Abstract Introduction Bufei Huayu Decoction (BFHY) is a clinical prescription with reported efficacy in enhancing the therapeutic outcomes of chemotherapeutic agents for non-small cell lung cancer (NSCLC). However, the underlying metabolic mechanism of BFHY's action remains unexplored. Objective The objective of this study is to investigate the global metabolic effects of cisplatin and cisplatin plus BFHY on NSCLC. Methods Three groups (NSCLC, cisplatin, and cisplatin + BFHY) underwent a serum metabolomics procedure based on UHPLC-QE-MS. Then, a pathway analysis was carried out using MetaboAnalyst 3.0 to elucidate the therapeutic action routes of cisplatin and cisplatin plus BFHY in NSCLC. Results In the subcutaneous NSCLC model, both cisplatin and cisplatin + BFHY reduced the tumor volume and caused cell death. In comparison to cisplatin alone, cisplatin + BFHY showed a stronger tumor-suppressing impact. Furthermore, the same 16 metabolic signaling pathways were shared by the cisplatin and cisplatin + BFHY treatments. These typical metabolites are mainly involved in amino acid metabolism, lipid mobilization, nucleic acid metabolism and carbohydrate metabolites. Conclusions Potential biomarkers and metabolic networks of cisplatin and cisplatin + BFHY's anti-tumor actions are revealed in our investigation. Keywords: Bufei huayu decoction, Non-small cell lung cancer, Metabolomics, Metabolic signaling pathways Graphical abstract [37]Image 1 [38]Open in a new tab 1. Introduction Due to its high morbidity and mortality, lung cancer, also known as pulmonary carcinoma, poses the greatest danger to human life and health on a worldwide scale. It may be further split into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) [[39]1,[40]2]. NSCLC accounts for around 85% of all cases of lung cancer [[41]3]. The clinical result of advanced stage NSCLC has remained unsatisfactory in recent years, despite the enormous advancements made in surgery, immunotherapy, chemotherapy, and radiotherapy, which significantly improved the prognosis of NSCLC [[42]4]. Unfortunately, the majority of NSCLC patients obtain a diagnosis of locally progressed or metastatic illness at an advanced stage, making surgery impossible and allowing for only palliative therapy, which lowers the 5-year overall survival rate [[43]5]. Patients with stage III and stage IV NSCLC are still treated mostly with chemotherapy and radiotherapy, but it might be challenging to find an acceptable course of treatment for these patients because of irradiation and chemotherapy resistance as well as negative effects on healthy cells or organs [[44]6]. Therefore, it is vital and essential to find better methods for enhancing NSCLC clinical effectiveness. For the treatment of a variety of solid malignancies, including lymphomas, melanoma, bladder cancer, and breast cancer, and NSCLC, cisplatin is one of the most effective and often used chemotherapeutic medicines [[45]7]. However, cisplatin has two intrinsic obstacles that restrict its use and effectiveness: side effects and drug resistance, Chinese traditional medicine (TCM) offers some benefits such as varied bioactivities, multi-targeting, low toxicity, and minimal side effects [[46]8]. Recent researches have shown that TCM treatments may considerably lessen the toxic side effects of different chemotherapy medications as well as chemotherapeutic resistance, and the TCM can also improve the therapeutic impact of chemotherapy drugs [[47]9,[48]10]. Additionally, TCM may inhibit the development or spread of lung tumors and enhance the quality of life for patients by tonifying and replenishing spleen and kidney, resolving stasis and detoxification [[49][11], [50][12], [51][13]]. Bufei Huayu Decoction (BFHY) is a product of clinical research that treats both common lung conditions and lung cancer [[52][14], [53][15], [54][16]]. It has been shown that Astragalus, Curcuma phaeocaulis Valeton, Curcuma phaeocaulis Valeton, and Hedyotis diffusa in BFHY have potent anti-tumor properties [[55]17,[56]18]. The mechanisms of BFHY's anti-NSCLC action have seldom been described, and such research has mostly focused on the therapeutic