Combination Therapy of Sini Decoction and Cyclophosphamide Inhibits Tumor Growth in an Orthotopic Lung Cancer Model

Abstract

Background:

Lung cancer represents a frequently seen respiratory system malignancy. Sini Decoction combined with cyclophosphamide is demonstrated to remarkably extend survival and improve quality of life of these patients; however, the associated anti-tumor mechanisms are largely unexplored.

Objective:

The present work focused on investigating the inhibition of tumor cells by Sini Decoction plus cyclophosphamide within the orthotopic lung cancer model and exploring the mechanisms in terms of exosome-based tumor hypoxic microenvironment modulation.

Methods:

A549-luc2-tdT-2 cells were implanted in left lung of nude mice for establishing the orthotopic lung cancer xenograft model. After 5 days, bioluminescence imaging was conducted for model validation. Mice were later randomized as 4 groups: model, Sini Decoction, cyclophosphamide, as well as Sini Decoction plus cyclophosphamide. Bioluminescence imaging was conducted to assess anti-tumor effects. Enzyme-linked immunosorbent assay (ELISA) was performed for measuring liver and kidney function indicators (ALT, AST, Cr) in serum. Additionally, RT-qPCR and immunohistochemistry were carried out for detecting hypoxia-related factor levels (HIF-1α, VEGF, PDGF-β) within lung tissue. Additionally, characterizations of the separated exosomes were completed with transmission electron microscopy, BCA protein assay, nanoparticle tracking analysis, and Western blotting. Exosomal miR-20a-5p was chosen based on TargetScan database for analysis, while RT-qPCR was completed for validation.

Results:

Sini Decoction plus cyclophosphamide dramatically suppressed tumor growth within the orthotopic lung cancer model while ameliorating hepatorenal toxicities, as evidenced by serum liver and kidney function indicators and bioluminescence imaging. Meanwhile, immunohistochemistry showed that the combination therapy markedly downregulated HIF-1α expression in lung tissue and suppressed the expression of its downstream target genes VEGF and PDGF-β, which was also confirmed by RT-qPCR. Additionally, bioinformatic analysis suggested that tumor-derived exosomal miR-20a-5p could target HIF-1α. Isolation and RT-qPCR analysis of lung tissue-derived exosomes demonstrated that the combination therapy significantly downregulated the expression of exosomal miR-20a-5p.

Conclusion:

This study indicates that the combination of Sini Decoction and cyclophosphamide can effectively inhibit lung cancer growth and alleviate hepatorenal toxicity. The mechanism may be associated with the amelioration of the tumor hypoxic microenvironment, inhibition of the HIF-1α-mediated tumor hypoxia signaling pathway, and regulation of exosomal miR-20a-5p expression.

Keywords

sini decoction cyclophosphamide lung cancer exosomes tumor hypoxic microenvironment

Introduction

According to statistics from the International Agency for Research on Cancer (IARC), there were nearly 20 million new cancer cases and 9.7 million cancer-related deaths globally in 2022. Lung cancer, a major contributor to cancer morbidity and mortality, occupies about 12.4% of new cancer cases and 18.7% of cancer-induced death cases globally.¹ In China, lung cancer ranks first across diverse cancers with regard to morbidity and mortality, making it extremely challenging to prevent and treat this disease.² Although great progress has been made in surgical treatment, chemotherapy, radiotherapy, immunotherapy, and targeted therapy, lung cancer patients still have unfavorable prognostic outcomes because of their different histological subtypes and disease stages.

In the complicated landscape of lung cancer research, dynamically regulating the tumor microenvironment (TME) has recently gained significant attention. Hypoxia, an important solid tumor hallmark, is an important factor driving tumor development.³ Hypoxia affects TME composition.⁴ Exosomes, a key TME component, connect tumor cells with the hypoxic microenvironment. In hypoxia, tumor cells can secrete rich exosomes to regulate interactions inside the TME.^5,6 Typically, exosomal miRNAs play a key role in regulating intercellular communication in the hypoxic TME, modulating tumor angiogenesis,⁷ enhancing tumor cell growth and migration,⁸ promoting immune evasion,⁹ and facilitating drug resistance.¹⁰ miRNAs are a class of endogenous small non-coding RNAs approximately 21 to 25 nucleotides in length.¹¹ Functioning as post-transcriptional regulators of gene expression, they exhibit high conservation, temporal specificity, and tissue specificity.¹² The pronounced heterogeneity of miRNAs suggests that their targeted regulatory effects may vary across different types of tumor cells.

This study examines miR-20a-5p, which exhibits aberrant expression in various cancers and is involved in tumorigenesis, progression, and metastasis. miR-20a-5p is among the core members of the miR-17-92 gene cluster, one of the most extensively studied oncogenic clusters.¹³ Studies have shown that extracellular vesicles derived from A549 cells subjected to intermittent hypoxia deliver miR-20a-5p to target the PTEN, thereby activating Akt phosphorylation and promoting M2 polarization of macrophages.¹⁴ Furthermore, miR-20a-5p is upregulated in tumors such as non-small cell lung cancer¹⁵ and triple-negative breast cancer,¹⁶ where it influences the tumor hypoxic microenvironment and participates in tumor cell proliferation and metastasis by regulating downstream target genes. Its high expression may serve as a potential marker for malignant tumor progression.¹⁷ HIF-1α is a key regulatory factor in the tumor hypoxic microenvironment, and its high expression creates conditions favorable for tumor cell proliferation and metastasis.¹⁸ VEGF and PDGF-β, as downstream target genes of HIF-1α, are involved in tumor angiogenesis and stromal remodeling, and their expression is positively correlated with that of HIF-1α.^19,20 Research indicates that HIF-1α can accelerate tumor cell proliferation by upregulating the expression of VEGF and PDGF-β.

