Study on the effect of Ursolic acid on liver cancer cells using z-vad-fmk and ROS inhibitors

Study on the effect of Ursolic acid on liver cancer cells using z-vad-fmk and ROS inhibitors | 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 Study on the effect of Ursolic acid on liver cancer cells using z-vad-fmk and ROS inhibitors 朦 徐, 金玉 吴, 志敏 黄, 爱桃 林, 庭娜 付, 美 施, 国梁 张 This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8619071/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Mar, 2026 Read the published version in Cytotechnology → Version 1 posted 7 You are reading this latest preprint version Abstract Objective: To investigate the effect of Ursolic acid (UA) on liver cancer cells through cell experiments, in order to inhibit cell proliferation and induce apoptosis. Method: By adding z-vad-fmk apoptosis inhibitor and ROS inhibitor NAC, CCK8, cell apoptosis, Western blot experiments, mitochondrial membrane potential detection, and ROS flow cytometry were conducted. Compared with the positive drug sorafenib, the inhibitory effect of UA on liver cancer cells and its promotion of apoptosis were studied. The results of CCK8 confirmed that when UA was at its optimal concentration, its inhibitory effect on liver cancer cell viability was greater than that of sorafenib; Through cell apoptosis experiments, it can be observed that under fluorescence microscopy, the fluorescence intensity in the UA+HepG2 group is significantly stronger than that in the sorafenib+HepG2 group; Western blot experiments showed that the relative expression levels of proteins in HepG2 were almost equal between the HepG2+UA and sorafenib+HepG2 groups; through ROS experiments, it was found that when the reactive oxygen species in the HepG2+UA group were lower than those in the sorafenib+HepG2 group, it indicated that the former had a stronger effect of UA on HepG2. Conclusion: UA has a significant inhibitory effect on proliferation and promotes apoptosis in liver cancer cells. Ursolic acid Hepatocellular carcinoma cells Cell proliferation apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Primary liver cancer (PLC) is malignant tumor with a mortality rate and incidence rate that are almost identical. Its malignancy is extremely high, and PLC ranks third as a cause of death among malignant tumors worldwide(Xu R et al. 2026 ). At present, the treatment for PLC mainly includes liver resection, radiotherapy and chemotherapy, radiofrequency ablation, immunotherapy, use of molecular targeted drugs, and liver transplantation, and their efficacy is not significant(Wang P et al. 2025 ). This also indicates the importance and urgency of continuing to research PLC treatment methods. Ursolic acid (UA) belongs to the pentacyclic triterpenoid class and is widely present in various plants. It has attracted attention to its anti-cancer effects in various experimental studies, including cell apoptosis, tumor growth inhibition, and autophagy. The existence of this effect indicates its potential to inhibit tumor cell proliferation and promote apoptosis. However, due to its inherent limitations such as rapid systemic clearance, poor water solubility, and low oral bioavailability, UA is subject to various restrictions in clinical use. Therefore, this has also become a new direction and necessity for our research. Enable it to break through its own limitations and fully utilize it in the treatment of liver cancer(Bokhtia RM et al. 2025 ). For z-vad fmk, which is an inhibitor of cell apoptosis, it was found in Zhang Shuqing's study of ZIKV infection that the combination of z-vad fmk with other substances can greatly increase the inhibition of ZIKV induced cell death(Zhang S et al. 2025 ); When Ye Chen studied APG, he learned that APG has anti-tumor properties, and z-vad-fmk can significantly weaken the effect of APG and accelerate the occurrence of tumors(Chen Y et al. 2025 ). Currently, numerous studies have shown that ROS at toxic levels play a role in promoting apoptosis and killing cancer cells in anti-cancer targets. NAC is often used as an antioxidant to inhibit ROS and prevent cancer cell death; but if the concentration of NAC is too low, it can also induce excessive oxidative stress and ROS production, thereby inducing cancer cell death (Jung EJ et al. 2025 ). Therefore, this article starts with z-vad-fmk and ROS inhibitors to investigate the inhibitory effect of UA on proliferation and promotion of apoptosis in liver cancer cells, providing a new possibility for new drug research. According to the preliminary experiments conducted by the research group, the optimal concentration and time of UA is 40 µ M UA for 24 hours(Xu M et al. 2025 ). 2. Experimental materials: 1.1 Cells: LO-2(normal human hepatocellular), HepG2(human hepatic carcinoma cell lines). 