Integrated UPLC-Q-TOF-MS, network pharmacology and experimental approach to evaluate the effects of blood stasis constitution ointment on lung cancer

Integrated UPLC-Q-TOF-MS, network pharmacology and experimental approach to evaluate the effects of blood stasis constitution ointment on lung cancer | 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 Integrated UPLC-Q-TOF-MS, network pharmacology and experimental approach to evaluate the effects of blood stasis constitution ointment on lung cancer Qiaozhi Wang, Juhe Wang, Chuanhao Dai, Tianming Lu, Xingjiang Xiong, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5924974/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Dec, 2025 Read the published version in Cancer Management and Research → Version 1 posted You are reading this latest preprint version Abstract Background Blood stasis constitution ointment (BSCO) is based on the classic formulas Xuefu Zhuyu and Buyang Huanwu decoctions described in Yilin Gaicuo by Wang Qingren of the Qing Dynasty. These formulas have been used to effectively treat blood stasis, which is identified as a pathological factor of lung cancer in traditional Chinese medicine theory. Aim of the study : To analyze the chemical components of BSCO, predict target pathways, and evaluate its effects on lung cancer through in vivo and in vitro experiments. Methods Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry was used to identify components of BSCO, and network pharmacology was used to predict their targets and signaling pathways associated with lung cancer. A Lewis lung cancer model was established in mice to evaluate the effects of BSCO by observing tissue morphology, whole animal imaging, and determination of serum biochemical indicators. The effects of BSCO on Lewis cancer cells in vitro were assessed using a CCK-8 cell proliferation assay. Results Twenty major chemical components of BSCO were identified, with 341 potential targets identified by network pharmacology. BSCO effectively inhibited tumor growth in the Lewis lung cancer mouse model and normalized serum markers of cancer to varying degrees. The IC 50 of BSCO on Lewis cell proliferation was 173 mg/mL. Low- and high-dose BSCO-containing drug serum inhibited proliferation of Lewis cells after 24 and 48 h incubation. Conclusion The results suggest that BSCO may exert anticancer effects through targets including GAPDH, AKT1, TP53, TNF, IL6, and the PI3K-Akt signaling pathway, providing a reference for its clinical application in the prevention and treatment of lung cancer. Blood stasis constitution ointment (BSCO) UPLC-Q-TOF-MS network pharmacology medicine food homology Chinese Medicine Constitution lung cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights UHPLC-Q-TOF-MS identified 20 components of BSCO. Network pharmacology suggested potential targets and pathways affected by BSCO in the treatment of lung cancer. BSCO effectively inhibited tumor growth in a Lewis lung cancer mouse model. BSCO increased serum levels of CAT, GSH-Px, and SOD, and decreased levels of PFK, GLUT1, HK2, MDA, and PK to varying degrees. 1. Introduction Lung cancer is still the leading cause of cancer deaths worldwide and the number of cases continues to rise in most countries 1 . It is characterized by a high mortality rate after diagnosis and it has been estimated by the International Agency for Research on Cancer (IARC) under the World Health Organization (WHO) that there were about 2.2 million new cases and 1.8 million deaths in 2020 2 . Therefore, screening and prevention have become important to reduce the mortality rate of lung cancer 3 . The pathogenesis of lung cancer is still unclear in modern medicine. Recently, it has been linked to the lung microbiome, and smoking is the main risk factor 4 , 5 . Most lung cancers are preceded by obvious precancerous lesions, such as hyperplasia, metaplasia, abnormal growth, and carcinoma in situ, which are mostly believed to be consequences of immune dysfunction. According to the theory of traditional Chinese medicine (TCM), lung cancer belongs to the category of "lung accumulation", and the main pathogenesis is "depression". It is primarily due to deficiency of lung qi, which leads to internal obstruction of phlegm and turbidity in the body, followed by invasion of the lungs by external pathogens. Ultimately, this leads to qi stagnation and dampness obstruction, internal stagnation of phlegm and turbidity, phlegm and blood stasis interconnections, and the pathogenesis of transforming toxins into cancers. Clinical treatment of lung cancer domestically and internationally includes surgery, radiotherapy, chemotherapy, ablation, targeted therapy, and immunotherapy 6 . Surgical treatment is preferred, but due to factors such as surgical risk, comorbidities, and patient willingness, many patients do not meet the criteria for standard surgical resection. Although radiotherapy and chemotherapy can inhibit the growth of cancer cells, they are accompanied by significant side effects. The development of the targeted therapeutic drug osimertinib and the immunotherapeutic drug pembrolizumab has provided new treatment options for lung cancer, but the survival rate is still not high 7 , 8 . New, effective drugs are required to improve clinical survival. Oral herbal medicines have also been shown to effectively inhibit the growth of lung cancer 9 . The Chinese medicine constitution is a comprehensive and relatively stable inherent trait that is based on innate and acquired characteristics of the human body during life processes. It includes morphological structure, physiological function, and psychological state, and represents individual characteristics of the human body that are adapted to the natural and social environment during growth and development 10 . The Chinese medicine constitution has been widely recognized internationally 11 . prescription According to the standard "Classification and Determination of Constitution in TCM" (ZYYXH/T157-2009) issued by the Chinese Society of Traditional Chinese Medicine, Chinese medicine constitutions are categorized into nine types: Qi-deficiency, Damp-heat, Yin-deficiency, Qi-stagnation, Yang-deficiency, Phlegm-dampness, Blood stasis, Special, and Balanced. TCM constitutions are associated with specific diseases and could be used to guide individualized prevention and treatment 12 . The constitution has both stability and variability, through intervention to adjust its bias, reflecting the adjustability of the constitution. Oral paste is the best choice for adjusting the constitution 13 . Experts from top institutions, such as the China Academy of Chinese Medical Sciences, Nanjing University of Chinese Medicine, and other leading international organizations, have combined TCM constitution and "Medicine and Food Homology" (MFH) theories, leveraging the research strengths of academic institutions and the clinical expertise of frontline hospitals. They have carefully formulated blood stasis constitution ointment (BSCO) based on selected classic TCM prescriptions. Based on the classic formulas Xuefu Zhuyu and Buyang Huanwu decoctions appearing in Yilin Gaicuo by Wang Qingren in 1830, BSCO consists of Panax ginseng C.A.Mey., Prunus persica (L.) Batsch, Hippophae rhamnoides L., Vigna umbellata (Thunb.) Ohwi & H.Ohashi, E’Jiao (Colla corii asini), oxhide gelatin and other herbs, and is made into a paste according to the law. Xuefu Zhuyu and Buyang Huanwu decoctions are well-known TCM prescriptions that are used to treat blood stasis syndrome. They are currently employed extensively in the management of cardiovascular disorders, and substantial therapeutic efficacy has been demonstrated 14 , 15 . According to TCM theory, one of the core elements of lung cancer is an unbalanced constitution, with blood stasis being the representative constitution 16 . Blood stasis can lead to a hypercoagulable state, which not only increases the risk of thrombosis in lung cancer patients, but also contributes to tumor proliferation, severely affecting quality of life and prognosis in these patients. Recent studies have shown the palliative effect of Xuefu Zhuyu and Buyang Huanwu decoctions on symptoms associated with lung cancer 17 , 18 . Based on the two prescriptions mentioned above, modifications were made to prepare BSCO, and its therapeutic effect on lung cancer was evaluated. The formulation addresses four aspects: Reinforce healthy qi, Correcting Imbalances, Eliminate pathogenic factors, and flavoring. Huangdi Neijing suggests that “When the healthy qi is strong within, pathogenic factors cannot act”. In this formula, Panax ginseng C.A.Mey. and oxhide gelatin are used to replenish qi and blood, assist in strengthening qi and eliminate pathogenic factors. They serve as the monarch medicinal components. Since yin and blood are interdependent, and Essence and blood share the same source, E'Jiao (Colla corii asini) and Tremella fuciformis Berk. are used to nourish yin, while Lycium chinense Mill. and Nelumbo nucifera Gaertn. are used to assist the postnatal foundation. These substances support the postnatal constitution and nourish the source of transformation, serving as the minister medicines. Prunus persica (L.) Batsch, Pueraria alopecuroides Craib, and Rosae rugosae Thunb. are used to circulate blood and transform stasis, alleviating deficiency in the middle jiao. Hordeum vulgare L. is used to soothe the liver and regulate qi, promoting movement of qi to enhance blood flow. Poria cocos ( Schw.) Wolf is used to transform fluid retention, Citrus reticulata Blanco to transform phlegm, and Crataegus pinnatifida Bunge and Hippophae rhamnoides L to transform indigestion. These ingredients work together to alleviate the Phlegm qi stagnation pattern, serving as the assistant medicines. Vigna umbellata (Thunb.) Ohwi & H.Ohashi is used to eliminate dampness and Circulate blood, while Allium macrostemon Bunge is used to activate yang and dissipate cold. Oligosaccharide maltose is used to strengthen the spleen and stomach, correct the bitter taste, provide shape and texture, and improve the flavor without the concern of raising blood sugar levels, making it more acceptable to the public. The formula considers food safety while enhancing therapeutic effect in the concentrated form of a paste. It is convenient for long-term use and has no obvious toxic side effects. Due to the high viscosity of the paste, it has a number of advantages, such as high concentration of effective components, rapid absorption, prolonged and lasting effects, and tangible therapeutic efficacy. The effective components regulate the body's yin-yang balance through the Meridian affinity of the medicine and reinforce healthy qi to strengthen the body and improve the constitution. They act on pathogenic characteristics of the blood stasis constitution comprehensively and fundamentally, thus serving the purpose of regulation and health care. It has promising industrial application prospects and holds significant social and economic value. "Oral Paste" is a type of Chinese medicinal paste that is primarily used for health preservation and wellness, and is also known as "nourishing ointment". These oral pastes are prescribed by experienced TCM practitioners who, based on the