In China, the use of traditional medicine for cancer treatment has a history spanning thousands of years. As a complementary and alternative therapy, it continues to be relevant in the management of lung cancer.²¹ Previous studies have indicated that the occurrence of tumors is closely associated with a Yang deficient constitution, and symptoms of Yang deficiency become more pronounced in patients after chemotherapy.²² The use of warming Yang agents in combination with chemotherapy has demonstrated efficacy in palliative care for patients with advanced lung cancer.²³ Such agents synergistically affect apoptosis, cytotoxicity and inhibition of tumor development. The associated mechanisms include arresting cell cycle, suppressing angiogenesis, and regulating gene expression levels.²⁴ Sini Decoction (SND), the famous Chinese medicinal prescription documented in the Pharmacopeia of the People’s Republic of China, is comprised of 3 medicines: Aconiti Lateralis Radix Praeparata, Zingiberis Rhizoma, and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle.²⁵ It functions to warm Yang and dispel cold, effectively alleviating symptoms of Yang deficiency in lung cancer patients such as cold intolerance, fatigue, poor appetite, loose stools, cough, wheezing, and excessive phlegm,²⁶ thereby contributing to prolonged survival. Clinically, the combination of Sini Decoction and cyclophosphamide has been shown to significantly improve the quality of life and prolong survival in lung cancer patients. Preliminary experiments indicate that Sini Decoction combined with cyclophosphamide can inhibit tumor growth and extend survival in C57BL/6 mice bearing subcutaneous Lewis lung carcinoma.²⁷ Furthermore, treatment of A549 cells with Sini Decoction alone has been found to suppress lung cancer cell growth while significantly reducing the expression of HIF-1α.²⁸ Therefore, this study aims to investigate the inhibitory effect of Sini Decoction combined with cyclophosphamide on tumor cells in an orthotopic lung cancer model, and to preliminarily observe its biological mechanisms from the perspective of exosome-mediated regulation of the tumor hypoxic microenvironment (Figure 1).

Figure 1.

Experimental flowchart of orthotopic lung cancer model establishment, group treatment, and sample analysis. The flowchart outlines the key procedures, beginning with the establishment of an orthotopic lung cancer xenograft model. Following successful modeling, mice were randomized into 4 groups: Model, SND, CTX, and SND + CTX, and received a 3-week treatment regimen with tumor growth monitored via bioluminescence imaging. Samples were subsequently collected for analysis. Serum levels of ALT, AST, and Cr were measured by ELISA. HIF-1α expression in lung tissue was detected by IHC. Exosomes were isolated and characterized by TEM, NTA, BCA protein quantification, and Western blot. Using the TargetScan database, miRNAs targeting the HIF-1α gene were predicted. RT-qPCR was performed to validate the expression of exosomal miR-20a-5p and to determine the expression levels of its potential downstream targets, including HIF-1α, VEGF, and PDGF-β.

Materials and Methods

Materials

Animals

A total of 32 male BALB/c-nu nude mice, specific pathogen-free (SPF) grade, aged 4 to 6 weeks and weighing 20 to 22 g, were used in this study (Sibeifu (Beijing) Biotechnology Co., Ltd.). They were divided into groups of 8 mice each. The Animal Quality Certificate number was SCXK (Jing) 2024 to 0001. Mice were housed in the Animal Research Center of Beijing University of Chinese Medicine under controlled conditions: temperature, 23°C ± 1°C; relative humidity, 45% to 70%. The Institutional License number for animal use was SYXK (Jing) 2023 to 0011. All animal procedures and experimental protocols were approved by the Animal Ethics Committee of Beijing University of Chinese Medicine.

Cell Line

The A549-luc2-tdT-2 cell line (Catalog number: 1101HUM-PUMC000629; RRID:CVCL_XE69) was purchased from the National Biomedical Experimental Cell Resource Bank.

Drugs

Sini Decoction: The formula consists of prepared Aconiti Lateralis Radix Praeparata (9 g; batch: 240909; Jiangyou, Sichuan), Zingiberis Rhizoma (6 g; batch: 240929; Wenshan, Yunnan), and honey-fried Glycyrrhizae Radix et Rhizoma (6 g; batch: 240902; Dingxi, Gansu). All herbs were purchased from the Third Affiliated Hospital of Beijing University of Chinese Medicine. About 210 g of raw herb was washed with clean water and decocted 3 times at a material-to-liquid ratio of 1:10. The first decoction lasted 1 hour, and the subsequent 2 were 0.5 hours each. The decoctions were combined, filtered through gauze, and centrifuged at 5000 rpm for 10 minutes to collect the supernatant. The supernatant was concentrated to 500 mL under reduced pressure at 40°C. The concentrated solution was dispensed into freeze-drying trays and pre-frozen at −80°C for 24 hours. After pre-freezing, the vacuum system was started, and primary drying was carried out by gradually raising the temperature to between −20°C and −10°C to remove most of the water. For secondary drying, the temperature was further increased to between 20°C and 30°C for 6 to 12 hours to eliminate residual moisture. The resulting freeze-dried cake was removed from the trays, pulverized into a fine powder, and sieved. The sieved powder was packaged to yield 33.2476 g of Sini Decoction freeze-dried powder. The powder yield was calculated as follows: Powder Yield (%) = [Weight of Lyophilized Powder/Weight of Raw Herbs] × 100% = [33.2476 g/210 g] × 100% = 15.83%.

Identification of Constituents in SND Lyophilized Powder

An aliquot (40 mg) of the SND lyophilized powder was extracted with 3 mL of 50% methanol (Thermo Fisher Scientific, Waltham, MA, USA; Cat# A456-1) via vortex mixing. The supernatant was collected for analysis. Chromatographic separation was performed using a Waters Acquity UPLC I-Class system equipped with an Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm). The mobile phase consisted of 0.1% formic acid in water (A; Thermo Fisher Scientific, Waltham, MA, USA; Cat# A117-50) and acetonitrile (B; Thermo Fisher Scientific, Waltham, MA, USA; Cat# A955-1). A gradient elution was applied at a flow rate of 0.3 mL/min with an injection volume of 2 μL. Data acquisition and processing were conducted using Xcalibur 2.1 software (Thermo Fisher). The compounds were identified by comparing their retention times, high-resolution exact mass, and MS/MS fragmentation patterns.

Cyclophosphamide for Injection (Batch No. 2K583A) was purchased from Baxter Oncology GmbH.