1.2 Drugs and Reagents: UA, z-vad-fmk apoptosis inhibitor, sorafenib (10µmol/L, cultured for 24h)(Yu W et al. 2020), ROS inhibitor NACare purchased from Anhui Zhongkang Biological Technology Co., Ltd., CCK8 assay kit (Lot No.: BJ05203090), Caspase-8 are purchased from BIOSS, β-Actin (Lot No.: 19C10509) is purchased from Zsbio, Caspase-3 (Lot No.: GR3241586-6), Caspase-9 (Lot No.: GR305416-13), Caspase-7 (Lot No.: 7). 3. Experimental protocol 3.1 Comparison of the effects of UA and sorafenib on cells by adding z-vad-fmk apoptosis inhibitor: 3.1.1 Groups are: HepG2, HepG2 + 0, 20, 40, 80µM z-vad-fmk apoptosis inhibitor. CCK8 experiment: HepG2 cells added with UA: the pancreatin digests the logarithmic-phase cells, which are processed and put in the incubator for being cultured (monolayer cells fully cover the bottom of the pore (96-pore flat plate)) over night under conditions of 5%CO2 and 37℃. It is then observed under an inverted microscope. HepG2 are divided into 5 groups. Each group is added with: 0µM, 20µM, 40µM, and 80µM z-vad-fmk apoptosis inhibitor respectively, and cultured for 12h, 24h, 48h and 72h. After that, each pore is added with 10ul CCK8 and continued the culture for another 4 hours. The absorbance of each pore is measured at OD450nm of the enzyme-linked immunosorbent assay. At the same time, blank pores (substratum, CCK8) are set as well. (Cell viability = (experimental pores - blank pores)/ (control pores - blank pores)). 3.1.2 Groups are: LO-2, HepG2, HepG2 + UA (40µmol/L, cultured for 24h), HepG2 + optimal concentration of z-vad-fmk apoptosis inhibitor, HepG2 + sorafenib (10µmol/L, cultured for 24h), HepG2 + UA (40µmol/L, cultured for 24h) + optimal concentration of z-vad-fmk apoptosis inhibitor, HepG2 + sorafenib (10µmol/L, cultured for 24h) + optimal concentration of z-vad-fmk apoptosis inhibitor. CCK8 experiment: Cells are LO-2 and HepG2. The pancreatin digests the two groups of logarithmic-phase cells, which are processed and put in incubator for being cultured (monolayer cells fully cover the bottom of the pores (96-pore flat plate)), overnight under conditions 5%CO2 and 37℃, and observed under an inverted microscope. HepG2 cells are divided into 6 groups as mentioned above. Each pore is added with 10ul CCK8 and continued the culture for another 4 hours. The absorbance of each pore is measured at OD450nm of the enzyme-linked immunosorbent assay. At the same time, blank pore (substratum, CCK8) are set. (Cell viability = (experimental pores - blank pores)/ (control pores - blank pores)). Cell apoptosis: Cells are centrifuged and collected, and then performed with centrifugal washing by cold PBS. A total of 1–10*10^5 cells are collected and diluted with double diluted water to take 500ul 1*Binding Buffer suspended cells. Then 5ulAnnexin V-FITC and 10ulPI are added as well for to conduct cytometry analysis. Western blot experiment: LO-2 and HepG2 cells are inoculated in 6-pore plates, and HepG2 cells are divided into 6 groups as mentioned above. Then, 600µl RIPA cell lysate is added for lysis and extraction of total tissue protein. SDS-PAGE gel preparation is performed according to the TRAKRA product catalog, concentrate gel (2ml) 10% and separate it (10ml). 5X SDS-PAGE protein sample loading buffer is added to the collected protein samples by 1:4 proportion. The filter paper and PVDF membrane are immersed in the membrane rotating buffer for 5min, and then performed with constant flow trarsmembrane through the membrane rotating device. After that, the protein membrane is rinsed in Western washing solution for 5 min, and then added with Western blocking solution (5% skim milk powder) for 2 hours. Then the filter paper and PVDF membrane are immersed in the membrane transfer buffer for 5min, and performed with the constant flow trarsmembrane through the membrane transfer device. After finishing the membrane transfer, the protein membrane is rinsed in Western washing solution for 5 min, and then put in Western blocking solution(5% skim milk powder) for 2 hours. After that, the samples are diluted with primary antibody diluent at 4℃ overnight in an appropriate proportion(see Table 1 ), and reacted with the secondary antibody(1:2000 dilution) marked by horseradish peroxidase for 1.2h. Finally the proteins are detected by ECL luminescence kit according to relevant instructions. Table 1 Primary antibody dilution ratios Antibody Name Dilution Ratio Species And Genus Caspase-3 1: 2000 Rabbit Caspase-9 1: 2000 Rabbit Caspase-7 1: 1000 Rabbit Caspase-8 1: 2000 Mouse 3.2 Comparison of the effects of UA and sorafenib on cells through the addition of ROS inhibitor NAC: 3.2.1 Groups are: HepG2, HepG2 + 0, 2.5, 5.0, 7.5µM ROS inhibitor NAC. CCK8 experiment: The process is the same as that of CCK8 experiment in 2.1. 