Mechanism Study of Xiaoyan Decoction in the Treatment of Non-small Cell Lung Cancer Through Glycometabolic Reprogramming

Mechanism Study of Xiaoyan Decoction in the Treatment of Non-small Cell Lung Cancer Through Glycometabolic Reprogramming | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanism Study of Xiaoyan Decoction in the Treatment of Non-small Cell Lung Cancer Through Glycometabolic Reprogramming xiaoqun Wang, yuting Li, suxuan OUYANG, haojian Zhang, linlin Zhao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8540053/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract [Objective] To investigate the effects of Xiaoyan Decoction ( XYT) on autophagy and glucose metabolism reprogramming in Non-small Cell Lung Cancer(NSCLC) cells and to explore its mechanism of action. [Methods] ① Screen the half maximal inhibitory concentration (IC50) of XYT-containing serum using Cell Counting Kit 8 (CCK8); ② After pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) and the activator Rapamycin (RAPA) and intervention with XYT, transmission electron microscopy was used to observe the overall autophagy status of A549 cells, and Western Blot was used to detect changes in Sequestosome1(P62) and Microtubule-associated protein 1 light chain 3(LC3); ③ The kit was used to detect glucose uptake and lactate production, Seahorse was used to assess cellular energy metabolism, and Western Blot was used to assess the expression of Hypoxia inducible factor-1α(HIF-1α) protein in A549 cells after XYT drug-containing serum intervention alone and in combination with RAPA intervention. [Results] ①XYT-containing serum can slow down cell proliferation in a concentration-dependent manner, with a 30% concentration of XYT-containing serum for 24 hours being the optimal concentration and duration of intervention. ②Following XYT intervention, the number of autophagosomes—a characteristic double-membrane structure of autophagy—significantly increased in A549 cells. Western blot analysis revealed a significant downregulation of P62 expression and a significant upregulation of LC3-II/LC3-I protein expression. ③After XYT intervention, glucose uptake and lactate production in A549 cells were significantly reduced, glycolytic rate was significantly slowed, and maximum glycolytic capacity was also significantly reduced. Western blot analysis showed a significant downregulation of HIF-1α expression. Additionally, Oxygen consumption rate(OCR) assay results indicated that O₂ consumption significantly increased after XYT intervention, with parameters such as basal respiration and maximal respiration significantly up-regulated. These effects were further enhanced by co-treatment with autophagy modulators 3-MA/RAPA. [Conclusion] XYT can inhibit A549 cell proliferation, upregulate cellular autophagy levels, regulate HIF-1α expression, shift the metabolic phenotype from glycolysis to aerobic oxidation, reprogram glucose metabolism, and thereby exert an inhibitory effect on cell proliferation. Xiaoyan Decoction Non-small cell lung cancer Autophagy Glycolytic reprogramming HIF-1α Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. INTRODUCTION Lung cancer remains the most prevalent malignancy globally. According to the latest U.S. cancer statistics report, approximately 2.04 million new cancer cases and 620,000 cancer-related deaths are projected in the United States for 2025. Lung cancer persists as the leading cause of cancer mortality, accounting for over 20% of all cancer deaths . 1 Histologically, lung cancer is classified into NSCLC and small cell lung cancer (SCLC). NSCLC constitutes approximately 83% of all lung cancer cases . 2 Alarmingly, about two-thirds of patients present with stage III/IV disease at diagnosis. The high malignancy, elevated risk of recurrence and metastasis, and limited therapeutic efficacy pose significant challenges in clinical management. Glycolysis, the process by which cells catabolize glucose to generate energy under hypoxic conditions, is notably hijacked by cancer cells. Otto Warburg observed that tumor cells preferentially rely on glycolysis for energy production even in the presence of adequate oxygen, a phenomenon termed the "Warburg effect" . 3 Substantial evidence confirms enhanced glycolytic activity in NSCLC cells, characterized by elevated expression of key glycolytic enzymes and increased lactate production . 4,5 Critically, specific glycolytic enzymes are implicated in lung cancer pathogenesis. For instance, Phospholipase C delta 3 (PLCD3) triggers the protein kinase C (PKC), subsequently stimulating the Rap1 pathway. This cascade induces glycolytic reprogramming, supplying the energy and metabolic substrates essential for lung cancer cell proliferation and dissemination. Moreover, PLCD3 promotes metastasis and invasion via Rap1 (Ras-related protein 1) pathway activation . 6 Conversely, Brain-specific angiogenesis inhibitor 1 (BAI1) inhibits the Warburg effect and glycolysis by upregulating stearoyl-CoA desaturase 1 (SCD1) and suppressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), thereby inducing metabolic reprogramming . 7 Consequently, targeting glycolysis has emerged as a promising therapeutic strategy in oncology research. HIF-1α serves as a master regulator of cellular energy metabolism . 8 It dimerizes with the hypoxia-inducible factor 1 beta subunit (HIF-1β), binds to hypoxia-response elements (HREs) in chromatin, and transactivates target genes involved in metabolic adaptation. Key transcriptional targets include pyruvate dehydrogenase kinase (PDK), pyruvate dehydrogenase (PDH), glucose transporters (GLUT1, GLUT3), monocarboxylate transporters (MCT1, MCT4), the mitochondrial pyruvate carrier (MPC), lactate dehydrogenase A (LDHA), adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), and glycolytic enzymes (e.g. Hexokinase1(HK1), Hexokinase2(HK2), phosphofructokinase (PFK), Pyruvate kinase M(PKM), alpha-enolase (ENO1)) . 9 Collectively, HIF-1α plays a pivotal role in mediating hypoxia adaptation and aerobic glycolysis (the Warburg effect) in cancer. Autophagy, a conserved catabolic process unique to eukaryotic cells, involves the sequestration of cytoplasmic components within double-membrane vesicles (autophagosomes) for lysosomal degradation. It is a fundamental cellular mechanism crucial for maintaining homeostasis, facilitating intracellular turnover, and responding to stress . 10 Autophagy exhibits a complex, context-dependent role in lung cancer progression, significantly influencing NSCLC pathophysiology. Liang et al. demonstrated that anlotinib induces protective autophagy in human lung cancer cells, evidenced by increased autophagosome formation. This autophagy enhances anlotinib's anti-angiogenic properties via the JAK2/STAT3/VEGFA signaling pathway, ultimately suppressing NSCLC cell proliferation and inducing apoptosis . 11 Similarly, Wang et al. reported that ginkgolide B induces Beclin-1-dependent autophagy in lung cancer cells. Furthermore, autophagy critically regulates glycolytic metabolism. Jiao et al. established a negative correlation between autophagy and glycolysis, mediated partly through the regulation of HK2, a key glycolytic enzyme . 12 Wang et al. 13 found that cadmium-induced glycolysis is autophagy-dependent, and the autophagy-glycolysis axis plays an important role in the proliferation of cadmium-treated A549 cells. In addition, HIF-1α, a protein associated with hypoxic adaptation, is stably expressed in cancer cells, thereby regulating autophagy and glycolysis . 14 Xiaoyan Decoction (Jinyao Zhizi Z20060786) is an empirical prescription developed by Professor Jia Yingjie, a national renowned traditional Chinese medicine physician. It consists of 7 Chinese medicinal herbs, including Astragalus membranaceus, Radix Pseudostellariae, Hedyotis diffusa, raw Oyster, Prunella vulgaris, Curcuma aromatica, and Curcuma longa. Previous studies have demonstrated that XYT can effectively inhibit tumor cell proliferation, reverse multidrug resistance, and induce cell apoptosis . 15 Notably, it also exerts a certain regulatory effect on cell autophagy , 16 but its underlying molecular mechanism remains to be further explored. This study aims to investigate whether XYT exerts anti-NSCLC effects by regulating cellular glucose metabolism reprogramming through the HIF-1α signaling pathway and autophagy. Firstly, the CCK-8 assay was used to evaluate the effect of XYT-containing serum on the viability of A549 cells, so as to determine the optimal concentration and action time of XYT-containing serum for intervening A549 cells. Secondly, autophagy inhibitor 3-MA and autophagy activator RAPA were applied to A549 cells. Transmission electron microscopy was used to observe the overall autophagic status of A549 cells, and Western blot was employed to detect the expression levels of autophagy-related factors P62 and LC3-II/LC3-I proteins, thereby investigating the effect of XYT on autophagy in A549 cells. To further clarify whether XYT regulates glucose metabolism reprogramming through autophagy, methods such as Western blot, Seahorse Glycolysis Stress Test Kit, Seahorse Mitochondrial Stress Test Kit, lactate production kit, and glucose uptake kit were used to detect the following after autophagy inhibition and activation: the expression levels of key glycolytic enzymes including HK2, 6-Phosphofructo-2-Kinase(PFKFB3), PKM, GLUT1, GLUT4, LDHA, and HIF-1α; lactate production and glucose uptake in A549 cells, so as to clarify the changes in glucose metabolism reprogramming. 2.MATERIALS AND METHODS 2.1 Experimental cells and animals The A549 cell line was purchased from Zhejiang Meisen Cell Technology Co., Ltd. (Product No.: CTCC-001-0036). Thirty male Balb/c nude mice (specific pathogen-free [SPF] grade), aged 4–5 weeks and weighing 16–18 g, were supplied by Beijing Huafukang Biotechnology Co., Ltd. (Animal Quality Certificate No.: 110322220103119965; License No.: SCXK(Jing)2019-0008). Mice were housed in the SPF-grade animal facility of Tianjin Guosheng Zhongyuan Technology Co., Ltd. The experimental protocol was approved by the Animal Ethics Committee of Yishengyuan Gene Technology (Tianjin) Co., Ltd. (Protocol No.: YSY-DWLL-2022133). 2.2 Experimental drugs and reagents The following Chinese herbal medicines were purchased from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (TJUTM). The decoction was prepared by the decoction pharmacy of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. Preparation of drug-containing serum for the elimination of rocks: 20 male SD rats, 6–8 weeks old, weighing 180–220 g, were randomly divided into the normal group and the traditional Chinese medicine group (n = 10). Rats in the normal group were gavaged with 2 mL of saline daily, while rats in the traditional Chinese medicine group were gavaged with 2 mL (12.25 g/kg) of the Chinese medicine solution of XYT daily for 7 consecutive days. After the last administration for 1h, rats in each group were anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital, and then blood was collected from the abdominal aorta. The blood was allowed to stand for 1h and then centrifuged at 3000 rpm for 10 min to collect the serum, which was then filtered on an ultra-clean bench using a 0.22 µm microporous filter to remove bacteria. The treated serum was divided and labeled as blank serum and Xieyan Tang drug-containing serum (hereinafter referred to as XYT drug-containing serum), and then stored in a refrigerator at -80°C for spare use. 2-Deoxyglucose(2-DG) and glutaraldehyde fixative were purchased from Shanghai Yuanye Company (batch no.: S11070, R20515); RPMI-1640 medium and hosphate buffer saline(PBS) were purchased from Cytiva Company, China (batch no.: AH30380359, AH29827185); penicillin-streptomycin solution, trypsin, lyophilization solution, Rainbow 180 broad-spectrum protein marker, BCA method protein quantitative analysis kit, and protein uploading buffer (2×) were purchased from Solarbio, Beijing, China, (batch no. P7630, T1300, 24800, PR1910, PC0020, P1019); fetal bovine serum was purchased from Sbjbio, China (batch no.: BC-SE-FBS07); PVDF membrane was purchased from Merck Millipore (batch no.: IPVH00010); ECL Chemiluminescent Substrate Kit was purchased from Biosharp, China (batch no.: BL520B); 10× TBST, electrophoresis solution and membrane transfer solution were purchased from Beyotime, Shanghai, China (batch nos. ST673, P0561, P0572); protease inhibitors and phosphatase inhibitors were purchased from Boster, Wuhan, China (batch nos. AR1183, AR1195); CCK-8 detection kit was purchased from Tongren Chemical Company, Japan (batch no. CK04); GLUT1 antibody, GLUT1 antibody, PFKFB3 antibody and LC3 antibody were purchased from CST (batch nos. 73015S, 13123S, 12741S); HK2 antibody, GLUT4 antibody, LDHA antibody, PKM antibody and SQSTM1/p62 antibody were purchased from Abcam (batch nos. ab104836, ab48547, ab101562, ab150377, ab150377, ab101562). MMP9 antibody and MMP2 antibody were purchased from Affinity (lot numbers: AF5228 and AF5330); β-actin antibody and HRP-labeled goat anti-rabbit IgG were purchased from Wuhan Proteintech (lot numbers: 20536-1-AP and SA00001-2); HiFiScript cDNA was used for the analysis of β-actin antibody and HRP-labeled goat anti-rabbit IgG. (batch no.: 20536-1-AP, SA00001-2); HiFiScript cDNA Synthesis Kit, UltraSYBR Mixture (Low ROX) were purchased from CW BIO, Jiangsu, China (batch no.: CW2569M, CW260M); DMSO was purchased from GENTIHOLD, China (batch no.: D8371); Seahorse XF Glycolytic Stress Test Kit, Seahorse XF Mitochondrial Stress Test Kit, Seahorse XF Basal Medium, XF 200mM Glutamine Solution, XF 100mM Sodium Pyruvate Solution, and 1.0M Glucose Solution were purchased from Agilent, USA (Lot Nos. 100, 103579-100, 103578-100, 103577-100); lactate and glucose test kits were purchased from Nanjing Jianjian Company, China (lot numbers: A019-2-1, A154-2-1). 2.3 Instruments A multifunctional microplate reader was purchased from Thermo Fisher Scientific (USA). A fully automated gel imaging analysis system (ZF-288) was purchased from Shanghai Jiapeng Technology Co., Ltd. (China). 2.4 Cell culture and treatment Cell culture A549 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (PS) under conditions of 37℃and 5% CO₂. Cell Revival ( 1 ) Pre-warm a water bath to 37℃.( 2 ) Retrieve the cryopreserved cells from − 80℃ and immediately immerse in a 37℃ water bath for thawing.( 3 ) After thawing, transfer the cell suspension into a 15 mL centrifuge tube, add 3 mL of complete medium, and centrifuge at 1000 rpm for 5 min.( 4 ) Discard the supernatant, resuspend the cell pellet in 1 mL of complete medium, and transfer into a 10 cm culture dish. Subsequently, add 8 mL of complete medium and place the dish in a constant-temperature incubator for continued culture.( 5 ) Observe cell viability under a microscope on the following day. Medium Change ( 1 ) Remove the cultured cells from the incubator and examine their adherence and growth status under a microscope.( 2 ) Transfer the culture dish to a sterile biosafety cabinet, aspirate the old medium, and gently wash the cells once with PBS.( 3 ) Add 8–10 mL of fresh complete medium.( 4 ) After microscopic examination, return the dish to the incubator for continuous culture and monitor the cells periodically. Cell Subculture : When cell confluence reaches approximately 80%-90% as observed under a microscope, perform subculture as follows:( 1 ) Transfer the cells from the incubator to the biosafety cabinet, aspirate the medium, and wash once with PBS.( 2 ) Add 2 mL of trypsin to the culture dish and incubate at 37℃ for 1–2 min. Monitor cell morphology dynamically under the microscope; digestion should be terminated when cells become rounded and appear brighter.( 3 ) Add 2 mL of complete medium to the dish, pipette repeatedly to detach the cells, and collect the cell suspension into a 15 mL centrifuge tube.( 4 ) Centrifuge at 1000 rpm for 5 min.( 5 ) Discard the supernatant, resuspend the pellet in 1 mL of complete medium, mix thoroughly by pipetting, and transfer to a new 10 cm culture dish. Then add 7–8 mL of complete medium and gently mix by drawing a “十” shape with the dish.( 6 ) Place the dish in a 37℃ incubator and inspect cell status daily. Cell Counting : ( 1 ) Following the same digestion procedure, resuspend the cell suspension repeatedly to ensure a uniform distribution of single cells for counting.