constitution and health of the individual, follow the holistic view and the principle of syndrome differentiation and treatment in TCM. They select single or multiple medicinal herbs to create a well-balanced formula, which is then processed through rigorous and specific techniques. The main purposes of these pastes are to nourish and strengthen the body, resist aging, prolong life, and prevent and treat diseases. On November 10, 2021, China's National Health and Wellness Commission issued the “Circular on the Issuance of the Provisions on the Management of the Catalog of Substances that are Traditionally Used as Both Food and Chinese Herbal Medicines”, which stipulates that food and medicinal substances refer to those substances that are traditionally used as food and included in the “Pharmacopoeia of the People's Republic of China”. Previously, the former Ministry of Health published the “Notice on Further Standardizing the Management of Health Food Raw Materials”, which made specific provisions on MFH substances, substances that can be used in health food, and substances prohibited in health food. According to the above provisions, the constitution, dietary therapy, and oral pastes are closely related. These MFH pastes are increasingly gaining recognition among the public. They are appreciated for their safety and convenience, with a sweet and palatable taste that encourages long-term compliance. In particular, they are highly effective for improving the constitution and offer lasting and stable therapeutic effects. Driven by the wave of traditional Chinese medicine health and wellness, consumption of these MFH pastes for body regulation and disease treatment is becoming increasingly popular in China. In summary, this research is based on the doctrine of constitution and medicinal paste, drawing on classic prescriptions and utilizing medicine food homology substances that meet safety requirements to create BSCO. This study utilized ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) combined with network pharmacology to identify the active components of BSCO and predict their biological targets. Additionally, the effects of BSCO on lung cancer were evaluated both in vitro and in vivo, providing evidence-based support for its efficacy. 2. Materials and methods 2.1. Reagents and instruments BSCO was obtained from Sichuan Chengdu Guzheng Baohe Health Management Co., Ltd., chromatographic grade methanol was purchased from Fisher Scientific, and pure water was from Hangzhou Wahaha Group Co., Ltd. Instruments: ACQUITY UPLC H-Class ultra-high performance liquid chromatography system and Vion IMS QTof high-resolution mass spectrometer (Waters Corporation, USA); ME155DU electronic balance (Mettler-Toledo Instruments); BioTek Synergy H1 Multifunctional Microplate Detector (Agilent); QX200™ Droplet Digital™ PCR System (Bio-RAD). 2.2. UPLC-Q-TOF-MS analysis 2.2.1. UPLC-Q-TOF-MS conditions An ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7µm) was used, with a mobile phase consisting of 0.1% aqueous formic acid solution (A) and methanol (B) and gradient elution (0–15 min, 25–65% B; 15–19 min, 65–80% B; 19–20 min, 80–25% B). The flow rate was set at 0.3 mL/min, and the column temperature was maintained at 35°C. Electrospray ionization (ESI) was used in positive ion mode scanning. The desolvation N 2 gas flow rate was set at 1000 L/h, with a desolvation gas temperature of 450 ℃. The cone gas flow rate was 50 L/h, the capillary voltage was 3.0 kV, the cone voltage was 30 V, and the ion source temperature was 120 ℃. 2.2.2. Preparation of test solution An appropriate amount of BSCO was weighed and placed in a 100 mL stoppered conical flask. Then, 30% methanol (50 mL) was added and the weight recorded. The test sample was subjected to 30 min of ultrasonic extraction at 300 W. After cooling, the weight was made up to the original weight with 30% methanol and the solution was mixed thoroughly. The sample solution was filtered through a 0.22 µm microporous filter membrane and the filtrate was collected for testing. 2.3. Network pharmacology analysis 2.3.1. BSCO active ingredients and target screening After identification of all active chemical components by UPLC-Q-TOF-MS, their structural data were uploaded to the SwissADME platform ( http://www.swissadme.ch ). Chemical components meeting at least two of Lipinski's Rule of Five (RO5) criteria were included in the active compounds, together with compounds for which in vivo activity studies had been reported in the literature. Other compounds were excluded. Chemical structures were obtained from the PubChem database ( https://pubchem.ncbi.nlm.nih.gov/ ). Potential targets of BSCO components were obtained from three databases: TCMSP ( https://old.tcmsp-e.com/tcmsp.php/ ) , ETCM2.0 ( http://www.tcmip.cn/ETCM2/front/ ) and Swiss Target Prediction ( http://www.swisstargetprediction.ch/ ). The targets obtained from the three databases were combined and duplicates removed to obtain the therapeutic targets of BSCO. The names of all targets were cross-checked using the UniProt online protein database ( https://www.uniprot.org/ ). 2.3.2. Identification of lung cancer specific targets Relevant targets were searched for using the keywords 'lung cancer' and 'lung cancer tumor' in four databases: Therapeutic Target Database (TTD, https://db.idrblab.net/ttd/ ) , GeneCards ( https://www.genecards.org/ ) , Online Mendelian Inheritance in Man (OMIM, https://omim.org/ ) and DisGeNET ( https://www.disgenet.org/ ). The targets obtained were combined and duplicates removed to obtain therapeutic targets for lung cancer. The names of all targets were cross-checked using the UniProt online protein database ( https://www.uniprot.org/ ). 2.3.3. Hub gene PPI network and pathway enrichment analysis The Venn online analysis platform ( http://jvenn.toulouse.ina.fr/app/example.html ) was used to draw a Venn diagram of the overlapping targets between drugs and diseases. The data were then input into the STRING database ( https://string-db.org/ ) to construct a protein-protein interaction (PPI) network. Cytoscape 3.10.2 was used to visualize the BSCO-lung cancer targets PPI network. The built-in data analysis function, Network Analyzer, was used to calculate topological parameters for each target in the network. The median node degree (Degree), median betweenness centrality (BC), and median closeness centrality (CC), each calculated twice, were used as evaluation criteria for the nodes. The obtained targets were imported into Cytoscape 3.10.2 and the hub gene PPI network was constructed. In parallel, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the Metascape database ( http://metascape.org/gp/index.html ). Pathways with P ≤ 0.01 according to the KEGG biological pathway enrichment analysis were selected. 2.4. Animal experiments 2.4.1. Animals and cell Male specific pathogen-free (SPF) Sprague Dawley (SD) rats (180–200 g) and male SPF C57BL/6J mice (18–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), permit number: SCXK (Jing) 2021–0011. The animals were fed in a temperature-controlled (25 ± 1 ℃) room. All experiments were approved by the Animal Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (NO.202313195) (NO.202313259). The cell line used Mouse-derived LLC (Lewis)-luc cells (mouse lung cancer cells with luciferase labeling), catalog number LZQ0009, Shanghai Zhongqiao Xinzhou Company, cultured in DMEM medium containing 10% fetal bovine serum. 2.4.2. Assessment of Lewis cell growth in mice The Lewis lung cancer mouse model was used to evaluate the therapeutic effect of BSCO on lung cancer, observing tissue morphology, in vivo imaging of the mice, and determination of multiple serum biochemical indicators 19 . 2.4.3. Experimental animals, grouping, and model construction Seventy-two C57BL/6 male mice weighing 20 ± 2 g were used for the experiments. The animals were housed at room temperature (25 ± 1 ℃), relative humidity of 60%, and normal alternation of day and night for 12 h, with free access to food and water. The animals were randomly divided into six groups of 12 animals: blank control, model, positive drug cyclophosphamide (Cytoxan, 0.2 mL/10 g), and BSCO low, medium, and high dose (0.2, 0.4, and 0.8 mL/10 g) groups. The mice were anesthetized and placed on the operating table in the right lateral position. The left armpit was shaved and disinfected with 75% ethanol. A 5 mm incision was made in the left axilla at the upper edge of the rib arch at the anterior line at about 1.5 cm. The skin and subcutaneous tissues were separated, and the chest wall was exposed until the pink lobes of the lungs could be seen moving due to respiration. Serum-free medium or phosphate buffered saline (50 µL) containing tumor cells (10 6 cells per mouse) was mixed with Matrigel matrix (50 µL) on ice and injected vertically into the left lung with a micro feeder at a depth of about 3 mm. After 20 s the needle was removed and the incision closed with 1 or 2 stitches. The cell line was mouse-derived LLC (Lewis)-luc cells (luciferase-labeled mouse lung cancer cells, No. LZQ0009, Shanghai Zhongqiao Xinzhou Company) cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum (FBS). After successful establishment of the model, the low, medium, and high dose BSCO groups were dosed by oral gavage once a day (0.2, 0.4, or 0.8 mL/10 g). The positive drug group was dosed by intraperitoneal injection with cyclophosphamide (0.2 mL/10 g) every other day. The model group received an equivalent volume of distilled water, and dosing was continued for 14 days. 2.4.4. Histomorphology observation After the mice had been euthanized, lung tissue (1 lobe) was taken and fixed in formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Morphological changes were observed by light microscopy (n = 3 in each group). The remaining lung tissues were frozen and stored. 2.4.5. Determination of serum biochemical indexes Blood samples were obtained 24 h after the final administration by removing the eyeballs. After resting at 4 ℃ for 30 min, the samples were centrifuged at 3500 r/min for 10 min and the supernatants stored at − 80 ℃ for analysis. The serum biochemical indexes determined in this study included: superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), hexokinase 2 (HK2), phosphofructokinase (PFK), malondialdehyde (MDA), glucose transporter protein 1 (GLUT1) and pyruvate kinase (PK). 2.4.6. Cell culture and evaluation of lung cancer cell growth Lung cancer Lewis cells were cultured in RPMI medium containing 10% FBS at 37°C under 5% CO 2 saturated humidity. The cells were trypsin digested and passaged when cell growth reached 70%~80%. Tumor cells in the logarithmic growth phase were incubated in 96-well plates for 12 h. After the cells had attached to the wall, they were treated with different concentrations of BSCO for 24, 48, and 72 h. The negative control group was Lewis cells cultured in culture medium. Cell viability was determined using the CCK-8 method. 