Network pharmacology and experimental methods to explore the mechanism of Poria extract in overcoming cisplatin-resistant ovarian cancer

Network pharmacology and experimental methods to explore the mechanism of Poria extract in overcoming cisplatin-resistant ovarian 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 Network pharmacology and experimental methods to explore the mechanism of Poria extract in overcoming cisplatin-resistant ovarian cancer Lei Dou, Lei Deng, Enting Lu, Fangmei Li, Rongjin Zhang, Fanyi Meng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8917089/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ovarian cancer is a common gynecological malignancy with a high incidence rate among female cancers. This study explored the mechanism of Poria extract in treating ovarian cancer through a combination of network pharmacology, bioinformatics analysis, and both in vitro and in vivo experimental validations. First, potential target genes associated with ovarian cancer and the active chemical components of Poria extract were identified using network pharmacology to determine possible therapeutic targets. Subsequently, these predictions were validated through cell culture and animal experiments. A total of 146 potential target sites were identified as being influenced by Poria extract's active components. Gene enrichment analysis indicated that dysregulated glycolysis metabolism and its related pathways are promising therapeutic targets for ovarian cancer. Experimental findings demonstrated that cisplatin resistance in ovarian cancer correlates with increased glycolytic activity. When administered in combination with cisplatin, Poria extract effectively suppresses glycolysis levels and the activity of its regulatory enzymes in A2780/DDP cells, while inducing an initial increase followed by a decrease in oxidative phosphorylation. The activation of ATP via oxidative phosphorylation leads to ROS accumulation, triggering mitochondrial autophagy and apoptosis. In vivo studies further confirmed that the combined treatment significantly inhibits tumor growth, as well as glycolysis and oxidative phosphorylation, in mice models. Overall, the combination of Poria extract and cisplatin can rebalance cellular energy metabolism and promote apoptosis in ovarian cancer cells. Poria extract ovarian cancer cisplatin resistance Network pharmacology Glucose metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction According to statistics, the incidence of ovarian cancer ranks the third in the female reproductive system malignant tumors, and its mortality is second only to breast cancer and cervical cancer. Globally, the incidence of ovarian cancer in developed countries is 9.1 per 100,000, while the incidence in developing countries is 5.0 per 100,000 [1] . At present, the increase of drug resistance in ovarian cancer seriously affects the treatment effect of ovarian cancer. There are many reasons for the occurrence of ovarian cancer drug resistance, such as individual differences in drug sensitivity, gene mutations, poor body immunity, cancer cell metastasis and so on [2,3] . Therefore, accurate treatment plans should be formulated according to the specific conditions of patients in the treatment process to avoid the occurrence of drug resistance and improve the treatment effect and survival rate of patients. Otto Warburg, a German biochemist, proposed that the way of glucose metabolism in tumor cells has changed from oxidative phosphorylation to aerobic glycolysis, namely the Warburg effect [4] . Tumor cells choose glycolysis as the main way of energy production. On the one hand, through glycolysis, they can obtain intermediate metabolites for the synthesis of fat, protein and nucleic acid to meet their active synthesis needs [5] . Secondly, lactic acid produced by glycolysis can provide an acidic environment for the growth and invasion of tumor cells, which is conducive to the survival and spread of tumor cells [6] . Therefore, understanding the relationship between tumors and oxidative phosphorylation and glycolysis will help us to understand the metabolic characteristics of tumors more deeply, and provide new ideas and methods for the diagnosis and treatment of tumors. There is mounting evidence that the application of network pharmacology and bioinformatics can accurately predict the interaction between active chemical components in medicines and specific disease targets. Poria extract is a fungal medicinal material that parasitizes the roots of pine trees. Poria extract is a highly valued substance with dual applications as both a medicinal ingredient and a food source, derived from the same botanical origin. It exhibits a range of biological activities, such as enhancing immune function, providing antioxidant effects, and modulating physiological processes. Studies have shown that Poria has anti-inflammatory and anti-tumor effects [7–9] . However, the anti-tumor mechanism of Poria extract is not fully understood, and there are still some limitations and challenges in clinical application. The aim of this study is to explore the molecular mechanism of Poria extract in reversing cisplatin resistance of ovarian cancer, and to lay a theoretical foundation for clinical application. Materials and methods Cell lines and cell culture Human ovarian cancer cell line A2780 and cisplatin-resistant ovarian cancer cell line A2780/DDP were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). The cells were grown in DMEM/high glucose (Hyclone, USA) with 10% fetal bovine serum (Hyclone, Logan, Utah, USA), 100 U mL-1 penicillin, and 100 mg mL-1 streptomycin (Genview, San Francisco, California, USA), and they were incubated at 37 ℃ and 5% CO 2 . The cells used in the experiments were all cleared for mycoplasma by mycoplasma scavengers and tested for mycoplasma to ensure contamination. Reagents and antibodies Poria extract was obtained from Liaoning University of Traditional Chinese Medicine. Cisplatin was procured from Sigma Aldrich Company (St. Louis, MO, USA). Primary antibodies included HIF-1α, HK2, PKM2, LDHA, ATP1, ATP5, Caspase 3/p17/p19, Caspase 9/p35, Bcl2, BAX, P glycoprotein, MRP, BCRP were obtained from Proteintech Company (Wuhan, Hubei, China). HRP-Goat anti-rabbit recombinant secondary antibody (H + L) was obtained from Proteintech Company (Wuhan, Hubei, China). Screening of ovarian cancer and Poria extract targets Utilizing the GeneCards database collection ( https://www.genecards.org/ ) of targets related to ovarian cancer. Utilizing TCMSP ( http://tcmspw.com/tcmsp.php ) and ETCM ( http://www.tcmip.cn/ETCM/index.php ) for the exploration of the chemical composition and targets of Poria extract. Target gene enrichment and network pharmacological analysis A Venn diagram was constructed, and the overlapping region was identified as a potential target for Poria extract treatment of ovarian cancer. Poria extract and ovarian cancer were found to have common targets for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, with all screenings showing p < 0.05. TCMSP filters the active components of Poria extract corresponding to common target genes and constructs a network pharmacological map, and the filtration criteria are: oral bioavailability (OB) ≥ 30%, drug similarity (DL) ≥ 0.18. Cell viability assay Parental A2780 cells or resistant A2780/DDP cells were seeded into 96-well plates at a density of 5000 cells/well. After 16 h of culture, the culture medium was removed. The cells were then treated with different concentrations of cisplatin (ranging from 0 to 300 µM) or Poria extract solution (ranging from 0 to 300 mg mL-1) for 24 h. Subsequently, 10 µL of MTT solution (Solarbio, Beijing, China) was added to each well and incubated at 37 ℃ for 4 h. The absorbance was measured using a microplate reader set to a wavelength of 570 nm. Cell proliferation was detected by EdU Cell slides were initially placed in 24-well plates, and either parental A2780 cells or drug-resistant A2780/DDP cells at a density of 2×10 4 cells/well were seeded into the plates. After 16 h of culture, the culture medium was removed. The cells were then treated with drug-free culture medium for 24 h followed by treatment with the drug for another 24 h. Subsequently, 300 µL of EdU-labeled culture medium (Beyotime, Shanghai, China) was added and incubated at 37 ºC in the dark for 2 h. The slides were then gently washed once with PBS using sterile forceps. Following this, the cells were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with Triton X-100 solution (0.1%) for an additional 5 minutes at room temperature. Nuclei staining was achieved by adding 300 µL of Hoechst 33342 solution and incubating in the dark at room temperature for a further period of time lasting up to approximately thirty minutes. Finally, experimental results were captured using a fluorescence microscope. Glucose, pyruvate and lactic acid assay After 24 h of incubation, A2780 cells, drug-resistant A2780/DDP cells, drug-free A2780/DDP cells, and drug-treated A2780/DDP cells were collected with a total cell count of 5×10 6 . The cells were lysed using low temperature ultrasonic waves and subsequently boiled in water for 10 minutes. The resulting cell lysate was then centrifuged at 8000 g for 10 minutes at room temperature, and the supernatant was retained. Following the instructions provided with the glucose, pyruvate, and lactic acid detection kit (mlbio, Shanghai, China), detection reagents were added to each sample. Subsequently, the absorbance value of each sample was measured at wavelengths of 505 nm, 520 nm, and 570 nm using an enzyme label reader. Mitochondrial transhydrogenase 2 (TH2) enzyme activity assay A total of 5×10 6 cells were homogenized using an ice-bath homogenizer, and the homogenate was then centrifuged at 600 g for 5 minutes at 4°C. The supernatant was removed and subjected to a further centrifugation at 110000 g for 10 minutes at 4°C. Subsequently, the resulting supernatant was combined with the reaction reagent (Sangon Biotecand, Shanghai, China) incubated in a water bath at 37°C for 5 minutes. The absorbance was measured using a microplate reader set to a wavelength of 375 nm. ATP content assay The protocol was carried out in accordance with the instructions of the ATP content detection kit (mlbio, Shanghai, China). A total of 5×10 6 cells were collected and added to 1 mL of acidic extract. The cells were then disrupted using an ice bath homogenizer. The homogenate was subsequently centrifuged at 8000 g for 10 minutes at 4 ℃. The resulting supernatant was combined with 1 mL of alkaline extract, thoroughly mixed, and then subjected to another round of centrifugation at 8000 g for 10 minutes at 4 ℃. The wavelength for enzyme label measurement was set to 700 nm. Hexokinase, pyruvate kinase and lactate dehydrogenase enzyme activities assay A total of 5×10 6 cells were added to 1mL of the extract, and the cells were disrupted by sonication and centrifuged at 8000 g for 10 min at 4 ℃. The supernatant