( 2 ) Clean the hemocytometer with an alcohol swab, allow it to air-dry, and place a coverslip over the counting chamber.( 3 ) Dilute 10 µL of the cell suspension with 90 µL PBS (10-fold dilution) and mix thoroughly.( 4 ) Gently load 10 µL of the diluted suspension at the edge of the coverslip.( 5 ) Under the microscope, count the cells in the four corner squares. Calculate the cell density using the formula:Cells per mL = (Total count in 4 squares ÷ 4)×10×10 4 .( 6 ) Repeat the counting three times and take the average value as the final cell density. Cell Cryopreservation ( 1 ) Harvest cells in the logarithmic growth phase using the same digestion procedure, followed by centrifugation at 1000 rpm for 5 min.( 2 ) Discard the supernatant, add 1 mL of cell freezing medium, gently resuspend the pellet, and transfer to a cryovial. Seal the vial with parafilm.( 3 ) Label the vial with cell information and the date of cryopreservation. Store the cells at -80℃ or in liquid nitrogen for long-term preservation. Treatment Working solutions were prepared: 100 mL of the autophagy activator RAPA(10 nM) and 10 mL of the 3-MA(3 µM). Cells were divided into six experimental groups:Control group: No drug treatment; XYT group: Treated with 30% XYT drug-containing serum for 24 h; 3-MA group: Treated with 3 µM 3-MA for 1 h ; XYT + 3-MA group: Pretreated with 3 µM 3-MA for 1 h, followed by treatment with 30% XYT serum for 24 h; RAPA group: Treated with 10 nM RAPA for 1 h ; XYT + RAPA group: Pretreated with 10 nM RAPA for 1 h, followed by treatment with 30% XYT serum for 24 h. 2.5 CCK-8 cell activity : A549 cells were digested routinely and then inoculated into 96-well plates at a concentration of 5×10 4 cells/mL. A549 cells were routinely digested and then inoculated into 96-well plates at a concentration of 5×10 4 cells/mL. The cells were cultured overnight in a cell culture incubator, and the growth status of the cells was observed on the next day. The 96-well plate was divided into the following 10 groups according to the vertical arrangement of one group, as shown in the following table. In each group, there were 6 replicate wells, and after discarding the overnight medium, each well was refilled with 100 µL of working solution (working solution configuration is shown in the table 1 below). After incubation for different times (12 h, 24 h, 48 h), the medium of each Table.1 Working Fluid Configuration Concentration Table Group Operating Fluid blank group Complete medium (without cells) control group Complete medium (with cells) blank serum group 0.2 mL blank drug-containing serum dissolved in 1.8 mL DMEM medium 10% serum-containing group 0.2 mL XYT-containing serum dissolved in 1.8 mL DMEM medium 15% serum-containing group 0.3 mL XYT-containing serum was dissolved in 1.7 mL DMEM medium 20% serum-containing group 0.4 mL XYT-containing serum was dissolved in 1.6 mL DMEM medium 25% serum-containing group 0.5 mL XYT-containing serum dissolved in 1.5 mL DMEM medium 30% serum-containing group 0.6 mL XYT-containing serum was dissolved in 1.4 mL DMEM medium 35% serum-containing group 0.7 mL XYT-containing serum was dissolved in 1.3 mL DMEM medium 40% serum-containing group 0.8 mL XYT-containing serum was dissolved in 1.2 mL DMEM medium well was discarded, and 10 µL of CCK-8 reagent and 90 µL of DMEM medium were added to the wells and incubated for 1–4 h. Finally, the absorbance A value was read at 450 nm using an enzyme counter, and the cell viability was calculated, cell viability=(A experimental group-A blank group)/(A control group-A blank group)×100%, and the experiment was repeated three times. The experiment was repeated 3 times. 2.6 Transmission electron microscopy This experiment was commissioned to Tianjin Guosheng Zhongyuan Technology Co., Ltd. for completion. 2.7 Western blot analysis Cell protein extraction: Cells were digested and collected, washed twice with PBS, and total proteins were collected by adding RIPA lysis buffer. After quantification using the BCA method, electrophoresis and membrane transfer were performed, followed by blocking with 5% non-fat milk for 1 hour. Primary antibodies were added respectively, including HK2 antibody, GLUT1 antibody, GLUT4 antibody, LDHA antibody, MMP9 antibody, MMP2 antibody (all diluted at 1:1000), PFKFB3 antibody, LC3 antibody (both diluted at 1:2000), β-actin antibody (diluted at 1:3000), SQSTM1/p62 antibody (diluted at 1:5000), PKM antibody, and HRP-labeled goat anti-rabbit IgG (both diluted at 1:10000). After overnight incubation, the membranes were incubated with secondary antibody diluent (diluted at 1:10000) for 1 hour at room temperature. After washing, images were captured using a chemiluminescence imaging system. The gray values of target protein bands were analyzed with Image J software. Each experiment was repeated three times, and the average gray value was calculated. β-actin was used as the internal reference to analyze the expression level of target proteins in each group. 2.8 Determination of extracellular lactic acid content After 24 hours of drug treatment, the culture medium of each group was collected, centrifuged, and the supernatant was taken. The lactic acid content was detected according to the kit instructions.Lactic acid content in the culture medium (mmol·L-1) = (ODsample - ODblank) / (ODstandard - ODblank) × Cstandard × N, where Cstandard is the lactic acid content of the standard, and N is the sample dilution factor.Lactic acid production content = lactic acid content in the culture medium after drug treatment - lactic acid content in the original culture medium.Relative lactic acid production rate (%) = (lactic acid production content in the experimental group / lactic acid production content in the control group) × 100%. 2.9 Determination of cellular glucose uptake After 24 hours of drug treatment, the culture medium of each group was collected, centrifuged, and the supernatant was taken. The glucose content was detected according to the kit instructions.Glucose concentration in the sample = ODsample / ODstandard × 5.Relative glucose consumption rate (%) = [(glucose concentration in the original culture medium - glucose concentration in the culture medium after drug treatment) / glucose concentration consumed in the control group] × 100%. 2.10 Detection of cellular energy metabolism A549 cells in the logarithmic growth phase were taken and seeded into an XFe 96-well plate, with 1×104 cells per well, and 80 µL of complete medium was added. The plate was placed in a 37°C incubator overnight. The Seahorse probe plate was hydrated using Seahorse XF calibration solution and incubated overnight in a Seahorse incubator, ensuring that bubbles at the bottom of the probe plate were eliminated. 2 mM glutamine, 1 mM sodium pyruvate, and 10 mM glucose were added to Seahorse XF DMEM medium. The original 80 µL system was supplemented to 250 µL with detection medium, 200 µL was removed to leave 50 µL, then 200 µL of detection medium was added and 200 µL was removed. This washing step was repeated twice, leaving 50 µL, and the plate was incubated at 37°C in a CO2-free environment for 1 hour. Oligomycin, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone(FCCP), and rotenone/antimycin A were sequentially added to the drug-adding wells to evaluate mitochondrial oxidative function of cells, while glucose, oligomycin, and 2-DG were sequentially added to the drug-adding wells to assess cellular glycolytic capacity. The cell culture plate was then loaded into the instrument for detection. 2.11 Statistical analysis methods The experimental results were processed using SPSS 26.0 software. Quantitative data with normal distribution were described as mean ± standard deviation. Independent sample t-test was used to compare differences between two groups, and a P-value < 0.05 was considered statistically significant. 3.RESULTS 3.1 Effect of Xiaoyan Decoction on A549 cell viability Firstly, the effect of XYT-containing serum on the viability of A549 cells was evaluated by the CCK-8 assay. A549 cells were treated with XYT-containing serum at different concentrations (10%, 15%, 20%, 25%, 30%, 35%, 40%) for 24 h, 48 h, and 72 h, respectively. The experimental data showed that XYT-containing serum could inhibit cell viability in a concentration-dependent manner (Fig. 1a). To further determine the optimal concentration and action time of XYT-containing serum for intervening A549 cells, the IC50 values at different intervention time points were analyzed (Table 2 , Fig. 1b). The results showed that the IC50 values of XYT-containing serum after 12 h, 24 h, and 48 h of intervention were 44.37 ± 2.94%, 30.37 ± 1.58%, and 45.41 ± 3.11%, respectively. Finally, 30% XYT-containing serum with 24 h of intervention was used as the intervention concentration and time for subsequent studies. Figure 1 The effect of XYT on the activity of A549 cells (a) Effect of XYT-containing serum on the activity of A549 cells. (b) IC50 values of XYT serum at different time. Table 2 IC50 values of XYT serum at different time( ±s, n = 3). Time IC50 (%) 12 H 44.37 ± 2.94 24 H 30.37 ± 1.58 48 H 45.41 ± 3.11 3.2 Induction of autophagy in A549 cells by Xiaoyan Decoction In this part of the study, autophagy inhibitor 3-MA and autophagy activator RAPA were applied to A549 cells to observe the effect of XYT on autophagy in A549 cells. Firstly, transmission electron microscopy was used to observe the overall autophagic status of A549 cells. As shown in Fig. 2, there were few autophagosomes in the control group. After intervention with XYT-containing serum and RAPA, the number of autophagosomes—marked by the characteristic double-membrane structure of autophagy—significantly increased, containing incompletely degraded cytoplasmic components. The number of autophagosomes was even greater after intervention with XYT combined with RAPA. The autophagic level in the 3-MA group was low, while the autophagic level in the XYT combined with 3-MA group was higher than that in the 3-MA group and the control group.Subsequently, Western blot was used to detect changes in autophagy-related factors at the molecular level. After A549 cells were intervened with XYT-containing serum alone or in combination with RAPA, Western blot was applied to detect the expression of P62 and LC3-II/LC3-I proteins. As shown in Fig. 3 a, compared with the control group, intervention with XYT-containing serum, RAPA, or XYT-containing serum combined with RAPA significantly downregulated the expression of P62 and significantly upregulated the expression of LC3-II/LC3-I protein, with statistically significant differences (P < 0.05 or P < 0.01). Compared with the XYT group, the expression level of P62 in the RAPA group and XYT+RAPA group was lower than that in the XYT group, while the expression level of LC3-II/LC3-I protein was higher than that in the XYT group, with statistically significant differences (P < 0.05 or P < 0.01). Compared with the RAPA group, the expression level of P62 in the XYT+RAPA group was significantly decreased, while the expression level of LC3-II/LC3-I was significantly increased, with a statistically significant difference (P < 0.05).In addition, Western blot was used to detect the expression of P62 and LC3-II/LC3-I proteins in A549 cells after intervention with XYT-containing serum alone or in combination with 3-MA. As shown in Fig. 3 b, compared with the control group, the XYT group showed a significant decrease in P62 expression (P < 0.01) and a significant increase in LC3-II/LC3-I expression (P < 0.05); the 3-MA group showed a significant upregulation in P62 expression (P < 0.05) and a significant downregulation in LC3-II/LC3-I expression (P 0.05) but a significant decrease in LC3-II/LC3-I expression (P < 0.01). Compared with the XYT group, the 3-MA group had a significantly higher P62 expression (P < 0.01) and a significantly lower LC3-II/LC3-I expression (P < 0.01); the XYT + 3-MA group had a significantly increased P62 expression (P < 0.01) and a significantly decreased LC3-II/LC3-I expression (P < 0.01), with all differences being statistically significant. Compared with the 3-MA group, the XYT + 3-MA group had a decreased P62 expression level (P < 0.01) and an increased LC3-II/LC3-I expression level (P < 0.05), with statistically significant differences.In this part of the study, we applied the autophagy inhibitor 3-MA and the autophagy activator RAPA in A549 cells to observe the effect of XYT on autophagy in A549 cells. Firstly, transmission electron microscopy was utilized to observe the overall situation of autophagy in A549 cells, followed by Western blot to detect the changes of autophagy-related factors at the molecular level. Figure 2 Transmission electron microscopy of A549 cell ultrastructure (×5000). red arrow:autophagosome 3.3 Xiaoyan Decoction-induced Cell Autophagy Inhibits Glucose Metabolism Reprogramming in A549 Cells In this part of the study, A549 cells were first treated with the autophagy activator RAPA and inhibitor 3-MA to observe the effects of XYT on glucose uptake and lactic acid production. The experimental results showed that compared with the control group, the glucose uptake and lactic acid production in the XYT group, RAPA group, XYT+RAPA group, and XYT + 3-MA group were significantly reduced (P < 0.05 or P 0.05). In addition, there were no significant differences in glucose uptake and lactic acid production between the RAPA group and the XYT group (P > 0.05), while the glucose consumption and lactic acid production in the 3-MA group were significantly higher than those in the XYT group (P < 0.05); compared with the XYT group, the XYT+RAPA group showed no significant difference in glucose uptake, and the XYT + 3-MA group showed no significant differences in glucose consumption and lactic acid production (P > 0.05), whereas the lactic acid production in the XYT+RAPA group was significantly lower than that in the XYT group (P < 0.05). Compared with the groups treated with autophagy regulators alone, the combination of the two drugs significantly downregulated cellular glucose uptake and extracellular lactic acid production (P < 0.05 or P < 0.01), as shown in Fig. 4 .Next, the Seahorse energy platform was used to evaluate glycolytic reserve capacity. In the experiment, a saturated concentration of glucose was first added, and the process of glucose being decomposed into pyruvate through glycolysis was observed, which could reflect the basal glycolytic rate. Subsequently, oligomycin was injected; as an ATP synthase inhibitor, it can impair mitochondrial respiration, shift energy metabolism toward glycolysis, and the increased extracellular acidification rate (ECAR) reflects the maximum glycolytic capacity of cells. As shown in Fig. 5 , compared with the control group, the glycolytic rate in the XYT group, RAPA group, and XYT+RAPA group was significantly slowed, and the maximum glycolytic capacity was also significantly reduced (P < 0.01). Further calculation showed that the glycolytic reserve in each intervention group was significantly lower than that in the control group, with statistically significant differences (P 0.05), while the maximum glycolytic capacity and glycolytic reserve in the XYT+RAPA group were significantly lower than those in the XYT group (P < 0.01). Compared with the RAPA group, the maximum glycolytic capacity and glycolytic reserve in the XYT+RAPA group were significantly decreased (P < 0.01). Similarly, Fig. 5 indicates that 3-MA intervention could not significantly downregulate the maximum glycolytic capacity or glycolytic reserve; however, compared with the individually use of XYT or 3-MA, the combination of 3-MA and XYT significantly downregulated the maximum glycolytic capacity and glycolytic reserve of cells (P < 0.01).Based on the above experimental results, it can be concluded that XYT not only weakens the glycolytic rate of lung cancer cells but also significantly reduces their glycolytic reserve capacity. Subsequently, Western Blot was used to evaluate the expression levels of key enzymes in the glycolysis process, and their changing trends were found to be consistent with the glycolytic levels. Finally, Western Blot was used to detect the changes in glycolysis-related molecules.Western blot was used to assess the expression levels of HK2, PFKFB3, PKM, GLUT1, GLUT4, LDHA, and HIF-1α proteins in A549 cells following intervention with XYT-containing serum alone or in combination with RAPA. As shown in Fig. 6, compared with the control group, the expression levels of HK2, PFKFB3, PKM, GLUT1, GLUT4, LDHA, and HIF-1α in the XYT group, RAPA group, and XYT+RAPA group were all significantly downregulated, with statistically significant differences (P < 0.05 or P < 0.01). Compared with the XYT group, the RAPA group exhibited significantly lower expression levels of PKM (P < 0.05), GLUT4 (P < 0.05), LDHA (P < 0.05), and HIF-1α (P 0.05). The XYT+RAPA group showed significantly lower expression levels of HK2 (P < 0.01), PFKFB3 (P < 0.05), PKM (P < 0.01), GLUT1 (P < 0.05), GLUT4 (P < 0.01), LDHA (P < 0.01), and HIF-1α (P < 0.01) compared with the XYT group. In comparison with the RAPA group, intervention with XYT+RAPA significantly reduced the expression levels of HK2 (P < 0.05), PFKFB3 (P < 0.05), PKM (P < 0.05), GLUT1 (P < 0.05), GLUT4 (P < 0.05), LDHA (P < 0.05), and HIF-1α (P < 0.05), with all differences being statistically significant.In addition, after A549 cells were intervened with XYT-containing serum alone or in combination with 3-MA, Western blot was performed to detect the expression of HK2, PFKFB3, PKM, GLUT1, GLUT4, LDHA, and HIF-1α proteins in the cells. As shown in Fig. 34, compared with the control group, XYT intervention significantly decreased the expression of HK2 (P < 0.01), PFKFB3 (P < 0.01), PKM (P < 0.01), GLUT1 (P < 0.05), GLUT4 (P < 0.01), LDHA (P < 0.05), and HIF-1α (P < 0.05). 