effectiveness of BFHY on NSCLC. Metabonomic technology, developed in the 1970s, offers a brand-new viewpoint for illness research [[57]19]. Recent metabolomic investigations have significantly aided in the diagnosis, treatment, and monitoring of many different incurable illnesses by helping to align the metabolic pathways involved [[58]20]. While several studies on NSCLC and its metabolites have come to light, there have not yet been any reports on how BFHY affects the metabolome of NSCLC [[59]21,[60]22]. This is the first study to assess the changes in metabolites in the serum of NSCLC mice following treatment with cisplatin or cisplatin combined with BFHY. Potential metabolic pathways and targets were sought by logical network prediction in an effort to comprehend the processes behind the therapeutic effects of BFHY on NSCLC. 2. Materials and methods 2.1. Preparation of Bufei Huayu decoction Eastern medicine Bufei Huayu Decoction (BFHY) formula contained 20 g of Codonopsis pilosula, 15 g of Astragalus, 15 g of Rhubarb, 10 g each of Aster and Chuanxiong, 12 g of Paeonia, 10 g each of Salvia miltiorrhiza, Peucedanum praeruptorum Dunn, Almond, Curcuma phaeocaulis Valeton, Triprism, and Scutellaria barbata, 15 g of Hedyotis diffusa, 10 g of Turtle shell, 6 g of Liquorice. 500 mL of water were decocted into 173 mL, split into two bags, sealed, then stored and transported at 4 °C. 2.2. Determination the active component in BFHY by UHPLC-QE-MS Samples were thawed on ice, vortexed for 30 s, and then centrifuged at 13,800 ( × g) at 4 °C for 15 min. Supernatant (300 μL) was transported into an EP tube, adding 1000 μL of extraction (methanol:water = 4:1, IS = 1000:10), vortexed for 30 s, and then ultrasonic ice water bathed for 5 min. Left to rest at −40 °C for 1 h, the samples were centrifuged at 13,800 ( × g) at 4 °C for 15 min. The supernatant was passed through a 0.22 μm filter membrane, then put the sample bottle into the machine for detection,. LC-MS/MS analysis conducted using an Agilent ultra-high performance liquid chromatography 1290 UPLC system equipped with a Waters UPLC BEH C18 column (1.7 μm 2.1 × 100 mm). The flow rate was set at 0.4 mL/min and the a sample injection volume of 5 μL was used. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A multi-step linear elution gradient program was employed as follows: 0–3.5 min, 95%–85% A; 3.5–6 min, 85–70% A; 6–6.5 min, 70%–70% A; 6.5–12 min, 70%–30% A; 12–12.5 min, 30%–30% A; 12.5–18 min, 30%–0% A; 18–25 min, 0%–0% A; 25–26 min, 0%–95% A; 26–30 min, 95%–95% A. An Q Exactive Focus mass spectrometer coupled with an Xcalibur software was employed to obtain the MS and MS/MS data based on the information-dependent acquisition (IDA) acquisition mode. Each acquisition cycle covered a mass range of 100–1500, and the top three ions were selected for MS/MS analysis. The instrument parameters were set as follows: sheath gas flow rate of 45 Arb, auxiliary gas flow rate of 15 Arb, capillary temperature of 400 °C, full MS resolution of 70,000, MS/MS resolution of 17,500, collision energy set at 15/30/45 in NCE mode, and spray voltage of 4.0 kV (positive) or −3.6 kV (negative). 2.3. Xenograft tumor inoculation We obtained 30 male BALB/c nude mice (four weeks old, 18–20 g) from Guangdong Medical Laboratory Animal Center. After a one-week of acclimation period, A549 cells were subcutaneously inoculated at a density of 5 × 10^6 cells/mouse. Body weight and tumor volume were measured every two days. Once the tumors reached a size of 100–120 mm^3, the mice were randomly divided into five groups of six animals each. The mice were treated with saline, cisplatin and different doses of BFHY (low, middle and high) in combination with cisplatin. The NSCLC group received saline via gavage for 15 consecutive days. The cisplatin treatment group received saline via gavage for 15 consecutive days, along with an intraperitoneal injection of 2 mg/kg cisplatin every other day [[61]23,[62]24]. The cisplatin + low, middle, and high-dose BFHY groups were treated with BFHY (0.225/0.45/0.9 mL) via gavage for 15 consecutive days, in addition to an intraperitoneal injection of 2 mg/kg cisplatin every other day. After collecting blood samples from the eyeball, the mice were euthanized by cervical dislocation following isoflurane (3%) anesthesia. The experiments were conducted in compliance with national legislation and ethical guidelines set by the relevant institution or local body responsible for overseeing animal experimentation. 