was mixed with the reaction reagent (mlbio, Shanghai, China), and the wavelength of the microplate reader was adjusted to 340 nm, 340 nm and 450 nm for measurement. Rreactive oxygen species assay A2780/DDP cells were treated for 24 h in both the drug-free control group and the drug experimental group. After treatment, cells were collected following pancreatic enzyme digestion. The cell concentration was adjusted to 1×10 7 mL-1 using DCFH-DA solution (final concentration of 10 µmol-1). Subsequently, the cells were incubated in a 37 ºC incubator for 20 minutes and then washed three times with serum-free cell culture solution to ensure complete removal of any DCFH-DA that had not entered the cells. The intensity of fluorescence before and after stimulation was measured using a fluorescent enzyme label under conditions set at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Cell apoptosis was detected by Annexin V-PE/7-AAD After the treatment of drug-free and drug-free experimental group with A2780/DDP cells for 24 h, the amount of pancreatic enzyme digestion was 4×10 5 cells. The cells were washed twice with pre-cooled PBS, centrifuged at 4 ℃ for 5 min at 300 g, and then the supernatant was discarded and added with 100 µL Binding Buffer. Annexin 5 µL V-PE and 5 µL 7-AAD staining solution (Beyotime, Shanghai, China) were added, incubated for 10 min at room temperature and protected from light, and detected by flow cytometry (Thermofisher Scientific, Waltham, Massachusetts, United States). Cell apoptosis was detected by TUNEL assay Cell climbs were initially placed in 24-well plates, followed by seeding of 2×10 5 A2780/DDP cells in each well. After 16 h of adherent culture, the cells in both the control group and the drug experimental groups were treated for 24 h. The slides were then gently grabbed with sterile forceps and washed once with PBS. Subsequently, the cells were fixed with 4% paraformaldehyde for 30 minutes, followed by incubation with a Triton X−100 solution (0.1%) for 5 minutes at room temperature to permeabilize the cells. Next, 50µL of TUNEL detection solution (Beyotime, Shanghai, China) was added and incubated at 37 ºC in the dark for 60 minutes, after which the experimental results were captured using a fluorescence microscope (Zeiss, Jena, German). Chemical constituents of Poria extract based on LC-MS/MS analysis The Poria extract powder was dissolved in 50% aqueous methanol, and the supernatant was retained after centrifugation. Using Q Exactive Orbitrap high resolution mass spectrometry to mass spectrum data collection, specific parameters setting is: The positive ion and negative ion modes were scanned separately. The ion source voltage was 3.2kV, Capillary temp was 320 ° C, Aux gas heater temp was 350 ° C, Sheath gas flow rate was 40L/min. The Aux gas flow rate was 15L/min. In vivo experiments Drug preparation: Poria extract was dissolved in saline to a final concentration of 1g mL-1. Cisplatin was dissolved in saline at a final concentration of 100 µg mL-1. Female BALB/c nude mice weighing 20 ± 2 g were randomly selected, 5 mice in each group, a total of 4 groups. Each nude mouse was subcutaneously inoculated with 0.2 mL A2780/DDP cells (1×10 7 cells mL-1) on the right side. Once the tumors reached a volume of 50–100 mm³, daily recordings were made of the mice's body weight, food consumption, tumor dimensions, and activity levels. The mice with successfully constructed transplanted tumors were randomly divided into four groups. Control group: normal saline 0.2 mL/10 g/d by gavage for 14 days. Cisplatin group: intraperitoneal injection of cisplatin 0.2 mL/10 g, twice a week for 14 days. Poria extract group: gavage of Poria extract 0.2 mL/10 g/ d for 14 days; At the same time, cisplatin was injected intraperitoneally 0.2 mL/10 g twice a week for 14 days. Following a 14-day period, the protein expression levels were analyzed using western blotting. All animal experiments were conducted at the School of Life Sciences and Health, Northeastern University. The experimental animal project received ethical approval from the Biological and Medical Ethics Committee of Northeastern University.(No. NEU-EC-2023A079S). Western blot The parental A2780 cells, drug-resistant A2780/DDP cells, A2780/DDP cells without drug treatment, and A2780/DDP cells treated with drug were lysed using RIPA solution to extract protein. Equal amounts of protein samples from each group were loaded for SDS-PAGE electrophoresis at 80V for 25 min and then at 120V for 50 min. The protein bands in the SDS-PAGE gel were transferred to a PVDF membrane at 25V and 1.3A for 14 minutes. The PVDF membranes containing proteins were washed with TBST, followed by overnight blocking with a 5% skim milk solution, a two-hour incubation with the primary antibody, and finally a one-hour incubation with the secondary antibody. Subsequently, the ECL solution (Vazyme, Shanghai, China) was used to develop the imaging. Statistical Analysis The experimental data were all repeated at least three times and expressed as mean ± SD, and all of the datas were analyzed with SPSS 19.0. The differences in cell cycle and proliferation, glycolytic parameters and oxidative phosphorylation between parental A2780 cells and resistant A2780/DDP cells were evaluated by two-tailed Student's t test. In A2780/DDP cells, the differences of cell cycle and proliferation, apoptosis, glycolysis and oxidative phosphorylation in different experimental groups were evaluated by Analysis of variance (ANOVA). P value lower than 0.05 was considered significant. Results Poria extract treatment of ovarian cancer target prediction and build network pharmacology A total of 10273 ovarian cancer targets were obtained from the GeneCards database. A total of 65 compounds were identified from the Poria extract through the TCMSP and ETCM databases, and these compounds corresponded to a total of 241 targets. The 241 targets corresponding to the 65 compounds of Poria extract and the 10273 targets of ovarian cancer were used for subsequent bioinformatics analysis. As shown in Fig. 1 -A, the targets for Poria extract and ovarian cancer have 146 intersections. Enrichment analysis was performed on the targets of Poria extract in the treatment of ovarian cancer. GO and KEGG correlation analysis showed that the top 20 enriched items (P < 0.05), Mainly includes the Glycolysis and Gluconeogenesis, Pyruvate metabolism, Carbon metabolism, Starch and sucrose metabolism, etc. (Fig. 1 -B and C). Furthermore, a network pharmacological map of Poria extract for the treatment of ovarian cancer was constructed (Fig. 1 -D). The compounds of Poria extract were identified by LC-MS/MS method, the top ten chemical constituents were Paeoniflorin (18.769%), Amygdalin (17.971%), Albiflorin (8.195%), Sucrose (5.809%), Pyrogallol (5.535%), Citric Acid (4.841%) and Benzoylpaeoniflorin (3.167%), Cinnamaldehyde (3.122%), Coumarin (2.790%), Pachymic acid (2.541%) (Supplementary Fig. 1 and table 1). Cisplatin resistance in ovarian cancer caused by glycolysis The imbalance between glycolysis and oxidative phosphorylation is a ubiquitous feature of cancer cells. As shown in Fig. 2 -A to F, the glycolysis level and the expression levels of key enzymes in the glycolysis pathway in cisplatin-resistant A2780/DDP cells were significantly higher than those in parental A2780 cells (p 0.05). In addition, the sensitivity of A2780 cells and A2780/DDP cells to cisplatin and Poria extract was determined. Both cisplatin and Poria extract inhibited the growth of A2780 and A2780/DDP cells in a concentration-dependent manner. The IC 50 value denotes the concentration of a drug at which its inhibitory effect on a specific biological process, such as cell proliferation or enzyme activity, reaches 50% within a defined period of time. The IC 50 of cisplatin for A2780 cells and A2780/DDP cells was 22.60 ± 0.14 µM and 39.48 ± 0.36 µM, respectively (Fig. 2 -G and H). The IC 50 of Poria extract on A2780 cells and A2780/DDP cells was 0.60 ± 0.14 mg mL−1 and 1.04 ± 0.31 mg mL−1 (Fig. 2 -I and J). Increasing drug concentrations produce toxic effects on cells, affecting cell viability and masking the molecular mechanism of drug action. To exclude this factor, we selected the IC 5 value, which is the concentration of drug required to inhibit cell growth by 5%, for subsequent studies. Cisplatin combined with Poria extract at IC 5 concentrations had no effect on A2780/DDP cell proliferation (Fig. 2 -K). Poria extract combined with cisplatin regulated glycolysis and oxidative phosphorylation levels in A2780/DDP cells The changes of glycolysis and oxidative phosphorylation in A2780/DDP cells treated with Poria extract alone, cisplatin alone, or Poria extract combined with cisplatin were measured. As shown in Fig. 3 -A to F, the combined treatment of the two drugs continuously inhibited glucose consumption, pyruvate production, lactate accumulation, and the activities of their related regulatory enzymes (p < 0.05). As shown in Fig. 3 -G, the protein expression levels of HK2, PKM2 and LDHA in A2780/DDP cells were significantly decreased after 24 h of drug treatment (p < 0.05). However, as shown in Fig. 3 -H and I, the ATP content and mitochondrial transhydrogenase 2 (TH2) in A2780/DDP cells increased at first and then decreased after drug combined intervention. The expressions of ATP1 and ATP5 protein in A2780/DDP cells were significantly inhibited by Poria extract alone or combined with drug intervention (p < 0.05) (Fig. 3 -J), and the inhibitory effect of combined drug intervention was better than that of Poria extract alone. Poria extract combined with cisplatin activates ROS to induce mitophagy in A2780/DDP cells Firstly, we found that Poria extract combined with cisplatin could promote a significant increase in ROS level in A2780/DDP cells (p < 0.01) (Fig. 4 -A). In addition, we found that the combined treatment of Poria extract and cisplatin could activate the NF-κB/TGF-β1/PINK1/Parkin signaling pathway in A2780/DDP cells (Fig. 4 -B). The NF-κB/TGF-β1/PINK1/Parkin signaling pathway is closely related to mitochondrial damage. In A2780/DDP cells, the presence of mitochondrial vesicles, autophagosomes and autolysosomes was detected by fluorescence and transmission electron microscopy (Fig. 4 -C and D). Poria extract combined with cisplatin reversed cisplatin resistance by inducing apoptosis in A2780/DDP cells Tunel fluorescence staining showed that Poria extract combined with cisplatin could significantly promote the apoptosis of A2780/DDP cells (p < 0.01) (Fig. 5 -A). In addition, flow cytometry showed that the combined treatment of Poria extract and cisplatin significantly promoted the sum of early and late apoptosis of A2780/DDP cells (p < 0.01) (Fig. 5 -B). We tested the apoptosis regulation related protein expression level, the results showed that combination of drugs intervention can promote A2780 / DDP cells Cleaved-Casepase 3, Cleaved-Casepase 9, and the expression of BAX protein, inhibition of Bcl-2 protein