3-MA intervention downregulated the expression of HK2 (P < 0.01), GLUT4 (P < 0.01), and LDHA (P 0.05). XYT + 3-MA intervention significantly reduced the expression of HK2 (P < 0.01), PFKFB3 (P < 0.01), PKM (P < 0.01), GLUT4 (P < 0.01), and LDHA (P 0.05). Compared with the XYT group, the 3-MA group had significantly lower expression levels of HK2 (P < 0.01), PFKFB3 (P < 0.05), PKM (P < 0.01), and GLUT1 (P < 0.01), and significantly higher HIF-1α (P 0.05). The XYT + 3-MA group showed significant changes in the expression levels of HK2 (P < 0.05), PFKFB3 (P < 0.01), PKM (P < 0.05), GLUT1 (P < 0.05), GLUT4 (P < 0.05), and LDHA (P 0.05). Compared with the 3-MA group, XYT + 3-MA intervention significantly reduced the expression levels of HK2 (P < 0.01), PFKFB3 (P < 0.01), PKM (P < 0.01), GLUT1 (P < 0.05), GLUT4 (P < 0.01), LDHA (P < 0.05), and HIF-1α (P < 0.05), with all differences being statistically significant. The respiration of normal cells includes aerobic respiration and anaerobic glycolysis. Previous studies have preliminarily confirmed the effect of XYT on the glycolytic level of lung cancer cells, so this part intends to further investigate its impact on aerobic respiration (i.e., OXPHOS). The cellular OCR was measured using the Seahorse platform. Firstly, the basal respiration of cells was detected. Subsequently, oligomycin was added to inhibit ATP synthesis in mitochondria, so as to evaluate the ATP level generated by mitochondria to meet the energy demand of cells under this condition. Then, FCCP was added, which can stimulate the maximum operation of the mitochondrial respiratory chain and promote the rapid oxidation of substrates to cope with this metabolic challenge, and at this time, the maximum value of oxygen consumption rate can be obtained. Finally, rotenone and antimycin A were added to inhibit the mitochondrial respiration process. The results of the OCR experiment (Fig. 7 ) showed that compared with the control group, the O₂ consumption in the XYT group, RAPA group, and XYT+RAPA group significantly increased, and parameters such as basal respiration and maximum respiration were significantly upregulated, with statistically significant differences (P < 0.05 or P 0.05), while the basal respiration (P < 0.01) and maximum respiration (P < 0.01) in the XYT+RAPA group showed an increasing trend with statistically significant differences. Compared with the RAPA group, the basal respiration (P < 0.01) and maximum respiration (P < 0.01) in the XYT+RAPA group significantly increased, with statistically significant differences. Similar conclusions can be drawn from the 3-MA intervention. These results indicate that XYT or RAPA intervention can alleviate the cumulative damage of mitochondria in A549 cells, which is manifested by an increase in the number of mitochondria and a partial recovery of OXPHOS capacity. Figure 6 Protein expression of glycolysis-related molecules in cells of each group ( ±s, n = 3). Note: Compared with the control group, *P < 0.05, **P < 0.01; compared with the XYT group, #P < 0.05, ##P < 0.01; compared with the RAPA group.△P < 0.05. 3 Discussion Lung cancer is one of the malignant tumors with high morbidity and mortality. Clinical treatments for NSCLC mainly include surgery, radiotherapy, chemotherapy, immunotherapy, and molecular targeted therapy . 17 However, recurrence and metastasis occur in approximately 20% of patients with early-stage lung cancer after surgery, and the 5-year survival rate of patients receiving chemotherapy alone is less than 5% . 18,19 In recent years, traditional Chinese medicine (TCM) has shown promising efficacy in the comprehensive treatment of lung cancer. It can improve the body’s immune function, inhibit lung cancer cell proliferation and metastasis, thereby significantly enhancing patients’ quality of life and prolonging their survival . 20 Director Jia Yingjie believes that lung cancer is characterized by "deficiency in origin," where primordial qi deficiency forms the pathological basis of lung cancer, while toxin, stasis, and phlegm are its pathological products . 21 XYT aligns with the core pathogenesis of lung cancer—deficiency, toxin, stasis, and phlegm—and is formulated under the guidance of the "turbidity-eliminating and constitution-nourishing" principle. Clinically, modified XYT combined with radiotherapy and chemotherapy has achieved favorable outcomes in treating lung cancer, effectively reducing metastasis and drug resistance. However, its molecular mechanism remains unclear, which prompted this preliminary investigation. The Warburg effect is a hallmark of tumor cells, characterized by high lactate production and glucose consumption, accompanied by enhanced glycolysis . 22 Tumor cells proliferate rapidly, consuming large amounts of oxygen, which easily leads to a hypoxic environment. The glycolytic pathway can enhance their hypoxia tolerance, thereby effectively avoiding apoptosis . 23 Intermediates of glycolysis, such as glucose-6-phosphate and pyruvate, serve as raw materials for biosynthesis in tumor cells, providing essential substances for their growth. Additionally, lactate produced during glycolysis acidifies the tumor microenvironment, disrupts the extracellular matrix, and facilitates tumor invasion and metastasis . 24 Further studies on TCM have revealed that some herbal medicines can regulate glycolysis by modulating related proteins in the glycolytic pathway, thereby influencing tumor progression. Research has shown that a TCM decoction inhibited the growth and epithelial-mesenchymal transition (EMT) of gastric cancer (GC) cells by reducing glycolysis in a PKM2-dependent manner . 25 Therefore, this study detected the glycolytic level and expression of related proteins in A549 cells after XYT treatment. The results suggested that XYT can inhibit lactate production and glucose consumption in A549 cells and downregulate the expression of glycolysis-related proteins. Autophagy, a conserved physiological catabolic process, is a unique mechanism evolved in eukaryotic cells and plays an indispensable role in maintaining cellular homeostasis . 26 It exerts a pivotal effect in metabolic reprogramming of tumor cells, with a close association with the tricarboxylic acid cycle, a core metabolic part. A large number of experimental results have demonstrated that metabolic processes such as glucose metabolism, lipid metabolism, and amino acid metabolism are closely linked to cancer progression, and the critical role of autophagy in these processes has become increasingly prominent . 27,28 Qin et al. 29 found that inhibiting autophagy can effectively promote glycolytic activity in gastric cancer cells through the ROS-NF-κB-HIF-1α pathway. In addition, FUNC1-mediated mitophagy suppresses the inflammatory response by clearing dysfunctional mitochondria, thereby exerting an inhibitory effect during the initial stage of hepatocellular carcinoma (HCC) development. 30 In this study, XYT was found to upregulate the expression of LC3-II/LC3-I and downregulate the expression of P62 in tumor tissues and cells. Consistent with these findings, transmission electron microscopy results also confirmed that XYT induced more autophagosomes in A549 cells. All these results indicate that XYT can effectively induce cellular autophagy. To further clarify whether XYT regulates glucose metabolism reprogramming through autophagy, we used methods such as Western blot, Seahorse Glycolysis Stress Test Kit, Seahorse Mitochondrial Stress Test Kit, lactate production kit, and glucose uptake kit in vitro to detect changes in glucose metabolism reprogramming after autophagy inhibition and activation. The results showed that, similar to RAPA, XYT-containing serum could downregulate the expression levels of key glycolytic enzymes such as HIF-1α, HK2, PFKFB3, PKM, GLUT1, GLUT4, and LDHA in A549 cells, reduce glycolytic reserve and maximum glycolytic capacity, decrease lactate production, and reduce glucose uptake. Similarly, after pretreatment of A549 cells with 3-MA, XYT-containing serum could also reduce the expression of key glycolytic enzymes, slow down glycolytic rate, decrease lactate production, and reduce glucose uptake. Moreover, the inhibitory effect of XYT combined with autophagy regulators was stronger than that of single-drug treatment. HIF-1α regulates a variety of tumor processes for adaptation, including metabolism, angiogenesis, invasion and cell proliferation. Studies showed that HIF-1α could modulate various EMT transcription factors, histone modifiers, enzymes (MMPs), chemokine receptors . 31 Research indicated that HIF-1α affects mitochondrial fission 1 protein (Fis1) and dynamin-related protein 1 (DRP1) related to mitochondrial dynamics, thereby inducing Parkin-associated mitophagy . 32 Studies have shown that HIF-1α can directly induce autophagy in tumor cells. In colorectal cancer cells, high expression of HIF-1α is associated with increased autophagy levels, and HIF-1α promotes autophagy by regulating the expression of autophagy-related genes, thereby helping tumor cells survive and proliferate in harsh environments such as hypoxia. Results suggested that liensinine can inhibit autophagy by targeting HIF-1 alpha/eNOS . 33 In this study, it was found that the expression of HIF-1α in A549 cells was significantly affected after XYT treatment, and the inhibitory effect of XYT combined with autophagy regulators was stronger than that of single-drug treatment. These findings further confirm that XYT can enhance the inhibitory effect of RAPA or 3-MA on cellular glycolysis through the HIF-1α signaling pathway and autophagy, thereby exerting an anti-tumor effect. 4.Conclusion This study demonstrates that Xiaoyan Decoction inhibits NSCLC progression through metabolic reprogramming via three key mechanisms: ( 1 ) inducing autophagy (increased autophagosomes, LC3-II/LC3-I ratio, decreased P62); ( 2 ) suppressing glycolysis (reduced glucose uptake, lactate production, glycolytic enzymes) while enhancing oxidative phosphorylation (elevated OCR); and ( 3 ) modulating the HIF-1α pathway. The coordinated regulation of the autophagy-HIF-1α-glycolysis axis by XYT provides novel insights into traditional Chinese medicine's anti-cancer mechanisms.However, our study also has its limitations.The experiments were confined to in vitro A549 cell model, lacking validation in animal tumors or patient-derived tissues.Clinical translatability remains unassessed, as dosing regimens and pharmacokinetics of XYT’s bioactive components in humans were not explored. Potential crosstalk with other oncogenic pathways (e.g., mTOR, Akt) was not fully dissected, leaving broader mechanistic networks incompletely mapped. Clinical prospective studies and further in vitro and ex vivo experiments through targeted metabolomics, target organ gene knockdown and other technologies can be carried out to explore the specific mechanism of action of this formula. Declarations Ethics approval The ethics approval is not applicable in this research. Consent to publish Not applicable. Conflicts of interests The authors maintain the utmost transparency and hereby declare the absence of any potential conflicts of interest pertaining to this study. Funding The author(s) disclosed receipt of the following financial support for the research,authorship, and/or publication of this article: This article was supported by Tianjin Education Commission scientific research project (grant number 2021KJ143). Author Contribution Xiaoqun Wang: Conceptualization, Writing – Original Draft;Yuting Li: Investigation,Formal Analysis, Data Curation;Suxuan Ouyang: Writing – Original Draft;Haojian Zhang: Writing – Review & Editing;Linlin Zhao: Writing – Review & Editing;Jing Zhang: Writing – Review & Editing;Fanming Kong: Supervision, Writing – Review & Editing (Corresponding Author);Yingjie Jia: Supervision, Writing – Review & Editing (Corresponding Author). Acknowledgements We thank Professor Yingjie Jia and Professor Fanming Kong for their guidance and assistance with this work. Data Availability All relevant data selected for the study are included in the article. References Siegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. Cancer statistics, 2025. CA Cancer J Clin. 2025;75(1):10–45. 10.3322/caac.21871 . Li Y, Liu T, Wang X, Jia Y, Cui H. Autophagy and Glycometabolic Reprograming in the Malignant Progression of Lung Cancer: A Review. Technol Cancer Res Treat. 2023;22:15330338231190545. 10.1177/15330338231190545 . Warburg OH. The classic: The chemical constitution of respiration ferment. Clin Orthop Relat Res. 2010;468(11):2833–9. 10.1007/s11999-010-1534-y . Wang K, Huang W, Chen R, et al. Di-methylation of CD147-K234 Promotes the Progression of NSCLC by Enhancing Lactate Export. Cell Metab. 2023;35(6):1084. 10.1016/j.cmet.2023.05.006 . Xu JQ, Fu YL, Zhang J, et al. Targeting glycolysis in non-small cell lung cancer: Promises and challenges. Front Pharmacol. 2022;13:1037341. 10.3389/fphar.2022.1037341 . Zhang L, Li M, Li X, et al. Deciphering the role of PLCD3 in lung cancer: A gateway to glycolytic reprogramming via PKC-Rap1 activation. Heliyon. 2024;10(17):e37063. 10.1016/j.heliyon.2024.e37063 . Liu L, Chai L, Ran J, Yang Y, Zhang L. BAI1 acts as a tumor suppressor in lung cancer A549 cells by inducing metabolic reprogramming via the SCD1/HMGCR module. Carcinogenesis. 2020;41(12):1724–34. 10.1093/carcin/bgaa036 . Song H, Qiu Z, Wang Y, et al. HIF-1α/YAP Signaling Rewrites Glucose/Iodine Metabolism Program to Promote Papillary Thyroid Cancer Progression. Int J Biol Sci. 2023;19(1):225–41. 10.7150/ijbs.75459 . Courtnay R, Ngo DC, Malik N, Ververis K, Tortorella SM, Karagiannis TC. Cancer metabolism and the Warburg effect: the role of HIF-1 and PI3K. Mol Biol Rep. 2015;42(4):841–51. 10.1007/s11033-015-3858-x . Nazio F, Bordi M, Cianfanelli V, Locatelli F, Cecconi F. Autophagy and cancer stem cells: molecular mechanisms and therapeutic applications. Cell Death Differ. 2019;26(4):690–702. 10.1038/s41418-019-0292-y . Liang L, Hui K, Hu C, et al. Autophagy inhibition potentiates the anti-angiogenic property of multikinase inhibitor anlotinib through JAK2/STAT3/VEGFA signaling in non-small cell lung cancer cells. J Exp Clin Cancer Res. 2019;38(1):71. 10.1186/s13046-019-1093-3 . Jiao L, Zhang HL, Li DD, et al. Regulation of glycolytic metabolism by autophagy in liver cancer involves selective autophagic degradation of HK2 (hexokinase 2). Autophagy. 2018;14(4):671–84. 10.1080/15548627.2017.1381804 . Wang X, Li Z, Gao Z, et al. Cadmium induces cell growth in A549 and HELF cells via autophagy-dependent glycolysis. Toxicol Vitro. 2020;66:104834. 10.1016/j.tiv.2020.104834 . Yang Y, Song B, Guo M, et al. p53-dependent HIF-1α /autophagy mediated glycolysis to support Cr(VI)-induced cell growth and cell migration. Ecotoxicol Environ Saf. 2024;272:116076. 10.1016/j.ecoenv.2024.116076 . Zheng X, Han Y, Gu L, Gao S, Lv Y, Li C. Study of the mechanism by which Xiaoyan decoction combined with E7449 regulates tumorigenesis in lung adenocarcinoma. J Cell Mol Med. 2024;28(12):e18467. 