2.4. Hematoxylin-eosin (HE) and TUNEL staining A 16-h paraformaldehyde fixation on tumor samples was followed by dehydration and paraffin embedding. Using a freezing microtome (Leica, RM 2016), the tissues were then sliced into 5 mm slices. The tumor slices were hydrated using a gradient of ethanol to distilled water after being dewaxed with xylene. According to the instructions in the manufacturer's handbook, tumor slices were stained using a HE staining kit (Solarbio, G1120). The photos (about 100 × ) were captured using an Olympus BX53 microscope. Tumor sections were stained using the One-Step TUNEL Apoptosis Assay Kit (Beyotime, C1088) in accordance with the manufacturer's protocol. The photographs (around 200 × ) were examined using an Olympus BX53 microscope. 2.5. Metabolomics analysis of serum Each serum sample (50 μL) obtained from NSCLC, cisplatin and cisplatin + high-dose BFHY was transferred to EP tubes. Subsequently, 200 μL of precooled (−20 °C) extraction solution (methanol:acetonitrile = 1:1) containing 5 μg/mL internal standard was added. The mixture was vortexed for 30 s, subjected to ultrasonic treatment for 10 min, and then incubated at −40 °C for 1 h. After incubation, the mixture was centrifuged at 13,800 g, for 15 min at 4 °C. The resulting supernatant was carefully transferred to a fresh glass vial for subsequent analysis. The quality control (QC) sample was prepared by combining equal aliquots of the supernatants from all the samples. LC-MS/MS analyses were performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) equipped with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm), coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). The mobile phase consisted of 25 mmol/L ammonium acetate, and 25 ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The autosampler temperature set at 4 °C, and the injection volume was 3 μL. The QE HFX mass spectrometer was chosen for its ability to acquire MS/MS spectra in IDA mode, controlled by the acquisition software (Xcalibur, Thermo). In this mode, the software continuously evaluates the full-scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate at 30 Arb, auxiliary gas flow rate at 25 Arb, capillary temperature at 350 °C, full MS resolution at 60,000, MS/MS resolution at 7,500, collision energy at 10/30/60 in NCE mode, and spray voltage at 3.6 kV (positive) or −3.2 kV (negative), respectively. ProteoWizard was used to convert the raw data to the mzXML format, and internal software based on XCMS and created in R was used to analyze the data for peak identification, extraction, alignment, and integration. Then, metabolite annotation was done using a proprietary MS2 database (BiotreeDB). The annotation threshold was set at 0.3. 2.6. KEGG pathway mapping Pathway enrichment studies were carried out to find possible biological processes of the differentially expressed metabolite (Variable Importance in the Projection (VIP) > 1.0 and p < 0.05) based on pathways ([63]http://www.genome.jp/kegg/pathway.html) and MetaboAnalyst ([64]http://www.metaboanalyst.ca/) database. The KEGG pathway enrichment analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID). 2.7. Statistical analysis Software from California, USA's GraphPad Prism (version 8.0), was used for the statistical analysis. The findings in this research are reported as the mean standard deviation and reflect the three times that each experiment was conducted. SPASS 18.0 (SPASS, Chicago, IL) used a one-way ANOVA analysis to assess group differences, followed by a Turkey post hoc test. It was deemed statistically significant if p < 0.05. 