy-8-(hydroxymethyl)-5a,5b,8,11a-tetramethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a,13b-hexadecahydrocyclopenta[a]chrysene-3a-carboxylic acid’. The molecular weight of 23-HBA is 472.7 g/mol, while it has a melting point of 305˚C. 23-HBA is soluble in dimethyl sulfoxide (DMSO), a commonly used solvent in the laboratory, at a concentration of 2 mg/ml. 23-HBA is sensitive to light, humidity and heat, and its sensitivity is an important point to consider during its handling and storage (27).

A total of >8,000 lung adenocarcinoma-related genes were screened through the DisGeNET (www.disgenet.org) and GeneCards (www.genecards.org) databases (28). Subsequently, the 23-HBA drug targets were identified through the Traditional Chinese Medicine Systems Pharmacology (TCMSP; old.tcmsp-e.com/tcmsp.p) and SwissTargetPrediction (www.swisstargetprediction.ch) databases. The 23-HBA potential targets and lung adenocarcinoma-related genes were mapped using Venny tool version 2.1.0 (bioinfogp.cnb.csic.es/tools/venny/). The total number of common targets was analyzed.

H1299 cells in the logarithmic growth phase were inoculated into 96-well plates, with 2,000 cells placed in each well. After 16-18 h, the cells completely attached to the wells, and were divided into two groups: Experimental group (drug group) and control group (treated with DMSO). Several drug doses (0, 40, 60, 80 and 100 µM) were examined to determine the optimal concentration for subsequent experiments. The cells were cultured with 23-HBA at 37˚C for 48 h. Subsequently, the supernatant was discarded, and 90 µl RPMI-1640 complete medium (Gibco; Thermo Fisher Scientific, Inc.) with 10 µl MTT solution were added to each well. The plates were incubated at 37˚C in the dark for 4 h, before the supernatant was discarded. Subsequently, 150 µl DMSO (Sigma Corporation) was added and gently mixed to dissolve the formazan crystals. An ELISA microplate detector (Biosharp Life Sciences) was used to measure the optical density values at a wavelength of 491 nm.

H1299 cells in the logarithmic growth phase were inoculated at a concentration of 1x10⁵ cells/well in a 6-well plate. After 16-18 h of culture, the cells were exposed to varying concentrations (0, 20, 40 and 60 µM) of 23-HBA. Following 48 h of treatment (37˚C), the supernatant was collected in a 2.0 ml Eppendorf tube. For cell collection, 300 µl trypsin (Sigma-Aldrich; Merck KGaA) was added to the cells and incubated at 37˚C for 3 min. When the cells became spherical in shape, an equivalent amount of RPMI-1640 complete was added to terminate digestion. The suspension was centrifuged (37˚C) at 300 x g, and the pellet was collected after 5 min. The single-cell suspension was washed with PBS (OriGene Technologies, Inc.) and then subjected to centrifugation under the same conditions. Subsequently, the suspension was fixed (4˚C) in 75% pre-cooled ethanol (500 µl) for a duration of 2 h to overnight (12 h), and was stored at 4˚C. Prior to staining, the fixed cells were washed with PBS. RNase A solution (100 µl) was added to the cell pellet, and the cells were incubated in a water bath at 37˚C for 30 min. Subsequently, 400 µl propidium iodide (Sigma-Aldrich; Merck KGaA) staining solution was added and thoroughly mixed, and the samples were incubated in the dark at 4˚C for 30 min. The cell cycle was determined using a flow cytometer (BD Biosciences) (30) and analyzed using FlowJo version 10.6.2 (Tree Star).

The targets associated with the pathogenesis of lung adenocarcinoma and the 23-HBA drug targets were imported into the STRING database (https://string-db.org/) to generate the PPI network of overlapping targets. Subsequently, the overlapping targets were imported into Cytoscape software version is 3.3 (https://cytoscape.org/) for visual analysis. Five core targets with a high degree of association between 23-HBA and lung adenocarcinoma were identified using the betweenness centrality (BC; the degree to which nodes are independent of each other) size sequencing (31), and the characteristics were analyzed. The characteristic is mainly manifested in: ACE mainly exists in vascular endothelial cells, while tumor cells have no blood vessels. Research on PTSG2 has been relatively clear and specific. MDM2 is highly expressed in tumors and has been proven to bind to p53. H1299 cells have a homozygous partial deletion of TP53 and therefore cannot express p53, and PPAR- and PPAR-γ belong to the same category and need not be studied. The median degree of freedom of the targets was 7.055, and the maximum degree of freedom was 18. The inclusion criteria for screening core targets were set at 15 and 18, and the best five core targets were selected based on the relevant parameters of each target (32).