10.1111/jcmm.18467 . Yang P, Xu W, Liu L. Study on the autophagy and resistance protein affected by Xiaoyan decoction in A549/DDP cells(in Chinese). Tianjin J Traditional Chin Med. 2016;33(06):358–62. Duma N, Santana-Davila R, Molina JR. Non-Small Cell Lung Cancer: Epidemiology, Screening, Diagnosis, and Treatment. Mayo Clin Proc . 2019;94(8):1623–1640. 10.1016/j.mayocp.2019.01.013 Chang JY, Mehran RJ, Feng L, et al. Stereotactic ablative radiotherapy for operable stage I non-small-cell lung cancer (revised STARS): long-term results of a single-arm, prospective trial with prespecified comparison to surgery. Lancet Oncol. 2021;22(10):1448–57. 10.1016/S1470-2045(21)00401-0 . Gettinger S, Horn L, Jackman D, et al. Five-Year Follow-Up of Nivolumab in Previously Treated Advanced Non-Small-Cell Lung Cancer: Results From the CA209-003 Study. J Clin Oncol. 2018;36(17):1675–84. 10.1200/JCO.2017.77.0412 . Li Z, Feiyue Z, Gaofeng L. Traditional Chinese medicine and lung cancer-From theory to practice. Biomed Pharmacother. 2021;137:111381. 10.1016/j.biopha.2021.111381 . Wang X, Liao D, Xu J, Kong F, Jia Y. Connotation of Eliminating Turbidity and Banking up Essence and Its Application in Differentiation and Treatment of Malignant Tumor(in Chinese). J Tradit Chin Med. 2023;64(06):545–9. Liberti MV, Locasale JW. The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem Sci. 2016;41(3):211–8. 10.1016/j.tibs.2015.12.001 . Kroemer G, Pouyssegur J. Tumor cell metabolism: cancer’s Achilles’ heel. Cancer Cell. 2008;13(6):472–82. 10.1016/j.ccr.2008.05.005 . Gatenby RA, Gawlinski ET, Gmitro AF, Kaylor B, Gillies RJ. Acid-mediated tumor invasion: a multidisciplinary study. Cancer Res. 2006;66(10):5216–23. 10.1158/0008-5472.CAN-05-4193 . Sun Q, Yuan M, Wang H, et al. PKM2 Is the Target of a Multi-Herb-Combined Decoction During the Inhibition of Gastric Cancer Progression. Front Oncol. 2021;11:767116. 10.3389/fonc.2021.767116 . Klassen A, Faccio AT, Canuto GAB, et al. Metabolomics: Definitions and Significance in Systems Biology. Adv Exp Med Biol. 2017;965:3–17. 10.1007/978-3-319-47656-8_1 . Ferro F, Servais S, Besson P, Roger S, Dumas JF, Brisson L. Autophagy and mitophagy in cancer metabolic remodelling. Semin Cell Dev Biol. 2020;98:129–38. 10.1016/j.semcdb.2019.05.029 . Kikuchi N, Soga T, Nomura M, et al. Comparison of the ischemic and non-ischemic lung cancer metabolome reveals hyper activity of the TCA cycle and autophagy. Biochem Biophys Res Commun. 2020;530(1):285–91. 10.1016/j.bbrc.2020.07.082 . Qin W, Li C, Zheng W, et al. Inhibition of autophagy promotes metastasis and glycolysis by inducing ROS in gastric cancer cells. Oncotarget. 2015;6(37):39839–54. 10.18632/oncotarget.5674 . Li W, Li Y, Siraj S, et al. FUN14 Domain-Containing 1-Mediated Mitophagy Suppresses Hepatocarcinogenesis by Inhibition of Inflammasome Activation in Mice. Hepatology. 2019;69(2):604–21. 10.1002/hep.30191 . Tam SY, Wu VWC, Law HKW. Hypoxia-Induced Epithelial-Mesenchymal Transition in Cancers: HIF-1α and Beyond. Front Oncol. 2020;10:486. 10.3389/fonc.2020.00486 . Pan WL, Ma RJ, Hou YX, Wang Y, Sun SC. HIF-1α regulates mitochondria function for oxidative stress and autophagy during oocyte maturation. AA. 2025;2(1):0–0. 10.48130/animadv-0025-0014 . Feng Z, Zhang S, Han Q, et al. Liensinine sensitizes colorectal cancer cells to oxaliplatin by targeting HIF-1α to inhibit autophagy. Phytomedicine. 2024;129:155647. 10.1016/j.phymed.2024.155647 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 Mar, 2026 Reviewers agreed at journal 09 Mar, 2026 Reviewers invited by journal 12 Feb, 2026 Submission checks completed at journal 08 Feb, 2026 First submitted to journal 04 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8540053","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":590290928,"identity":"7661094b-0ec4-4fc3-bb10-ea209c2d1e88","order_by":0,"name":"xiaoqun Wang","email":"","orcid":"","institution":"First Teaching Hospital of Tianjin University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"xiaoqun","middleName":"","lastName":"Wang","suffix":""},{"id":590290929,"identity":"5a818bc2-67ca-4bb0-a909-34c016b9dcef","order_by":1,"name":"yuting Li","email":"","orcid":"","institution":"First Teaching Hospital of Tianjin University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"yuting","middleName":"","lastName":"Li","suffix":""},{"id":590290930,"identity":"380297cd-6c2c-402d-833e-7451efd404bc","order_by":2,"name":"suxuan OUYANG","email":"","orcid":"","institution":"First Teaching Hospital of Tianjin University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"suxuan","middleName":"","lastName":"OUYANG","suffix":""},{"id":590290931,"identity":"9e4e1657-1258-402c-a9f0-598906c360eb","order_by":3,"name":"haojian Zhang","email":"","orcid":"","institution":"First Teaching Hospital of Tianjin University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"haojian","middleName":"","lastName":"Zhang","suffix":""},{"id":590290932,"identity":"d2fe0f07-29a6-4fe2-ac0b-fa229c36039e","order_by":4,"name":"linlin Zhao","email":"","orcid":"","institution":"First Teaching Hospital of Tianjin University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"linlin","middleName":"","lastName":"Zhao","suffix":""},{"id":590290933,"identity":"3a1b2ef0-eae5-4d7d-987f-ce0bc3bd4338","order_by":5,"name":"jing Zhang","email":"","orcid":"","institution":"First Teaching Hospital of Tianjin University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"jing","middleName":"","lastName":"Zhang","suffix":""},{"id":590290934,"identity":"115dcafd-c0ab-4e62-9dfc-338342aee9f2","order_by":6,"name":"fanming Kong","email":"","orcid":"","institution":"First Teaching Hospital of Tianjin University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"fanming","middleName":"","lastName":"Kong","suffix":""},{"id":590290935,"identity":"b4138d92-038d-4b3a-b6b3-14b8be7713dd","order_by":7,"name":"yingjie Jia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYBACxmYgkcDAwMzGwNj4IKHChoGNBC3MzQYPzqQR1oIE2NsEH7YcJqyOuZ35mcTDHbXsfOyNbQyJDeft+aSbHzD8qNiGx2FsZhKJZ44zs/EcbHuQuON2YpvMMQPGnjO38fkFqKXtGDObRGK7QeKZ2wlsEgkGzIxt+LSwf4NokX/YBmScs2eTSP9AQAsPyJYaoC2MIC0HgGQOIVt4ii2AKoF+SWw2SDiTnAjUUnAQn18M+49vvPmzrS5Zvv34w4c/Kuzs5Wekb3zwowKPlgYGFgkGhsPJKKIHcKoHAnlg1HxgYKizw6doFIyCUTAKRjgAAH/1VyAeBp0gAAAAAElFTkSuQmCC","orcid":"","institution":"First Teaching Hospital of Tianjin University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"yingjie","middleName":"","lastName":"Jia","suffix":""}],"badges":[],"createdAt":"2026-01-07 10:23:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8540053/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8540053/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103013706,"identity":"511ae814-017f-4371-ae3d-a7efd15ae42e","added_by":"auto","created_at":"2026-02-19 16:02:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34393,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of XYT on the activity of A549 cells\u003cstrong\u003e (a)\u003c/strong\u003eEffect of XYT-containing serum on the activity of A549 cells.\u003cstrong\u003e(b)\u003c/strong\u003e IC50 values of XYT serum at different time.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8540053/v1/1c0acff970e72cf808ea651d.jpg"},{"id":103013707,"identity":"95de243c-ef27-48c6-8614-d4a8fddbee8d","added_by":"auto","created_at":"2026-02-19 16:02:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70733,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscopy of A549 cell ultrastructure (×5000). red arrow:autophagosome\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8540053/v1/fbf44ae613290690daf39b1a.jpg"},{"id":103013710,"identity":"d2f04773-a043-4fa2-afd0-3b276b2939c2","added_by":"auto","created_at":"2026-02-19 16:02:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":80756,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of autophagy marker proteins in cells of each group \u003cstrong\u003e(a) \u003c/strong\u003eP62 and LC3-II/LC3-I protein expression after RAPA intervention.\u003cstrong\u003e (b)\u003c/strong\u003e P62 and LC3-II/LC3-I protein expression after 3-MA intervention. Note: Compared with the control group, *P\u0026lt;0.05, **P\u0026lt;0.01; compared with the XYT group, #P\u0026lt;0.05, ##P\u0026lt;0.01; compared with the RAPA/3-MAgroup, △P\u0026lt;0.05, △△P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8540053/v1/1ec9e4ba6fe6d03bc8cd2b49.jpg"},{"id":103013711,"identity":"970592ce-8939-48b9-872a-25d32abb477c","added_by":"auto","created_at":"2026-02-19 16:02:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57334,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in glucose uptake and lactate production in various groups of cells.\u003cstrong\u003e(a)\u003c/strong\u003e Glucose uptake in cells in each group.\u003cstrong\u003e(b)\u003c/strong\u003e Extracellular lactate production in each group.Note: Compared with the control group, *P\u0026lt;0.05, **P\u0026lt;0.01; compared with the XYT group, #P\u0026lt;0.05; compared with the RAPA/3-MA group, △P\u0026lt;0.05, △△P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8540053/v1/4688e1c73251c9b522571398.jpg"},{"id":103050277,"identity":"28f4279b-1b43-43f5-b0e5-a499909a8e11","added_by":"auto","created_at":"2026-02-20 07:49:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98354,"visible":true,"origin":"","legend":"\u003cp\u003eGlycolytic stress test curves for each group of cells. Note: Compared with the control group, **P\u0026lt;0.01; compared with the XYT group, ##P\u0026lt;0.01; compared with the RAPA/3-MA group, △△P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8540053/v1/e6cdad429330e49d8168fb1c.jpg"},{"id":103013712,"identity":"3b18bcb7-bed2-48fb-bc8a-208c02bacb78","added_by":"auto","created_at":"2026-02-19 16:02:55","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":141532,"visible":true,"origin":"","legend":"\u003cp\u003eProtein expression of glycolysis-related molecules in cells of each group (±s，n=3). Note: Compared with the control group, *P\u0026lt;0.05, **P\u0026lt;0.01; compared with the XYT group, #P\u0026lt;0.05, ##P\u0026lt;0.01; compared with the RAPA group.△P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8540053/v1/05ee7a2fc7f470d8b4e78e6f.jpg"},{"id":103049571,"identity":"f80deb98-e233-4f3b-82c2-50ef3315142c","added_by":"auto","created_at":"2026-02-20 07:42:45","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":132606,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial stress test curves for each group of cells. Note: Compared with the control group, *P\u0026lt;0.05, **P\u0026lt;0.01; compared with the XYT group, ##P\u0026lt;0.01; compared with the RAPA/3-MA group, ΔΔP\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8540053/v1/6896153c1d73b4aa642d1a3f.jpg"},{"id":103051204,"identity":"95223952-25d1-4c0d-b088-1bbef6bcb060","added_by":"auto","created_at":"2026-02-20 07:58:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1497725,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8540053/v1/74f9fc8f-a6e9-4bef-8357-7e20f892c0bc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanism Study of Xiaoyan Decoction in the Treatment of Non-small Cell Lung Cancer Through Glycometabolic Reprogramming","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eLung cancer remains the most prevalent malignancy globally. According to the latest U.S. cancer statistics report, approximately 2.04\u0026nbsp;million new cancer cases and 620,000 cancer-related deaths are projected in the United States for 2025. Lung cancer persists as the leading cause of cancer mortality, accounting for over 20% of all cancer deaths .\u003csup\u003e1\u003c/sup\u003e Histologically, lung cancer is classified into NSCLC and small cell lung cancer (SCLC). NSCLC constitutes approximately 83% of all lung cancer cases .\u003csup\u003e2\u003c/sup\u003e Alarmingly, about two-thirds of patients present with stage III/IV disease at diagnosis. The high malignancy, elevated risk of recurrence and metastasis, and limited therapeutic efficacy pose significant challenges in clinical management.\u003c/p\u003e \u003cp\u003eGlycolysis, the process by which cells catabolize glucose to generate energy under hypoxic conditions, is notably hijacked by cancer cells. Otto Warburg observed that tumor cells preferentially rely on glycolysis for energy production even in the presence of adequate oxygen, a phenomenon termed the \"Warburg effect\" .\u003csup\u003e3\u003c/sup\u003e Substantial evidence confirms enhanced glycolytic activity in NSCLC cells, characterized by elevated expression of key glycolytic enzymes and increased lactate production .\u003csup\u003e4,5\u003c/sup\u003e Critically, specific glycolytic enzymes are implicated in lung cancer pathogenesis. For instance,\u003c/p\u003e \u003cp\u003ePhospholipase C delta 3 (PLCD3) triggers the protein kinase C (PKC), subsequently stimulating the Rap1 pathway. This cascade induces glycolytic reprogramming, supplying the energy and metabolic substrates essential for lung cancer cell proliferation and dissemination. Moreover, PLCD3 promotes metastasis and invasion via Rap1 (Ras-related protein 1) pathway activation .\u003csup\u003e6\u003c/sup\u003e Conversely, Brain-specific angiogenesis inhibitor 1 (BAI1) inhibits the Warburg effect and glycolysis by upregulating stearoyl-CoA desaturase 1 (SCD1) and suppressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), thereby inducing metabolic reprogramming .\u003csup\u003e7\u003c/sup\u003e Consequently, targeting glycolysis has emerged as a promising therapeutic strategy in oncology research.\u003c/p\u003e \u003cp\u003eHIF-1α serves as a master regulator of cellular energy metabolism .\u003csup\u003e8\u003c/sup\u003e It dimerizes with the hypoxia-inducible factor 1 beta subunit (HIF-1β), binds to hypoxia-response elements (HREs) in chromatin, and transactivates target genes involved in metabolic adaptation. Key transcriptional targets include pyruvate dehydrogenase kinase (PDK), pyruvate dehydrogenase (PDH), glucose transporters (GLUT1, GLUT3), monocarboxylate transporters (MCT1, MCT4), the mitochondrial pyruvate carrier (MPC), lactate dehydrogenase A (LDHA), adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), and glycolytic enzymes (e.g. Hexokinase1(HK1), Hexokinase2(HK2), phosphofructokinase (PFK), Pyruvate kinase M(PKM), alpha-enolase (ENO1)) .\u003csup\u003e9\u003c/sup\u003e Collectively, HIF-1α plays a pivotal role in mediating hypoxia adaptation and aerobic glycolysis (the Warburg effect) in cancer.\u003c/p\u003e \u003cp\u003eAutophagy, a conserved catabolic process unique to eukaryotic cells, involves the sequestration of cytoplasmic components within double-membrane vesicles (autophagosomes) for lysosomal degradation. It is a fundamental cellular mechanism crucial for maintaining homeostasis, facilitating intracellular turnover, and responding to stress .\u003csup\u003e10\u003c/sup\u003e Autophagy exhibits a complex, context-dependent role in lung cancer progression, significantly influencing NSCLC pathophysiology. Liang et al. demonstrated that anlotinib induces protective autophagy in human lung cancer cells, evidenced by increased autophagosome formation. This autophagy enhances anlotinib's anti-angiogenic properties via the JAK2/STAT3/VEGFA signaling pathway, ultimately suppressing NSCLC cell proliferation and inducing apoptosis .\u003csup\u003e11\u003c/sup\u003e Similarly, Wang et al. reported that ginkgolide B induces Beclin-1-dependent autophagy in lung cancer cells. Furthermore, autophagy critically regulates glycolytic metabolism. Jiao et al. established a negative correlation between autophagy and glycolysis, mediated partly through the regulation of HK2, a key glycolytic enzyme .\u003csup\u003e12\u003c/sup\u003e Wang et al.\u003csup\u003e13\u003c/sup\u003e found that cadmium-induced glycolysis is autophagy-dependent, and the autophagy-glycolysis axis plays an important role in the proliferation of cadmium-treated A549 cells. In addition, HIF-1α, a protein associated with hypoxic adaptation, is stably expressed in cancer cells, thereby regulating autophagy and glycolysis .