expression (Fig. 5 -C). Compared with cisplatin alone, Poria extract combined with cisplatin significantly increased the content of cisplatin in A2780/DDP cells (p < 0.01) (Fig. 5 -D). Finally, the expression levels of drug resistance regulatory proteins in A2780/DDP cells were detected, and the results showed that the combined treatment of Poria extract and cisplatin significantly inhibited the expression of P-gp, MRP and BCRP proteins (p < 0.05) (Fig. 5 -E). Poria extract combined with cisplatin significantly inhibited tumor energy metabolism and growth in vivo A2780/DDP xenograft mouse model was established. As shown in Fig. 6 -A, when the mice with transplanted tumors were treated with Poria extract and cisplatin for 14 days, the tumor size in the armpit of the mice was significantly smaller than that of the drug treatment group alone. The body weight of the mice with transplanted tumors in the combined drug treatment group was also significantly higher than that in the drug treatment group alone (p < 0.01). As shown in Fig. 6 -B, the tumor volume of the combined drug treatment group was significantly reduced from day 7 (p < 0.01). The effect of Poria extract combined with cisplatin on glycolysis and oxidative phosphorylation levels in A2780/DDP cells showed that the combined drug treatment group could inhibit the expression of HIF-1α, HK2, PKM2, ATP1 and ATP5 proteins (Fig. 6 -C and D). The contents of ATP and lactate in tumor tissues were inhibited in the combined treatment group (p < 0.05) (Fig. 6 -E and F). Discussion There is a close relationship between tumors and oxidative phosphorylation and glycolysis. Oxidative phosphorylation is the main way in which cells produce ATP through oxidative reactions, which is an efficient way to generate energy. However, in tumor cells, the mode of glucose metabolism is changed from oxidative phosphorylation to aerobic glycolysis [10,11] . Aerobic glycolysis, a way in which cells produce energy by breaking down glucose under hypoxic conditions, produces a smaller amount of ATP but at a faster rate. ATP synthase is a large protein complex located in the inner mitochondrial membrane that plays a key role in oxidative phosphorylation and is responsible for catalyzing the phosphorylation of ADP to ATP. ATP synthase is composed of several subunits, of which ATP1 and ATP5 are important subunits directly involved in ADP phosphorylation to generate ATP [12] . HK2, PKM2 and LDHA are the key enzymes in the different stages of glycolytic pathway from glucose to pyruvate and finally to lactate [13] . Together, they play an important role in the regulation of the process of glycolysis and energy metabolism of cells. Energy production and growth of tumor cells can be inhibited by inhibiting key enzymes of glycolysis, such as hexokinase 2 (HK2) or lactate dehydrogenase A (LDHA) [14,15] . In addition, a deeper understanding of tumor metabolism, by activating oxidative phosphorylation or restoring mitochondrial function, offers the possibility to develop new cancer therapies. The molecular mechanism of drug resistance in ovarian cancer is a complex process involving changes in multiple aspects. For example, the abnormalities of signaling pathways and metabolic enzymes, epithelial-mesenchymal transition (EMT), abnormal expression of miRNA, the role of cancer stem cells (CSC), and changes in the immune microenvironment can cause drug resistance of ovarian cancer [16–18] . Recent studies have found that the balance between oxidative phosphorylation and glycolysis plays a crucial role in the development and drug resistance of ovarian cancer. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial chaperone protein from Hsp90 family [19] . Up-regulation of TRAP1 expression increased aerobic glycolysis in ovarian cancer cells [20] . When TRAP1 expression is low or silenced, the oxygen consumption of ovarian cancer cells increases, leading to a high dependence on oxidative phosphorylation and is associated with platinum resistance [21] . Abnormal activation of PI3K/AKT/mTOR pathway plays a key role in the formation of drug resistance in ovarian cancer [22] . The key enzymes in glucose metabolism of ovarian cancer cells, such as hexokinase, 6-phosphofructokinase and lactate dehydrogenase, can promote the growth of ovarian cancer cells and reduce the sensitivity to chemotherapy drugs [23–25] . When ovarian cancer cells are attacked by chemotherapeutic drugs, they will increase glycolysis and glutamine metabolism to produce more energy and reducing power, thus resisting the pressure of drugs. In this study, the cisplatin sensitive ovarian cancer cell line A2780 and cisplatin resistance cell line A2780/DDP were used as research objects. The results showed that compared with A2780 cells, the activities of glucose metabolism regulatory enzymes such as hexokinase (HK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) were increased in A2780/DDP cells, leading to increased glycolysis level. However, there was no significant difference in oxidative phosphorylation between A2780 and DDP cells. The increased glycolysis level in A2780/DDP cells is closely related to the cisplatin resistance phenotype. Our previous study demonstrated that Guizhi Fuling capsule inhibits ovarian cancer metastasis by targeting the PA2G4/PI3K/AKT/GSK-3β signaling pathway both in vivo and in vitro [26] . Composed of Cinnamomum cassia and Poria extract, the capsule contains hundreds of active ingredients as identified by LC-MS/MS analysis. To elucidate the specific active ingredients responsible for its anti-ovarian cancer effects, bioinformatics prediction revealed a significant overlap between the active components of Poria extract and the therapeutic targets associated with ovarian cancer. Poria extract is a kind of precious food and medicinal material with the same origin of food and medicine. It has the functions of enhancing immunity, anti-oxidation and regulating body function. In this study, we identified Poria extract through database searches and identified 146 common targets for ovarian cancer. Subsequently, we constructed a network pharmacology model. Functional enrichment analysis of these common targets revealed that the dysregulation of glycolysis is a key factor in Poria extract treatment for ovarian cancer. We predicted that glycolysis would be the key point in the treatment of ovarian cancer by Poria extract, and found that the abnormality of glycolysis was a significant feature of cisplatin resistance in ovarian cancer. Therefore, we tested whether Poria extract could also exert inhibitory effect on cisplatin-resistant ovarian cancer. ELISA and Western blot were used to confirm that Poria extract combined with cisplatin could reduce the level of glycolysis by inhibiting the enzyme activity and protein expression of glucose metabolism regulatory enzymes in A2780/DDP cells. Studies have demonstrated that Poria extract exerts therapeutic effects on primary dysmenorrhea in rats through the regulation of multiple metabolic pathways, including the TCA cycle, gluconeogenesis, glycolysis, and the pentose phosphate pathway [27] . Studies found that Poria extract can activate the PI3K/AKT/mTOR signaling pathway, thereby decreasing levels of follicle-stimulating hormone, testosterone, and fasting insulin, while increasing blood glucose levels, ultimately improving ovulatory dysfunction in PCOS rats with insulin resistance [28] . The results of our study are consistent with the existing published articles. The metabolic shift from efficient aerobic oxidative phosphorylation to enhanced glycolysis in tumor cells is referred to as the "Warburg effect." This phenomenon is primarily driven by mitochondrial dysfunction, the diversion of TCA cycle intermediates for biosynthetic purposes, and elevated levels of ROS, all of which contribute to reduced efficiency or inhibition of aerobic oxidative phosphorylation. In addition, the ATP content of A2780/DDP cells increased at first and then decreased during the treatment of Poria extract combined with cisplatin. In our study, we observed that the level of oxidative phosphorylation(ATP content), was comparable between A2780 cells and A2780/DDP cells. This suggests that glycolysis was elevated in A2780/DDP cells, while oxidative phosphorylation was not further suppressed. During the treatment of A2780/DDP cells with Poria extract in combination with cisplatin, ATP levels exhibited an initial increase followed by a subsequent decrease, returning toward normal levels after 24 hours. We further investigated whether this transient enhancement of oxidative phosphorylation could induce alterations in certain biological parameters of A2780/DDP cells. The results show that elevated ATP could induce ROS, activate the NF-κB/TGF-β1/PINK1/Parkin signaling pathway, and promote mitophagy in A2780/DDP cells. We found that Poria extract combined with cisplatin could promote the apoptosis of A2780/DDP cells as detected by TUNEL and flow cytometry. Finally, we carried out in vivo experiments to verify that Poria extract combined with cisplatin could inhibit the tumor growth, glucose metabolism and oxidative phosphorylase expression in tumor-bearing mice. In summary, the relationship between tumors and oxidative phosphorylation and glycolysis is a complex and worthy area of further study. By regulating the expression of related genes or key enzymes in energy metabolism pathways, the balance of oxidative phosphorylation and glycolysis of tumor cells can be reshuffled, thereby enhancing their sensitivity to chemotherapy drugs. By in-depth study of the mechanisms and effects of this metabolic shift, we can better understand tumor growth and development and develop more effective treatments. Conclusion This study showed that imbalance of glycolysis and oxidative phosphorylation is a prominent feature of cisplatin resistant ovarian cancer cell line A2780/DDP. Poria extract combined with cisplatin can reshape the balance of glycolysis and oxidative phosphorylation, induce ROS production, and promote mitophagy and apoptosis in A2780/DDP cells. Poria extract improve the curative effect of cisplatin for the treatment provides a theoretical basis for cisplatin resistance ovarian cancer. Declarations CRediT authorship contribution statement Lei Dou: Writing-review & editing, Writing-original draft, Methodology, Investigation. Lei Deng: Writing-review & editing, Writing-original draft, Methodology, Investigation, Conceptualization. Enting Lu: Writing-original draft, Methodology, Investigation. Fangmei Li: Writing-review & editing, Writing-original draft, Methodology. Fanyi Meng: Writing-review & editing, Writing-original draft, Methodology. Xinyang Chen: Writing-review & editing, Writing-original draft, Methodology. Yin Li: Writing-review & editing, Writing original draft, Supervision, Methodology. Yi Zhang: Writing-review & editing, Writing original draft, Supervision, Resources, Conceptualization. Ye Sun: Writing-review & editing, Writing original