Primary Reagents and Instruments

Serum levels of alanine aminotransferase (ALT; Cat# JM-11587M2, RRID: AB_3094650), aspartate aminotransferase (AST; Cat# JM-03113M2, RRID: AB_3094649), and creatinine (Cr; Cat# JM-03154M2, RRID: AB_731730) were measured using ELISA kits (Jingmei Biological, China). Protein concentrations were determined using a BCA protein assay kit (Cat# Omp-03; Omiget, China). Reverse transcription was performed using a reverse transcription kit (Cat# Omn-22; Omiget, China). Exosomes were isolated and purified using an exosome extraction and purification kit (Cat# Ome-01E; Omiget, China). The following primary antibodies were used: HIF-1α (Cat# HA721997; RRID: AB_2799095), VEGF (Cat# ET1604-28; RRID: AB_10579183), PDGF-β (Cat# HA722003; RRID: AB_2783647), TSG101 (CST, Cat# 72312; RRID: AB_2924771), CD63 (Bioswamp, Cat# PAB48050; RRID: AB_2924771), and CD9 (CST, Cat# 13174; RRID: AB_2798139). Secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology (Cat# ZB-2301; RRID: AB_2747412; Cat# ZB-2305, RRID: AB_2747415).

Paraffin sections were prepared using a Leica RM2016 microtome. RT-qPCR detection was performed using a Bio-Rad CFX-96 system. Western blot-related equipment was from Bio-Rad, and imaging was conducted using a Tanon 4800 Multi system. Transmission electron microscopy was performed with a Hitachi HT7800. A Thermo Scientific Multiskan FC microplate reader was used. All other equipment was standard laboratory apparatus.

Model Establishment and Grouping

An orthotopic lung cancer xenograft model was established using a modified intrapulmonary injection method with A549-luc2-tdT-2 cells stably expressing red luciferase. After trypsin digestion, cells were resuspended. Nude mice were anesthetized via intraperitoneal injection of sodium pentobarbital (5 mg/kg) and fixed on the mouse board. At the left axillary front line, (1-1.5 cm) above the left costal margin, a vertical incision was made, and a percutaneous puncture (3 mm) was performed. 50 μL of cell suspension (1 × 10⁵ cells) was slowly injected into the left lung tissue. After removing the needle, the wound was disinfected with iodophor and animals were allowed to recover.

Following successful model establishment, the nude mice were randomly divided into 4 groups (n = 8): Model (control) group; Sini Decoction (SND) group; Cyclophosphamide (CTX) group; and Sini Decoction combined with Cyclophosphamide (SND + CTX) group.

Drug Administration

The human equivalent dose for nude mice was calculated using the body surface area normalization method:

\begin{array}{l} Mouse dose (mg / kg) = Human dose (mg / kg) \times \\ (Human K_{m} / Mouse K_{m}) \end{array}

Note: The K_m factor is approximately 37 for humans and 3 for mice.

Sini Decoction lyophilized powder (with a yield of 15.83%) was prepared as a 68.34 mg/mL suspension in normal saline. Cyclophosphamide was prepared as a 10 mg/mL injectable suspension in normal saline.

Model group: Received daily oral gavage of normal saline 10 mL/kg body weight and weekly intraperitoneal injection of normal saline 10 mL/kg body weight. SND group: Received daily oral gavage of the Sini Decoction lyophilized powder suspension at a dose of 683.4 mg/kg body weight. CTX group: Received weekly intraperitoneal injection of the cyclophosphamide suspension at a dose of 100 mg/kg body weight. SND + CTX group: Received both the daily oral gavage of Sini Decoction suspension (same as the SND group) and the weekly intraperitoneal injection of cyclophosphamide (same as the CTX group). Gavage was administered once daily for 21 consecutive days, and intraperitoneal injections were administered once weekly for a total of 3 times. On the 21st day after intragastric administration, the nude mice were fasted for 12 hours and only allowed to drink water freely. All the nude mice were euthanized on the 22nd day after intragastric administration, and lung tumor tissues and blood samples were collected and analyzed. All the operations were carried out strictly in accordance with the “Guide for the Care and Use of Laboratory Animals” (eighth edition, 2011) of the National Institutes of Health of the United States.

Assay Methods and Parameters

Bioluminescence Imaging

On days 7, 14, and 21 post-treatment, tumor growth in the lungs was monitored using a bioluminescence imaging system. Three mice from each group were intraperitoneally injected with the luciferase substrate D-Luciferin (0.1-0.15 mL), anesthetized, and placed in the imaging chamber. The reaction of substrate with A549 cells expressing red luciferase produced bioluminescent signals. Automatic image capturing was completed with suitable exposure. Alterations of tumor growth were examined through comparing fluorescence intensity.

Detection of Serum Liver and Kidney Function Indices (ELISA)

ELISA kits were employed for measuring creatinine (Cr), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) contents in serum. Serum samples under appropriate dilution and standards were introduced into pre-coated plates. Serum samples were diluted 5 times before the test. The specific operation was as follows: 40 μL of sample diluent was added to each well first, then 10 μL of the sample to be tested was added, and gently mixed. The standard proteins provided by the kit were diluted according to the following gradients to establish the standard curve: AST standard (initial concentration: 240 ng/L); Dilution final concentration: 120, 60, 30, 15, and 7.5 ng/L; Dilution method: Equal parts of 150 μL of the previous concentration standard + 150 μL of standard diluent were used for stepwise dilution. ALT standard (initial concentration: 160 ng/L); Dilution final concentration: 80, 40, 20, 10, and 5 ng/L; Dilution method: The same as above, stepwise dilution. Cr standard (initial concentration: 240 μmol/L); Dilution final concentration: 120, 60, 30, 15, and 7.5 μmol/L; Dilution method: The same as above, stepwise dilution. Following incubation and rinsing, the enzyme-labeled secondary antibody along with the substrate was introduced in sequence for initiating colorimetric reaction. About 50 μL of secondary antibody was added to each well. A microplate reader was utilized to determine optical density (OD) at 450 nm. Later, Cr, ALT and AST contents within every sample were determined by the standard curve.