\u003csup\u003e14\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eXiaoyan Decoction (Jinyao Zhizi Z20060786) is an empirical prescription developed by Professor Jia Yingjie, a national renowned traditional Chinese medicine physician. It consists of 7 Chinese medicinal herbs, including Astragalus membranaceus, Radix Pseudostellariae, Hedyotis diffusa, raw Oyster, Prunella vulgaris, Curcuma aromatica, and Curcuma longa. Previous studies have demonstrated that XYT can effectively inhibit tumor cell proliferation, reverse multidrug resistance, and induce cell apoptosis .\u003csup\u003e15\u003c/sup\u003e Notably, it also exerts a certain regulatory effect on cell autophagy ,\u003csup\u003e16\u003c/sup\u003e but its underlying molecular mechanism remains to be further explored.\u003c/p\u003e \u003cp\u003eThis study aims to investigate whether XYT exerts anti-NSCLC effects by regulating cellular glucose metabolism reprogramming through the HIF-1α signaling pathway and autophagy. Firstly, the CCK-8 assay was used to evaluate the effect of XYT-containing serum on the viability of A549 cells, so as to determine the optimal concentration and action time of XYT-containing serum for intervening A549 cells. Secondly, autophagy inhibitor 3-MA and autophagy activator RAPA were applied to A549 cells. Transmission electron microscopy was used to observe the overall autophagic status of A549 cells, and Western blot was employed to detect the expression levels of autophagy-related factors P62 and LC3-II/LC3-I proteins, thereby investigating the effect of XYT on autophagy in A549 cells. To further clarify whether XYT regulates glucose metabolism reprogramming through autophagy, methods such as Western blot, Seahorse Glycolysis Stress Test Kit, Seahorse Mitochondrial Stress Test Kit, lactate production kit, and glucose uptake kit were used to detect the following after autophagy inhibition and activation: the expression levels of key glycolytic enzymes including HK2, 6-Phosphofructo-2-Kinase(PFKFB3), PKM, GLUT1, GLUT4, LDHA, and HIF-1α; lactate production and glucose uptake in A549 cells, so as to clarify the changes in glucose metabolism reprogramming.\u003c/p\u003e"},{"header":"2.MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental cells and animals\u003c/h2\u003e \u003cp\u003eThe A549 cell line was purchased from Zhejiang Meisen Cell Technology Co., Ltd. (Product No.: CTCC-001-0036). Thirty male Balb/c nude mice (specific pathogen-free [SPF] grade), aged 4\u0026ndash;5 weeks and weighing 16\u0026ndash;18 g, were supplied by Beijing Huafukang Biotechnology Co., Ltd. (Animal Quality Certificate No.: 110322220103119965; License No.: SCXK(Jing)2019-0008). Mice were housed in the SPF-grade animal facility of Tianjin Guosheng Zhongyuan Technology Co., Ltd. The experimental protocol was approved by the Animal Ethics Committee of Yishengyuan Gene Technology (Tianjin) Co., Ltd. (Protocol No.: YSY-DWLL-2022133).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental drugs and reagents\u003c/h2\u003e \u003cp\u003eThe following Chinese herbal medicines were purchased from the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (TJUTM). The decoction was prepared by the decoction pharmacy of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. Preparation of drug-containing serum for the elimination of rocks: 20 male SD rats, 6\u0026ndash;8 weeks old, weighing 180\u0026ndash;220 g, were randomly divided into the normal group and the traditional Chinese medicine group (n\u0026thinsp;=\u0026thinsp;10). Rats in the normal group were gavaged with 2 mL of saline daily, while rats in the traditional Chinese medicine group were gavaged with 2 mL (12.25 g/kg) of the Chinese medicine solution of XYT daily for 7 consecutive days. After the last administration for 1h, rats in each group were anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital, and then blood was collected from the abdominal aorta. The blood was allowed to stand for 1h and then centrifuged at 3000 rpm for 10 min to collect the serum, which was then filtered on an ultra-clean bench using a 0.22 \u0026micro;m microporous filter to remove bacteria. The treated serum was divided and labeled as blank serum and Xieyan Tang drug-containing serum (hereinafter referred to as XYT drug-containing serum), and then stored in a refrigerator at -80\u0026deg;C for spare use. 2-Deoxyglucose(2-DG) and glutaraldehyde fixative were purchased from Shanghai Yuanye Company (batch no.: S11070, R20515); RPMI-1640 medium and hosphate buffer saline(PBS) were purchased from Cytiva Company, China (batch no.: AH30380359, AH29827185); penicillin-streptomycin solution, trypsin, lyophilization solution, Rainbow 180 broad-spectrum protein marker, BCA method protein quantitative analysis kit, and protein uploading buffer (2\u0026times;) were purchased from Solarbio, Beijing, China, (batch no. P7630, T1300, 24800, PR1910, PC0020, P1019); fetal bovine serum was purchased from Sbjbio, China (batch no.: BC-SE-FBS07); PVDF membrane was purchased from Merck Millipore (batch no.: IPVH00010); ECL Chemiluminescent Substrate Kit was purchased from Biosharp, China (batch no.: BL520B); 10\u0026times; TBST, electrophoresis solution and membrane transfer solution were purchased from Beyotime, Shanghai, China (batch nos. ST673, P0561, P0572); protease inhibitors and phosphatase inhibitors were purchased from Boster, Wuhan, China (batch nos. AR1183, AR1195); CCK-8 detection kit was purchased from Tongren Chemical Company, Japan (batch no. CK04); GLUT1 antibody, GLUT1 antibody, PFKFB3 antibody and LC3 antibody were purchased from CST (batch nos. 73015S, 13123S, 12741S); HK2 antibody, GLUT4 antibody, LDHA antibody, PKM antibody and SQSTM1/p62 antibody were purchased from Abcam (batch nos. ab104836, ab48547, ab101562, ab150377, ab150377, ab101562). MMP9 antibody and MMP2 antibody were purchased from Affinity (lot numbers: AF5228 and AF5330); β-actin antibody and HRP-labeled goat anti-rabbit IgG were purchased from Wuhan Proteintech (lot numbers: 20536-1-AP and SA00001-2); HiFiScript cDNA was used for the analysis of β-actin antibody and HRP-labeled goat anti-rabbit IgG. (batch no.: 20536-1-AP, SA00001-2); HiFiScript cDNA Synthesis Kit, UltraSYBR Mixture (Low ROX) were purchased from CW BIO, Jiangsu, China (batch no.: CW2569M, CW260M); DMSO was purchased from GENTIHOLD, China (batch no.: D8371); Seahorse XF Glycolytic Stress Test Kit, Seahorse XF Mitochondrial Stress Test Kit, Seahorse XF Basal Medium, XF 200mM Glutamine Solution, XF 100mM Sodium Pyruvate Solution, and 1.0M Glucose Solution were purchased from Agilent, USA (Lot Nos. 100, 103579-100, 103578-100, 103577-100); lactate and glucose test kits were purchased from Nanjing Jianjian Company, China (lot numbers: A019-2-1, A154-2-1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Instruments\u003c/h2\u003e \u003cp\u003eA multifunctional microplate reader was purchased from Thermo Fisher Scientific (USA). A fully automated gel imaging analysis system (ZF-288) was purchased from Shanghai Jiapeng Technology Co., Ltd. (China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell culture and treatment\u003c/h2\u003e \u003cp\u003e \u003cb\u003eCell culture\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA549 cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (PS) under conditions of 37℃and 5% CO₂.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell Revival\u003c/strong\u003e \u003cp\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Pre-warm a water bath to 37℃.(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Retrieve the cryopreserved cells from \u0026minus;\u0026thinsp;80℃ and immediately immerse in a 37℃ water bath for thawing.(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) After thawing, transfer the cell suspension into a 15 mL centrifuge tube, add 3 mL of complete medium, and centrifuge at 1000 rpm for 5 min.(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Discard the supernatant, resuspend the cell pellet in 1 mL of complete medium, and transfer into a 10 cm culture dish. Subsequently, add 8 mL of complete medium and place the dish in a constant-temperature incubator for continued culture.(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) Observe cell viability under a microscope on the following day.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMedium Change\u003c/strong\u003e \u003cp\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Remove the cultured cells from the incubator and examine their adherence and growth status under a microscope.(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Transfer the culture dish to a sterile biosafety cabinet, aspirate the old medium, and gently wash the cells once with PBS.(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Add 8\u0026ndash;10 mL of fresh complete medium.(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) After microscopic examination, return the dish to the incubator for continuous culture and monitor the cells periodically.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCell Subculture\u003c/b\u003e: When cell confluence reaches approximately 80%-90% as observed under a microscope, perform subculture as follows:(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Transfer the cells from the incubator to the biosafety cabinet, aspirate the medium, and wash once with PBS.(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Add 2 mL of trypsin to the culture dish and incubate at 37℃ for 1\u0026ndash;2 min. Monitor cell morphology dynamically under the microscope; digestion should be terminated when cells become rounded and appear brighter.(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Add 2 mL of complete medium to the dish, pipette repeatedly to detach the cells, and collect the cell suspension into a 15 mL centrifuge tube.(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Centrifuge at 1000 rpm for 5 min.(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) Discard the supernatant, resuspend the pellet in 1 mL of complete medium, mix thoroughly by pipetting, and transfer to a new 10 cm culture dish. Then add 7\u0026ndash;8 mL of complete medium and gently mix by drawing a \u0026ldquo;十\u0026rdquo; shape with the dish.(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) Place the dish in a 37℃ incubator and inspect cell status daily.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell Counting\u003c/b\u003e: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Following the same digestion procedure, resuspend the cell suspension repeatedly to ensure a uniform distribution of single cells for counting.(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Clean the hemocytometer with an alcohol swab, allow it to air-dry, and place a coverslip over the counting chamber.(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Dilute 10 \u0026micro;L of the cell suspension with 90 \u0026micro;L PBS (10-fold dilution) and mix thoroughly.(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Gently load 10 \u0026micro;L of the diluted suspension at the edge of the coverslip.(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) Under the microscope, count the cells in the four corner squares. Calculate the cell density using the formula:Cells per mL = (Total count in 4 squares\u0026thinsp;\u0026divide;\u0026thinsp;4)\u0026times;10\u0026times;10\u003csup\u003e4\u003c/sup\u003e.(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) Repeat the counting three times and take the average value as the final cell density.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell Cryopreservation\u003c/strong\u003e \u003cp\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Harvest cells in the logarithmic growth phase using the same digestion procedure, followed by centrifugation at 1000 rpm for 5 min.(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Discard the supernatant, add 1 mL of cell freezing medium, gently resuspend the pellet, and transfer to a cryovial. Seal the vial with parafilm.(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Label the vial with cell information and the date of cryopreservation. Store the cells at -80℃ or in liquid nitrogen for long-term preservation.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTreatment\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWorking solutions were prepared: 100 mL of the autophagy activator RAPA(10 nM) and 10 mL of the 3-MA(3 \u0026micro;M). Cells were divided into six experimental groups:Control group: No drug treatment; XYT group: Treated with 30% XYT drug-containing serum for 24 h; 3-MA group: Treated with 3 \u0026micro;M 3-MA for 1 h ; XYT\u0026thinsp;+\u0026thinsp;3-MA group: Pretreated with 3 \u0026micro;M 3-MA for 1 h, followed by treatment with 30% XYT serum for 24 h; RAPA group: Treated with 10 nM RAPA for 1 h ; XYT\u0026thinsp;+\u0026thinsp;RAPA group: Pretreated with 10 nM RAPA for 1 h, followed by treatment with 30% XYT serum for 24 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.5 CCK-8 cell activity\u003c/b\u003e: A549 cells were digested routinely and then inoculated into 96-well plates at a concentration of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL. A549 cells were routinely digested and then inoculated into 96-well plates at a concentration of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL. The cells were cultured overnight in a cell culture incubator, and the growth status of the cells was observed on the next day. The 96-well plate was divided into the following 10 groups according to the vertical arrangement of one group, as shown in the following table. In each group, there were 6 replicate wells, and after discarding the overnight medium, each well was refilled with 100 \u0026micro;L of working solution (working solution configuration is shown in the table 1 below). After incubation for different times (12 h, 24 h, 48 h), the medium of each\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.1\u003c/b\u003e Working Fluid Configuration Concentration Table\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOperating Fluid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eblank group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eComplete medium (without cells)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003econtrol group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eComplete medium (with cells)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eblank serum group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2 mL blank drug-containing serum dissolved in 1.8 mL DMEM medium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10% serum-containing group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2 mL XYT-containing serum dissolved in 1.8 mL DMEM medium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15% serum-containing group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.3 mL XYT-containing serum was dissolved in 1.7 mL DMEM medium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20% serum-containing group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4 mL XYT-containing serum was dissolved in 1.6 mL DMEM medium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25% serum-containing group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5 mL XYT-containing serum dissolved in 1.5 mL DMEM medium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30% serum-containing group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.6 mL XYT-containing serum was dissolved in 1.4 mL DMEM medium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35% serum-containing group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7 mL XYT-containing serum was dissolved in 1.3 mL DMEM medium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40% serum-containing group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8 mL XYT-containing serum was dissolved in 1.2 mL DMEM medium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ewell was discarded, and 10 \u0026micro;L of CCK-8 reagent and 90 \u0026micro;L of DMEM medium were added to the wells and incubated for 1\u0026ndash;4 h. Finally, the absorbance A value was read at 450 nm using an enzyme counter, and the cell viability was calculated, cell viability=(A experimental group-A blank group)/(A control group-A blank group)\u0026times;100%, and the experiment was repeated three times. The experiment was repeated 3 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Transmission electron microscopy\u003c/h2\u003e \u003cp\u003eThis experiment was commissioned to Tianjin Guosheng Zhongyuan Technology Co., Ltd. for completion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Western blot