draft, Supervision, Methodology, Investigation, Conceptualization. Authors Declarations Ethical Approval and Consent to participate This study was conducted at the School of Life Sciences and Health at Northeastern University in accordance with the Declaration of Helsinki and approved by the local Council of Governance. Their care during research on animals follows ARRIVE guidelines and is conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 8023, as revised in 1978). Patient consent for publication Not applicable. Data Availability The datasets during and/or analysed during the current study available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Acknowledgements The author thanks the Science and Technology Department of Liaoning Province and Science and Technology Bureau of Shenyang City for funding this study. This work was financially supported by the Liaoning Province Applied Basic Research Program (Grant 2022JH2/101300039) and Shenyang Science and Technology Plan (22-321-33-08). The above funds were used to carry out the research related consumables and reagents, detection, carrier synthesis and publication costs. References Penny SM (2020) Ovarian Cancer: An Overview. Radiol Technol 91:561–575 Yang L, Xie HJ, Li YY, Wang X, Liu XX, Mai J (2022) Molecular mechanisms of platinum-based chemotherapy resistance in ovarian cancer (Review). Oncol Rep 47:82–92 Wang L, Wang X, Zhu X, Zhong L, Jiang Q, Wang Y, Tang Q, Li Q, Zhang C, Wang H, Zou D (2024) Drug resistance in ovarian cancer: from mechanism to clinical trial. Mol Cancer 23:66–91 Kobayashi Y, Banno K, Kunitomi H, Takahashi T, Takeda T, Nakamura K, Tsuji K, Tominaga E, Aoki D (2019) Warburg effect in Gynecologic cancers. J Obstet Gynaecol Res 45:542–548 Liberti MV, Locasale JW (2016) The Warburg Effect: How Does it Benefit Cancer Cells. 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Int J Biochem Cell Biol 95:9–16 Papadaki C, Manolakou S, Lagoudaki E, Pontikakis S, Ierodiakonou D, Vogiatzoglou K, Messaritakis I, Trypaki M, Giannikaki L, Sfakianaki M, Kalykaki A, Mavroudis D, Tzardi M, Souglakos J (2020) Correlation of PKM2 and CD44 Protein Expression with Poor Prognosis in Platinum-Treated Epithelial Ovarian Cancer: A Retrospective Study, Cancers (Basel). 12:1013 Xintaropoulou C, Ward C, Wise A, Queckborner S, Turnbull A, Michie CO, Williams ARW, Rye T, Gourley C, Langdon SP (2018) Expression of glycolytic enzymes in ovarian cancers and evaluation of the glycolytic pathway as a strategy for ovarian cancer treatment. BMC Cancer 18:636 Dou L, Yan Y, Lu E, Li F, Tian D, Deng L, Zhang X, Zhang R, Li Y, Zhang Y, Sun Y (2025) Composition analysis and mechanism of Guizhi Fuling capsule in anti-cisplatin-resistant ovarian cancer. Transl Oncol 52:102244 Xiong Z, Lang L, Gao X, Xiao W, Wang Z, Zhao L (2019) An integrative urinary metabolomic study of the therapeutic effect of Guizhi Fuling capsule on primary dysmenorrheal rats based 1H NMR and UPLC-MS. J Pharm Biomed Anal 164:750–758 Liu M, Zhu H, Zhu Y, Hu X (2021) Guizhi Fuling Wan reduces autophagy of granulosa cell in rats with polycystic ovary syndrome via restoring the PI3K/AKT/mTOR signaling pathway. J Ethnopharmacol 270:113821 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigureandtable.docx fulluncroppedGelsandBlotsimages.pdf Cite Share Download PDF Status: Posted 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. 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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-8917089","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":595166579,"identity":"a9f160bf-cca4-4995-9adb-0b01d8f56d81","order_by":0,"name":"Lei Dou","email":"","orcid":"","institution":"The First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Dou","suffix":""},{"id":595166580,"identity":"2fb9befd-54c1-4b69-aaa7-704e1df8c352","order_by":1,"name":"Lei Deng","email":"","orcid":"","institution":"The First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Deng","suffix":""},{"id":595166581,"identity":"52433c89-a930-459a-b386-687a2a23cfc8","order_by":2,"name":"Enting Lu","email":"","orcid":"","institution":"The First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Enting","middleName":"","lastName":"Lu","suffix":""},{"id":595166582,"identity":"f7fa9923-e4f2-46fb-b837-0410b37916cd","order_by":3,"name":"Fangmei Li","email":"","orcid":"","institution":"The First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fangmei","middleName":"","lastName":"Li","suffix":""},{"id":595166583,"identity":"1f4a86e3-4fda-48cc-9974-1354843b8f40","order_by":4,"name":"Rongjin Zhang","email":"","orcid":"","institution":"The First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rongjin","middleName":"","lastName":"Zhang","suffix":""},{"id":595166584,"identity":"bad80695-96e8-42b3-9575-db2216dfff86","order_by":5,"name":"Fanyi Meng","email":"","orcid":"","institution":"The First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fanyi","middleName":"","lastName":"Meng","suffix":""},{"id":595166585,"identity":"47a972f6-f602-4ade-9e47-fe9f197e4d87","order_by":6,"name":"Xinyang Chen","email":"","orcid":"","institution":"The First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinyang","middleName":"","lastName":"Chen","suffix":""},{"id":595166586,"identity":"115e1325-fc93-4dab-abd4-fc9b70959bb9","order_by":7,"name":"Yin Li","email":"","orcid":"","institution":"Peking University","correspondingAuthor":false,"prefix":"","firstName":"Yin","middleName":"","lastName":"Li","suffix":""},{"id":595166587,"identity":"14fd9347-a28f-4c8a-80f1-bd0b8f5d1365","order_by":8,"name":"Yi Zhang","email":"","orcid":"","institution":"The First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":595166588,"identity":"48bd7551-e05d-44b4-922b-7059e8a1361d","order_by":9,"name":"Ye Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYNCCCijNQ7yWMyRrYWwjRYv8jNxjEh/n1SWubT/A+OBtG4O8OSEtBjfy0iRnbjucuO1MArPh3DYGw50NhLRI5JhJ8247kLjtBgObNG8bQ4LBAYIOA2r5O6cOpIX9N1FaGG4AtTA2MINtYSZKi8GZN8aWPccOG287k9gsOeechOEGgg5rzzG88aOmTnbb8cMHP7wps5En7DAGBhYJIOHYwMDYAKQlCKsHAuYPQMKeKKWjYBSMglEwMgEA52pAhTH27lwAAAAASUVORK5CYII=","orcid":"","institution":"Department of Pathogen Biology, Shenyang Medical College","correspondingAuthor":true,"prefix":"","firstName":"Ye","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2026-02-19 11:27:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8917089/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8917089/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103506689,"identity":"0a26d3ad-175c-4535-af1d-47039ff1cf5d","added_by":"auto","created_at":"2026-02-26 13:38:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1082481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrediction and Network Pharmacology for Poria extract Treatment of Ovarian Cancer. \u003c/strong\u003eVenn diagram of ovarian cancer target genes and Poria extract target genes (A). GO enrichment analysis of common target gene of ovarian cancer and Poria extract (B). KEGG enrichment analysis of common target gene of ovarian cancer and Poria extract (C). Network pharmacological map of Poria extract treatment of ovarian cancer (D).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8917089/v1/316943a5a44cb4b0ccdc76d4.png"},{"id":103354664,"identity":"08cd13b3-3591-44b9-a5c2-accb97a98bb0","added_by":"auto","created_at":"2026-02-24 18:06:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1067640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA2780 cells and A2780 / DDP cells biological characteristics differences and Poria extract and cisplatin on cell growth inhibition.\u003c/strong\u003e Glucose consumption and lactate accumulation assay(A). The enzyme activities of hexokinase (HK) (B), pyruvate kinase (PK) (C)and lactate dehydrogenase (LDH) assay (D). ATP content assay (E). Enzyme activity of Mitochondrial hydrogenase-2 (TH-2) assay (F). Effect of cisplatin on viability and IC\u003csub\u003e50\u003c/sub\u003e of A2780 and A2780/DDP cells (G and H). Effect of Poria extract on viability and IC\u003csub\u003e50\u003c/sub\u003e of A2780 and A2780/DDP cells (I and J). The effect of Poria extract combined with cisplatin on A2780/DDP cell viability was determined by Edu method (K). The experiments were repeated three times, and the results are expressed as the mean ± standard deviation. *= P \u0026lt;0.05, **= P \u0026lt;0.01, vs control.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8917089/v1/374290081ea46ac906b3d1c0.png"},{"id":103354670,"identity":"299cdd81-070e-4321-b022-bd2fcfadcc52","added_by":"auto","created_at":"2026-02-24 18:06:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":686730,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Poria extract combined with cisplatin on glycolysis and oxidative phosphorylation in A2780/DDP cells.\u003c/strong\u003e Glucose consumption (A), pyruvate accumulation (B) and lactic acid accumulation assay (C). HK enzyme (D), PK enzyme (E) and LDH enzyme (F) activity assay. Expression of key enzyme proteins in glucose metabolism pathway (G). ATP content (H) and enzyme activity of Mitochondrial hydrogenase-2 (TH-2) assay (I). Expression of key enzyme proteins in oxidative phosphorylation pathway (J). The experimental results were performed in triplicate, and the results are expressed as mean ± standard deviation. β-actin was the internal reference. ImageJ software statistics protein gray value, and GraphPad Prism 8 was used for statistical analysis of the data. *= P \u0026lt;0.05, **= P \u0026lt;0.01, vs control.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8917089/v1/baeb2a9ad8a7de1d888b8acb.png"},{"id":103506343,"identity":"c93d4e45-b4e7-4a9b-bd81-b93218b03f2a","added_by":"auto","created_at":"2026-02-26 13:35:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2259712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePoria extract combined with cisplatin induces mitochondrial autophagy in A2780/DDP cells.\u003c/strong\u003e ROS assay (A). Effect of Poria extract combined with cisplatin on NF-κB/TGF-β1/PINK1/Parkin signaling pathway in A2780/DDP cells (B). Mitochondrial autophagy of A2780/DDP cells was detected by fluorescence (C) and electron microscopy (D).The experiment was repeated three times, and the results are expressed as mean ± standard deviation. β-actin was the internal reference. ImageJ software statistics protein gray value, and GraphPad Prism 8 was used for statistical analysis of the data. *= P \u0026lt;0.05, **= P \u0026lt;0.01, vs control.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8917089/v1/aa4ced5e86fb8cb285dbf4bb.png"},{"id":103506373,"identity":"96bf7113-cb04-4e42-b29e-fed46494d1ca","added_by":"auto","created_at":"2026-02-26 13:35:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1224429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePoria extract combined with cisplatin induces apoptosis in A2780/DDP cells.