2.4.7. Serum pharmacology With reference to the reported method for serum pharmacology of traditional Chinese medicine 20 , rats were randomly divided into blank control (n = 15), high-dose (n = 5), low-dose (n = 5), and cyclophosphamide control (n = 5) groups. The blank control group received an equal volume of saline by oral gavage for 7 d. Blood was collected under sterile conditions 1 h after the last gavage, and the serum was separated and filtered to remove bacteria. Lewis cells in the logarithmic growth phase were divided into five groups: normal control (10% normal culture serum + RPMI − 640 medium); blank serum (10% blank serum + RPMI-1640 medium); positive control group (10% cyclophosphamide group serum + RPMI-1640 medium); BSCO low-dose group (10% low-dose group serum + RPMI-1640 medium); BSCO high-dose group (10% high-dose group serum + RPMI1640 medium). After culture for 24, 48 or 72 h, cell proliferation was analyzed using the CCK-8 method. 2.5. Statistical analysis Data were analyzed using GraphPad Prism 5.0 software and expressed as mean ± standard deviation. Comparisons between groups were performed by one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns indicates no significant difference. 3. Results 3.1. Identification of the main chemical components of BSCO by UPLC-Q-TOF-MS The base peak chromatogram of BSCO is shown in Fig. 1. Using the accurate relative molecular mass data, information on secondary fragment ions, and literature data, a total of 20 chemical components were identified, as detailed in Table 1 . 3.2. Identification of overlap of BSCO targets and lung cancer A total of 17 active ingredients of BSCO were obtained using the SwissADME platform and relevant literature. Alpha-lactose, sucrose, and purine are excluded. These active ingredients were then searched in the Swiss Target Prediction, ETCM 2.0, and TCMSP databases to identify 460 therapeutic targets of BSCO. Additionally, by retrieving data from GeneCards, TTD, OMIM, and DisGeNET databases, a total of 5184 lung cancer-related genes were identified, among which 341 genes overlapped with the BSCO therapeutic targets. A Venn diagram was created and is shown in Fig. 2 A. 3.3. Hub gene PPI network analysis of BSCO lung cancer targets The PPI network constructed from the STRING database consisted of 341 overlapping targets, including 341 nodes representing the targets and 6,779 edges representing interactions. The data were imported into Cytoscape for analysis, where 51 hub genes were identified using topological parameter values. All nodes were ranked from high to low based on degree, with their sizes representing the degree and their colors ranging from dark to light. The results showed that the nodes representing glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein kinase B1 (AKT1), tumor protein p53 (TP53), tumor necrosis factor (TNF), and interleukin 6 (IL6) had larger areas and darker colors, indicating that these targets were of greater importance (Fig. 2 B). 3.4. GO and KEGG pathway enrichment analyses Using the Metascape database, 3,572 GO terms were enriched when applying a filter of P < 0.05. Among these, 2,694 entries were related to biological processes (BP), including response to hormone, cellular response to nitrogen compound, and cellular response to hormone stimulus. The cellular component (CC) category contained 161 entries, particularly membrane raft, membrane microdomain, and neuronal cell body. The molecular function (MF) category included 327 entries, such as phosphotransferase activity, alcohol group as acceptor, kinase activity, and protein kinase activity. The top 10 entries in each category were analyzed and plotted (Fig. 2 C). Similarly, KEGG enrichment analysis conducted using Metascape with a filter of P < 0.05 identified 230 pathways. Relevant information for the top 20 enriched pathways was imported through a bioinformatics platform to generate a KEGG enrichment pathway diagram. The results indicated that BSCO treatment of lung cancer primarily involves pathways such as Pathways in Cancer, Proteoglycans in Cancer, and the PI3K-Akt signaling pathway (Fig. 2 D). 3.5. Histopathological morphology changes in the lungs of Lewis lung cancer mice The results of H&E staining showed that lung tissue from mice in the model group had blurred cell edges, large and heterogeneous nuclei, and disturbed cell arrangement accompanied by partial alveolar rupture. Compared with the model group, lung tissue from the BSCO low-, medium-, and high-dose groups showed varying degrees of cell necrosis accompanied by hemorrhage, and structural damage to the lung tissue was ameliorated. In the positive cyclophosphamide(CTX) group, the cells were locally necrotic accompanied by mild hemorrhage, and a small number of necrotic cell fragments were seen, as shown in Fig. 3 . 3.6. Effects of BSCO on tumor growth in mice Imaging results showed that fluorescence values in the model group were elevated and significantly higher than those in the BSCO-treated groups ( P < 0.05, P < 0.01), suggesting that BSCO slowed the growth of LLC cells in mice. The positive control drug, cyclophosphamide, significantly inhibited the growth of LLC cells in mice compared with that in the model group ( P < 0.01), as shown in Fig. 4 . 3.7. Lung cancer-related serum indexes Compared with the blank control group (Control), serum levels of CAT, GSH-Px, and SOD were significantly lower in the model mice (Model, P < 0.001), while levels of PFK, GLUT1, HK2, MDA, and PK were significantly elevated ( P < 0.001). These data indicated successful establishment of the model. Compared with the model group, treatment with BSCO increased the levels of CAT, GSH-Px, and SOD to varying degrees, and decreased the levels of PFK, GLUT1, HK2, MDA, and PK, with the high dose being the most effective ( P < 0.001, Fig. 5 ). The results suggest that BSCO has a certain ameliorative effect on Lewis lung cancer in mice. 3.8. Effect of BSCO and drug-containing serum on lung cancer cell viability Serum from rats dosed with low and high doses of BSCO, as well as cyclophosphamide, did not affect Lewis cell viability compared to the blank group when incubated for 72 h, with more apoptotic cells observed under the microscope in all groups at this time point. The 24 and 48 h data showed that low and high doses of BSCO as well as cyclophosphamide significantly inhibited the viability of Lewis cells (Fig. 6 C). 4. Discussion In recent years, an increasing number of traditional Chinese medicine and herbal therapies have been shown to be effective with few side effects, and traditional Chinese medicine is gradually aligning with modern medicine 21 . We first analyzed the main chemical components of BSCO using UPLC-Q-TOF-MS. Subsequently, through network pharmacology and in vitro and in vivo experiments, we identified the key targets and pathways associated with BSCO treatment of lung cancer, and confirmed its efficacy against this disease. Our analysis suggested that the primary active component may be caffeic acid phenethyl ester (CAPE), a phenolic acid compound with significant potential as an anticancer agent. CAPE exerts its effects in cancer progression through the phosphoinositide 3-kinase/protein kinase B (PI3K-Akt) and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways, which is consistent with predictions from network pharmacology KEGG analysis 22 , 23 . The identified key targets included GAPDH, AKT1, TP53, TNF, and IL6. TP53 plays a role in preventing carcinogenesis, and the most common genetic alteration in various human cancers is mutation of TP53 24 . It is also speculated that BSCO may exert its anticancer effects through the PI3K-Akt signaling pathway. This pathway is crucial for cancer cell survival and affects tumor proliferation, apoptosis, and autophagy 24 . The PI3K-Akt signaling pathway plays a pivotal role in regulating various cellular processes by activating downstream effectors, significantly contributing to tumor proliferation, invasion, and metastasis. One key aspect of Akt activation is its ability to enhance glucose uptake in cancer cells by modulating the expression of GLUT1 25 . In addition to increasing glucose uptake, Akt regulates several key glycolytic enzymes, including HK2 and PFK, through both post-translational and transcriptional mechanisms, which ultimately promotes the activation of PFK1 26 . This was confirmed in subsequent experiments. The activation of both PFK1 and GLUT1 facilitates cell proliferation and tumorigenesis 27 . As a result, targeting the PI3K-Akt pathway to control tumor metabolism has emerged as a potential therapeutic strategy for treating cancer. 28 . In this study, the efficacy of BSCO on lung cancer was evaluated from the cellular and serum pharmacological levels. BSCO was shown to inhibit proliferation of Lewis lung cancer cells with an IC 50 of 173 mg/mL. The viability of Lewis cells treated with serum from rats dosed with high and low doses of BSCO, and cyclophosphamide, was reduced after incubation for 24 and 48 h compared with the control. The effect of high dose BSCO serum was greater than that of low dose BSCO serum. The levels of SOD, GSH-Px, CAT, HK2, PFK, MDA, GLUT1, and PK serum markers were restored towards normal by BSCO in Lewis lung carcinoma mice, with the best effect seen at the high dose. BSCO regulates cancer cell carbon metabolism and glucose uptake by influencing key metabolic pathways. The histopathological morphology of mouse lung tissue was observed by H&E staining and showed that BSCO inhibited the growth of LLC tumor cells in mice. This result was confirmed by fluorescence imaging of lung cancer mice. In summary, this study identified compounds contained in BSCO and network pharmacology enabled identification of potential molecular targets, signaling pathways, and protein interactions regulated by those components. Experimental methods were employed to validate these findings both in vitro and in vivo. The results confirmed that BSCO exerts significant anti-cancer effects on lung cancer cells, providing valuable insights into its therapeutic potential. 5. Conclusion This study identified 20 major chemical components of BSCO. Network pharmacology suggested that BSCO may exert anticancer effects through targets including GAPDH, AKT1, TP53, TNF, and IL6, and the PI3K-Akt signaling pathway. Experimental data demonstrated effective inhibition of lung cancer cell proliferation in vitro and in vivo. Moreover, BSCO significantly restored serum biochemical indicators closely related to lung cancer and inhibited the growth of lung tumors, providing a reference for its clinical application in the prevention and treatment of lung cancer. Abbreviations BSCO Blood stasis constitution ointment; UHPLC-Q-TOF-MS Ultra-High Performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry; PPI Protein-Protein Interaction; GO Geneontology; KEGG Kyoto encyclopedia of genes and genomes; BP Biological Process; CC Cellular Component; MF Molecular Function; HE Hematoxylin-Eosin staining; PI3K-Akt signaling pathway Phosphoinositide 3-Kinase/Protein Kinase B pathway; GAPDH Glyceraldehyde-3-phosphate dehydrogenase; AKT1 Protein kinase B1; TP53 Tumor protein p53; TNF Tumor Necrosis Factor; IL6 Interleukin 6; SOD superoxide dismutase; GSH-Px glutathione peroxidase; CAT catalase; HK2 hexokinase 2; PFK phosphofructokinase; MDA malondialdehyde ; GLUT1 glucose transporter protein 1; PK pyruvate kinase. Declarations Competing interests The authors declare no conflicts of interest. Clinical trial number not applicable. Author Contribution Maobo Du and Hu You designed the study and helped coordinate support and funding. Qiaozhi Wang conducted research and wrote the manuscripts. Chuanhao Dai and Tianming Lu participated in the experiments. Juhe Wang and Shuo Shen analyzed the data. Xingjiang Xiong and Qiuyan Guo guided the experiment. Acknowledgement This research was financially supported by National Intangible Cultural Heritage Project [IX-4(1)]，China Academy of Chinese Medical Sciences Science and Technology Innovation Project (CI2021A04313). References Lortet-Tieulent J, Renteria E, Sharp L, Weiderpass E, Comber H, Baas P, Bray F, Coebergh JW, Soerjomataram I. Convergence of Decreasing Male and Increasing Female Incidence Rates in Major Tobacco-Related Cancers in Europe in 1988–2010. Eur J Cancer. 