analysis\u003c/h2\u003e \u003cp\u003eCell protein extraction: Cells were digested and collected, washed twice with PBS, and total proteins were collected by adding RIPA lysis buffer. After quantification using the BCA method, electrophoresis and membrane transfer were performed, followed by blocking with 5% non-fat milk for 1 hour. Primary antibodies were added respectively, including HK2 antibody, GLUT1 antibody, GLUT4 antibody, LDHA antibody, MMP9 antibody, MMP2 antibody (all diluted at 1:1000), PFKFB3 antibody, LC3 antibody (both diluted at 1:2000), β-actin antibody (diluted at 1:3000), SQSTM1/p62 antibody (diluted at 1:5000), PKM antibody, and HRP-labeled goat anti-rabbit IgG (both diluted at 1:10000). After overnight incubation, the membranes were incubated with secondary antibody diluent (diluted at 1:10000) for 1 hour at room temperature. After washing, images were captured using a chemiluminescence imaging system. The gray values of target protein bands were analyzed with Image J software. Each experiment was repeated three times, and the average gray value was calculated. β-actin was used as the internal reference to analyze the expression level of target proteins in each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Determination of extracellular lactic acid content\u003c/h2\u003e \u003cp\u003eAfter 24 hours of drug treatment, the culture medium of each group was collected, centrifuged, and the supernatant was taken. The lactic acid content was detected according to the kit instructions.Lactic acid content in the culture medium (mmol\u0026middot;L-1) = (ODsample - ODblank) / (ODstandard - ODblank) \u0026times; Cstandard \u0026times; N, where Cstandard is the lactic acid content of the standard, and N is the sample dilution factor.Lactic acid production content\u0026thinsp;=\u0026thinsp;lactic acid content in the culture medium after drug treatment - lactic acid content in the original culture medium.Relative lactic acid production rate (%) = (lactic acid production content in the experimental group / lactic acid production content in the control group) \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Determination of cellular glucose uptake\u003c/h2\u003e \u003cp\u003eAfter 24 hours of drug treatment, the culture medium of each group was collected, centrifuged, and the supernatant was taken. The glucose content was detected according to the kit instructions.Glucose concentration in the sample\u0026thinsp;=\u0026thinsp;ODsample / ODstandard \u0026times; 5.Relative glucose consumption rate (%) = [(glucose concentration in the original culture medium - glucose concentration in the culture medium after drug treatment) / glucose concentration consumed in the control group] \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Detection of cellular energy metabolism\u003c/h2\u003e \u003cp\u003eA549 cells in the logarithmic growth phase were taken and seeded into an XFe 96-well plate, with 1\u0026times;104 cells per well, and 80 \u0026micro;L of complete medium was added. The plate was placed in a 37\u0026deg;C incubator overnight. The Seahorse probe plate was hydrated using Seahorse XF calibration solution and incubated overnight in a Seahorse incubator, ensuring that bubbles at the bottom of the probe plate were eliminated. 2 mM glutamine, 1 mM sodium pyruvate, and 10 mM glucose were added to Seahorse XF DMEM medium. The original 80 \u0026micro;L system was supplemented to 250 \u0026micro;L with detection medium, 200 \u0026micro;L was removed to leave 50 \u0026micro;L, then 200 \u0026micro;L of detection medium was added and 200 \u0026micro;L was removed. This washing step was repeated twice, leaving 50 \u0026micro;L, and the plate was incubated at 37\u0026deg;C in a CO2-free environment for 1 hour. Oligomycin, Carbonyl cyanide\u003c/p\u003e \u003cp\u003e4-(trifluoromethoxy)phenylhydrazone(FCCP), and rotenone/antimycin A were sequentially added to the drug-adding wells to evaluate mitochondrial oxidative function of cells, while glucose, oligomycin, and 2-DG were sequentially added to the drug-adding wells to assess cellular glycolytic capacity. The cell culture plate was then loaded into the instrument for detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis methods\u003c/h2\u003e \u003cp\u003eThe experimental results were processed using SPSS 26.0 software. Quantitative data with normal distribution were described as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Independent sample t-test was used to compare differences between two groups, and a P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3.RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of Xiaoyan Decoction on A549 cell viability\u003c/h2\u003e \u003cp\u003eFirstly, the effect of XYT-containing serum on the viability of A549 cells was evaluated by the CCK-8 assay. A549 cells were treated with XYT-containing serum at different concentrations (10%, 15%, 20%, 25%, 30%, 35%, 40%) for 24 h, 48 h, and 72 h, respectively. The experimental data showed that XYT-containing serum could inhibit cell viability in a concentration-dependent manner (Fig.\u0026nbsp;1a). To further determine the optimal concentration and action time of XYT-containing serum for intervening A549 cells, the IC50 values at different intervention time points were analyzed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;1b). The results showed that the IC50 values of XYT-containing serum after 12 h, 24 h, and 48 h of intervention were 44.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94%, 30.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58%, and 45.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.11%, respectively. Finally, 30% XYT-containing serum with 24 h of intervention was used as the intervention concentration and time for subsequent studies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1\u003c/b\u003e The effect of XYT on the activity of A549 cells \u003cb\u003e(a)\u003c/b\u003eEffect of XYT-containing serum on the activity of A549 cells.\u003cb\u003e(b)\u003c/b\u003e IC50 values of XYT serum at different time.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIC50 values of XYT serum at different time(\u003cspan class=\"InlineEquation\"\u003e\u003c/span\u003e\u0026plusmn;s, n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIC50 (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e44.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e48 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e45.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Induction of autophagy in A549 cells by Xiaoyan Decoction\u003c/h2\u003e \u003cp\u003eIn this part of the study, autophagy inhibitor 3-MA and autophagy activator RAPA were applied to A549 cells to observe the effect of XYT on autophagy in A549 cells. Firstly, transmission electron microscopy was used to observe the overall autophagic status of A549 cells. As shown in Fig.\u0026nbsp;2, there were few autophagosomes in the control group. After intervention with XYT-containing serum and RAPA, the number of autophagosomes\u0026mdash;marked by the characteristic double-membrane structure of autophagy\u0026mdash;significantly increased, containing incompletely degraded cytoplasmic components. The number of autophagosomes was even greater after intervention with XYT combined with RAPA. The autophagic level in the 3-MA group was low, while the autophagic level in the XYT combined with 3-MA group was higher than that in the 3-MA group and the control group.Subsequently, Western blot was used to detect changes in autophagy-related factors at the molecular level. After A549 cells were intervened with XYT-containing serum alone or in combination with RAPA, Western blot was applied to detect the expression of P62 and LC3-II/LC3-I proteins. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, compared with the control group, intervention with XYT-containing serum, RAPA, or XYT-containing serum combined with RAPA significantly downregulated the expression of P62 and significantly upregulated the expression of LC3-II/LC3-I protein, with statistically significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the XYT group, the expression level of P62 in the RAPA group and XYT+RAPA group was lower than that in the XYT group, while the expression level of LC3-II/LC3-I protein was higher than that in the XYT group, with statistically significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the RAPA group, the expression level of P62 in the XYT+RAPA group was significantly decreased, while the expression level of LC3-II/LC3-I was significantly increased, with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).In addition, Western blot was used to detect the expression of P62 and LC3-II/LC3-I proteins in A549 cells after intervention with XYT-containing serum alone or in combination with 3-MA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, compared with the control group, the XYT group showed a significant decrease in P62 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and a significant increase in LC3-II/LC3-I expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); the 3-MA group showed a significant upregulation in P62 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and a significant downregulation in LC3-II/LC3-I expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01); the XYT\u0026thinsp;+\u0026thinsp;3-MA group showed no significant change in P62 expression (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) but a significant decrease in LC3-II/LC3-I expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the XYT group, the 3-MA group had a significantly higher P62 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and a significantly lower LC3-II/LC3-I expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01); the XYT\u0026thinsp;+\u0026thinsp;3-MA group had a significantly increased P62 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and a significantly decreased LC3-II/LC3-I expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with all differences being statistically significant. Compared with the 3-MA group, the XYT\u0026thinsp;+\u0026thinsp;3-MA group had a decreased P62 expression level (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and an increased LC3-II/LC3-I expression level (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with statistically significant differences.In this part of the study, we applied the autophagy inhibitor 3-MA and the autophagy activator RAPA in A549 cells to observe the effect of XYT on autophagy in A549 cells. Firstly, transmission electron microscopy was utilized to observe the overall situation of autophagy in A549 cells, followed by Western blot to detect the changes of autophagy-related factors at the molecular level.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2\u003c/b\u003e Transmission electron microscopy of A549 cell ultrastructure (\u0026times;5000).\u003c/p\u003e \u003cp\u003ered arrow:autophagosome\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Xiaoyan Decoction-induced Cell Autophagy Inhibits Glucose Metabolism Reprogramming in A549 Cells\u003c/h2\u003e \u003cp\u003eIn this part of the study, A549 cells were first treated with the autophagy activator RAPA and inhibitor 3-MA to observe the effects of XYT on glucose uptake and lactic acid production. The experimental results showed that compared with the control group, the glucose uptake and lactic acid production in the XYT group, RAPA group, XYT+RAPA group, and XYT\u0026thinsp;+\u0026thinsp;3-MA group were significantly reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01); although the levels of glucose uptake and lactic acid production in the 3-MA group were lower than those in the control group, the differences were not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In addition, there were no significant differences in glucose uptake and lactic acid production between the RAPA group and the XYT group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while the glucose consumption and lactic acid production in the 3-MA group were significantly higher than those in the XYT group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); compared with the XYT group, the XYT+RAPA group showed no significant difference in glucose uptake, and the XYT\u0026thinsp;+\u0026thinsp;3-MA group showed no significant differences in glucose consumption and lactic acid production (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), whereas the lactic acid production in the XYT+RAPA group was significantly lower than that in the XYT group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with the groups treated with autophagy regulators alone, the combination of the two drugs significantly downregulated cellular glucose uptake and extracellular lactic acid production (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e.Next, the Seahorse energy platform was used to evaluate glycolytic reserve capacity. In the experiment, a saturated concentration of glucose was first added, and the process of glucose being decomposed into pyruvate through glycolysis was observed, which could reflect the basal glycolytic rate. Subsequently, oligomycin was injected; as an ATP synthase inhibitor, it can impair mitochondrial respiration, shift energy metabolism toward glycolysis, and the increased extracellular acidification rate (ECAR) reflects the maximum glycolytic capacity of cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e, compared with the control group, the glycolytic rate in the XYT group, RAPA group, and XYT+RAPA group was significantly slowed, and the maximum glycolytic capacity was also significantly reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Further calculation showed that the glycolytic reserve in each intervention group was significantly lower than that in the control group, with statistically significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the XYT group, the RAPA group showed no significant differences in glycolytic rate, maximum glycolytic capacity, or glycolytic reserve (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while the maximum glycolytic capacity and glycolytic reserve in the XYT+RAPA group were significantly lower than those in the XYT group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the RAPA group, the maximum glycolytic capacity and glycolytic reserve in the XYT+RAPA group were significantly decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e indicates that 3-MA intervention could not significantly downregulate the maximum glycolytic capacity or glycolytic reserve; however, compared with the individually use of XYT or 3-MA, the combination of 3-MA and XYT significantly downregulated the maximum glycolytic capacity and glycolytic reserve of cells (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).Based on the above experimental results, it can be concluded that XYT not only weakens the glycolytic rate of lung cancer cells but also significantly reduces their glycolytic reserve capacity. Subsequently, Western Blot was used to evaluate the expression levels of key enzymes in the glycolysis process, and their changing trends were found to be consistent with the glycolytic levels. Finally, Western Blot was used to detect the changes in glycolysis-related molecules.Western blot was used to assess the expression levels of HK2, PFKFB3, PKM, GLUT1, GLUT4, LDHA, and HIF-1α proteins in A549 cells following intervention with XYT-containing serum alone or in combination with RAPA. As shown in Fig.