\u003c/strong\u003e Apoptosis of A2780/DDP cells was detected by TUNEL (A) and flow cytometry (B). Western blot assay was used to detect apoptosis-related proteins in A2780/DDP cells (C). High performance liquid chromatography (HPLC) method to detect the contents of intracellular cisplatin (D). Western blot method to detect multidrugresistanceproteins (MRPs) in A2780/DDP cells (E). The experiment was repeated three times, and the results are expressed as mean ± standard deviation. β-actin was the internal reference. ImageJ software statistics protein gray value, and GraphPad Prism 8 was used for statistical analysis of the data. *= P \u0026lt;0.05, **= P \u0026lt;0.01, vs control.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8917089/v1/bed3e503d5f1356cc2f42366.png"},{"id":103354669,"identity":"8a2fd473-f560-49da-935e-174e092f39ce","added_by":"auto","created_at":"2026-02-24 18:06:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1549575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo experiment Poria extract combined cisplatin antitumor effect. \u003c/strong\u003eEffect of Poria extract combined with cisplatin on the body weight of tumor-bearing mice (A). Effect of Poria extract combined with cisplatin on subcutaneous tumors (B). Effects of Poria extract combined with cisplatin on the expression of regulatory enzymes related to glycolysis (C) and oxidative phosphorylation (D) in tumor tissues of mice. The experiment was repeated three times, and the results are expressed as mean ± standard deviation. β-actin was the internal reference. ImageJ software statistics protein gray value, and GraphPad Prism 8 was used for statistical analysis of the data. *= P \u0026lt;0.05, **= P \u0026lt;0.01, vs control.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8917089/v1/4adcd320719fdf324b9b26c0.png"},{"id":106461071,"identity":"22ce148e-7230-407e-b6eb-e09b99fca568","added_by":"auto","created_at":"2026-04-08 19:55:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8528081,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8917089/v1/791de76b-5e22-4957-80e5-9e3932b1e957.pdf"},{"id":103354665,"identity":"eea78a4d-3245-42fa-94a0-1d68c445f40f","added_by":"auto","created_at":"2026-02-24 18:06:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1153202,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigureandtable.docx","url":"https://assets-eu.researchsquare.com/files/rs-8917089/v1/4d8170e9d609824deb43a287.docx"},{"id":103506174,"identity":"6a06a9ed-a69b-44c1-b8b2-63d396f1abaa","added_by":"auto","created_at":"2026-02-26 13:34:24","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5111565,"visible":true,"origin":"","legend":"","description":"","filename":"fulluncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8917089/v1/c6df22e0378279c094ba01eb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Network pharmacology and experimental methods to explore the mechanism of Poria extract in overcoming cisplatin-resistant ovarian cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccording to statistics, the incidence of ovarian cancer ranks the third in the female reproductive system malignant tumors, and its mortality is second only to breast cancer and cervical cancer. Globally, the incidence of ovarian cancer in developed countries is 9.1 per 100,000, while the incidence in developing countries is 5.0 per 100,000\u003csup\u003e[1]\u003c/sup\u003e. At present, the increase of drug resistance in ovarian cancer seriously affects the treatment effect of ovarian cancer. There are many reasons for the occurrence of ovarian cancer drug resistance, such as individual differences in drug sensitivity, gene mutations, poor body immunity, cancer cell metastasis and so on\u003csup\u003e[2,3]\u003c/sup\u003e. Therefore, accurate treatment plans should be formulated according to the specific conditions of patients in the treatment process to avoid the occurrence of drug resistance and improve the treatment effect and survival rate of patients.\u003c/p\u003e \u003cp\u003eOtto Warburg, a German biochemist, proposed that the way of glucose metabolism in tumor cells has changed from oxidative phosphorylation to aerobic glycolysis, namely the Warburg effect\u003csup\u003e[4]\u003c/sup\u003e. Tumor cells choose glycolysis as the main way of energy production. On the one hand, through glycolysis, they can obtain intermediate metabolites for the synthesis of fat, protein and nucleic acid to meet their active synthesis needs\u003csup\u003e[5]\u003c/sup\u003e. Secondly, lactic acid produced by glycolysis can provide an acidic environment for the growth and invasion of tumor cells, which is conducive to the survival and spread of tumor cells\u003csup\u003e[6]\u003c/sup\u003e. Therefore, understanding the relationship between tumors and oxidative phosphorylation and glycolysis will help us to understand the metabolic characteristics of tumors more deeply, and provide new ideas and methods for the diagnosis and treatment of tumors.\u003c/p\u003e \u003cp\u003eThere is mounting evidence that the application of network pharmacology and bioinformatics can accurately predict the interaction between active chemical components in medicines and specific disease targets. Poria extract is a fungal medicinal material that parasitizes the roots of pine trees. Poria extract is a highly valued substance with dual applications as both a medicinal ingredient and a food source, derived from the same botanical origin. It exhibits a range of biological activities, such as enhancing immune function, providing antioxidant effects, and modulating physiological processes. Studies have shown that Poria has anti-inflammatory and anti-tumor effects\u003csup\u003e[7\u0026ndash;9]\u003c/sup\u003e. However, the anti-tumor mechanism of Poria extract is not fully understood, and there are still some limitations and challenges in clinical application. The aim of this study is to explore the molecular mechanism of Poria extract in reversing cisplatin resistance of ovarian cancer, and to lay a theoretical foundation for clinical application.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture\u003c/h2\u003e \u003cp\u003eHuman ovarian cancer cell line A2780 and cisplatin-resistant ovarian cancer cell line A2780/DDP were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). The cells were grown in DMEM/high glucose (Hyclone, USA) with 10% fetal bovine serum (Hyclone, Logan, Utah, USA), 100 U mL-1 penicillin, and 100 mg mL-1 streptomycin (Genview, San Francisco, California, USA), and they were incubated at 37 ℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells used in the experiments were all cleared for mycoplasma by mycoplasma scavengers and tested for mycoplasma to ensure contamination.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents and antibodies\u003c/h3\u003e\n\u003cp\u003ePoria extract was obtained from Liaoning University of Traditional Chinese Medicine. Cisplatin was procured from Sigma Aldrich Company (St. Louis, MO, USA). Primary antibodies included HIF-1α, HK2, PKM2, LDHA, ATP1, ATP5, Caspase 3/p17/p19, Caspase 9/p35, Bcl2, BAX, P glycoprotein, MRP, BCRP were obtained from Proteintech Company (Wuhan, Hubei, China). HRP-Goat anti-rabbit recombinant secondary antibody (H\u0026thinsp;+\u0026thinsp;L) was obtained from Proteintech Company (Wuhan, Hubei, China).\u003c/p\u003e\n\u003ch3\u003eScreening of ovarian cancer and Poria extract targets\u003c/h3\u003e\n\u003cp\u003eUtilizing the GeneCards database collection (\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) of targets related to ovarian cancer. Utilizing TCMSP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tcmspw.com/tcmsp.php\u003c/span\u003e\u003cspan address=\"http://tcmspw.com/tcmsp.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and ETCM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.tcmip.cn/ETCM/index.php\u003c/span\u003e\u003cspan address=\"http://www.tcmip.cn/ETCM/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the exploration of the chemical composition and targets of Poria extract.\u003c/p\u003e\n\u003ch3\u003eTarget gene enrichment and network pharmacological analysis\u003c/h3\u003e\n\u003cp\u003eA Venn diagram was constructed, and the overlapping region was identified as a potential target for Poria extract treatment of ovarian cancer. Poria extract and ovarian cancer were found to have common targets for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, with all screenings showing p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. TCMSP filters the active components of Poria extract corresponding to common target genes and constructs a network pharmacological map, and the filtration criteria are: oral bioavailability (OB)\u0026thinsp;\u0026ge;\u0026thinsp;30%, drug similarity (DL)\u0026thinsp;\u0026ge;\u0026thinsp;0.18.\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eParental A2780 cells or resistant A2780/DDP cells were seeded into 96-well plates at a density of 5000 cells/well. After 16 h of culture, the culture medium was removed. The cells were then treated with different concentrations of cisplatin (ranging from 0 to 300 \u0026micro;M) or Poria extract solution (ranging from 0 to 300 mg mL-1) for 24 h. Subsequently, 10 \u0026micro;L of MTT solution (Solarbio, Beijing, China) was added to each well and incubated at 37 ℃ for 4 h. The absorbance was measured using a microplate reader set to a wavelength of 570 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell proliferation was detected by EdU\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCell slides were initially placed in 24-well plates, and either parental A2780 cells or drug-resistant A2780/DDP cells at a density of 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well were seeded into the plates. After 16 h of culture, the culture medium was removed. The cells were then treated with drug-free culture medium for 24 h followed by treatment with the drug for another 24 h. Subsequently, 300 \u0026micro;L of EdU-labeled culture medium (Beyotime, Shanghai, China) was added and incubated at 37 \u0026ordm;C in the dark for 2 h. The slides were then gently washed once with PBS using sterile forceps. Following this, the cells were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with Triton X-100 solution (0.1%) for an additional 5 minutes at room temperature. Nuclei staining was achieved by adding 300 \u0026micro;L of Hoechst 33342 solution and incubating in the dark at room temperature for a further period of time lasting up to approximately thirty minutes. Finally, experimental results were captured using a fluorescence microscope.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGlucose, pyruvate and lactic acid assay\u003c/h2\u003e \u003cp\u003eAfter 24 h of incubation, A2780 cells, drug-resistant A2780/DDP cells, drug-free A2780/DDP cells, and drug-treated A2780/DDP cells were collected with a total cell count of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e. The cells were lysed using low temperature ultrasonic waves and subsequently boiled in water for 10 minutes. The resulting cell lysate was then centrifuged at 8000 g for 10 minutes at room temperature, and the supernatant was retained. Following the instructions provided with the glucose, pyruvate, and lactic acid detection kit (mlbio, Shanghai, China), detection reagents were added to each sample. Subsequently, the absorbance value of each sample was measured at wavelengths of 505 nm, 520 nm, and 570 nm using an enzyme label reader.