2015;51(9):1144–63. https://doi.org/10.1016/j.ejca.2013.10.014 . Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. 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Overall Survival with Osimertinib in Resected Mutated NSCLC. N Engl J Med. 2023;389(2):137–47. https://doi.org/10.1056/NEJMoa2304594 . Chen S, Flower A, Ritchie A, Liu J, Molassiotis A, Yu H, Lewith G. Oral Chinese Herbal Medicine (CHM) as an Adjuvant Treatment during Chemotherapy for Non-Small Cell Lung Cancer: A Systematic Review. Lung Cancer. 2010;68(2):137–45. https://doi.org/10.1016/j.lungcan.2009.11.008 . Wong W, Lam CLK, Wong VT, Yang ZM, Ziea ETC, Kwan AK. L. Validation of the Constitution in Chinese Medicine Questionnaire: Does the Traditional Chinese Medicine Concept of Body Constitution Exist? Evid Based Complement Alternat Med 2013, 2013 , 481491. https://doi.org/10.1155/2013/481491 Cho W, Kim JH, Jeong M, Kim M-S, Lee J, Son H, Cheon C, Park S, Ko S-G. Pattern Identification of Lung Cancer Patients Based on Body Constitution Questionnaires (BCQ) and Glycoproteomics for Precision Medicine. Medicine. 2019;98(24):e16035. https://doi.org/10.1097/MD.0000000000016035 . Liang X, Wang Q, Jiang Z, Li Z, Zhang M, Yang P, Wang X, Wang Y, Qin Y, Li T, Zhang T, Wang Y, Sun J, Li Y, Luo H, Li L. Clinical Research Linking Traditional Chinese Medicine Constitution Types with Diseases: A Literature Review of 1639 Observational Studies. J Tradit Chin Med. 2020;40(4):690–702. https://doi.org/10.19852/j.cnki.jtcm.2020.04.019 . You H. Nine Types of Constitution and Health Preserving Pills (Second Edition). China Traditional Chinese Medicine Publishing House: Beijing, 2019. Wang X, Xing X, Huang P, Zhang Z, Zhou Z, Liang L, Yao R, Wu X, Yang LA. Chinese Classical Prescription Xuefu Zhuyu Decoction in the Treatment of Coronary Heart Disease: An Overview. Heliyon. 2024;10(7):e28919. https://doi.org/10.1016/j.heliyon.2024.e28919 . Xu Y, Chen B, Yi J, Tian F, Liu Y, Ouyang Y, Yuan C, Liu B. Buyang Huanwu Decoction Alleviates Cerebral Ischemic Injury through Modulating Caveolin-1-Mediated Mitochondrial Quality Control. Front Pharmacol. 2023;14:1137609. https://doi.org/10.3389/fphar.2023.1137609 . Han Zhiqiang; Deng Dehou, Feng Y, Zhang Y. Jin Ying; Cheng Bin; Zhang Aiqin. Clinical Study on the Correlation between Traditional Chinese Medicine Blood Stasis Constitution and Lung Cancer and the Polymorphism of the CHRNB4 rs7178270 Gene. China J Traditional Chin Med Pharm. 2020;38(6):90–93269. https://doi.org/10.13193/j.issn.1673-7717.2020.06.022 . Feng Y, Dai L, Zhang Y, Sun S, Cong S, Ling S, Zhang H. Buyang Huanwu Decoction Alleviates Blood Stasis, Platelet Activation, and Inflammation and Regulates the HMGB1/NF-κB Pathway in Rats with Pulmonary Fibrosis. J Ethnopharmacol. 2024;319:117088. https://doi.org/10.1016/j.jep.2023.117088 . Li H, Zhang W, Lou Q, Chang Y, Lin Z, Lou L. XueFu ZhuYu Decoction Alleviates Cardiopulmonary Bypass-Induced NLRP3 Inflammasome-Dependent Pyroptosis by Inhibiting IkB-α/NF-κB Pathway in Acute Lung Injury Rats. Evidence-Based Complementary and Alternative Medicine 2022, 2022 , 1–15. https://doi.org/10.1155/2022/6248870 Liu P, Zhao L, Senovilla L, Kepp O, Kroemer G. In Vivo Imaging of Orthotopic Lung Cancer Models in Mice. Methods Mol Biol. 2021;2279:199–212. https://doi.org/10.1007/978-1-0716-1278-1_16 . Ge J, Wang D, He R, Zhu H, Wang Y, He S. Medicinal Herb Research: Serum Pharmacological Method and Plasma Pharmacological Method. Biol Pharm Bull. 2010;33(9):1459–65. https://doi.org/10.1248/bpb.33.1459 . Peng L, Zhang K, Li Y, Chen L, Gao H, Chen H. Real-World Evidence of Traditional Chinese Medicine (TCM) Treatment on Cancer: A Literature-Based Review. Evidence-Based Complementary and Alternative Medicine 2022, 2022 , 1–10. https://doi.org/10.1155/2022/7770380 Elumalai P, Muninathan N, Megalatha ST, Suresh A, Kumar KS, Jhansi N, Kalaivani K, Krishnamoorthy G. An Insight into Anticancer Effect of Propolis and Its Constituents: A Review of Molecular Mechanisms. Evidence-Based Complementary and Alternative Medicine 2022, 2022 , 1–14. https://doi.org/10.1155/2022/5901191 Mirzaei S, Gholami MH, Zabolian A, Saleki H, Farahani MV, Hamzehlou S, Far FB, Sharifzadeh SO, Samarghandian S, Khan H, Aref AR, Ashrafizadeh M, Zarrabi A, Sethi G. Caffeic Acid and Its Derivatives as Potential Modulators of Oncogenic Molecular Pathways: New Hope in the Fight against Cancer. Pharmacol Res. 2021;171:105759. https://doi.org/10.1016/j.phrs.2021.105759 . Huang R, Dai Q, Yang R, Duan Y, Zhao Q, Haybaeck J, Yang ZA, Review. PI3K/AKT/mTOR Signaling Pathway and Its Regulated Eukaryotic Translation Initiation Factors May Be a Potential Therapeutic Target in Esophageal Squamous Cell Carcinoma. Front Oncol. 2022;12:817916. https://doi.org/10.3389/fonc.2022.817916 . Hosios AM, Manning BD. Cancer Signaling Drives Cancer Metabolism: AKT and the Warburg Effect. Cancer Res. 2021;81(19):4896–8. https://doi.org/10.1158/0008-5472.CAN-21-2647 . Hoxhaj G, Manning BD. The PI3K–AKT Network at the Interface of Oncogenic Signalling and Cancer Metabolism. Nat Rev Cancer. 2020;20(2):74–88. https://doi.org/10.1038/s41568-019-0216-7 . Lee J-H, Liu R, Li J, Wang Y, Tan L, Li X-J, Qian X, Zhang C, Xia Y, Xu D, Guo W, Ding Z, Du L, Zheng Y, Chen Q, Lorenzi PL, Mills GB, Jiang T, Lu Z. EGFR-Phosphorylated Platelet Isoform of Phosphofructokinase 1 Promotes PI3K Activation. Mol Cell. 2018;70(2):197–e2107. https://doi.org/10.1016/j.molcel.2018.03.018 . Zhang W, Cai S, Qin L, Feng Y, Ding M, Luo Z, Shan J, Di L. Alkaloids of Aconiti Lateralis Radix Praeparata Inhibit Growth of Non-Small Cell Lung Cancer by Regulating PI3K/Akt-mTOR Signaling and Glycolysis. Commun Biol. 2024;7(1):1118. https://doi.org/10.1038/s42003-024-06801-6 . Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1MainchemicalcomponentsofBSCO.docx Cite Share Download PDF Status: Published Journal Publication published 08 Dec, 2025 Read the published version in Cancer Management and Research → Version 1 posted 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-5924974","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":411330928,"identity":"e0bd3ebb-5261-4428-9528-b9c4cd7a9f84","order_by":0,"name":"Qiaozhi Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIie3RsWrDMBCA4RMGeTFNxyuG+hVkDMGDSV/ljEBZQikUSrIkKgV1yQP4MfIIoabJIjqn0EGh4NljhwztHIrtMYP+7eC+4TgAn+8yYw7nuEoSJ10riskQEojc5pRqatLqQckhhF8vzJxgW7o4at+Y7tsX+2kDnwbvma4pK8Q2gLB+33QS69Sx+sDHkBn6nomvK4iUOnSR8YH2GT4he36JKJuJJgCMxj2kNPGJI9O7URvnoma6n0iOaLDUFiiGIeTONoFAi1lagUrXQkned8vN6/T498rlbYIg3c+pmIzCetdJAOhs5t3r/xGfz+fznfcLC3VPiXqheewAAAAASUVORK5CYII=","orcid":"","institution":"China Academy of Chinese Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Qiaozhi","middleName":"","lastName":"Wang","suffix":""},{"id":411330930,"identity":"2d6e2e68-64bb-45e8-802c-6c4e0abd39a2","order_by":1,"name":"Juhe Wang","email":"","orcid":"","institution":"Gu Zheng Bao He Internet Hospital","correspondingAuthor":false,"prefix":"","firstName":"Juhe","middleName":"","lastName":"Wang","suffix":""},{"id":411330931,"identity":"d1cdcb19-6121-48c4-9e37-d1059ebb55f1","order_by":2,"name":"Chuanhao Dai","email":"","orcid":"","institution":"China Academy of Chinese Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chuanhao","middleName":"","lastName":"Dai","suffix":""},{"id":411330932,"identity":"e549500f-e372-48a7-9c4e-cad86ab283f5","order_by":3,"name":"Tianming Lu","email":"","orcid":"","institution":"China Academy of Chinese Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tianming","middleName":"","lastName":"Lu","suffix":""},{"id":411330933,"identity":"1ffc54bf-eab7-4877-ac30-73140028152f","order_by":4,"name":"Xingjiang Xiong","email":"","orcid":"","institution":"Gu Zheng Bao He Internet Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xingjiang","middleName":"","lastName":"Xiong","suffix":""},{"id":411330934,"identity":"b6461ba4-7638-4b70-9d5e-4946a2cd6bb2","order_by":5,"name":"Shuo Shen","email":"","orcid":"","institution":"China Academy of Chinese Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Shen","suffix":""},{"id":411330935,"identity":"ac800db3-c4e8-4c14-a53a-32112ae2e2b1","order_by":6,"name":"Qiuyan Guo","email":"","orcid":"","institution":"China Academy of Chinese Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qiuyan","middleName":"","lastName":"Guo","suffix":""},{"id":411330936,"identity":"72c2fa55-a0cf-4d4b-9731-3abd395da704","order_by":7,"name":"Hu You","email":"","orcid":"","institution":"Gu Zheng Bao He Internet Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hu","middleName":"","lastName":"You","suffix":""},{"id":411330937,"identity":"5e0de37d-fbd6-4106-a0d0-077666dff213","order_by":8,"name":"Maobo Du","email":"","orcid":"","institution":"China Academy of Chinese Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Maobo","middleName":"","lastName":"Du","suffix":""}],"badges":[],"createdAt":"2025-01-29 14:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5924974/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5924974/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.2147/CMAR.S563551","type":"published","date":"2025-12-09T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75706807,"identity":"ebb5ba41-59c0-40ef-8db3-b4ec07bffd23","added_by":"auto","created_at":"2025-02-07 10:25:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":339115,"visible":true,"origin":"","legend":"\u003cp\u003eBase peak chromatogram of BSCO in positive ion mode\u003c/p\u003e","description":"","filename":"Onlinefig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5924974/v1/9e4cff498aeb88580b8121bf.png"},{"id":75707200,"identity":"2e38f8f8-56cc-40e3-9c0a-232b9d625145","added_by":"auto","created_at":"2025-02-07 10:33:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1121256,"visible":true,"origin":"","legend":"\u003cp\u003eNetwork pharmacology analysis of BSCO in lung cancer. (A) Venn diagram of BSCO drug targets and lung cancer targets. (B) PPI network interaction diagram of BSCO and lung cancer shared hub targets. (C) GO enrichment analysis based on overlapping genes. (D) KEGG enrichment analysis based on overlapping genes.