\u0026nbsp;6, compared with the control group, the expression levels of HK2, PFKFB3, PKM, GLUT1, GLUT4, LDHA, and HIF-1α in the XYT group, RAPA group, and XYT+RAPA group were all significantly downregulated, with statistically significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the XYT group, the RAPA group exhibited significantly lower expression levels of PKM (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), GLUT4 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), LDHA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and HIF-1α (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with statistically significant differences, while there were no statistically significant differences in the expression levels of HK2, PFKFB3, and GLUT1 in the RAPA group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The XYT+RAPA group showed significantly lower expression levels of HK2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PFKFB3 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), PKM (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), GLUT1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), GLUT4 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), LDHA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and HIF-1α (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared with the XYT group. In comparison with the RAPA group, intervention with XYT+RAPA significantly reduced the expression levels of HK2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), PFKFB3 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), PKM (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), GLUT1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), GLUT4 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), LDHA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and HIF-1α (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with all differences being statistically significant.In addition, after A549 cells were intervened with XYT-containing serum alone or in combination with 3-MA, Western blot was performed to detect the expression of HK2, PFKFB3, PKM, GLUT1, GLUT4, LDHA, and HIF-1α proteins in the cells. As shown in Fig.\u0026nbsp;34, compared with the control group, XYT intervention significantly decreased the expression of HK2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PFKFB3 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PKM (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), GLUT1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), GLUT4 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), LDHA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and HIF-1α (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). 3-MA intervention downregulated the expression of HK2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), GLUT4 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and LDHA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while there were no significant changes in the expression of HIF-1α, PFKFB3, PKM, and GLUT1 (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). XYT\u0026thinsp;+\u0026thinsp;3-MA intervention significantly reduced the expression of HK2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PFKFB3 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PKM (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), GLUT4 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and LDHA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with no significant changes in the expression of GLUT1 and HIF-1α (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Compared with the XYT group, the 3-MA group had significantly lower expression levels of HK2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PFKFB3 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), PKM (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and GLUT1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and significantly higher HIF-1α (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while there were no statistically significant differences in the expression levels of GLUT4 and LDHA (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The XYT\u0026thinsp;+\u0026thinsp;3-MA group showed significant changes in the expression levels of HK2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), PFKFB3 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PKM (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), GLUT1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), GLUT4 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and LDHA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared with the XYT group, with no significant difference in HIF-1α (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Compared with the 3-MA group, XYT\u0026thinsp;+\u0026thinsp;3-MA intervention significantly reduced the expression levels of HK2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PFKFB3 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PKM (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), GLUT1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), GLUT4 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), LDHA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and HIF-1α (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with all differences being statistically significant.\u003c/p\u003e \u003cp\u003eThe respiration of normal cells includes aerobic respiration and anaerobic glycolysis. Previous studies have preliminarily confirmed the effect of XYT on the glycolytic level of lung cancer cells, so this part intends to further investigate its impact on aerobic respiration (i.e., OXPHOS). The cellular OCR was measured using the Seahorse platform. Firstly, the basal respiration of cells was detected. Subsequently, oligomycin was added to inhibit ATP synthesis in mitochondria, so as to evaluate the ATP level generated by mitochondria to meet the energy demand of cells under this condition. Then, FCCP was added, which can stimulate the maximum operation of the mitochondrial respiratory chain and promote the rapid oxidation of substrates to cope with this metabolic challenge, and at this time, the maximum value of oxygen consumption rate can be obtained. Finally, rotenone and antimycin A were added to inhibit the mitochondrial respiration process.\u003c/p\u003e \u003cp\u003eThe results of the OCR experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003e) showed that compared with the control group, the O₂ consumption in the XYT group, RAPA group, and XYT+RAPA group significantly increased, and parameters such as basal respiration and maximum respiration were significantly upregulated, with statistically significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the XYT group, the basal respiration and maximum respiration in the RAPA group slightly increased, but there was no statistical difference (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while the basal respiration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and maximum respiration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the XYT+RAPA group showed an increasing trend with statistically significant differences. Compared with the RAPA group, the basal respiration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and maximum respiration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the XYT+RAPA group significantly increased, with statistically significant differences. Similar conclusions can be drawn from the 3-MA intervention. These results indicate that XYT or RAPA intervention can alleviate the cumulative damage of mitochondria in A549 cells, which is manifested by an increase in the number of mitochondria and a partial recovery of OXPHOS capacity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 6\u003c/b\u003e Protein expression of glycolysis-related molecules in cells of each group ( \u0026plusmn;s, n\u0026thinsp;=\u0026thinsp;3). Note: Compared with the control group, *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; compared with the XYT group, #P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ##P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; compared with the RAPA group.△P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Discussion","content":"\u003cp\u003eLung cancer is one of the malignant tumors with high morbidity and mortality. Clinical treatments for NSCLC mainly include surgery, radiotherapy, chemotherapy, immunotherapy, and molecular targeted therapy .\u003csup\u003e17\u003c/sup\u003e However, recurrence and metastasis occur in approximately 20% of patients with early-stage lung cancer after surgery, and the 5-year survival rate of patients receiving chemotherapy alone is less than 5% .\u003csup\u003e18,19\u003c/sup\u003e In recent years, traditional Chinese medicine (TCM) has shown promising efficacy in the comprehensive treatment of lung cancer. It can improve the body\u0026rsquo;s immune function, inhibit lung cancer cell proliferation and metastasis, thereby significantly enhancing patients\u0026rsquo; quality of life and prolonging their survival .\u003csup\u003e20\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDirector Jia Yingjie believes that lung cancer is characterized by \"deficiency in origin,\" where primordial qi deficiency forms the pathological basis of lung cancer, while toxin, stasis, and phlegm are its pathological products .\u003csup\u003e21\u003c/sup\u003e XYT aligns with the core pathogenesis of lung cancer\u0026mdash;deficiency, toxin, stasis, and phlegm\u0026mdash;and is formulated under the guidance of the \"turbidity-eliminating and constitution-nourishing\" principle. Clinically, modified XYT combined with radiotherapy and chemotherapy has achieved favorable outcomes in treating lung cancer, effectively reducing metastasis and drug resistance. However, its molecular mechanism remains unclear, which prompted this preliminary investigation.\u003c/p\u003e \u003cp\u003eThe Warburg effect is a hallmark of tumor cells, characterized by high lactate production and glucose consumption, accompanied by enhanced glycolysis .\u003csup\u003e22\u003c/sup\u003e Tumor cells proliferate rapidly, consuming large amounts of oxygen, which easily leads to a hypoxic environment. The glycolytic pathway can enhance their hypoxia tolerance, thereby effectively avoiding apoptosis .\u003csup\u003e23\u003c/sup\u003e Intermediates of glycolysis, such as glucose-6-phosphate and pyruvate, serve as raw materials for biosynthesis in tumor cells, providing essential substances for their growth. Additionally, lactate produced during glycolysis acidifies the tumor microenvironment, disrupts the extracellular matrix, and facilitates tumor invasion and metastasis .\u003csup\u003e24\u003c/sup\u003e Further studies on TCM have revealed that some herbal medicines can regulate glycolysis by modulating related proteins in the glycolytic pathway, thereby influencing tumor progression. Research has shown that a TCM decoction inhibited the growth and epithelial-mesenchymal transition (EMT) of gastric cancer (GC) cells by reducing glycolysis in a PKM2-dependent manner .\u003csup\u003e25\u003c/sup\u003e Therefore, this study detected the glycolytic level and expression of related proteins in A549 cells after XYT treatment. The results suggested that XYT can inhibit lactate production and glucose consumption in A549 cells and downregulate the expression of glycolysis-related proteins.\u003c/p\u003e \u003cp\u003eAutophagy, a conserved physiological catabolic process, is a unique mechanism evolved in eukaryotic cells and plays an indispensable role in maintaining cellular homeostasis .\u003csup\u003e26\u003c/sup\u003e It exerts a pivotal effect in metabolic reprogramming of tumor cells, with a close association with the tricarboxylic acid cycle, a core metabolic part. A large number of experimental results have demonstrated that metabolic processes such as glucose metabolism, lipid metabolism, and amino acid metabolism are closely linked to cancer progression, and the critical role of autophagy in these processes has become increasingly prominent .\u003csup\u003e27,28\u003c/sup\u003e Qin et al. \u003csup\u003e29\u003c/sup\u003e found that inhibiting autophagy can effectively promote glycolytic activity in gastric cancer cells through the ROS-NF-κB-HIF-1α pathway. In addition, FUNC1-mediated mitophagy suppresses the inflammatory response by clearing dysfunctional mitochondria, thereby exerting an inhibitory effect during the initial stage of hepatocellular carcinoma (HCC) development.\u003csup\u003e30\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, XYT was found to upregulate the expression of LC3-II/LC3-I and downregulate the expression of P62 in tumor tissues and cells. Consistent with these findings, transmission electron microscopy results also confirmed that XYT induced more autophagosomes in A549 cells. All these results indicate that XYT can effectively induce cellular autophagy. To further clarify whether XYT regulates glucose metabolism reprogramming through autophagy, we used methods such as Western blot, Seahorse Glycolysis Stress Test Kit, Seahorse Mitochondrial Stress Test Kit, lactate production kit, and glucose uptake kit in vitro to detect changes in glucose metabolism reprogramming after autophagy inhibition and activation. The results showed that, similar to RAPA, XYT-containing serum could downregulate the expression levels of key glycolytic enzymes such as HIF-1α, HK2, PFKFB3, PKM, GLUT1, GLUT4, and LDHA in A549 cells, reduce glycolytic reserve and maximum glycolytic capacity, decrease lactate production, and reduce glucose uptake. Similarly, after pretreatment of A549 cells with 3-MA, XYT-containing serum could also reduce the expression of key glycolytic enzymes, slow down glycolytic rate, decrease lactate production, and reduce glucose uptake. Moreover, the inhibitory effect of XYT combined with autophagy regulators was stronger than that of single-drug treatment.\u003c/p\u003e \u003cp\u003eHIF-1α regulates a variety of tumor processes for adaptation, including metabolism, angiogenesis, invasion and cell proliferation. Studies showed that HIF-1α could modulate various EMT transcription factors, histone modifiers, enzymes (MMPs), chemokine receptors .\u003csup\u003e31\u003c/sup\u003e Research indicated that HIF-1α affects mitochondrial fission 1 protein (Fis1) and dynamin-related protein 1 (DRP1) related to mitochondrial dynamics, thereby inducing Parkin-associated mitophagy .\u003csup\u003e32\u003c/sup\u003e Studies have shown that HIF-1α can directly induce autophagy in tumor cells. In colorectal cancer cells, high expression of HIF-1α is associated with increased autophagy levels, and HIF-1α promotes autophagy by regulating the expression of autophagy-related genes, thereby helping tumor cells survive and proliferate in harsh environments such as hypoxia. Results suggested that liensinine can inhibit autophagy by targeting HIF-1 alpha/eNOS .\u003csup\u003e33\u003c/sup\u003e In this study, it was found that the expression of HIF-1α in A549 cells was significantly affected after XYT treatment, and the inhibitory effect of XYT combined with autophagy regulators was stronger than that of single-drug treatment. These findings further confirm that XYT can enhance the inhibitory effect of RAPA or 3-MA on cellular glycolysis through the HIF-1α signaling pathway and autophagy, thereby exerting an anti-tumor effect.\u003c/p\u003e"},{"header":"4.Conclusion","content":"\u003cp\u003eThis study demonstrates that Xiaoyan Decoction inhibits NSCLC progression through metabolic reprogramming via three key mechanisms: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) inducing autophagy (increased autophagosomes, LC3-II/LC3-I ratio, decreased P62); (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) suppressing glycolysis (reduced glucose uptake, lactate production, glycolytic enzymes) while enhancing oxidative phosphorylation (elevated OCR); and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) modulating the HIF-1α pathway. The coordinated regulation of the autophagy-HIF-1α-glycolysis axis by XYT provides novel insights into traditional Chinese medicine's anti-cancer mechanisms.However, our study also has its limitations.The experiments were confined to in vitro A549 cell model, lacking validation in animal tumors or patient-derived tissues.Clinical translatability remains unassessed, as dosing regimens and pharmacokinetics of XYT\u0026rsquo;s bioactive components in humans were not explored. Potential crosstalk with other oncogenic pathways (e.g., mTOR, Akt) was not fully dissected, leaving broader mechanistic networks incompletely mapped. Clinical prospective studies and further in vitro and ex vivo experiments through targeted metabolomics, target organ gene knockdown and other technologies can be carried out to explore the specific mechanism of action of this formula.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003eThe ethics approval is not applicable in this research.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to publish\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflicts of interests\u003c/h2\u003e \u003cp\u003eThe authors maintain the utmost transparency and hereby declare the absence of any potential conflicts of interest pertaining to this study.