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMitochondrial transhydrogenase 2 (TH2) enzyme activity assay\u003c/h3\u003e\n\u003cp\u003eA total of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were homogenized using an ice-bath homogenizer, and the homogenate was then centrifuged at 600 g for 5 minutes at 4\u0026deg;C. The supernatant was removed and subjected to a further centrifugation at 110000 g for 10 minutes at 4\u0026deg;C. Subsequently, the resulting supernatant was combined with the reaction reagent (Sangon Biotecand, Shanghai, China) incubated in a water bath at 37\u0026deg;C for 5 minutes. The absorbance was measured using a microplate reader set to a wavelength of 375 nm.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eATP content assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol was carried out in accordance with the instructions of the ATP content detection kit (mlbio, Shanghai, China). A total of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were collected and added to 1 mL of acidic extract. The cells were then disrupted using an ice bath homogenizer. The homogenate was subsequently centrifuged at 8000 g for 10 minutes at 4 ℃. The resulting supernatant was combined with 1 mL of alkaline extract, thoroughly mixed, and then subjected to another round of centrifugation at 8000 g for 10 minutes at 4 ℃. The wavelength for enzyme label measurement was set to 700 nm.\u003c/p\u003e\n\u003ch3\u003eHexokinase, pyruvate kinase and lactate dehydrogenase enzyme activities assay\u003c/h3\u003e\n\u003cp\u003eA total of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were added to 1mL of the extract, and the cells were disrupted by sonication and centrifuged at 8000 g for 10 min at 4 ℃. The supernatant was mixed with the reaction reagent (mlbio, Shanghai, China), and the wavelength of the microplate reader was adjusted to 340 nm, 340 nm and 450 nm for measurement.\u003c/p\u003e\n\u003ch2\u003eRreactive oxygen species assay\u003c/h2\u003e\n\u003cp\u003eA2780/DDP cells were treated for 24 h in both the drug-free control group and the drug experimental group. After treatment, cells were collected following pancreatic enzyme digestion. The cell concentration was adjusted to 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e mL-1 using DCFH-DA solution (final concentration of 10 \u0026micro;mol-1). Subsequently, the cells were incubated in a 37 \u0026ordm;C incubator for 20 minutes and then washed three times with serum-free cell culture solution to ensure complete removal of any DCFH-DA that had not entered the cells. The intensity of fluorescence before and after stimulation was measured using a fluorescent enzyme label under conditions set at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCell apoptosis was detected by Annexin V-PE/7-AAD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the treatment of drug-free and drug-free experimental group with A2780/DDP cells for 24 h, the amount of pancreatic enzyme digestion was 4\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells. The cells were washed twice with pre-cooled PBS, centrifuged at 4 ℃ for 5 min at 300 g, and then the supernatant was discarded and added with 100 \u0026micro;L Binding Buffer. Annexin 5 \u0026micro;L V-PE and 5 \u0026micro;L 7-AAD staining solution (Beyotime, Shanghai, China) were added, incubated for 10 min at room temperature and protected from light, and detected by flow cytometry (Thermofisher Scientific, Waltham, Massachusetts, United States).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell apoptosis was detected by TUNEL assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell climbs were initially placed in 24-well plates, followed by seeding of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e A2780/DDP cells in each well. After 16 h of adherent culture, the cells in both the control group and the drug experimental groups were treated for 24 h. The slides were then gently grabbed with sterile forceps and washed once with PBS. Subsequently, the cells were fixed with 4% paraformaldehyde for 30 minutes, followed by incubation with a Triton X\u0026minus;100 solution (0.1%) for 5 minutes at room temperature to permeabilize the cells. Next, 50\u0026micro;L of TUNEL detection solution (Beyotime, Shanghai, China) was added and incubated at 37 \u0026ordm;C in the dark for 60 minutes, after which the experimental results were captured using a fluorescence microscope (Zeiss, Jena, German).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical constituents of Poria extract based on LC-MS/MS analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Poria extract powder was dissolved in 50% aqueous methanol, and the supernatant was retained after centrifugation. Using Q Exactive Orbitrap high resolution mass spectrometry to mass spectrum data collection, specific parameters setting is: The positive ion and negative ion modes were scanned separately. The ion source voltage was 3.2kV, Capillary temp was 320 \u0026deg; C, Aux gas heater temp was 350 \u0026deg; C, Sheath gas flow rate was 40L/min. The Aux gas flow rate was 15L/min.\u003c/p\u003e\n\u003ch2\u003eIn vivo experiments\u003c/h2\u003e\n\u003cp\u003eDrug preparation: Poria extract was dissolved in saline to a final concentration of 1g mL-1. Cisplatin was dissolved in saline at a final concentration of 100 \u0026micro;g mL-1. Female BALB/c nude mice weighing 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g were randomly selected, 5 mice in each group, a total of 4 groups. Each nude mouse was subcutaneously inoculated with 0.2 mL A2780/DDP cells (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells mL-1) on the right side. Once the tumors reached a volume of 50\u0026ndash;100 mm\u0026sup3;, daily recordings were made of the mice\u0026apos;s body weight, food consumption, tumor dimensions, and activity levels. The mice with successfully constructed transplanted tumors were randomly divided into four groups. Control group: normal saline 0.2 mL/10 g/d by gavage for 14 days. Cisplatin group: intraperitoneal injection of cisplatin 0.2 mL/10 g, twice a week for 14 days. Poria extract group: gavage of Poria extract 0.2 mL/10 g/ d for 14 days; At the same time, cisplatin was injected intraperitoneally 0.2 mL/10 g twice a week for 14 days. Following a 14-day period, the protein expression levels were analyzed using western blotting. All animal experiments were conducted at the School of Life Sciences and Health, Northeastern University. The experimental animal project received ethical approval from the Biological and Medical Ethics Committee of Northeastern University.(No. NEU-EC-2023A079S).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe parental A2780 cells, drug-resistant A2780/DDP cells, A2780/DDP cells without drug treatment, and A2780/DDP cells treated with drug were lysed using RIPA solution to extract protein. Equal amounts of protein samples from each group were loaded for SDS-PAGE electrophoresis at 80V for 25 min and then at 120V for 50 min. The protein bands in the SDS-PAGE gel were transferred to a PVDF membrane at 25V and 1.3A for 14 minutes. The PVDF membranes containing proteins were washed with TBST, followed by overnight blocking with a 5% skim milk solution, a two-hour incubation with the primary antibody, and finally a one-hour incubation with the secondary antibody. Subsequently, the ECL solution (Vazyme, Shanghai, China) was used to develop the imaging.\u003c/p\u003e\n\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n\u003cp\u003eThe experimental data were all repeated at least three times and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and all of the datas were analyzed with SPSS 19.0. The differences in cell cycle and proliferation, glycolytic parameters and oxidative phosphorylation between parental A2780 cells and resistant A2780/DDP cells were evaluated by two-tailed Student\u0026apos;s t test. In A2780/DDP cells, the differences of cell cycle and proliferation, apoptosis, glycolysis and oxidative phosphorylation in different experimental groups were evaluated by Analysis of variance (ANOVA). P value lower than 0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePoria extract treatment of ovarian cancer target prediction and build network pharmacology\u003c/h2\u003e \u003cp\u003eA total of 10273 ovarian cancer targets were obtained from the GeneCards database. A total of 65 compounds were identified from the Poria extract through the TCMSP and ETCM databases, and these compounds corresponded to a total of 241 targets. The 241 targets corresponding to the 65 compounds of Poria extract and the 10273 targets of ovarian cancer were used for subsequent bioinformatics analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003e-A, the targets for Poria extract and ovarian cancer have 146 intersections. Enrichment analysis was performed on the targets of Poria extract in the treatment of ovarian cancer. GO and KEGG correlation analysis showed that the top 20 enriched items (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Mainly includes the Glycolysis and Gluconeogenesis, Pyruvate metabolism, Carbon metabolism, Starch and sucrose metabolism, etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003e-B and C). Furthermore, a network pharmacological map of Poria extract for the treatment of ovarian cancer was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e1\u003c/span\u003e-D).\u003c/p\u003e \u003cp\u003eThe compounds of Poria extract were identified by LC-MS/MS method, the top ten chemical constituents were Paeoniflorin (18.769%), Amygdalin (17.971%), Albiflorin (8.195%), Sucrose (5.809%), Pyrogallol (5.535%), Citric Acid (4.841%) and Benzoylpaeoniflorin (3.167%), Cinnamaldehyde (3.122%), Coumarin (2.790%), Pachymic acid (2.541%) (Supplementary Fig.\u0026nbsp;1 and table 1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCisplatin resistance in ovarian cancer caused by glycolysis\u003c/h2\u003e \u003cp\u003eThe imbalance between glycolysis and oxidative phosphorylation is a ubiquitous feature of cancer cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003e-A to F, the glycolysis level and the expression levels of key enzymes in the glycolysis pathway in cisplatin-resistant A2780/DDP cells were significantly higher than those in parental A2780 cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but there was no significant difference in oxidative phosphorylation level and oxidative phosphorylation regulatory enzymes between the two cells (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In addition, the sensitivity of A2780 cells and A2780/DDP cells to cisplatin and Poria extract was determined. Both cisplatin and Poria extract inhibited the growth of A2780 and A2780/DDP cells in a concentration-dependent manner. The IC\u003csub\u003e50\u003c/sub\u003e value denotes the concentration of a drug at which its inhibitory effect on a specific biological process, such as cell proliferation or enzyme activity, reaches 50% within a defined period of time. The IC\u003csub\u003e50\u003c/sub\u003e of cisplatin for A2780 cells and A2780/DDP cells was 22.