Immunohistochemical (IHC) Analysis of Lung Tissues

To detect the expression level of HIF-1α protein in lung tissue, paraffin-embedded lung tissue sections were subjected to dewaxing, rehydration, and antigen retrieval. The sections were sequentially immersed in eco-friendly dewaxing solutions I, II, and III for 10 minutes each for dewaxing, followed by hydration in anhydrous ethanol I, II, and III for 5 minutes each, and then rinsed with distilled water. The sections were heated in antigen retrieval solution (pH 7.4), and after cooling, washed 3 times with PBS (Cat# ZLI-9062; ZSGB-BIO, China), 5 minutes each. To block endogenous peroxidase activity, the sections were incubated with 3% hydrogen peroxide for 25 minutes at room temperature in the dark, followed by 3 washes with PBS. Blocking was performed by applying 3% BSA at room temperature for 30 minutes. After removing the blocking solution, the sections were incubated with anti-HIF-1α primary antibody overnight at 4°C. The next day, after 3 washes with PBS, the sections were incubated with HRP-conjugated secondary antibody for 50 minutes at room temperature. Following PBS washes, DAB chromogen solution (Cat. No. ZLI-9019; ZSGB-BIO, China) was applied, and the staining development was monitored under a microscope (1-3 minutes). The sections were counterstained with hematoxylin for 3 minutes, differentiated, blued, and rinsed with running water. After gradient dehydration and clearing, the sections were mounted with neutral balsam, and images were acquired under a light microscope. ImageJ software was utilized for semi-quantitatively analyzing positive staining within the selected fields of view.²⁹

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

The TRIzol approach was utilized for extracting total lung tissue RNA, with the quality and content being determined by a spectrophotometer. RNA aliquots were prepared in cDNA through reverse-transcription with the reverse transcription kit. cDNA was later utilized as a template for amplification using SYBR Green Premix reagent by the real-time fluorescence quantitative PCR system. The genes analyzed in the present work encode for HIF-1α, VEGF, PDGF-β, and β-actin (control). The specificity of primer sequences was confirmed. The following thermal cycling conditions were applied: 2 minutes of initial denaturation under 95°C; 15 seconds under 95°C and 30 seconds under 60°C for 40 cycles. The amplified product specificity was verified through melt curve analysis. Finally, the 2^(−ΔΔCt) approach was employed for determining target gene expression.

Isolation and Characterization of Exosomes

Isolation and Transmission Electron Microscopy (TEM)

After exosome isolation, the exosome purification kit was employed for purification. Then, exosome suspension (10 μL) was added into the 200-mesh copper grid for 5 minutes of standing. After addition of 2% uranyl acetate to achieve negative staining, the samples were dried for a 10-minute period. TEM was later conducted for grid observation and imaging at magnifications of 100, 200, and 500 nm.

Nanoparticle Tracking Analysis (NTA)

After isolation, exosomes were examined with the Particle Metrix ZetaView PMX110 instrument under 405 nm laser irradiation for determining particle content. PBS was added to dilute the exosome suspension to 1 × 10⁷ to 1 × 10⁹ particles/mL. Upon later irradiation, particle scattering was determined, while scattered particles were counted to determine the concentration. Thereafter, the particle Brownian motion trajectories were evaluated by size distribution and quality.

Exosomal Protein Measurement

To prepare the working solution, BCA reagent A and B were completely mixed at the ratio of 50:1. Samples and diluted standards were introduced into the 96-well plate, and later working solution was poured into every well (200 μL). Following 30 minutes of incubation under 37°C, the microplate reader was utilized for measuring absorbance at 562 nm. Protein content was determined by plotting the standard curve.

Exosomal Marker Protein Detection

RIPA lysis buffer was added for exosome lysis. Protein aliquots received SDS-PAGE for separation before transfer on the PVDF membrane. The membrane was blocked, followed by overnight primary antibody incubation under 4°C (including TSG101, CD63, and CD9) and corresponding HRP-labeled secondary antibody probing under ambient temperature. The dilution ratios were as follows: anti-TSG101 at 1:1000, anti-CD63 at 1:1000, and anti-CD9 at 1:1000. The secondary antibodies, goat anti-rabbit IgG (HRP) and goat anti-mouse IgG (HRP), were both used at a dilution of 1:5000. The chemiluminescence imaging system was adopted for signal visualization and capturing.

Bioinformatics Analysis and Verification of Exosomal miRNA Expression

For selecting functional miRNAs that might regulate HIF-1α, we adopted the TargetScan database for analysis. After importing HIF-1α gene, its longest transcript isoform was selected for maximizing coverage of possible regulatory sites. To filter predicted outcomes, conservation (PhastCons score ≥ 0.8) as well as binding efficacy (context++ score ≤ −0.3) was applied. At last, we chose miR-20a-5p to be the potential molecule for later verification.

Expression Verification by RT-qPCR

The TRIzol approach was utilized for extracting total exosomal RNA, which was later synthesized in cDNA via reverse-transcription. cDNA was later utilized to be a template to detect miR-20a-5p expression by amplification using SYBR Green Premix reagent with the real-time quantitative PCR instrument. Data were normalized with U6 snRNA being an internal control. The thermal cycling conditions included 2 minutes of initial denaturation under 95°C; 15 seconds under 95°C and 30-second under 60°C for 40 cycles. The melt curve was analyzed to verify product specificity. The 2^(−ΔΔCt) approach was adopted for determining miR-20a-5p expression. Detailed RT-qPCR procedures are provided in Supplemental Methods.

Statistical Analysis

SPSS 23.0 was adopted for statistical analysis. Results were represented by mean ± standard deviation (Mean ± SD). Meanwhile, Shapiro-Wilk test was utilized for assessing normality, whereas Levene’s test for evaluating homogeneity of variance. One-way ANOVA was used for data meeting normality and homogeneity. We conducted Dunnett’s post hoc test to compare treatment groups with the model group. For pairwise comparisons among all groups, Tukey’s post hoc test was employed. If data violated parametric assumptions, the non-parametric Kruskal-Wallis test was performed, followed by Dunn’s test for post hoc comparisons. P < .05 was considered statistically significant. All statistical graphs were generated using GraphPad Prism 8.0.2.

Results

Identification of Constituents in SND Lyophilized Powder

The chemical composition of SND lyophilized powder was characterized using UPLC-LTQ-Orbitrap in both positive and negative ion modes (Figure 2). A total of 13 compounds were identified (Table 1) by comparing their retention times, high-resolution exact mass measurements, and characteristic MS/MS fragmentation patterns.

Figure 2.