The mRNA expression profile of PPAR-γ in various tumor types, including lung adenocarcinoma, was assessed using the GEPIA database (http://gepia.cancer-pku.cn).

Western blot analysis was performed to compare the expression of PPAR-γ between normal and lung cancer cells and explore the characteristics of this protein (33). BEAS-2B, A549, H1975 and H1299 cells with robust growth profiles were collected. These cells were digested with trypsin, and the cell concentration was adjusted to 1x10⁶ cells/ml. Subsequently, the cells were inoculated into a 6-well plate (1x10⁶ cells per well) and cultured overnight. After a further 48-h incubation, the cells were digested with trypsin, and the cell precipitate was collected through centrifugation (200 x g, 37˚C, 3 min). RIPA buffer (Beyotime Institute of Biotechnology) and PMSF (Beijing Solarbio Science & Technology Co., Ltd.) were used to lyse cells (30 min with a 10 min mixing interval), followed by centrifugation (300 x g, 37˚C, 10 min). The supernatant was collected, and 5X SDS (Xilong Scientific Co., Ltd.) solution was added to the supernatant at a 1:4 ratio. The mixture was boiled at 95˚C for 10 min and stored at -20˚C. The protein concentration was determined using the bicinchoninic acid method. A total of 40 µg protein/lane were separated using 5% SDS-PAGE transferred to PVDF. The membranes were incubated with primary anti-GAPDH (rabbit anti-human GAPDH; cat. no. GTX100118; 1:1,000-5,000 dilution; Bioworld Technology) and PPAR-γ antibodies (PPAR-γ rabbit polyAb; cat. no. HY-P80872; 5 µg/ml; Protein Technology) at 4˚C for 6 h, followed by secondary anti-rabbit antibodies (goat anti-rabbit IgG-HRP; cat. no. L27A9; 2 µg/ml; Bioworld Technology) at 25˚C for 30 min. Following washing with 10% SDS, the membranes were incubated with ECL reagent (Biosharp Life Sciences) for signal development. The absorbance values of each band were calculated using ImageJ software (version 64; National Institutes of Health). GAPDH served as the internal reference. The entire experiment was repeated three times. Subsequently, H1299 cells were selected for subsequent experiments, and 23-HBA with various concentrations (0, 20, 40 and 60 µM) was applied to the cells at 37˚C for 2 h to analyze the expression of PPAR-γ.

KEGG analysis was performed using the R (version 4.1.2) package ‘Clusterprofiler’ (https://bioconductor.org), and pathways with P<0.05 and an association with cancer were screened (34). This process resulted in the identification of 12 pathways, which were then visualized and further screened for pathways exhibiting significant enrichment. It was visualized by using KEGG analysis; a graph is automatically generated when a gene is imported into the website for visualization purposes. GO enrichment analysis was conducted using the R package ‘Clusterprofiler’, and P<0.05 was used as the level of statistical significance. The relevant biological processes (BPs), cellular components (CCs) and molecular functions (MFs) were analyzed based on their P-value. Processes displaying high enrichment were selected for subsequent analyses.

The target genes enriched in this pathway were identified through databases, such as KEGG (https://www.kegg.jp), to explore the mechanism of the PPAR signaling pathway. The mechanism of the PPAR pathway and its association with reactive oxygen species (ROS) were investigated by searching sources, such as PubMed (https://www.pubmed.ncbi.nlm.nih.gov) and Web of Science (https://clarivate.com.cn/solutions/web-of-science/), to predict the mechanism associated with the PPAR signaling pathway.

H1299 cells stably cultured for several generations were used and diluted to 8x10⁴ cells/ml after digestion. Of this cell suspension, 1 ml was added to a 6-well plate in addition to 1 ml of complete culture medium. After observing that the cells were almost completely attached to the wall, the old culture medium and was removed and 2 ml of 0, 20, 40 or 60 µmol/l 23-HBA culture medium was added to each well and incubated for 48 h. A ROS detection reagent (Beyotime Institute of Biotechnology) was employed for mitochondrial ROS detection. Dichlorodihydrofluorescein diacetate (DCFH-DA) was diluted with serum-free medium at a ratio of 1:1,000 to achieve a final concentration of 10 µmol/l. After the cell culture medium was discarded, an appropriate volume of diluted DCFH-DA was added to adequately cover the cells. The cells were then incubated in a 37˚C incubator for 20 min, followed by washing three times with serum-free medium to fully eliminate the DCFH-DA that did not enter the cells. Subsequently, the cells were observed directly using a laser confocal microscope (excitation wavelength, 488 nm; emission wavelength, 525 nm; Shanghai Tianneng Life Science Co., Ltd.).