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe author(s) disclosed receipt of the following financial support for the research,authorship, and/or publication of this article: This article was supported by Tianjin Education Commission scientific research project (grant number 2021KJ143).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXiaoqun Wang: Conceptualization, Writing \u0026ndash; Original Draft;Yuting Li: Investigation,Formal Analysis, Data Curation;Suxuan Ouyang: Writing \u0026ndash; Original Draft;Haojian Zhang: Writing \u0026ndash; Review \u0026amp; Editing;Linlin Zhao: Writing \u0026ndash; Review \u0026amp; Editing;Jing Zhang: Writing \u0026ndash; Review \u0026amp; Editing;Fanming Kong: Supervision, Writing \u0026ndash; Review \u0026amp; Editing (Corresponding Author);Yingjie Jia: Supervision, Writing \u0026ndash; Review \u0026amp; Editing (Corresponding Author).\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank Professor Yingjie Jia and Professor Fanming Kong for their guidance and assistance with this work.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll relevant data selected for the study are included in the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. Cancer statistics, 2025. CA Cancer J Clin. 2025;75(1):10\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21871\u003c/span\u003e\u003cspan address=\"10.3322/caac.21871\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Liu T, Wang X, Jia Y, Cui H. Autophagy and Glycometabolic Reprograming in the Malignant Progression of Lung Cancer: A Review. Technol Cancer Res Treat. 2023;22:15330338231190545. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/15330338231190545\u003c/span\u003e\u003cspan address=\"10.1177/15330338231190545\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarburg OH. The classic: The chemical constitution of respiration ferment. Clin Orthop Relat Res. 2010;468(11):2833\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11999-010-1534-y\u003c/span\u003e\u003cspan address=\"10.1007/s11999-010-1534-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K, Huang W, Chen R, et al. Di-methylation of CD147-K234 Promotes the Progression of NSCLC by Enhancing Lactate Export. Cell Metab. 2023;35(6):1084. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmet.2023.05.006\u003c/span\u003e\u003cspan address=\"10.1016/j.cmet.2023.05.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu JQ, Fu YL, Zhang J, et al. Targeting glycolysis in non-small cell lung cancer: Promises and challenges. Front Pharmacol. 2022;13:1037341. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fphar.2022.1037341\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2022.1037341\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Li M, Li X, et al. Deciphering the role of PLCD3 in lung cancer: A gateway to glycolytic reprogramming via PKC-Rap1 activation. Heliyon. 2024;10(17):e37063. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.heliyon.2024.e37063\u003c/span\u003e\u003cspan address=\"10.1016/j.heliyon.2024.e37063\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu L, Chai L, Ran J, Yang Y, Zhang L. BAI1 acts as a tumor suppressor in lung cancer A549 cells by inducing metabolic reprogramming via the SCD1/HMGCR module. Carcinogenesis. 2020;41(12):1724\u0026ndash;34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/carcin/bgaa036\u003c/span\u003e\u003cspan address=\"10.1093/carcin/bgaa036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong H, Qiu Z, Wang Y, et al. HIF-1α/YAP Signaling Rewrites Glucose/Iodine Metabolism Program to Promote Papillary Thyroid Cancer Progression. Int J Biol Sci. 2023;19(1):225\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7150/ijbs.75459\u003c/span\u003e\u003cspan address=\"10.7150/ijbs.75459\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCourtnay R, Ngo DC, Malik N, Ververis K, Tortorella SM, Karagiannis TC. Cancer metabolism and the Warburg effect: the role of HIF-1 and PI3K. Mol Biol Rep. 2015;42(4):841\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11033-015-3858-x\u003c/span\u003e\u003cspan address=\"10.1007/s11033-015-3858-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNazio F, Bordi M, Cianfanelli V, Locatelli F, Cecconi F. Autophagy and cancer stem cells: molecular mechanisms and therapeutic applications. Cell Death Differ. 2019;26(4):690\u0026ndash;702. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41418-019-0292-y\u003c/span\u003e\u003cspan address=\"10.1038/s41418-019-0292-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang L, Hui K, Hu C, et al. Autophagy inhibition potentiates the anti-angiogenic property of multikinase inhibitor anlotinib through JAK2/STAT3/VEGFA signaling in non-small cell lung cancer cells. J Exp Clin Cancer Res. 2019;38(1):71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13046-019-1093-3\u003c/span\u003e\u003cspan address=\"10.1186/s13046-019-1093-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiao L, Zhang HL, Li DD, et al. Regulation of glycolytic metabolism by autophagy in liver cancer involves selective autophagic degradation of HK2 (hexokinase 2). Autophagy. 2018;14(4):671\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/15548627.2017.1381804\u003c/span\u003e\u003cspan address=\"10.1080/15548627.2017.1381804\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Li Z, Gao Z, et al. Cadmium induces cell growth in A549 and HELF cells via autophagy-dependent glycolysis. Toxicol Vitro. 2020;66:104834. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tiv.2020.104834\u003c/span\u003e\u003cspan address=\"10.1016/j.tiv.2020.104834\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Song B, Guo M, et al. p53-dependent HIF-1α /autophagy mediated glycolysis to support Cr(VI)-induced cell growth and cell migration. Ecotoxicol Environ Saf. 2024;272:116076. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ecoenv.2024.116076\u003c/span\u003e\u003cspan address=\"10.1016/j.ecoenv.2024.116076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng X, Han Y, Gu L, Gao S, Lv Y, Li C. Study of the mechanism by which Xiaoyan decoction combined with E7449 regulates tumorigenesis in lung adenocarcinoma. J Cell Mol Med. 2024;28(12):e18467. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jcmm.18467\u003c/span\u003e\u003cspan address=\"10.1111/jcmm.18467\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang P, Xu W, Liu L. Study on the autophagy and resistance protein affected by Xiaoyan decoction in A549/DDP cells(in Chinese). Tianjin J Traditional Chin Med. 2016;33(06):358\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuma N, Santana-Davila R, Molina JR. Non-Small Cell Lung Cancer: Epidemiology, Screening, Diagnosis, and Treatment. \u003cem\u003eMayo Clin Proc\u003c/em\u003e. 2019;94(8):1623\u0026ndash;1640. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.mayocp.2019.01.013\u003c/span\u003e\u003cspan address=\"10.1016/j.mayocp.2019.01.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang JY, Mehran RJ, Feng L, et al. Stereotactic ablative radiotherapy for operable stage I non-small-cell lung cancer (revised STARS): long-term results of a single-arm, prospective trial with prespecified comparison to surgery. Lancet Oncol. 2021;22(10):1448\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1470-2045(21)00401-0\u003c/span\u003e\u003cspan address=\"10.1016/S1470-2045(21)00401-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGettinger S, Horn L, Jackman D, et al. Five-Year Follow-Up of Nivolumab in Previously Treated Advanced Non-Small-Cell Lung Cancer: Results From the CA209-003 Study. J Clin Oncol. 2018;36(17):1675\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/JCO.2017.77.0412\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2017.77.0412\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Feiyue Z, Gaofeng L. Traditional Chinese medicine and lung cancer-From theory to practice. Biomed Pharmacother. 2021;137:111381. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biopha.2021.111381\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2021.111381\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Liao D, Xu J, Kong F, Jia Y. Connotation of Eliminating Turbidity and Banking up Essence and Its Application in Differentiation and Treatment of Malignant Tumor(in Chinese). J Tradit Chin Med. 2023;64(06):545\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiberti MV, Locasale JW. The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem Sci. 2016;41(3):211\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tibs.2015.12.001\u003c/span\u003e\u003cspan address=\"10.1016/j.tibs.2015.12.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKroemer G, Pouyssegur J. Tumor cell metabolism: cancer\u0026rsquo;s Achilles\u0026rsquo; heel. Cancer Cell. 2008;13(6):472\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ccr.2008.05.005\u003c/span\u003e\u003cspan address=\"10.1016/j.ccr.2008.05.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGatenby RA, Gawlinski ET, Gmitro AF, Kaylor B, Gillies RJ. Acid-mediated tumor invasion: a multidisciplinary study. Cancer Res. 2006;66(10):5216\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/0008-5472.CAN-05-4193\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-05-4193\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Q, Yuan M, Wang H, et al. PKM2 Is the Target of a Multi-Herb-Combined Decoction During the Inhibition of Gastric Cancer Progression. Front Oncol. 2021;11:767116. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2021.767116\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2021.767116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlassen A, Faccio AT, Canuto GAB, et al. Metabolomics: Definitions and Significance in Systems Biology. Adv Exp Med Biol. 2017;965:3\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-319-47656-8_1\u003c/span\u003e\u003cspan address=\"10.1007/978-3-319-47656-8_1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerro F, Servais S, Besson P, Roger S, Dumas JF, Brisson L. Autophagy and mitophagy in cancer metabolic remodelling. Semin Cell Dev Biol. 2020;98:129\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.semcdb.2019.05.029\u003c/span\u003e\u003cspan address=\"10.1016/j.semcdb.2019.05.029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKikuchi N, Soga T, Nomura M, et al. Comparison of the ischemic and non-ischemic lung cancer metabolome reveals hyper activity of the TCA cycle and autophagy. Biochem Biophys Res Commun. 2020;530(1):285\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbrc.2020.07.082\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2020.07.082\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin W, Li C, Zheng W, et al. Inhibition of autophagy promotes metastasis and glycolysis by inducing ROS in gastric cancer cells. Oncotarget. 2015;6(37):39839\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/oncotarget.5674\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.5674\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, Li Y, Siraj S, et al. FUN14 Domain-Containing 1-Mediated Mitophagy Suppresses Hepatocarcinogenesis by Inhibition of Inflammasome Activation in Mice. Hepatology. 2019;69(2):604\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/hep.30191\u003c/span\u003e\u003cspan address=\"10.1002/hep.30191\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTam SY, Wu VWC, Law HKW. Hypoxia-Induced Epithelial-Mesenchymal Transition in Cancers: HIF-1α and Beyond. Front Oncol. 2020;10:486. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fonc.2020.00486\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2020.00486\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan WL, Ma RJ, Hou YX, Wang Y, Sun SC. HIF-1α regulates mitochondria function for oxidative stress and autophagy during oocyte maturation. AA. 2025;2(1):0\u0026ndash;0. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.48130/animadv-0025-0014\u003c/span\u003e\u003cspan address=\"10.48130/animadv-0025-0014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng Z, Zhang S, Han Q, et al. Liensinine sensitizes colorectal cancer cells to oxaliplatin by targeting HIF-1α to inhibit autophagy. Phytomedicine. 2024;129:155647. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.phymed.2024.155647\u003c/span\u003e\u003cspan address=\"10.1016/j.phymed.2024.155647\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Xiaoyan Decoction, Non-small cell lung cancer, Autophagy, Glycolytic reprogramming, HIF-1α","lastPublishedDoi":"10.21203/rs.3.rs-8540053/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8540053/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003e[Objective]\u003c/h2\u003e \u003cp\u003eTo investigate the effects of Xiaoyan Decoction ( XYT) on autophagy and glucose metabolism reprogramming in Non-small Cell Lung Cancer(NSCLC) cells and to explore its mechanism of action.\u003c/p\u003e\u003ch2\u003e[Methods]\u003c/h2\u003e \u003cp\u003e① Screen the half maximal inhibitory concentration (IC50) of XYT-containing serum using Cell Counting Kit 8 (CCK8); ② After pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) and the activator Rapamycin (RAPA) and intervention with XYT, transmission electron microscopy was used to observe the overall autophagy status of A549 cells, and Western Blot was used to detect changes in Sequestosome1(P62) and Microtubule-associated protein 1 light chain 3(LC3); ③ The kit was used to detect glucose uptake and lactate production, Seahorse was used to assess cellular energy metabolism, and Western Blot was used to assess the expression of Hypoxia inducible factor-1α(HIF-1α) protein in A549 cells after XYT drug-containing serum intervention alone and in combination with RAPA intervention.\u003c/p\u003e\u003ch2\u003e[Results]\u003c/h2\u003e \u003cp\u003e①XYT-containing serum can slow down cell proliferation in a concentration-dependent manner, with a 30% concentration of XYT-containing serum for 24 hours being the optimal concentration and duration of intervention. ②Following XYT intervention, the number of autophagosomes\u0026mdash;a characteristic double-membrane structure of autophagy\u0026mdash;significantly increased in A549 cells. Western blot analysis revealed a significant downregulation of P62 expression and a significant upregulation of LC3-II/LC3-I protein expression. ③After XYT intervention, glucose uptake and lactate production in A549 cells were significantly reduced, glycolytic rate was significantly slowed, and maximum glycolytic capacity was also significantly reduced. Western blot analysis showed a significant downregulation of HIF-1α expression. Additionally, Oxygen consumption rate(OCR) assay results indicated that O₂ consumption significantly increased after XYT intervention, with parameters such as basal respiration and maximal respiration significantly up-regulated. These effects were further enhanced by co-treatment with autophagy modulators 3-MA/RAPA.\u003c/p\u003e\u003ch2\u003e[Conclusion]\u003c/h2\u003e \u003cp\u003eXYT can inhibit A549 cell proliferation, upregulate cellular autophagy levels, regulate HIF-1α expression, shift the metabolic phenotype from glycolysis to aerobic oxidation, reprogram glucose metabolism, and thereby exert an inhibitory effect on cell proliferation.\u003c/p\u003e","manuscriptTitle":"Mechanism Study of Xiaoyan Decoction in the Treatment of Non-small Cell Lung Cancer Through Glycometabolic Reprogramming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-19 16:02:50","doi":"10.21203/rs.3.rs-8540053/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-18T12:01:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"323613708962738287009641309821259889427","date":"2026-03-09T11:48:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-12T12:56:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-09T02:26:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Cell International","date":"2026-02-04T08:41:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"81d41067-ae70-4fd6-916a-5880743cfaf5","owner":[],"postedDate":"February 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-19T16:02:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-19 16:02:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8540053","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8540053","identity":"rs-8540053","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}