Mass spectrometry chromatograms of SND lyophilized powder formulation components. (A) Positive ion mode. scan range 0–25.01 minutes, characteristic main peak retention time at 10.21 minutes, primarily corresponding to alkaloids and nitrogen-containing compounds in SND. Abscissa: retention time (min); Ordinate: relative abundance. (B) Negative ion mode. scan range 0–25.01 minutes, characteristic main peak retention time at 16.05 minutes, primarily corresponding to organic acids and phenolic acids in SND. Abscissa: retention time (min); Ordinate: relative abundance.

Table 1.

Identification of chemical constituents from SND lyophilized powder extract (screening in the positive and negative ion modes).

No.	retention time (min)	Additive ions	Observed molecular mass (Da)	Error (ppm)	Theoretical m/z	Chemical formula	Ingredient name	Fragment ions	Source
1	12.62	[M + H]+	616.3168	−8.404839097	616.31162	C₃₃H₄₅NO₁₀	Hypaconitine	590.3051, 556.2985, 460.2015	Aconiti Lateralis Radix Praeparata
2	10.68	[M + H]+	604.3156	−6.619101801	604.3116	C₃₂H₄₅NO₁₀	Benzoylaconine	556.2012, 524.2264, 338.1747, 105.1759	Aconiti Lateralis Radix Praeparata
3	10.63	[M + H]+	574.303	−3.380979317	574.3010583	C₃₁H₄₃NO₉	Benzoylhypaconine	542.2667, 524.3504, 510.3142	Aconiti Lateralis Radix Praeparata
4	17.34	[M + H]+	295.19184	−4.926312204	295.1903858	C₁₇H₂₆O₄	6-Gingerol	177.0927, 164.0826, 151.1117, 137.0618	Zingiberis Rhizoma
5	17.34	[M + H]+	293.177	−7.723380375	293.1747357	C₁₇H₂₄O₄	6-Shogaol	177.0927, 163.1132, 135.1179, 111.0814, 109.0775	Zingiberis Rhizoma
6	17.14	[M + H]+	137.1333	−6.000766685	137.1324771	C₁₀H₁₆	β-Phellandrene	121.102	Zingiberis Rhizoma
7	17.14	[M + H]+	137.1333	−6.000766685	137.1324771	C₁₀H₁₆	Camphene	121.1023	Zingiberis Rhizoma
8	8.82	[M + H]+	257.0807	0.526682589	257.0808354	C₁₅H₁₂O₄	Liquiritigenin	137.0237	Glycyrrhizae Radix et Rhizoma
9	7.18	[M − H]−	417.11887	0.551401266	417.1191	C₂₁H₂₂O₉	Liquiritin	255.0441	Glycyrrhizae Radix et Rhizoma
10	8.68	[M − H]−	549.1597	3.030256877	549.1613641	C₂₆H₃₀O₁₃	Liquiritin apioside	255.0663	Glycyrrhizae Radix et Rhizoma
11	13.03	[M − H]−	821.3952	1.593383933	821.3965088	C₄₂H₆₂O₁₆	Isoliquiritin	113.1432	Glycyrrhizae Radix et Rhizoma
12	10.14	[M − H]−	417.1181	2.410342722	417.1191054	C₂₁H₂₂O₉	Glycyrrhizic acid	255.066	Glycyrrhizae Radix et Rhizoma
13	12.9	[M − H]−	837.3888	3.132824061	837.3914234	C₄₂H₆₂O₁₇	Licorice saponin G2	351.0075	Glycyrrhizae Radix et Rhizoma

Establishment and Validation of the Orthotopic Lung Cancer Model

To establish a stable model for lung cancer research, a modified orthotopic intrapulmonary injection technique was employed to inoculate A549-luc2-tdT-2 cells stably expressing red luciferase into the left lung of nude mice. On day 5 post-implantation, bioluminescence imaging revealed specific fluorescent signal enrichment in the left lung region of all nude mice. Spatial distribution and signal intensity were the same among animals in groups, while fluorescent signals could not be measured within non-target organs (Figure 3A). Such results demonstrated that tumor cells were successfully implanted and their growth in lungs was observed, verifying that the model was successfully established.

Figure 3.

In vivo antitumor efficacy of SND + CTX in an orthotopic lung cancer model. (A) An orthotopic lung cancer model was successfully established. Bioluminescence imaging showed specific fluorescence signal accumulation in the left lung region of all nude mice, with consistent signal intensity across groups prior to treatment. After 7 days of treatment, the tumor fluorescence signal increased, with no significant differences observed between groups. By day 14, the signal in the Model group continued to intensify, whereas signals in all treatment groups had declined. On day 21, the signal in the Model group peaked. All treatment groups exhibited significantly lower signals compared to the Model group, with the SND + CTX group showing the lowest fluorescence intensity. The observed decrease in fluorescence signal intensity was based on qualitative visual evaluation. (B) Anatomical observation revealed multiple, firm gray-white nodules on the lung surface in the Model group. The number of nodules was reduced in the SND group. In the CTX group, both the number and size of nodules were further decreased, accompanied by localized fibrotic scars. The SND + CTX group showed a substantial reduction in tumor nodules.

SND + CTX Regulates Tumor Growth in the Orthotopic Lung Cancer Model

According to dynamic bioluminescence imaging analysis, relative to the baseline upon modeling, tumor fluorescence signal intensity was enhanced in each group after a 7-day treatment process, and there was no obvious between-group difference. On the 14th day, the Model group still had increasing signal, whereas those in the 3 treatment groups mildly decreased. On the 21st day, the tumor signal of Model group was the highest. The treatment groups showed remarkably decreased signals relative to Model group, and the SND + CTX group displayed the lowest fluorescence signal (Figure 3A). Notably, the observed decrease in fluorescence signal intensity was based on qualitative visual evaluation.

The above imaging results were further verified by terminal in situ anatomical analysis. Several grayish-white, firm nodules could be seen on lung tissue surface in model group. The nodule number in SND group decreased relative to model group. In CTX group, nodule size and number further declined, accompanied by local fibrotic scar formation. The remarkable pathological improvement was observed in SND + CTX group, featured by the substantially reduced tumor nodules and retained lung tissue structure (Figure 3B).

Effects of SND + CTX on Hepatorenal Toxicity

For evaluating how diverse treatments affected liver and lung functions of nude mice, serum Cr, ALT, and AST contents were determined.