3. Results 3.1. The active constituents of Bufei Huayu decoction were determined by LC-MS Under typical positive and negative ion modes, the representative total ion chromatogram (TIC) of the active components of BFHY was produced ([65]Fig. 1A). By using LC-MS, 355 metabolites in the positive ion mode and 222 metabolites in the negative ion mode were found, respectively. Furthermore, 53 components in total were found in both positive and negative ion modes ([66]Fig. 1B). The top five active ingredients in mass spectrometry of BFHY in positive ion mode were Guanine, 6-[2-(1,3-benzodioxol-5-y1)ethy1]-4-methoxypyran-2-one, Baicalein, Byakangelicin, and Oleamide, while in negative ion mode were 2-Methylmaleate, 2,3-Dihydroxybenzoic acid, Cirsimaritin, Dihydroferulic acid, and Ethyl ferulate ([67]Fig. 1C). Fig. 1. [68]Fig. 1 [69]Open in a new tab Determination of the active component in Bufei Huayu Decoction (BFHY) by UHPLC-QE-MS. (A) Total ion current (TIC) of the active component in BFHY. (B) Venn diagram of the active component in BFHY identified under positive ion mode (POS) and negative ion mode (NEG). (C) The top five active components in BFHY are identified under POS and NEG. 3.2. BFHY enhanced anti-tumor efficacy of cisplatin on NSCLC Then, the anti-tumor effect of BFHY and cisplatin in vivo was investigated. The body weight of the cisplatin, cisplatin + low-dose BFHY group, cisplatin + middle-dose BFHY and cisplatin + high-dose BFHY groups was significantly lower compared to those of the NSCLC group ([70]Fig. 2A). Notably, there was an aggravated loss of body weight observed in the cisplatin + high-dose BFHY groups starting from the fifth day after the administration of cisplatin and BFHY ([71]Fig. 2A). As expected, the tumor volume in the cisplatin group was significantly lower compared to the NSCLC group starting on Day 8; while the cisplatin + high-dose BFHY treatment effectively suppressed tumor growth starting from Day 5, resulting in a significantly smaller tumor size compared to cisplatin treatment alone ([72]Fig. 2B–D). These data indicated that BFHY enhanced the anti-tumor efficacy of cisplatin. Fig. 2. [73]Fig. 2 [74]Open in a new tab Cisplatin and cisplatin plus BFHY exert anti-tumor efficacy on NSCLC. (A) Body weight analysis of the subcutaneous NSCLC model mice. (B, C, D) The tumor size was measured, and the tumor volume was calculated. Results are expressed as means ± SD (n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001). (E) H&E staining image of the NSCLC tumor tissue ( × 100). (F) TUNEL staining of NSCLC tissues ( × 200, Green fluorescence indicates dead cells). In addition, H&E staining indicated that tumors in the NSCLC group exhibited a small amount of necrotic cells. Cisplatin treatment increased intratumoral necrotic areas, while cisplatin + high-dose BFHY treatment resulted in overall tumor necrosis ([75]Fig. 2E). The TUNEL assay demonstrated limited TUNEL-positive cells in the NSCLC group. Cisplatin treatment increased intratumoral necrotic areas with a limited number of apoptotic cells, whereas cisplatin + high-dose BFHY treatment significantly promoted apoptosis in tumor tissues ([76]Fig. 2F). 3.3. BFHY induced different metabolic changes from cisplatin alone treatment Non-targeted metabolomics profiling of the serum was conducted to investigate the mechanism of BFHY in combination with cisplatin. OPLS-DA results revealed distinct separation of samples from all groups in the experiment (Fig. 1S). [77]Fig. 3A and B showed distinct separation of samples from the NSCLC and cisplatin groups, as well as the cisplatin and cisplatin + high-dose BFHY groups, indicating significant differences in their metabolic profiles. Regarding changes in the levels of differentially expressed metabolites, a total of 521 upregulated metabolites and 804 downregulated metabolites were identified between the cisplatin and NSCLC groups. Similarly, between the cisplatin + high-dose BFHY and cisplatin groups, 1148 upregulated metabolites and 863 downregulated metabolites were identified (VIP >1.0 and Student's t-test (p-value) < 0.05, [78]Fig. 3C and D, [79]Supplemental Table 1 and [80]Supplemental Table 2). Fig. 3. [81]Fig. 3 [82]Open in a new tab BFHY induced different metabolic changes from cisplatin alone treatment. (A) Orthogonal partial least squares discriminant analysis (OPLS-DA) diagrams of serum samples from cisplatin and NSCLC model mice. Blue squares correspond to data from cisplatin-treated mice, and purple squares correspond to data from NSCLC mice. (B) OPLS-DA diagrams of serum samples from cisplatin and cisplatin plus BFHY-treated NSCLC model mice. Blue squares correspond to data from cisplatin-treated mice, and red circles correspond to data from cisplatin plus BFHY-treated mice. (C) Volcanic maps show a distinguishable metabolite expression pattern between cisplatin and NSCLC model mice. (D) Volcanic maps show a distinguishable metabolites expression pattern between cisplatin versus cisplatin plus BFHY-treated NSCLC model mice. Red circles represent upregulated metabolites, blue circles represent downregulated metabolites, and gray circles represent no significant change. 3.4. Enrichment analysis of metabolic pathways Information about metabolites was obtained from the KEGG ([83]http://www.kegg.jp/) and PubChem ([84]http://pubchem.ncbi.nlm.nih.gov) databases. A total of 23 pathways were identified from the different metabolites between the cisplatin and NSCLC groups ([85]Fig. 4A), and 35 pathways were identified from the different metabolites between the cisplatin + high-dose BFHY and cisplatin groups ([86]Fig. 4B). Among them, 16 metabolic pathways were found to be common between the cisplatin/NSCLC and cisplatin + high-dose BFHY/cisplatin. These pathways include Tryptophan metabolism, Taurine and hypotaurine metabolism, Steroid hormone biosynthesis, Sphingolipid metabolism, Pyrimidine metabolism, Purine metabolism, Propanoate metabolism, Pantothenate and CoA biosynthesis, Limonene and pinene degradation, Citrate cycle (TCA cycle), Butanoate metabolism, Biosynthesis of unsaturated fatty acids, beta-Alanine metabolism, Arginine and proline metabolism, Aminoacyl-tRNA biosynthesis, and Alanine, aspartate and glutamate metabolism ([87]Fig. 4A–B). Fig. 4. [88]Fig. 4 [89]Open in a new tab Enrichment analysis of metabolic pathways. (A) Results of KEGG pathway enrichment analysis of differential metabolites between cisplatin and NSCLC model mice. (B) Results of KEGG pathway enrichment analysis of differential metabolites between cisplatin and cisplatin plus BFHY-treated NSCLC model mice. (C) The level of key metabolites related to the common pathways between cisplatin/NSCLC and cisplatin plus BFHY/cisplatin. Key metabolites related to the common pathways between cisplatin/NSCLC and cisplatin + high-dose BFHY/cisplatin were quantified in the metabolomic analysis. Cisplatin treatment significantly increased the expression of Dihydrouracil, Sphinganine, Penllic acid, and Sphingsine while suppressing the levels of Uracil and succinic acid compared to the NSCLC group ([90]Fig. 4C). Furthermore, cisplatin + high-dose BFHY treatment effectively suppressed the expression of Dihydrouracil, Sphinganine, Sphingosine, Uracil, and succinic acid while increasing the level of Penllic acid compared to cisplatin treatment alone ([91]Fig. 4C). 4. Discussion BFHY, a traditional Chinese medicine herbal formula, has been utilized in the treatment of NSCLC in combination with various chemotherapy drugs such as the gemcitabine Platinum regimen [[92]16], vinorelbine plus cisplatin [[93]16], and gefitinib [[94]15]. In this current study, we aimed to identify the active components of BFHY using UHPLC-QE-MS. Our previous research demonstrated that BFHY significantly improved the overall clinical response, enhanced cellular immune function and quality of life, and reduced chemotherapy-induced side effects [[95]16,[96]25]. However, the underlying mechanism of BFHY's anti-tumor effect in NSCLC remains poorly understood. Metabolomics, as an omics science in systems biology, plays a crucial role in enhancing our understanding of metabolic pathways and the mechanism of action of drugs [[97]26,[98]27]. In this study, we conducted a serum metabolomics evaluation of NSCLC and the treatment of cisplatin and cisplatin plus BFHY using a UHPLC-QE-MS metabolomics approach. Cisplatin and cisplatin plus BFHY significantly reduced tumor volume and induced cell death in tumor tissues compared to the subcutaneous