\u003c/p\u003e","description":"","filename":"Onlinefig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5924974/v1/c200a707fdbe7e08e3d52182.png"},{"id":75706808,"identity":"3efbc755-a969-4762-be0f-63d5fd4504f7","added_by":"auto","created_at":"2025-02-07 10:25:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2384834,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of BSCO on histopathological morphology in the lungs of Lewis lung cancer mice (H\u0026amp;E, ×200)\u003c/p\u003e","description":"","filename":"Onlinefig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5924974/v1/9bfee4be371f8828c07ccbb2.png"},{"id":75706809,"identity":"eaaf6103-29eb-4efb-9724-3f99bb7d60ae","added_by":"auto","created_at":"2025-02-07 10:25:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2702489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of BSCO on tumors in lung cancer mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(Data are shown as the mean ± SD; * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, **** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 vs. the model group, n = 6）\u003c/p\u003e","description":"","filename":"Onlinefig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5924974/v1/b950b2270f7b6b6820067b28.png"},{"id":75706806,"identity":"d70c40e7-d042-4901-b9e6-ee24264dbfc9","added_by":"auto","created_at":"2025-02-07 10:25:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":740267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of BSCO on serum indexes of Lewis lung cancer in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(n = 6, data expressed as mean ± standard deviation # \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ### \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. Control; * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. Model)\u003c/p\u003e","description":"","filename":"Onlinefig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-5924974/v1/7db73347a46df82a10c64c99.png"},{"id":75706816,"identity":"1c9b13b4-02e7-41b1-8a24-2d66bda8534f","added_by":"auto","created_at":"2025-02-07 10:25:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":489029,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BSCO and drug-containing serum on Lewis cell viability\u003c/p\u003e\n\u003cp\u003eBSCO at 100–200\u0026nbsp;mg/mL showed significant cytotoxicity towards Lewis cells at 24\u0026nbsp;h (\u003cem\u003eP\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.05), and the IC\u003csub\u003e50\u003c/sub\u003e was calculated to be 173\u0026nbsp;mg/mL (Figs. 6A, 6B).\u003c/p\u003e","description":"","filename":"Onlinefig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-5924974/v1/9036036f021c0c255be752bc.png"},{"id":99256488,"identity":"21de7d7c-f6f7-413c-8765-e274aedf8484","added_by":"auto","created_at":"2025-12-30 22:32:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2234241,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5924974/v1/6d9a4e52-8f04-455b-8a6b-dd78c7b38c6d.pdf"},{"id":75706822,"identity":"00ee1d13-683a-43d4-ace3-22bf71ab51ae","added_by":"auto","created_at":"2025-02-07 10:25:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":123482,"visible":true,"origin":"","legend":"","description":"","filename":"Table1MainchemicalcomponentsofBSCO.docx","url":"https://assets-eu.researchsquare.com/files/rs-5924974/v1/629bcc05b370f24a4d55c9c1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated UPLC-Q-TOF-MS, network pharmacology and experimental approach to evaluate the effects of blood stasis constitution ointment on lung cancer","fulltext":[{"header":"Highlights","content":"\u003cp\u003eUHPLC-Q-TOF-MS identified 20 components of BSCO.\u003c/p\u003e\u003cp\u003eNetwork pharmacology suggested potential targets and pathways affected by BSCO in the treatment of lung cancer.\u003c/p\u003e\u003cp\u003eBSCO effectively inhibited tumor growth in a Lewis lung cancer mouse model.\u003c/p\u003e\u003cp\u003eBSCO increased serum levels of CAT, GSH-Px, and SOD, and decreased levels of PFK, GLUT1, HK2, MDA, and PK to varying degrees.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eLung cancer is still the leading cause of cancer deaths worldwide and the number of cases continues to rise in most countries \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It is characterized by a high mortality rate after diagnosis and it has been estimated by the International Agency for Research on Cancer (IARC) under the World Health Organization (WHO) that there were about 2.2\u0026nbsp;million new cases and 1.8\u0026nbsp;million deaths in 2020 \u003csup\u003e2\u003c/sup\u003e. Therefore, screening and prevention have become important to reduce the mortality rate of lung cancer \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe pathogenesis of lung cancer is still unclear in modern medicine. Recently, it has been linked to the lung microbiome, and smoking is the main risk factor \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Most lung cancers are preceded by obvious precancerous lesions, such as hyperplasia, metaplasia, abnormal growth, and carcinoma in situ, which are mostly believed to be consequences of immune dysfunction. According to the theory of traditional Chinese medicine (TCM), lung cancer belongs to the category of \"lung accumulation\", and the main pathogenesis is \"depression\". It is primarily due to deficiency of lung qi, which leads to internal obstruction of phlegm and turbidity in the body, followed by invasion of the lungs by external pathogens. Ultimately, this leads to qi stagnation and dampness obstruction, internal stagnation of phlegm and turbidity, phlegm and blood stasis interconnections, and the pathogenesis of transforming toxins into cancers.\u003c/p\u003e \u003cp\u003eClinical treatment of lung cancer domestically and internationally includes surgery, radiotherapy, chemotherapy, ablation, targeted therapy, and immunotherapy \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Surgical treatment is preferred, but due to factors such as surgical risk, comorbidities, and patient willingness, many patients do not meet the criteria for standard surgical resection. Although radiotherapy and chemotherapy can inhibit the growth of cancer cells, they are accompanied by significant side effects. The development of the targeted therapeutic drug osimertinib and the immunotherapeutic drug pembrolizumab has provided new treatment options for lung cancer, but the survival rate is still not high \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. New, effective drugs are required to improve clinical survival. Oral herbal medicines have also been shown to effectively inhibit the growth of lung cancer \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Chinese medicine constitution is a comprehensive and relatively stable inherent trait that is based on innate and acquired characteristics of the human body during life processes. It includes morphological structure, physiological function, and psychological state, and represents individual characteristics of the human body that are adapted to the natural and social environment during growth and development \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The Chinese medicine constitution has been widely recognized internationally \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. prescription\u003c/p\u003e \u003cp\u003eAccording to the standard \"Classification and Determination of Constitution in TCM\" (ZYYXH/T157-2009) issued by the Chinese Society of Traditional Chinese Medicine, Chinese medicine constitutions are categorized into nine types: Qi-deficiency, Damp-heat, Yin-deficiency, Qi-stagnation, Yang-deficiency, Phlegm-dampness, Blood stasis, Special, and Balanced. TCM constitutions are associated with specific diseases and could be used to guide individualized prevention and treatment \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The constitution has both stability and variability, through intervention to adjust its bias, reflecting the adjustability of the constitution. Oral paste is the best choice for adjusting the constitution \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eExperts from top institutions, such as the China Academy of Chinese Medical Sciences, Nanjing University of Chinese Medicine, and other leading international organizations, have combined TCM constitution and \"Medicine and Food Homology\" (MFH) theories, leveraging the research strengths of academic institutions and the clinical expertise of frontline hospitals. They have carefully formulated blood stasis constitution ointment (BSCO) based on selected classic TCM prescriptions.\u003c/p\u003e \u003cp\u003eBased on the classic formulas Xuefu Zhuyu and Buyang Huanwu decoctions appearing in Yilin Gaicuo by Wang Qingren in 1830, BSCO consists of \u003cem\u003ePanax ginseng\u003c/em\u003e C.A.Mey., \u003cem\u003ePrunus persica\u003c/em\u003e (L.) Batsch, \u003cem\u003eHippophae rhamnoides\u003c/em\u003e L., \u003cem\u003eVigna umbellata\u003c/em\u003e (Thunb.) Ohwi \u0026amp; H.Ohashi, E\u0026rsquo;Jiao (Colla corii asini), oxhide gelatin and other herbs, and is made into a paste according to the law.\u003c/p\u003e \u003cp\u003eXuefu Zhuyu and Buyang Huanwu decoctions are well-known TCM prescriptions that are used to treat blood stasis syndrome. They are currently employed extensively in the management of cardiovascular disorders, and substantial therapeutic efficacy has been demonstrated \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccording to TCM theory, one of the core elements of lung cancer is an unbalanced constitution, with blood stasis being the representative constitution \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Blood stasis can lead to a hypercoagulable state, which not only increases the risk of thrombosis in lung cancer patients, but also contributes to tumor proliferation, severely affecting quality of life and prognosis in these patients. Recent studies have shown the palliative effect of Xuefu Zhuyu and Buyang Huanwu decoctions on symptoms associated with lung cancer \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Based on the two prescriptions mentioned above, modifications were made to prepare BSCO, and its therapeutic effect on lung cancer was evaluated.\u003c/p\u003e \u003cp\u003eThe formulation addresses four aspects: Reinforce healthy qi, Correcting Imbalances, Eliminate pathogenic factors, and flavoring. Huangdi Neijing suggests that \u0026ldquo;When the healthy qi is strong within, pathogenic factors cannot act\u0026rdquo;. In this formula, \u003cem\u003ePanax ginseng\u003c/em\u003e C.A.Mey. and oxhide gelatin are used to replenish qi and blood, assist in strengthening qi and eliminate pathogenic factors. They serve as the monarch medicinal components. Since yin and blood are interdependent, and Essence and blood share the same source, E'Jiao (Colla corii asini) and \u003cem\u003eTremella fuciformis\u003c/em\u003e Berk. are used to nourish yin, while \u003cem\u003eLycium chinense\u003c/em\u003e Mill. and \u003cem\u003eNelumbo nucifera\u003c/em\u003e Gaertn. are used to assist the postnatal foundation. These substances support the postnatal constitution and nourish the source of transformation, serving as the minister medicines. \u003cem\u003ePrunus persica\u003c/em\u003e (L.) Batsch, \u003cem\u003ePueraria alopecuroides\u003c/em\u003e Craib, and \u003cem\u003eRosae rugosae\u003c/em\u003e Thunb. are used to circulate blood and transform stasis, alleviating deficiency in the middle jiao. \u003cem\u003eHordeum vulgare\u003c/em\u003e L. is used to soothe the liver and regulate qi, promoting movement of qi to enhance blood flow. \u003cem\u003ePoria cocos (\u003c/em\u003eSchw.) Wolf is used to transform fluid retention, \u003cem\u003eCitrus reticulata\u003c/em\u003e Blanco to transform phlegm, and \u003cem\u003eCrataegus pinnatifida\u003c/em\u003e Bunge and \u003cem\u003eHippophae rhamnoides\u003c/em\u003e L to transform indigestion. These ingredients work together to alleviate the Phlegm qi stagnation pattern, serving as the assistant medicines. \u003cem\u003eVigna umbellata\u003c/em\u003e (Thunb.) Ohwi \u0026amp; H.Ohashi is used to eliminate dampness and Circulate blood, while \u003cem\u003eAllium macrostemon\u003c/em\u003e Bunge is used to activate yang and dissipate cold. Oligosaccharide maltose is used to strengthen the spleen and stomach, correct the bitter taste, provide shape and texture, and improve the flavor without the concern of raising blood sugar levels, making it more acceptable to the public.