The target genes enriched in the PPAR signaling pathway were obtained through the GEPIA database (36). Kaplan-Meier curve analysis was then used to examine the effect of these target genes on the survival rate of patients with lung adenocarcinoma, with an aim to predict the clinical prognosis of these patients in association with the expression levels of the PPAR pathway target genes.

Data are presented as the mean ± SD of three experimental repeats. R (version 4.1.2) were utilized for the statistical analysis and required in the present study. The primary prognostic value was assessed using Kaplan-Meier curve analysis and the log-rank test, as well as Cox regression analysis. Other statistical analyses were performed in GraphPad Prism 6 software (Dotmatics). Comparisons among multiple groups were performed using one-way ANOVA followed by Tukey's post-hoc test, while Student's t-test comparing the experimental group with the control group one by one was used for comparisons between two groups. P<0.05 was considered do indicate a statistically significant difference.

Initially, genes associated with the pathogenesis of lung adenocarcinoma in the DisGeNET and Genecards databases and 23-HBA target genes in the SwissTargetPrediction and TCMSP databases were examined. Subsequently, a Venn diagram was constructed, revealing 92 overlapping genes (Fig. 1A). Further analysis demonstrated that these overlapping genes constituted 87.6% of the genes associated with 23-HBA (Fig. 1B). This finding provides evidence that the target genes of 23-HBA are closely linked to the pathogenesis of lung adenocarcinoma.

First, the H1299 cell line, which was selected due to being cost-effective and producing reliable results, was cultured for MTT assays. The cells were divided into two groups, the control group and the experimental group, and various drug concentrations (0, 20, 40, 60, 80 and 100 µM) were used to explore the IC₅₀ value of 23-HBA. The cell survival rate exhibited a steady decline with increasing concentrations of 23-HBA (Fig. 2A). At a drug concentration of 60 µM, which is close to the IC₅₀ value, the cell survival rate was 54.4%. This finding provided evidence that 23-HBA had an inhibitory effect on the viability of H1299 cells. Therefore, in the follow-up experiments, drug doses between 0-60 µM were considered to be representative of the effect of 23-HBA. Microscopic analysis (fluorescence microscope) after treatment of the cells with 0-60 µM 23-HBA revealed a significant decrease in the number of adherent cells with an increase in drug concentration, accompanied by an increase in the number of floating dying cells (Fig. 2B). Finally, changes in the cell cycle of H1299 cells treated with various concentrations of 23-HBA were analyzed using flow cytometry (Fig. 2C), and the distribution of cells in each phase of the cell cycle was quantified (Fig. 2D). The proportion of H1299 cells in the G₁ phase notably increased with increasing concentrations of 23-HBA, and, apart from the 20 µm group, this difference was statistically significant compared with the control group. However, fewer cells were in G₂ and S phases. This finding indicated that 23-HBA affects H1299 cells by arresting the cell cycle in the G₁ phase. All these results collectively demonstrated that 23-HBA inhibited the proliferation of lung adenocarcinoma H1299 cells.

Firstly, the genes from the Venn diagram were imported into Cytoscape software, and topological parameter diagrams were created on the basis of the BC association of each gene for preliminary gene selection (Fig. 3A). Second, the related genes with lung adenocarcinoma pathogenesis genes and targets of the 23-HBA-associated domain were imported into STRING software to generate a PPI network diagram (Fig. 3B), which allowed for the analysis of gene associations to identify closely acting genes and exclude marginal genes of the network. Finally, the five most relevant genes associated with lung adenocarcinoma pathogenesis genes and targets of the 23-HBA-associated domain, prostaglandin-endoperoxide synthase 2, PPAR-γ, angiotensin I-converting enzyme (ACE), PPAR-α and mouse double minute 2 (MDM2), were selected and sorted in accordance with their association (Fig. 3C). ACE is a zinc metallopeptidase that is widely distributed in endothelial and epithelial cells. It plays a crucial role in the renin-angiotensin-aldosterone system. Consequently, ACE is mainly found in vascular endothelial cells (37). For PTSG2, the related studies have been relatively clear and specific, so we excluded it from further analysis. MDM2, as a key negative regulator of p53, exhibits high expression in tumors and serves an important role in tumor initiation and development. MDM2 has been demonstrated to bind to p53, thereby blocking its tumor-suppressor transactivation domain (38). Furthermore, MDM2 functions as an E3 ligase, marking p53 for degradation by the proteasome. H1299 cells possess a homozygous partial deletion of TP53(39), rendering them unable to express the tumor-suppressor protein p53, which contributes to their proliferative phenotype. Therefore, MDM2 was excluded from further analysis. PPAR-α and PPAR-γ belong to the same category. PPAR-γ has been rarely studied in the literature and it represents a novel finding. Therefore, it was selected for exploration in the present study.