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u0026micro;M and 39.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003e-G and H). The IC\u003csub\u003e50\u003c/sub\u003e of Poria extract on A2780 cells and A2780/DDP cells was 0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 mg mL\u0026minus;1 and 1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 mg mL\u0026minus;1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003e-I and J). Increasing drug concentrations produce toxic effects on cells, affecting cell viability and masking the molecular mechanism of drug action. To exclude this factor, we selected the IC\u003csub\u003e5\u003c/sub\u003e value, which is the concentration of drug required to inhibit cell growth by 5%, for subsequent studies. Cisplatin combined with Poria extract at IC\u003csub\u003e5\u003c/sub\u003e concentrations had no effect on A2780/DDP cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003e-K).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePoria extract combined with cisplatin regulated glycolysis and oxidative phosphorylation levels in A2780/DDP cells\u003c/h2\u003e \u003cp\u003eThe changes of glycolysis and oxidative phosphorylation in A2780/DDP cells treated with Poria extract alone, cisplatin alone, or Poria extract combined with cisplatin were measured. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e-A to F, the combined treatment of the two drugs continuously inhibited glucose consumption, pyruvate production, lactate accumulation, and the activities of their related regulatory enzymes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e-G, the protein expression levels of HK2, PKM2 and LDHA in A2780/DDP cells were significantly decreased after 24 h of drug treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e-H and I, the ATP content and mitochondrial transhydrogenase 2 (TH2) in A2780/DDP cells increased at first and then decreased after drug combined intervention. The expressions of ATP1 and ATP5 protein in A2780/DDP cells were significantly inhibited by Poria extract alone or combined with drug intervention (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e-J), and the inhibitory effect of combined drug intervention was better than that of Poria extract alone.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePoria extract combined with cisplatin activates ROS to induce mitophagy in A2780/DDP cells\u003c/h2\u003e \u003cp\u003eFirstly, we found that Poria extract combined with cisplatin could promote a significant increase in ROS level in A2780/DDP cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e-A). In addition, we found that the combined treatment of Poria extract and cisplatin could activate the NF-κB/TGF-β1/PINK1/Parkin signaling pathway in A2780/DDP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e-B). The NF-κB/TGF-β1/PINK1/Parkin signaling pathway is closely related to mitochondrial damage. In A2780/DDP cells, the presence of mitochondrial vesicles, autophagosomes and autolysosomes was detected by fluorescence and transmission electron microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e4\u003c/span\u003e-C and D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePoria extract combined with cisplatin reversed cisplatin resistance by inducing apoptosis in A2780/DDP cells\u003c/h2\u003e \u003cp\u003eTunel fluorescence staining showed that Poria extract combined with cisplatin could significantly promote the apoptosis of A2780/DDP cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e-A). In addition, flow cytometry showed that the combined treatment of Poria extract and cisplatin significantly promoted the sum of early and late apoptosis of A2780/DDP cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e-B). We tested the apoptosis regulation related protein expression level, the results showed that combination of drugs intervention can promote A2780 / DDP cells Cleaved-Casepase 3, Cleaved-Casepase 9, and the expression of BAX protein, inhibition of Bcl-2 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e-C). Compared with cisplatin alone, Poria extract combined with cisplatin significantly increased the content of cisplatin in A2780/DDP cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e-D). Finally, the expression levels of drug resistance regulatory proteins in A2780/DDP cells were detected, and the results showed that the combined treatment of Poria extract and cisplatin significantly inhibited the expression of P-gp, MRP and BCRP proteins (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e-E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePoria extract combined with cisplatin significantly inhibited tumor energy metabolism and growth in vivo\u003c/h2\u003e \u003cp\u003eA2780/DDP xenograft mouse model was established. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003e-A, when the mice with transplanted tumors were treated with Poria extract and cisplatin for 14 days, the tumor size in the armpit of the mice was significantly smaller than that of the drug treatment group alone. The body weight of the mice with transplanted tumors in the combined drug treatment group was also significantly higher than that in the drug treatment group alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003e-B, the tumor volume of the combined drug treatment group was significantly reduced from day 7 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The effect of Poria extract combined with cisplatin on glycolysis and oxidative phosphorylation levels in A2780/DDP cells showed that the combined drug treatment group could inhibit the expression of HIF-1α, HK2, PKM2, ATP1 and ATP5 proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003e-C and D). The contents of ATP and lactate in tumor tissues were inhibited in the combined treatment group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003e-E and F).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere is a close relationship between tumors and oxidative phosphorylation and glycolysis. Oxidative phosphorylation is the main way in which cells produce ATP through oxidative reactions, which is an efficient way to generate energy. However, in tumor cells, the mode of glucose metabolism is changed from oxidative phosphorylation to aerobic glycolysis\u003csup\u003e[10,11]\u003c/sup\u003e. Aerobic glycolysis, a way in which cells produce energy by breaking down glucose under hypoxic conditions, produces a smaller amount of ATP but at a faster rate. ATP synthase is a large protein complex located in the inner mitochondrial membrane that plays a key role in oxidative phosphorylation and is responsible for catalyzing the phosphorylation of ADP to ATP. ATP synthase is composed of several subunits, of which ATP1 and ATP5 are important subunits directly involved in ADP phosphorylation to generate ATP\u003csup\u003e[12]\u003c/sup\u003e. HK2, PKM2 and LDHA are the key enzymes in the different stages of glycolytic pathway from glucose to pyruvate and finally to lactate\u003csup\u003e[13]\u003c/sup\u003e. Together, they play an important role in the regulation of the process of glycolysis and energy metabolism of cells. Energy production and growth of tumor cells can be inhibited by inhibiting key enzymes of glycolysis, such as hexokinase 2 (HK2) or lactate dehydrogenase A (LDHA)\u003csup\u003e[14,15]\u003c/sup\u003e. In addition, a deeper understanding of tumor metabolism, by activating oxidative phosphorylation or restoring mitochondrial function, offers the possibility to develop new cancer therapies.\u003c/p\u003e \u003cp\u003eThe molecular mechanism of drug resistance in ovarian cancer is a complex process involving changes in multiple aspects. For example, the abnormalities of signaling pathways and metabolic enzymes, epithelial-mesenchymal transition (EMT), abnormal expression of miRNA, the role of cancer stem cells (CSC), and changes in the immune microenvironment can cause drug resistance of ovarian cancer\u003csup\u003e[16\u0026ndash;18]\u003c/sup\u003e. Recent studies have found that the balance between oxidative phosphorylation and glycolysis plays a crucial role in the development and drug resistance of ovarian cancer. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial chaperone protein from Hsp90 family\u003csup\u003e[19]\u003c/sup\u003e. Up-regulation of TRAP1 expression increased aerobic glycolysis in ovarian cancer cells\u003csup\u003e[20]\u003c/sup\u003e. When TRAP1 expression is low or silenced, the oxygen consumption of ovarian cancer cells increases, leading to a high dependence on oxidative phosphorylation and is associated with platinum resistance\u003csup\u003e[21]\u003c/sup\u003e. Abnormal activation of PI3K/AKT/mTOR pathway plays a key role in the formation of drug resistance in ovarian cancer\u003csup\u003e[22]\u003c/sup\u003e. The key enzymes in glucose metabolism of ovarian cancer cells, such as hexokinase, 6-phosphofructokinase and lactate dehydrogenase, can promote the growth of ovarian cancer cells and reduce the sensitivity to chemotherapy drugs\u003csup\u003e[23\u0026ndash;25]\u003c/sup\u003e. When ovarian cancer cells are attacked by chemotherapeutic drugs, they will increase glycolysis and glutamine metabolism to produce more energy and reducing power, thus resisting the pressure of drugs. In this study, the cisplatin sensitive ovarian cancer cell line A2780 and cisplatin resistance cell line A2780/DDP were used as research objects. The results showed that compared with A2780 cells, the activities of glucose metabolism regulatory enzymes such as hexokinase (HK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) were increased in A2780/DDP cells, leading to increased glycolysis level. However, there was no significant difference in oxidative phosphorylation between A2780 and DDP cells. The increased glycolysis level in A2780/DDP cells is closely related to the cisplatin resistance phenotype.