\u003c/p\u003e \u003cp\u003eThe formula considers food safety while enhancing therapeutic effect in the concentrated form of a paste. It is convenient for long-term use and has no obvious toxic side effects. Due to the high viscosity of the paste, it has a number of advantages, such as high concentration of effective components, rapid absorption, prolonged and lasting effects, and tangible therapeutic efficacy. The effective components regulate the body's yin-yang balance through the Meridian affinity of the medicine and reinforce healthy qi to strengthen the body and improve the constitution. They act on pathogenic characteristics of the blood stasis constitution comprehensively and fundamentally, thus serving the purpose of regulation and health care. It has promising industrial application prospects and holds significant social and economic value.\u003c/p\u003e \u003cp\u003e\"Oral Paste\" is a type of Chinese medicinal paste that is primarily used for health preservation and wellness, and is also known as \"nourishing ointment\". These oral pastes are prescribed by experienced TCM practitioners who, based on the constitution and health of the individual, follow the holistic view and the principle of syndrome differentiation and treatment in TCM. They select single or multiple medicinal herbs to create a well-balanced formula, which is then processed through rigorous and specific techniques. The main purposes of these pastes are to nourish and strengthen the body, resist aging, prolong life, and prevent and treat diseases.\u003c/p\u003e \u003cp\u003eOn November 10, 2021, China's National Health and Wellness Commission issued the \u0026ldquo;Circular on the Issuance of the Provisions on the Management of the Catalog of Substances that are Traditionally Used as Both Food and Chinese Herbal Medicines\u0026rdquo;, which stipulates that food and medicinal substances refer to those substances that are traditionally used as food and included in the \u0026ldquo;Pharmacopoeia of the People's Republic of China\u0026rdquo;. Previously, the former Ministry of Health published the \u0026ldquo;Notice on Further Standardizing the Management of Health Food Raw Materials\u0026rdquo;, which made specific provisions on MFH substances, substances that can be used in health food, and substances prohibited in health food.\u003c/p\u003e \u003cp\u003eAccording to the above provisions, the constitution, dietary therapy, and oral pastes are closely related. These MFH pastes are increasingly gaining recognition among the public. They are appreciated for their safety and convenience, with a sweet and palatable taste that encourages long-term compliance. In particular, they are highly effective for improving the constitution and offer lasting and stable therapeutic effects. Driven by the wave of traditional Chinese medicine health and wellness, consumption of these MFH pastes for body regulation and disease treatment is becoming increasingly popular in China.\u003c/p\u003e \u003cp\u003eIn summary, this research is based on the doctrine of constitution and medicinal paste, drawing on classic prescriptions and utilizing medicine food homology substances that meet safety requirements to create BSCO. This study utilized ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) combined with network pharmacology to identify the active components of BSCO and predict their biological targets. Additionally, the effects of BSCO on lung cancer were evaluated both in vitro and in vivo, providing evidence-based support for its efficacy.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagents and instruments\u003c/h2\u003e \u003cp\u003eBSCO was obtained from Sichuan Chengdu Guzheng Baohe Health Management Co., Ltd., chromatographic grade methanol was purchased from Fisher Scientific, and pure water was from Hangzhou Wahaha Group Co., Ltd.\u003c/p\u003e \u003cp\u003eInstruments: ACQUITY UPLC H-Class ultra-high performance liquid chromatography system and Vion IMS QTof high-resolution mass spectrometer (Waters Corporation, USA); ME155DU electronic balance (Mettler-Toledo Instruments); BioTek Synergy H1 Multifunctional Microplate Detector (Agilent); QX200\u0026trade; Droplet Digital\u0026trade; PCR System (Bio-RAD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. UPLC-Q-TOF-MS analysis\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. UPLC-Q-TOF-MS conditions\u003c/h2\u003e \u003cp\u003eAn ACQUITY UPLC BEH C18 column (2.1 \u0026times; 50 mm, 1.7\u0026micro;m) was used, with a mobile phase consisting of 0.1% aqueous formic acid solution (A) and methanol (B) and gradient elution (0\u0026ndash;15 min, 25\u0026ndash;65% B; 15\u0026ndash;19 min, 65\u0026ndash;80% B; 19\u0026ndash;20 min, 80\u0026ndash;25% B). The flow rate was set at 0.3 mL/min, and the column temperature was maintained at 35\u0026deg;C. Electrospray ionization (ESI) was used in positive ion mode scanning. The desolvation N\u003csub\u003e2\u003c/sub\u003e gas flow rate was set at 1000 L/h, with a desolvation gas temperature of 450 ℃. The cone gas flow rate was 50 L/h, the capillary voltage was 3.0 kV, the cone voltage was 30 V, and the ion source temperature was 120 ℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Preparation of test solution\u003c/h2\u003e \u003cp\u003eAn appropriate amount of BSCO was weighed and placed in a 100 mL stoppered conical flask. Then, 30% methanol (50 mL) was added and the weight recorded. The test sample was subjected to 30 min of ultrasonic extraction at 300 W. After cooling, the weight was made up to the original weight with 30% methanol and the solution was mixed thoroughly. The sample solution was filtered through a 0.22 \u0026micro;m microporous filter membrane and the filtrate was collected for testing.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Network pharmacology analysis\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. BSCO active ingredients and target screening\u003c/h2\u003e \u003cp\u003eAfter identification of all active chemical components by UPLC-Q-TOF-MS, their structural data were uploaded to the SwissADME platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissadme.ch\u003c/span\u003e\u003cspan address=\"http://www.swissadme.ch\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Chemical components meeting at least two of Lipinski's Rule of Five (RO5) criteria were included in the active compounds, together with compounds for which in vivo activity studies had been reported in the literature. Other compounds were excluded. Chemical structures were obtained from the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Potential targets of BSCO components were obtained from three databases: TCMSP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://old.tcmsp-e.com/tcmsp.php/\u003c/span\u003e\u003cspan address=\"https://old.tcmsp-e.com/tcmsp.php/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, ETCM2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.tcmip.cn/ETCM2/front/\u003c/span\u003e\u003cspan address=\"http://www.tcmip.cn/ETCM2/front/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and Swiss Target Prediction (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://www.swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e The targets obtained from the three databases were combined and duplicates removed to obtain the therapeutic targets of BSCO. The names of all targets were cross-checked using the UniProt online protein database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Identification of lung cancer specific targets\u003c/h2\u003e \u003cp\u003eRelevant targets were searched for using the keywords 'lung cancer' and 'lung cancer tumor' in four databases: Therapeutic Target Database (TTD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"https://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, Online Mendelian Inheritance in Man (OMIM, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003cspan address=\"https://omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and DisGeNET (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.disgenet.org/\u003c/span\u003e\u003cspan address=\"https://www.disgenet.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e The targets obtained were combined and duplicates removed to obtain therapeutic targets for lung cancer. The names of all targets were cross-checked using the UniProt online protein database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Hub gene PPI network and pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eThe Venn online analysis platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://jvenn.toulouse.ina.fr/app/example.html\u003c/span\u003e\u003cspan address=\"http://jvenn.toulouse.ina.fr/app/example.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to draw a Venn diagram of the overlapping targets between drugs and diseases. The data were then input into the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to construct a protein-protein interaction (PPI) network. Cytoscape 3.10.2 was used to visualize the BSCO-lung cancer targets PPI network. The built-in data analysis function, Network Analyzer, was used to calculate topological parameters for each target in the network. The median node degree (Degree), median betweenness centrality (BC), and median closeness centrality (CC), each calculated twice, were used as evaluation criteria for the nodes. The obtained targets were imported into Cytoscape 3.10.2 and the hub gene PPI network was constructed.\u003c/p\u003e \u003cp\u003eIn parallel, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the Metascape database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://metascape.org/gp/index.html\u003c/span\u003e\u003cspan address=\"http://metascape.org/gp/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Pathways with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 according to the KEGG biological pathway enrichment analysis were selected.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Animal experiments\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Animals and cell\u003c/h2\u003e \u003cp\u003eMale specific pathogen-free (SPF) Sprague Dawley (SD) rats (180\u0026ndash;200 g) and male SPF C57BL/6J mice (18\u0026ndash;22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), permit number: SCXK (Jing) 2021\u0026ndash;0011. The animals were fed in a temperature-controlled (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃) room. All experiments were approved by the Animal Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (NO.202313195) (NO.202313259).