One-way ANOVA revealed significant differences in ALT, AST, and Cr levels among the groups (ALT: F(3,28) = 181.803; AST: F(3,28) = 316.499; Cr: F(3,28) = 184.365, P < .001; Figure 4A-C). According to post hoc Tukey’s test, Cr, ALT, and AST contents remarkably decreased in SND, CTX, and SND + CTX groups relative to Model group (P < .001). The CTX and SND + CTX groups also showed markedly reduced ALT level relative to SND group (P < .001). Additionally, the SND + CTX group demonstrated evidently decreased AST contents compared with SND or CTX group (P < .001). However, the SND and CTX groups did not show any significant difference (P > .05).

Figure 4.

Effect of SND + CTX on hepatic and renal function serum biomarkers. Serum biomarkers of hepatic and renal function were measured using ELISA, including (A) alanine aminotransferase (ALT), (B) aspartate aminotransferase (AST), and (C) creatinine (Cr) levels. Data are presented as mean ± SD (n = 8). Statistical significance was analyzed by one-way ANOVA followed by Tukey’s post hoc test. ^***P < .001 versus Model group; ^###P < 0.001 versus SND group; ^&&&P < 0.001 versus CTX group.

For renal function, Cr levels were lower in the CTX and SND + CTX groups compared with the SND group (P < .001), with no significant difference between the CTX and SND + CTX groups (P > .05).

These results indicate that SND, CTX, and SND + CTX treatments all significantly reversed the abnormal hepatic and renal function indices induced by the tumor model. Furthermore, the combination regimen demonstrated an additive effect that was superior to either monotherapy in improving both hepatic and renal function.

Inhibitory Effect of SND + CTX on Key Signaling in the Tumor Hypoxic Microenvironment

For investigating the regulation of SND and CTX against the hypoxic TME, HIF-1α along with its downstream targets VEGF and PDGF-β were examined for their levels.

Based on IHC analysis, HIF-1α showed primary cytoplasmic expression within lung tissue cells. Relative to Model group, HIF-1α protein levels decreased in treatment groups. In the 100× whole-field view, SND, CTX, and SND + CTX groups displayed sequentially reduced positive expression. Similar results were obtained under 400× magnification (Figure 5A). RT-qPCR verified the above results for HIF-1α (Figure 5B) and was also conducted for VEGF, and PDGF-β (Figure 5C). One-way ANOVA of HIF-1α, VEGF, and PDGF-β revealed statistically significant differences among groups (HIF-1α: F(3,20) = 297.293, P < .001; VEGF: F(3,20) = 801.971, P < .001; PDGF-β: F(3,20) = 3622.507, P < .001). According to post hoc Dunnett’s test, HIF-1α, VEGF, and PDGF-β levels of diverse treatment groups apparently decreased compared with Model group (P < .01). Additionally, CTX and SND + CTX groups had more potent inhibition against HIF-1α than SND group, as evidenced by Tukey’s multiple comparisons (P < .001). No statistically significant difference was observed between the CTX and the SND + CTX group. The SND + CTX group had markedly reduced expression of VEGF and PDGF-β relative to SND or CTX group (P < .001; Figure 5B and C).

Figure 5.

Effect of SND + CTX on hypoxia-related factors HIF-1α, VEGF, and PDGF-β in lung tissue. (A) Immunohistochemical staining of HIF-1α in lung tissues from each group. At 100× magnification (whole-field view), HIF-1α was primarily expressed in the cytoplasm of lung tissue cells. At 400× magnification (high-power field), the expression pattern was largely consistent with that observed at 100×. (B) Expression level of HIF-1α in lung tissue detected by RT-qPCR. Data are presented as mean ± SD (n = 8). Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test. ^**P < .01, ^***P < .001 versus Model group; ^###P < 0.001 versus SND group. (C) Expression levels of VEGF and PDGF-β in lung tissue detected by RT-qPCR. Data are presented as mean ± SD (n = 8). Statistical significance was analyzed by one-way ANOVA followed by Tukey’s post hoc test. ^***P < .001 versus Model group; ^###P < 0.001 versus SND group; ^&&&P < 0.001 versus CTX group.

SND and CTX efficiently inhibited key signals within the hypoxic TME, which was validated by down-regulated HIF-1α and inhibited VEGF and PDGF-β. Typically, CTX and SND + CTX exerted an enhanced effect on inhibiting HIF-1α relative to SND alone, and their combination synergistically suppressed downstream effector molecules.

Exosome Isolation and Characterization in Lung Tumor Tissues

For investigating the effect of exosomes on regulating the hypoxic TME upon diverse treatments, we separated exosomes in lung tissues from diverse groups and comprehensively characterized them.

TEM illustrated the clear exosomal shape. At 100 nm, exosomes were hemispherical with a concave side (Figure 6A), but they were saucer-shaped with a clear lipid bilayer at 200 nm (Figure 6B), and had punctate structures at 500 nm, with no appearance of bilayer features (Figure 6C).

Figure 6.

Isolation and identification of exosomes from lung tumor tissue. (A) Under electron microscopy, exosomes exhibited a hemispherical shape with a concave side (scale bar: 100 nm). (B) The exosomes displayed a saucer-like morphology, with the lipid bilayer membrane clearly visible (scale bar: 200 nm). (C) At this magnification, exosomes appeared as punctate structures, and the detailed lipid bilayer membrane was not discernible (scale bar: 500 nm). (D) Nanoparticle tracking analysis revealed the size distribution of exosomes from each group, which aligned with characteristic exosomal profiles. (E) Western blot analysis confirmed the expression of exosomal marker proteins TSG101 (65 kDa), CD63 (65 kDa), and CD9 (25 kDa).

As revealed by NTA plus BCA protein measurement, exosomes in the treatment groups had the same particle concentration and size distribution as typical exosomal profiles (Figure 6D).

Western blotting verified the levels of exosomal marker proteins. A dominant band associated with exosome biogenesis-associated protein TSG101 could be detected at about 65 kDa, and the transmembrane marker CD63 was observed at about 65 kDa, while the surface marker CD9 at around 25 kDa (Figure 6E), verifying that exosomes were smoothly isolated.

Taken together, size distribution, morphology, protein measurement, and marker analysis verified that lung tissue-derived exosomes were successfully isolated, laying a certain foundation for investigating biological mechanisms underlying the associated bioactive components.