\u003c/p\u003e \u003cp\u003eOur previous study demonstrated that Guizhi Fuling capsule inhibits ovarian cancer metastasis by targeting the PA2G4/PI3K/AKT/GSK-3β signaling pathway both in vivo and in vitro\u003csup\u003e[26]\u003c/sup\u003e. Composed of Cinnamomum cassia and Poria extract, the capsule contains hundreds of active ingredients as identified by LC-MS/MS analysis. To elucidate the specific active ingredients responsible for its anti-ovarian cancer effects, bioinformatics prediction revealed a significant overlap between the active components of Poria extract and the therapeutic targets associated with ovarian cancer. Poria extract is a kind of precious food and medicinal material with the same origin of food and medicine. It has the functions of enhancing immunity, anti-oxidation and regulating body function. In this study, we identified Poria extract through database searches and identified 146 common targets for ovarian cancer. Subsequently, we constructed a network pharmacology model. Functional enrichment analysis of these common targets revealed that the dysregulation of glycolysis is a key factor in Poria extract treatment for ovarian cancer. We predicted that glycolysis would be the key point in the treatment of ovarian cancer by Poria extract, and found that the abnormality of glycolysis was a significant feature of cisplatin resistance in ovarian cancer. Therefore, we tested whether Poria extract could also exert inhibitory effect on cisplatin-resistant ovarian cancer. ELISA and Western blot were used to confirm that Poria extract combined with cisplatin could reduce the level of glycolysis by inhibiting the enzyme activity and protein expression of glucose metabolism regulatory enzymes in A2780/DDP cells. Studies have demonstrated that Poria extract exerts therapeutic effects on primary dysmenorrhea in rats through the regulation of multiple metabolic pathways, including the TCA cycle, gluconeogenesis, glycolysis, and the pentose phosphate pathway\u003csup\u003e[27]\u003c/sup\u003e. Studies found that Poria extract can activate the PI3K/AKT/mTOR signaling pathway, thereby decreasing levels of follicle-stimulating hormone, testosterone, and fasting insulin, while increasing blood glucose levels, ultimately improving ovulatory dysfunction in PCOS rats with insulin resistance\u003csup\u003e[28]\u003c/sup\u003e. The results of our study are consistent with the existing published articles. The metabolic shift from efficient aerobic oxidative phosphorylation to enhanced glycolysis in tumor cells is referred to as the \"Warburg effect.\" This phenomenon is primarily driven by mitochondrial dysfunction, the diversion of TCA cycle intermediates for biosynthetic purposes, and elevated levels of ROS, all of which contribute to reduced efficiency or inhibition of aerobic oxidative phosphorylation. In addition, the ATP content of A2780/DDP cells increased at first and then decreased during the treatment of Poria extract combined with cisplatin. In our study, we observed that the level of oxidative phosphorylation(ATP content), was comparable between A2780 cells and A2780/DDP cells. This suggests that glycolysis was elevated in A2780/DDP cells, while oxidative phosphorylation was not further suppressed. During the treatment of A2780/DDP cells with Poria extract in combination with cisplatin, ATP levels exhibited an initial increase followed by a subsequent decrease, returning toward normal levels after 24 hours. We further investigated whether this transient enhancement of oxidative phosphorylation could induce alterations in certain biological parameters of A2780/DDP cells. The results show that elevated ATP could induce ROS, activate the NF-κB/TGF-β1/PINK1/Parkin signaling pathway, and promote mitophagy in A2780/DDP cells. We found that Poria extract combined with cisplatin could promote the apoptosis of A2780/DDP cells as detected by TUNEL and flow cytometry. Finally, we carried out in vivo experiments to verify that Poria extract combined with cisplatin could inhibit the tumor growth, glucose metabolism and oxidative phosphorylase expression in tumor-bearing mice.\u003c/p\u003e \u003cp\u003eIn summary, the relationship between tumors and oxidative phosphorylation and glycolysis is a complex and worthy area of further study. By regulating the expression of related genes or key enzymes in energy metabolism pathways, the balance of oxidative phosphorylation and glycolysis of tumor cells can be reshuffled, thereby enhancing their sensitivity to chemotherapy drugs. By in-depth study of the mechanisms and effects of this metabolic shift, we can better understand tumor growth and development and develop more effective treatments.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study showed that imbalance of glycolysis and oxidative phosphorylation is a prominent feature of cisplatin resistant ovarian cancer cell line A2780/DDP. Poria extract combined with cisplatin can reshape the balance of glycolysis and oxidative phosphorylation, induce ROS production, and promote mitophagy and apoptosis in A2780/DDP cells. Poria extract improve the curative effect of cisplatin for the treatment provides a theoretical basis for cisplatin resistance ovarian cancer.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLei Dou:\u003c/strong\u003e Writing-review \u0026amp; editing, Writing-original draft, Methodology, Investigation.\u0026nbsp;\u003cstrong\u003eLei Deng:\u0026nbsp;\u003c/strong\u003eWriting-review \u0026amp; editing, Writing-original draft, Methodology, Investigation, Conceptualization.\u0026nbsp;\u003cstrong\u003eEnting Lu:\u003c/strong\u003e Writing-original draft, Methodology, Investigation.\u0026nbsp;\u003cstrong\u003eFangmei Li:\u003c/strong\u003eWriting-review \u0026amp; editing, Writing-original draft, Methodology.\u0026nbsp;\u003cstrong\u003eFanyi Meng:\u0026nbsp;\u003c/strong\u003eWriting-review \u0026amp; editing, Writing-original draft, Methodology.\u0026nbsp;\u003cstrong\u003eXinyang Chen:\u003c/strong\u003eWriting-review \u0026amp; editing, Writing-original draft, Methodology.\u003cstrong\u003eYin Li:\u003c/strong\u003e Writing-review \u0026amp; editing, Writing original draft, Supervision, Methodology.\u0026nbsp;\u003cstrong\u003eYi Zhang:\u0026nbsp;\u003c/strong\u003eWriting-review \u0026amp; editing, Writing original draft, Supervision, Resources, Conceptualization.\u0026nbsp;\u003cstrong\u003eYe Sun:\u0026nbsp;\u003c/strong\u003eWriting-review \u0026amp; editing, Writing original draft, Supervision, Methodology, Investigation, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted at the School of Life Sciences and Health at Northeastern University in accordance with the Declaration of Helsinki and approved by the local Council of Governance. Their care during research on animals follows ARRIVE guidelines and is conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 8023, as revised in 1978).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets during and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author thanks the Science and Technology Department of Liaoning Province and Science and Technology Bureau of Shenyang City for funding this study. This work was financially supported by the Liaoning Province Applied Basic Research Program (Grant 2022JH2/101300039) and Shenyang Science and Technology Plan (22-321-33-08). The above funds were used to carry out the research related consumables and reagents, detection, carrier synthesis and publication costs.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePenny SM (2020) Ovarian Cancer: An Overview. Radiol Technol 91:561\u0026ndash;575\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, Xie HJ, Li YY, Wang X, Liu XX, Mai J (2022) Molecular mechanisms of platinum-based chemotherapy resistance in ovarian cancer (Review). Oncol Rep 47:82\u0026ndash;92\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Wang X, Zhu X, Zhong L, Jiang Q, Wang Y, Tang Q, Li Q, Zhang C, Wang H, Zou D (2024) Drug resistance in ovarian cancer: from mechanism to clinical trial. 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BMC Cancer 18:636\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDou L, Yan Y, Lu E, Li F, Tian D, Deng L, Zhang X, Zhang R, Li Y, Zhang Y, Sun Y (2025) Composition analysis and mechanism of Guizhi Fuling capsule in anti-cisplatin-resistant ovarian cancer. Transl Oncol 52:102244\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong Z, Lang L, Gao X, Xiao W, Wang Z, Zhao L (2019) An integrative urinary metabolomic study of the therapeutic effect of Guizhi Fuling capsule on primary dysmenorrheal rats based 1H NMR and UPLC-MS. J Pharm Biomed Anal 164:750\u0026ndash;758\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Zhu H, Zhu Y, Hu X (2021) Guizhi Fuling Wan reduces autophagy of granulosa cell in rats with polycystic ovary syndrome via restoring the PI3K/AKT/mTOR signaling pathway. J Ethnopharmacol 270:113821\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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":"Poria extract, ovarian cancer, cisplatin resistance, Network pharmacology, Glucose metabolism","lastPublishedDoi":"10.21203/rs.3.rs-8917089/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8917089/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOvarian cancer is a common gynecological malignancy with a high incidence rate among female cancers. This study explored the mechanism of Poria extract in treating ovarian cancer through a combination of network pharmacology, bioinformatics analysis, and both in vitro and in vivo experimental validations. First, potential target genes associated with ovarian cancer and the active chemical components of Poria extract were identified using network pharmacology to determine possible therapeutic targets. Subsequently, these predictions were validated through cell culture and animal experiments. A total of 146 potential target sites were identified as being influenced by Poria extract's active components. Gene enrichment analysis indicated that dysregulated glycolysis metabolism and its related pathways are promising therapeutic targets for ovarian cancer. Experimental findings demonstrated that cisplatin resistance in ovarian cancer correlates with increased glycolytic activity. When administered in combination with cisplatin, Poria extract effectively suppresses glycolysis levels and the activity of its regulatory enzymes in A2780/DDP cells, while inducing an initial increase followed by a decrease in oxidative phosphorylation. The activation of ATP via oxidative phosphorylation leads to ROS accumulation, triggering mitochondrial autophagy and apoptosis. In vivo studies further confirmed that the combined treatment significantly inhibits tumor growth, as well as glycolysis and oxidative phosphorylation, in mice models. Overall, the combination of Poria extract and cisplatin can rebalance cellular energy metabolism and promote apoptosis in ovarian cancer cells.\u003c/p\u003e","manuscriptTitle":"Network pharmacology and experimental methods to explore the mechanism of Poria extract in overcoming cisplatin-resistant ovarian cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 18:05:55","doi":"10.21203/rs.3.rs-8917089/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":"1da35015-5320-4b5c-a7b9-d48b3cdb6311","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-08T19:54:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 18:05:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8917089","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8917089","identity":"rs-8917089","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}