\u003c/p\u003e \u003cp\u003eThe cell line used Mouse-derived LLC (Lewis)-luc cells (mouse lung cancer cells with luciferase labeling), catalog number LZQ0009, Shanghai Zhongqiao Xinzhou Company, cultured in DMEM medium containing 10% fetal bovine serum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Assessment of Lewis cell growth in mice\u003c/h2\u003e \u003cp\u003eThe Lewis lung cancer mouse model was used to evaluate the therapeutic effect of BSCO on lung cancer, observing tissue morphology, in vivo imaging of the mice, and determination of multiple serum biochemical indicators \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Experimental animals, grouping, and model construction\u003c/h2\u003e \u003cp\u003eSeventy-two C57BL/6 male mice weighing 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g were used for the experiments. The animals were housed at room temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃), relative humidity of 60%, and normal alternation of day and night for 12 h, with free access to food and water. The animals were randomly divided into six groups of 12 animals: blank control, model, positive drug cyclophosphamide (Cytoxan, 0.2 mL/10 g), and BSCO low, medium, and high dose (0.2, 0.4, and 0.8 mL/10 g) groups.\u003c/p\u003e \u003cp\u003eThe mice were anesthetized and placed on the operating table in the right lateral position. The left armpit was shaved and disinfected with 75% ethanol. A 5 mm incision was made in the left axilla at the upper edge of the rib arch at the anterior line at about 1.5 cm. The skin and subcutaneous tissues were separated, and the chest wall was exposed until the pink lobes of the lungs could be seen moving due to respiration. Serum-free medium or phosphate buffered saline (50 \u0026micro;L) containing tumor cells (10\u003csup\u003e6\u003c/sup\u003e cells per mouse) was mixed with Matrigel matrix (50 \u0026micro;L) on ice and injected vertically into the left lung with a micro feeder at a depth of about 3 mm. After 20 s the needle was removed and the incision closed with 1 or 2 stitches. The cell line was mouse-derived LLC (Lewis)-luc cells (luciferase-labeled mouse lung cancer cells, No. LZQ0009, Shanghai Zhongqiao Xinzhou Company) cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum (FBS). After successful establishment of the model, the low, medium, and high dose BSCO groups were dosed by oral gavage once a day (0.2, 0.4, or 0.8 mL/10 g). The positive drug group was dosed by intraperitoneal injection with cyclophosphamide (0.2 mL/10 g) every other day. The model group received an equivalent volume of distilled water, and dosing was continued for 14 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4. Histomorphology observation\u003c/h2\u003e \u003cp\u003eAfter the mice had been euthanized, lung tissue (1 lobe) was taken and fixed in formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H\u0026amp;E). Morphological changes were observed by light microscopy (n\u0026thinsp;=\u0026thinsp;3 in each group). The remaining lung tissues were frozen and stored.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.4.5. Determination of serum biochemical indexes\u003c/h2\u003e \u003cp\u003eBlood samples were obtained 24 h after the final administration by removing the eyeballs. After resting at 4 ℃ for 30 min, the samples were centrifuged at 3500 r/min for 10 min and the supernatants stored at \u0026minus;\u0026thinsp;80 ℃ for analysis. The serum biochemical indexes determined in this study included: superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), hexokinase 2 (HK2), phosphofructokinase (PFK), malondialdehyde (MDA), glucose transporter protein 1 (GLUT1) and pyruvate kinase (PK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.4.6. Cell culture and evaluation of lung cancer cell growth\u003c/h2\u003e \u003cp\u003eLung cancer Lewis cells were cultured in RPMI medium containing 10% FBS at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e saturated humidity. The cells were trypsin digested and passaged when cell growth reached 70%~80%. Tumor cells in the logarithmic growth phase were incubated in 96-well plates for 12 h. After the cells had attached to the wall, they were treated with different concentrations of BSCO for 24, 48, and 72 h. The negative control group was Lewis cells cultured in culture medium. Cell viability was determined using the CCK-8 method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.4.7. Serum pharmacology\u003c/h2\u003e \u003cp\u003eWith reference to the reported method for serum pharmacology of traditional Chinese medicine \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, rats were randomly divided into blank control (n\u0026thinsp;=\u0026thinsp;15), high-dose (n\u0026thinsp;=\u0026thinsp;5), low-dose (n\u0026thinsp;=\u0026thinsp;5), and cyclophosphamide control (n\u0026thinsp;=\u0026thinsp;5) groups. The blank control group received an equal volume of saline by oral gavage for 7 d. Blood was collected under sterile conditions 1 h after the last gavage, and the serum was separated and filtered to remove bacteria. Lewis cells in the logarithmic growth phase were divided into five groups: normal control (10% normal culture serum\u0026thinsp;+\u0026thinsp;RPMI\u0026thinsp;\u0026minus;\u0026thinsp;640 medium); blank serum (10% blank serum\u0026thinsp;+\u0026thinsp;RPMI-1640 medium); positive control group (10% cyclophosphamide group serum\u0026thinsp;+\u0026thinsp;RPMI-1640 medium); BSCO low-dose group (10% low-dose group serum\u0026thinsp;+\u0026thinsp;RPMI-1640 medium); BSCO high-dose group (10% high-dose group serum\u0026thinsp;+\u0026thinsp;RPMI1640 medium). After culture for 24, 48 or 72 h, cell proliferation was analyzed using the CCK-8 method.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using GraphPad Prism 5.0 software and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Comparisons between groups were performed by one-way ANOVA. * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; ns indicates no significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Identification of the main chemical components of BSCO by UPLC-Q-TOF-MS\u003c/h2\u003e\n \u003cp\u003eThe base peak chromatogram of BSCO is shown in Fig. 1. Using the accurate relative molecular mass data, information on secondary fragment ions, and literature data, a total of 20 chemical components were identified, as detailed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ch2 align=\"char\" class=\"colspec\"\u003e3.2. Identification of overlap of BSCO targets and lung cancer\u003c/h2\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003cp\u003eA total of 17 active ingredients of BSCO were obtained using the SwissADME platform and relevant literature. Alpha-lactose, sucrose, and purine are excluded. These active ingredients were then searched in the Swiss Target Prediction, ETCM 2.0, and TCMSP databases to identify 460 therapeutic targets of BSCO. Additionally, by retrieving data from GeneCards, TTD, OMIM, and DisGeNET databases, a total of 5184 lung cancer-related genes were identified, among which 341 genes overlapped with the BSCO therapeutic targets. A Venn diagram was created and is shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Hub gene PPI network analysis of BSCO lung cancer targets\u003c/h2\u003e\n \u003cp\u003eThe PPI network constructed from the STRING database consisted of 341 overlapping targets, including 341 nodes representing the targets and 6,779 edges representing interactions. The data were imported into Cytoscape for analysis, where 51 hub genes were identified using topological parameter values. All nodes were ranked from high to low based on degree, with their sizes representing the degree and their colors ranging from dark to light. The results showed that the nodes representing glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein kinase B1 (AKT1), tumor protein p53 (TP53), tumor necrosis factor (TNF), and interleukin 6 (IL6) had larger areas and darker colors, indicating that these targets were of greater importance (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. GO and KEGG pathway enrichment analyses\u003c/h2\u003e\n \u003cp\u003eUsing the Metascape database, 3,572 GO terms were enriched when applying a filter of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Among these, 2,694 entries were related to biological processes (BP), including response to hormone, cellular response to nitrogen compound, and cellular response to hormone stimulus. The cellular component (CC) category contained 161 entries, particularly membrane raft, membrane microdomain, and neuronal cell body. The molecular function (MF) category included 327 entries, such as phosphotransferase activity, alcohol group as acceptor, kinase activity, and protein kinase activity. The top 10 entries in each category were analyzed and plotted (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eSimilarly, KEGG enrichment analysis conducted using Metascape with a filter of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 identified 230 pathways. Relevant information for the top 20 enriched pathways was imported through a bioinformatics platform to generate a KEGG enrichment pathway diagram. The results indicated that BSCO treatment of lung cancer primarily involves pathways such as Pathways in Cancer, Proteoglycans in Cancer, and the PI3K-Akt signaling pathway (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Histopathological morphology changes in the lungs of Lewis lung cancer mice\u003c/h2\u003e\n \u003cp\u003eThe results of H\u0026amp;E staining showed that lung tissue from mice in the model group had blurred cell edges, large and heterogeneous nuclei, and disturbed cell arrangement accompanied by partial alveolar rupture. Compared with the model group, lung tissue from the BSCO low-, medium-, and high-dose groups showed varying degrees of cell necrosis accompanied by hemorrhage, and structural damage to the lung tissue was ameliorated. In the positive cyclophosphamide(CTX) group, the cells were locally necrotic accompanied by mild hemorrhage, and a small number of necrotic cell fragments were seen, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. Effects of BSCO on tumor growth in mice\u003c/h2\u003e\n \u003cp\u003eImaging results showed that fluorescence values in the model group were elevated and significantly higher than those in the BSCO-treated groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting that BSCO slowed the growth of LLC cells in mice. The positive control drug, cyclophosphamide, significantly inhibited the growth of LLC cells in mice compared with that in the model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), as shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7. Lung cancer-related serum indexes\u003c/h2\u003e\n \u003cp\u003eCompared with the blank control group (Control), serum levels of CAT, GSH-Px, and SOD were significantly lower in the model mice (Model, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while