NSCLC model. Interestingly, cisplatin plus BFHY exhibited a more pronounced tumor-suppressive effect compared to cisplatin alone. Moreover, metabolomics analysis revealed that BFHY enhanced the tumor-suppressive role of cisplatin by modulating the levels of metabolites: reduced the increase levels of cisplatin induced three metabolites (Dihydrouracil, Sphinganine, Sphingosine), facilitated the inhibition effect of cisplatin on Uracil and succinic acid, as well as the stimulation effect of cisplatin on Penllic acid. Additionally, cisplatin and cisplatin plus BFHY treatment shared 16 same metabolic signaling pathways compared to NSCLC model. Our findings demonstrated that the metabolic changes induced by cisplatin and cisplatin plus BFHY in the subcutaneous NSCLC model are predominantly associated with abnormal amino acid metabolism, including Tryptophan metabolism, Taurine and hypotaurine metabolism, Propanoate metabolism, beta-Alanine metabolism, Arginine and proline metabolism, Aminoacyl-tRNA biosynthesis, and Alanine, aspartate, and glutamate metabolism. Amino acid metabolism, as a principal component of nitrogen metabolism, has been implicated in various diseases [[99]28]. Tryptophan metabolism plays a central role in physiology and pathophysiology by affecting nicotinamide metabolism and glycolysishas [[100]29]. Impairment of the tryptophan metabolic pathway has been linked to the developmental programming of hypertension and kidney disease [[101]30]. Taurine and hypotaurine, functioning as non-enzymatic antioxidants, exert cytoprotective properties by preventing oxidative damage to tissues. Dysfunction in taurine and hypotaurine metabolism has been associated with lung toxicity [[102]31]. Furthermore, alterations in Alanine, aspartate, and glutamate metabolism have been observed in lung injuries caused by air pollutants [[103]32]. Our present study suggested that anti-tumor effect of cisplatin and cisplatin plus BFHY in NSCLC are mediated by modulating the balance of amino acid metabolism. Lipid mobilization, another distinctive trait of metabolic changes in NSCLC, was identified following cisplatin and cisplatin plus BFHY treatment. These changes involve pathways such as Steroid hormone biosynthesis, Sphingolipid metabolism, and Biosynthesis of unsaturated fatty acids. Lung lipid metabolism is relevant to both infantile and adult pulmonary diseases [[104]33]. All sex steroid hormone receptors are observed in lung tissue, which indicates a potential role of sex steroids in lung diseases, including lung cancer, pulmonary fibrosis, and pulmonary hypertension, where sex differences in incidence, morbidity, and mortality have been observed, although the role of sex steroids hormone are still not well-understood [[105]34]. Sphingolipids, essential components of cell membranes, play a regulatory role in cell apoptosis and proliferation [[106]35]. Their involvement in the initiation and persistence of chronic obstructive pulmonary disease has been recognized [[107]36]. Unsaturated fatty acids, characterized by double bonds, possess protective effects against the toxicity of saturated fatty acids. The total intake of polyunsaturated fatty acids, a subgroup of dietary unsaturated fatty acids, has shown an inverse association with lung cancer risk [[108]37]. Therefore, BFHY treatment improves the therapeutic effects of cisplatin on NSCLC by modulating lipid mobilization. Changes observed following cisplatin and cisplatin plus BFHY treatment are also mostly associated with the regulation of nucleic acid metabolism, including Pyrimidine metabolism and Purine metabolism. Nucleotide metabolism plays a crucial role in producing purine and pyrimidine molecules for cellular bioenergetics, RNA synthesis, and DNA replication. Abnormalities in nucleotide metabolism can provide robust support for uncontrolled tumor growth [[109]38]. Agents that suppress nucleotide synthesis and incorporation into DNA are commonly used to inhibit tumor growth, induce DNA damage, and promote cell death [[110]39]. Therefore, the enhancement of the therapeutic effect of cisplatin on NSCLC by BFHY may be attributed to the increased