The GEPIA database was utilized to analyze the expression of the target gene PPAR-γ in various tumor types (40), including lung adenocarcinoma (Fig. 4A). The results demonstrated that PPAR-γ was significantly downregulated in lung adenocarcinoma compared with normal tissues (Fig. 4B), while being highly expressed in most tumors, thereby indicating that PPAR-γ may function as a tumor-suppressor gene. Western blot assay was performed to measure PPAR-γ expression in normal cells (BEAS-2B) and multiple lung adenocarcinoma cell lines (A549, H1975 and H1299), to further confirm its role as a tumor suppressor. The results showed that PPAR-γ was highly expressed in normal cells, whereas its expression was notably lower in the lung adenocarcinoma cell lines (Fig. 4C). Subsequently, western blotting was conducted to examine the protein expression of PPAR-γ after treatment of the cells with different concentrations of 23-HBA. The results indicated that the expression of PPAR-γ in the 23-HBA treatment groups was higher than that in the control group (41), with statistical significance observed at a drug concentration of 60 µM (Fig. 4D). These results confirmed that 23-HBA treatment increases the expression of PPAR-γ. Given the earlier conclusion that 23-HBA can inhibit the survival of lung adenocarcinoma cells, PPAR-γ may act as a tumor suppressor, which is consistent with the results from the GEPIA database.

GO analysis was conducted to investigate the mode of action of 23-HBA on lung adenocarcinoma (42). P<0.05 was used as the screening criterion for selected functional terms in the categories BP, MF and CC (Fig. 5A-C). The results showed that most of the enriched BP terms require the consumption of ATP, which is primarily produced by mitochondria. From Fig. 5A, it can be concluded that most enriched BP terms are associated with regulation of inflammatory response, which require the consumption of ATP, such as lipid transporting and fatty acid metabolic processing. The most important mechanisms of action in the category MF are associated with nuclear receptor activity, which also contains ligand-activated transcription factor activity and carboxylic acid binding. The most dominant mechanism of action in the category CC is membrane raft, which also contains membrane microdomain and integral component of presynaptic membrane. Therefore, the mechanism of action of the PPAR pathway is related to mitochondria due to increasing the content of mitochondrial ROS, thereby inhibiting cell growth. Further investigation through PubMed into the PPAR signaling pathway revealed that it induces physiological changes, including inflammation and alterations in the mitochondrial membrane potential (43). Therefore, the mechanism by which 23-HBA inhibits the viability and/or proliferation of lung adenocarcinoma cells may be related to mitochondrial ROS. Subsequently, the mitochondrial ROS levels were detected, and the results showed that the production of intracellular mitochondrial ROS notably increased with increasing concentrations of 23-HBA (Fig. 5D), which is consistent with the aforementioned mechanism.

The PPAR signaling pathway is considered to be an important signal transduction pathway in several conditions, such as inflammation and cancer (44). Previous studies have demonstrated the inhibitory effects of 23-HBA and its derivatives on inflammation, as well as liver, lung and breast cancer (45,46). The KEGG analysis revealed that the pathway exhibiting the highest degree of enrichment was for the ‘PPAR signaling pathway’ (Fig. 6A). Investigation of the research mechanism within this pathway resulted in the identification of target genes associated with it, including CYP27, LPL, PGAR/ANGPTL4 and UBC (Fig. 6B). Subsequently, the GEPIA database was used to analyze the association of the expression levels of these target genes with the survival of patients with lung adenocarcinoma. The results indicated that high (ANGPTL4 and UBC) or low (CYP27 and LPL) expression levels of these genes were significantly associated with a lower patient survival (Fig. 6C-F), thereby demonstrating the clinical prognostic significance of the PPAR pathway.

Collectively, the results of the present study demonstrated that 23-HBA inhibits cell proliferation by blocking lung adenocarcinoma cells in the G₁ phase. The results indicated that PPAR-γ may function as a tumor-suppressor gene in lung adenocarcinoma. Further analysis unveiled the relationship among this finding, the PPAR signaling pathway and mitochondrial ROS. The relationship is that the survival rate of tumor cells decreases with the increase of drug concentration, and at the same time, the expression of ROS increases with the increase of drug concentration. Finally, a clinical prognosis association was demonstrated between the expression levels of PPAR pathway-related genes and the survival of patients with lung adenocarcinoma (Fig. 7).