levels of PFK, GLUT1, HK2, MDA, and PK were significantly elevated (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These data indicated successful establishment of the model. Compared with the model group, treatment with BSCO increased the levels of CAT, GSH-Px, and SOD to varying degrees, and decreased the levels of PFK, GLUT1, HK2, MDA, and PK, with the high dose being the most effective (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The results suggest that BSCO has a certain ameliorative effect on Lewis lung cancer in mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003e3.8. Effect of BSCO and drug-containing serum on lung cancer cell viability\u003c/h2\u003e\n \u003cp\u003eSerum from rats dosed with low and high doses of BSCO, as well as cyclophosphamide, did not affect Lewis cell viability compared to the blank group when incubated for 72 h, with more apoptotic cells observed under the microscope in all groups at this time point. The 24 and 48 h data showed that low and high doses of BSCO as well as cyclophosphamide significantly inhibited the viability of Lewis cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn recent years, an increasing number of traditional Chinese medicine and herbal therapies have been shown to be effective with few side effects, and traditional Chinese medicine is gradually aligning with modern medicine \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. We first analyzed the main chemical components of BSCO using UPLC-Q-TOF-MS. Subsequently, through network pharmacology and in vitro and in vivo experiments, we identified the key targets and pathways associated with BSCO treatment of lung cancer, and confirmed its efficacy against this disease. Our analysis suggested that the primary active component may be caffeic acid phenethyl ester (CAPE), a phenolic acid compound with significant potential as an anticancer agent. CAPE exerts its effects in cancer progression through the phosphoinositide 3-kinase/protein kinase B (PI3K-Akt) and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways, which is consistent with predictions from network pharmacology KEGG analysis \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe identified key targets included GAPDH, AKT1, TP53, TNF, and IL6. TP53 plays a role in preventing carcinogenesis, and the most common genetic alteration in various human cancers is mutation of TP53 \u003csup\u003e24\u003c/sup\u003e. It is also speculated that BSCO may exert its anticancer effects through the PI3K-Akt signaling pathway. This pathway is crucial for cancer cell survival and affects tumor proliferation, apoptosis, and autophagy \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The PI3K-Akt signaling pathway plays a pivotal role in regulating various cellular processes by activating downstream effectors, significantly contributing to tumor proliferation, invasion, and metastasis. One key aspect of Akt activation is its ability to enhance glucose uptake in cancer cells by modulating the expression of GLUT1 \u003csup\u003e25\u003c/sup\u003e. In addition to increasing glucose uptake, Akt regulates several key glycolytic enzymes, including HK2 and PFK, through both post-translational and transcriptional mechanisms, which ultimately promotes the activation of PFK1 \u003csup\u003e26\u003c/sup\u003e. This was confirmed in subsequent experiments. The activation of both PFK1 and GLUT1 facilitates cell proliferation and tumorigenesis \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. As a result, targeting the PI3K-Akt pathway to control tumor metabolism has emerged as a potential therapeutic strategy for treating cancer. \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, the efficacy of BSCO on lung cancer was evaluated from the cellular and serum pharmacological levels. BSCO was shown to inhibit proliferation of Lewis lung cancer cells with an IC\u003csub\u003e50\u003c/sub\u003e of 173 mg/mL. The viability of Lewis cells treated with serum from rats dosed with high and low doses of BSCO, and cyclophosphamide, was reduced after incubation for 24 and 48 h compared with the control. The effect of high dose BSCO serum was greater than that of low dose BSCO serum.\u003c/p\u003e \u003cp\u003eThe levels of SOD, GSH-Px, CAT, HK2, PFK, MDA, GLUT1, and PK serum markers were restored towards normal by BSCO in Lewis lung carcinoma mice, with the best effect seen at the high dose. BSCO regulates cancer cell carbon metabolism and glucose uptake by influencing key metabolic pathways.\u003c/p\u003e \u003cp\u003eThe histopathological morphology of mouse lung tissue was observed by H\u0026amp;E staining and showed that BSCO inhibited the growth of LLC tumor cells in mice. This result was confirmed by fluorescence imaging of lung cancer mice.\u003c/p\u003e \u003cp\u003eIn summary, this study identified compounds contained in BSCO and network pharmacology enabled identification of potential molecular targets, signaling pathways, and protein interactions regulated by those components. Experimental methods were employed to validate these findings both in vitro and in vivo. The results confirmed that BSCO exerts significant anti-cancer effects on lung cancer cells, providing valuable insights into its therapeutic potential.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study identified 20 major chemical components of BSCO. Network pharmacology suggested that BSCO may exert anticancer effects through targets including GAPDH, AKT1, TP53, TNF, and IL6, and the PI3K-Akt signaling pathway. Experimental data demonstrated effective inhibition of lung cancer cell proliferation in vitro and in vivo. Moreover, BSCO significantly restored serum biochemical indicators closely related to lung cancer and inhibited the growth of lung tumors, providing a reference for its clinical application in the prevention and treatment of lung cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBSCO Blood stasis constitution ointment; UHPLC-Q-TOF-MS Ultra-High Performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry; PPI Protein-Protein Interaction; GO Geneontology; KEGG Kyoto encyclopedia of genes and genomes; BP Biological Process; CC Cellular Component; MF Molecular Function; HE Hematoxylin-Eosin staining; PI3K-Akt signaling pathway Phosphoinositide 3-Kinase/Protein Kinase B pathway; GAPDH Glyceraldehyde-3-phosphate dehydrogenase; AKT1 Protein kinase B1; TP53 Tumor protein p53; TNF Tumor Necrosis Factor; IL6 Interleukin 6; \u0026nbsp;SOD superoxide dismutase; GSH-Px glutathione peroxidase; CAT catalase; HK2 hexokinase 2; PFK phosphofructokinase; MDA malondialdehyde ; GLUT1 glucose transporter protein 1; PK pyruvate kinase.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eClinical trial number\u003c/h2\u003e\n\u003cp\u003enot applicable.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eMaobo Du and Hu You designed the study and helped coordinate support and funding. Qiaozhi Wang conducted research and wrote the manuscripts. Chuanhao Dai and Tianming Lu participated in the experiments. Juhe Wang and Shuo Shen analyzed the data. Xingjiang Xiong and Qiuyan Guo guided the experiment.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThis research was financially supported by National Intangible Cultural Heritage Project [IX-4(1)]，China Academy of Chinese Medical Sciences Science and Technology Innovation Project (CI2021A04313).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLortet-Tieulent J, Renteria E, Sharp L, Weiderpass E, Comber H, Baas P, Bray F, Coebergh JW, Soerjomataram I. Convergence of Decreasing Male and Increasing Female Incidence Rates in Major Tobacco-Related Cancers in Europe in 1988\u0026ndash;2010. 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Commun Biol. 2024;7(1):1118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s42003-024-06801-6\u003c/span\u003e\u003cspan address=\"10.1038/s42003-024-06801-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Blood stasis constitution ointment (BSCO), UPLC-Q-TOF-MS, network pharmacology, medicine food homology, Chinese Medicine Constitution, lung cancer","lastPublishedDoi":"10.21203/rs.3.rs-5924974/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5924974/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBlood stasis constitution ointment (BSCO) is based on the classic formulas Xuefu Zhuyu and Buyang Huanwu decoctions described in Yilin Gaicuo by Wang Qingren of the Qing Dynasty. These formulas have been used to effectively treat blood stasis, which is identified as a pathological factor of lung cancer in traditional Chinese medicine theory.\u003c/p\u003e\u003ch2\u003eAim of the study\u003c/h2\u003e \u003cp\u003e: To analyze the chemical components of BSCO, predict target pathways, and evaluate its effects on lung cancer through in vivo and in vitro experiments.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eUltra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry was used to identify components of BSCO, and network pharmacology was used to predict their targets and signaling pathways associated with lung cancer. A Lewis lung cancer model was established in mice to evaluate the effects of BSCO by observing tissue morphology, whole animal imaging, and determination of serum biochemical indicators. The effects of BSCO on Lewis cancer cells in vitro were assessed using a CCK-8 cell proliferation assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTwenty major chemical components of BSCO were identified, with 341 potential targets identified by network pharmacology. BSCO effectively inhibited tumor growth in the Lewis lung cancer mouse model and normalized serum markers of cancer to varying degrees. The IC\u003csub\u003e50\u003c/sub\u003e of BSCO on Lewis cell proliferation was 173 mg/mL. Low- and high-dose BSCO-containing drug serum inhibited proliferation of Lewis cells after 24 and 48 h incubation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe results suggest that BSCO may exert anticancer effects through targets including GAPDH, AKT1, TP53, TNF, IL6, and the PI3K-Akt signaling pathway, providing a reference for its clinical application in the prevention and treatment of lung cancer.\u003c/p\u003e","manuscriptTitle":"Integrated UPLC-Q-TOF-MS, network pharmacology and experimental approach to evaluate the effects of blood stasis constitution ointment on lung cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 10:25:48","doi":"10.21203/rs.3.rs-5924974/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"704538ca-b2bc-420e-baf5-c468c6998564","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-30T22:32:16+00:00","versionOfRecord":{"articleIdentity":"rs-5924974","link":"https://doi.org/10.2147/CMAR.S563551","journal":{"identity":"cancer-management-and-research","isVorOnly":true,"title":"Cancer Management and Research"},"publishedOn":"2025-12-09 00:00:00","publishedOnDateReadable":"December 9th, 2025"},"versionCreatedAt":"2025-02-07 10:25:48","video":"","vorDoi":"10.2147/CMAR.S563551","vorDoiUrl":"https://doi.org/10.2147/CMAR.S563551","workflowStages":[]},"version":"v1","identity":"rs-5924974","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5924974","identity":"rs-5924974","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}