regulation of nucleic acid metabolism. Changes induced by cisplatin and cisplatin plus BFHY treatment affected carbohydrate metabolites, including Propanoate metabolism, Pantothenate and CoA biosynthesis, Limonene and pinene degradation, Citrate cycle, and Butanoate metabolism. The presence of the Citrate cycle in this study indicates mitochondrial dysfunction resulting from the treatment of cisplatin and cisplatin plus BFHY. Mitochondrial dysfunction is implicated in various diseases [[111]40], and targeting mitochondrial function with anti-tumor drugs can ultimately lead to cell death [[112]40]. BFHY enhanced the therapeutic effects of cisplatin on NSCLC by inducing mitochondrial disorders, suggesting that disrupting mitochondrial function may be a contributing factor to the anti-tumor mechanism of BFHY in combination with Cisplatin. Clinically, cisplatin is often used to treat NSCLC; nevertheless, toxic side effects and platinum-based chemoresistance restrict the therapeutic impact. BFHY may greatly enhance the therapeutic results of cisplatin when used as a clinical experience prescription for the treatment of NSCLC. In this work, we found that BFHY regulates amino acid metabolism, lipid mobilization, nucleic acid metabolism, and carbohydrate metabolites to alleviate the toxic side effects or chemoresistance of cisplatin. To further investigate the role of BFHY, we will investigate the significant metabolite that differentiate cisplatin and cisplatin + BFHY group in the future study. 5. Conclusions Cisplatin plus BFHY treatment exhibited a more pronounced therapeutic effect on NSCLC compared to Cisplatin alone through the regulation of amino acid metabolism, lipid mobilization, nucleic acid metabolism, and carbohydrate metabolites. Author contribution statement Yuan Feng: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper. Ying Jiang, Ying Zhou, Zhan-hua Li, Qi-qian Yang, Jin-feng Mo, Yu-yan Wen, Li-ping Shen: Performed the experiments; Analyzed and interpreted the data; Wrote the paper. Data availability statement Data will be made available on request. Funding This work was supported by the National Natural Science Foundation of China [grant number 81960804] and Guangxi Natural Science Foundation project [grant number 2023GXNSFAA026096]. Ethics approval statement All animal experiments were performed in accordance with the ethics committee of the Forevergen Biosciences Animal Center. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Footnotes ^Appendix A Supplementary data to this article can be found online at [113]https://doi.org/10.1016/j.heliyon.2023.e19155. Contributor Information Yuan Feng, Email: fyhaha@163.com, fengy@gxtcmu.edu.cn. Ying Jiang, Email: jyshjnk@163.com, jiangy2004@gxtcmu.edu.cn. Appendix A. Supplementary data The following are the Supplementary data to this article: Multimedia component 1 [114]mmc1.xlsx^ (239.9KB, xlsx) Multimedia component 2 [115]mmc2.xlsx^ (361.5KB, xlsx) Multimedia component 3 [116]mmc3.pdf^ (368.7KB, pdf) Multimedia component 4 [117]mmc4.pdf^ (520.5KB, pdf) Multimedia component 5 [118]mmc5.pdf^ (361.9KB, pdf) Multimedia component 6 [119]mmc6.pdf^ (302KB, pdf) Multimedia component 7 [120]mmc7.pdf^ (607.4KB, pdf) Multimedia component 8 [121]mmc8.pdf^ (565.7KB, pdf) Multimedia component 9 [122]mmc9.pdf^ (356.4KB, pdf) Multimedia component 10 [123]mmc10.pdf^ (606.8KB, pdf) Multimedia component 11 [124]mmc11.pdf^ (524.9KB, pdf) Multimedia component 12 [125]mmc12.pdf^ (528.9KB, pdf) Multimedia component 13 [126]mmc13.pdf^ (404.5KB, pdf) Multimedia component 14 [127]mmc14.pdf^ (303KB, pdf) Multimedia component 15 [128]mmc15.pdf^ (577.7KB, pdf) Multimedia component 16 [129]mmc16.pdf^ (508.7KB, pdf) Multimedia component 17 [130]mmc17.pdf^ (465.2KB, pdf) figs1. [131]figs1 [132]Open in a new tab Orthogonal partial least squares discriminant analysis (OPLS-DA) diagrams of serum samples from all samples. Blue squares correspond to data from cisplatin-treated mice, and purple squares correspond to data from NSCLC mice, red circles correspond to data from cisplatin plus BFHY-treated mice, and orange circles correspond to data from quality control (QC). References