Screening and Verification of Exosomal miR-20a-5p Expression

For elucidating the possible molecular mechanism related to exosome-induced modulation, bioinformatics analysis was carried out for selecting miRNAs that might target HIF-1α. The TargetScan database was utilized to analyze the 3′-UTR in HIF-1α (Ensembl transcript ENST00000371953), suggesting the presence of a conserved 7-mer seed-match site within miR-20a-5p. Moreover, the context++ score (−0.35) along with evolutionary conservation (PhastCons ≥ 0.8) demonstrated a high probability of functional binding (Figure 7A and B), revealing that miR-20a-5p might modulate HIF-1α level.

Figure 7.

Screening and expression validation of exosomal miR-20a-5p. (A) To elucidate the potential molecular mechanism of exosome-mediated regulation, the exosomal miRNA miR-20a-5p targeting HIF-1α was predicted based on the TargetScan database. (B) The predicted miRNA-mRNA recognition site is located primarily within the 3′-UTR of HIF-1α (Ensembl transcript ENST00000371953). miR-20a-5p contains a highly conserved 7-mer seed-match site.(C) The expression level of miR-20a-5p in lung tissue-derived exosomes was detected by RT-qPCR. Data are presented as mean ± SD (n = 8). Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test. ^**P < .01, ^***P < .001 versus Model group; ^###P < 0.001 versus SND group; ^&P < 0.05 versus CTX group.

Subsequently, the level of miR-20a-5p in exosomes derived from lung tissue was measured by RT-qPCR. One-way ANOVA showed a significant difference among groups (Figure 7C; F(3,20) = 154.896, P < .001). According to Dunnett’s test, relative to Model group, the SND group had reduced miR-20a-5p expression (P < .01), while CTX and SND + CTX groups had even more significant downregulation (P < .001). Both the CTX and SND + CTX groups showed significantly lower miR-20a-5p levels than the SND group (P < .001), and the SND + CTX group exhibited lower expression than the CTX group (P < .05).

Bioinformatic prediction suggested miR-20a-5p as a potential regulator of HIF-1α. RT-qPCR results confirmed that SND and CTX treatments, particularly the combination regimen, significantly downregulated the expression of this miRNA in lung tissue-derived exosomes.

Discussion

Lung cancer is one of the most common malignant tumors,³⁰ making research and the improvement of treatment strategies of critical importance. The combination of Traditional Chinese Medicine (TCM) and chemotherapy, which can reduce toxicity and enhance efficacy, holds promising therapeutic potential.³¹ In the complex progression from the continuous activation of proto-oncogenes to the loss of function of tumor suppressor genes leading to the formation of primary tumors, and further to the development of distant metastases,³² the role of the TME has garnered increasing attention.³³ Mouse lung tumor models with lung adenocarcinoma as an endpoint are widely used to evaluate the efficacy of lung cancer drugs. Meanwhile, the ability of human xenografts to faithfully recapitulate the relevant biological characteristics of primary human tumors, combined with the non-invasive and reproducible nature of in vivo bioluminescent imaging, makes A549-luc2-tdT-2 cells and BALB/c-nu nude mice the preferred combination.

To more closely mimic the complexity of the TME in vivo, this study employed an orthotopic xenograft model. A human lung cancer orthotopic xenograft model was established by surgically implanting A549-luc2-tdT-2 cells into the left lung of nude mice, enabling non-invasive dynamic monitoring via bioluminescence imaging.³⁴ On day 5 post-modeling, stable fluorescent signals were detected in the lung tissues of all groups through bioluminescence imaging, indicating the model was uniform and effective. Follow-up imaging was performed on days 7, 14, and 21 of treatment. Bioluminescence imaging throughout the treatment course demonstrated that the tumor signal intensity in the SND + CTX group remained the lowest among all groups, a finding highly consistent with the tumor burden assessment based on in situ anatomical examination of lung tissues.

Cyclophosphamide, as one of the most widely used anti-tumor drugs, can inhibit tumor cell proliferation but also carries various adverse effects, including hepatorenal toxicity.³⁵ Therefore, serum levels of ALT, AST, and Cr were measured in each group. The serum markers for hepatorenal injury in the SND + CTX group were significantly lower than those in the model group. This result indicates that the hepatorenal injury caused by SND + CTX is less severe or shows better recovery compared to the individual treatment groups. While inhibiting tumor growth, SND + CTX inhibited tumor development without markedly increasing hepatorenal functional impairment, which thus enhanced the safety of combination treatment.

Hypoxia plays a key role in driving solid tumor occurrence, metastasis, and treatment resistance.³⁶ As A549 cells colonize and grow, the local hypoxic microenvironment is created in the lung, which can be demonstrated by HIF-1α up-regulation in model group according to RT-qPCR and IHC analyses. Rapid solid tumor proliferation can cause insufficient blood supply, which is the major factor promoting the hypoxic TME. The persistently increasing HIF-1α expression induces stromal remodeling and tumor angiogenesis.³⁷ Activated VEGF expression increases, which stimulates endothelial cell growth and invasion, enhances neovascularization, and alleviates the hypoxia. VEGF, an important factor in angiogenesis, is under the direct modulation by HIF-1α.³⁸ Simultaneously, the continuous secretion of PDGF-β, regulated by HIF-1α, stimulates the remodeling of the extracellular matrix.³⁹ The significantly reduced expression levels of VEGF and PDGF-β in both the SND and CTX groups suggest that both SND and CTX can inhibit tumor angiogenesis and stromal remodeling. The further decrease in VEGF and PDGF-β expression in the SND + CTX group indicates a stronger synergistic effect of the combination therapy compared to either monotherapy. As downstream proteins of HIF-1α, VEGF, and PDGF-β work in concert, creating a positive feedback loop of “angiogenesis-stromal remodeling” that further exacerbates tumor hypoxia and malignant progression.⁴⁰ Inhibiting or delaying this process is key to suppressing tumor cell proliferation and metastasis.

To explore novel mechanistic drivers, this study provides preliminary observations on the biological mechanism of SND + CTX from the perspective of exosome-mediated regulation of the tumor hypoxic microenvironment (Figure 8). Exosomal miRNAs play pleiotropic regulatory roles in tumor angiogenesis,⁴¹ metastasis,⁴² and drug resistance.⁴³ In this study, the morphological characteristics of exosomes from each group showed no significant variation under TEM. NTA indicated that the particle size of exosomes across groups ranged from 115.9 to 131.5 nm. Combined with Western blot detection of the exosome marker proteins TSG101, CD63, and CD9, these results confirmed the successful extraction of exosomes.⁴⁴

Figure 8.

The mechanism by which SNT + CTX improves the hypoxic microenvironment of tumors and inhibits tumor growth. SND + CTX combination therapy alleviates the local hypoxic microenvironment in lung tumor tissue, reduces the expression of HIF-1α, and thereby downregulates the levels of its downstream target genes VEGF and PDGF-β, subsequently inhibiting tumor angiogenesis and stromal remodeling. Concurrently, the attenuated hypoxic stimulus also leads to downregulation of exosomal miR-20a-5p expression. Although miR-20a-5p can target and suppress HIF-1α, in the persistently hypoxic microenvironment, this inhibitory effect is overridden by the strong hypoxic drive, and the broader improvement in the tumor microenvironment induced by the combination therapy directly affects the expression of miRNA–target interactions.

The oncogenic role of miR-20a-5p and its host miR-17-92 gene cluster in various cancers has been established, and HIF-1α is closely associated with cancer progression.⁴⁵ However, another study indicated that the miR-17-92 cluster exerts cellular context-dependent pro- and anti-cancer effects by targeting multiple tumor angiogenesis-inducing genes such as TGFBR2, HIF1α, and VEGFA.⁴⁶ Within the tumor hypoxic microenvironment, HIF-1α is continuously activated, promoting the secretion of tumor-derived exosomes.⁴⁷ We found that tumor-derived exosomal miR-20a-5p correlates with HIF-1α expression and tumor burden, which aligns with the RT-qPCR results from the model group. Previous reports indicate that the miR-17-92 gene cluster (including miR-20a-5p) can target and inhibit HIF-1α expression, but this inhibition is oxygen-dependent.⁴⁸ Under normoxic conditions, HIF-1α expression is suppressed, whereas it can still be induced under hypoxia. Screening results from the TargetScan database reveal that miR-20a-5p can directly bind to the 3′ untranslated region (UTR) of HIF-1α mRNA via a highly conserved “7mer-m8” site, providing evidence for the direct targeting and inhibition of HIF-1α by miR-20a-5p. In this study, the RT-qPCR expression level of miR-20a-5p in the SND + CTX group was lower than that in the model group and the monotherapy groups. The downregulation of miR-20a-5p expression corresponded with the reduced expression level of HIF-1α. However, this may not result from a direct interaction between the 2 but rather from the remodeling of the tumor hypoxic microenvironment by the combination therapy. The co-directional decrease of both molecules reflects the therapeutic alleviation of hypoxia and does not indicate a loss of the inhibitory function of miR-20a-5p on HIF-1α. Although miR-20a-5p can target and suppress HIF-1α, in the persistently hypoxic in vivo microenvironment, the inhibitory regulation of HIF-1α by miR-20a-5p is dominated by the strong activating effect of hypoxia. The broader alterations in the tumor microenvironment induced by the combination therapy directly affect the expression of miRNA–target interactions.The treatment alleviated tumor hypoxia, leading to decreased HIF-1α expression. Consequently, its trans-activation effect on target genes was also attenuated, resulting in the downregulation of miR-20a-5p expression and a weakening of its inhibitory function on HIF-1α. This is one possible mechanism. Although we observed a correlation between miR-20a-5p levels in tumor-derived exosomes and tumorigenesis, the current understanding of miR-20a-5p’s role in the mechanism of combined therapy for lung cancer remains limited.

This study has several limitations. The investigation focused solely on miRNA-20a-5p, without conducting a systematic screening of other exosomal miRNAs. Additionally, the specific molecular pathways through which the Sini Decoction and cyclophosphamide combination regulate exosome secretion have not yet been validated through in vitro experiments. Further studies are warranted to address these aspects in greater depth.

Conclusion

In this work, Sini Decoction plus cyclophosphamide efficiently suppresses tumor cell development within the orthotopic lung cancer model and enhances hepatorenal safety. Its associated mechanism is probably related to ameliorating the hypoxic TME, inhibiting HIF-1α-related tumor hypoxia pathway, and regulating exosomal miR-20a-5p level. These findings have revealed a new exosome-mediated mechanism and have provided new ideas for the combined treatment of lung cancer using traditional Chinese and Western medicine.

Supplemental Material

sj-docx-1-ict-10.1177_15347354261450965 – Supplemental material for Combination Therapy of Sini Decoction and Cyclophosphamide Inhibits Tumor Growth in an Orthotopic Lung Cancer Model

Supplemental material, sj-docx-1-ict-10.1177_15347354261450965 for Combination Therapy of Sini Decoction and Cyclophosphamide Inhibits Tumor Growth in an Orthotopic Lung Cancer Model by Dacheng Pang, Shuo Zhang, Tian Tian and Tong Wang in Integrative Cancer Therapies

Footnotes

Acknowledgements

AI-Assisted Language Editing: The authors acknowledge the use of DeepSeek Translator for language polishing and translation assistance in the preparation of this manuscript. The authors are solely responsible for the final content, interpretation, and conclusions of the work.

ORCID iD

Dacheng Pang

Ethical Considerations

The authors declare that the experimental protocols of this study were approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine (Ethical Approval Number: BUCM-2024091201-3198; Institutional License Number: SYXK (Jing) 2023-0011).

Consent to Participate

All participants provided written informed consent prior to participating.

Author Contributions

Tian Tian and Tong Wang conceived and designed the project. Dacheng Pang performed the experiments, Shuo Zhang performed the data analysis. Dacheng Pang wrote and revised the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors declare that this study and the writing and publication of this paper were supported by the following funding sources: the National Natural Science Foundation of China (No. 82204963) and the Key project of ‘Jie Bang Gua Shuai’ of Beijing University of Chinese Medicine Fundamental Research Funds in 2024 (No. 2024-JYB-JBZD-036).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The datasets generated and analyzed during the current study are not publicly available due to the ongoing preparation of a doctoral dissertation and project confidentiality policies. However, they will be available from the corresponding author upon reasonable request following the completion of the dissertation and the expiration of the confidentiality period (confidentiality period: 5 years).

Supplemental Material

Supplemental material for this article is available online.

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