3.2.2 Groups are: LO-2, HepG2, HepG2 + UA(40µmol/L, cultured for 24h), HepG2 + optimal concentration of ROS inhibitor NAC, HepG2 + sorafenib (10µmol/L, cultured for 24h), HepG2 + UA(40µmol/L, cultured for 24h) + optimal concentration of ROS inhibitor NAC, HepG2 + sorafenib(10µmol/L, cultured for 24h) + optimal concentration of ROS inhibitor NAC. Mitochondrial membrane potential detection(JC-1): Cells of each group are collected, and washed twice with PBS. Part of the cells taken to make them resuspended in 0.5ml substratum by 500ul JC-1 staining working solution. The cells are cultured in incubator with conditions of 37℃ and 5% CO2 for 20min, and then collected based on centrifugation at 600g for 4min. After that, the JC-1 staining buffer solution is taken to wash cells twice, and then proper amount of JC-1 staining buffer is used again to make the cells resuspended for conducting flow cytometry. ROS: Cells: HepG2 cells are divided into 5 groups, which are HepG2, 0, 20, 40, and 80µM UA. The cells in each of the above groups are collected to regulate the cell concentration to (1–10)x10^6/ml. Then they are add with 10umol/L DCFH-DA, cultured for 20min under 37℃ condition, and mixed every 3-5min to make it fully act. Meanwhile the non-stained cells are set as control. After that, the cells are washed twice with PBS to conduct flow cytometry. 4. Results 4.1 Comparison of the effects of UA and sorafenib on cells by adding z-vad-fmk apoptosis inhibitor: It can be known by CCK8 experiment that: the cell viability enhances gradually with the concentration increase of z-vad-fmk apoptosis inhibitor, and becomes basically stable when the concentration reaches 80µM ( P < 0.01). The lowest inhibition effect of cell viability appears when the cells are cultured for 24h (P < 0.01) (Fig. 1 ). UA (40µM, cultured for 24h) has a greater inhibitory effect on the viability of liver cancer cells than that of the sorafenib (10µmol/L, cultured for 24h) (Fig. 2 ). From cell apoptosis experiment, it can be observed that the intracellular fluorescence intensity of Group C is significantly greater than that of Group E under the fluorescence microscope (Fig. 3 ). From Western blot experiment (Fig. 4 ), it can be observed that when UA is added to HepG2(40µM, cultured for 24h), the relative protein expression in HepG2 is almost the same as that of the status of adding sorafenib to HepG2(10µmol/L, cultured for 24h) ( P < 0.05). 4.2 Comparison of the effects of UA and sorafenib on cells through the addition of ROS inhibitor NAC: According to the results of CCK8 experiment, the cell viability enhances gradually with the concentration increase of ROS inhibitor NAC. And the inhibition effect of drug on cell viability becomes basically stable it reaches 7.5µM ( P < 0.01). The lowest inhibition effect on cell viability appears when the cells are cultured for 48h ( P < 0.01) (Fig. 5 ). It can be observed from mitochondrial membrane potential detection (JC-1) experiment that, under the fluorescence microscope, the intracellular fluorescence intensity of Group C is significantly weaker than that of Group E (Fig. 6 ). It can be known from ROS experiment that: When the UA(40 µM, cultured for 24 hours) is added to HepG2, the DCF fluorescence is lower than that of adding sorafenib (10µmol/L, cultured for 24h) to HepG2. This means that, for HepG2, the reactive oxygen species at the time when adding UA(40 µM, cultured for 24 hours) is lower than that adding sorafenib (10µmol/L,cultured for 24 hours). And this further proves that UA has a stronger effect on HepG2 than sorafenib (10µmol/L, cultured for 24h) (Fig. 7 ). 5. Discussion Research has shown that UA is present in many medicinal plants and has low toxicity. It has a wide range of pharmacological effects, including regulating immunity, protecting the liver, anti-tumor effects, etc. It can promote cell apoptosis, reverse tumor drug resistance, inhibit tumor cell proliferation, and increase the efficacy of chemotherapy in cancer treatment(Huang X et al. 2025 ). For liver cancer, studies by Liu T and Yee Y(Liu T et al. 2017 ;Yie Y et al. 2015 ) have shown that UA can inhibit the growth of liver cancer cells through the Stat3 pathway or by reducing AMPK α - mediated DNA methyltransferase 1. In the Yin Jie Fan8 (Fan YJ et al. 2024 ) study, it was shown that UA can significantly reduce liver enzyme indicators in liver cancer mouse models, inhibit the invasion and migration of liver cancer cells, and also reduce the resistance of liver cancer cells to sorafenib. This study mainly demonstrated the inhibitory effect of UA on proliferation and promoting apoptosis of liver cancer cells by adding z-vad-fmk inhibitor and ROS inhibitor NAC, and using sorafenib as a positive control group. After adding the inhibitor, it can be seen that UA has a greater effect on liver cancer cells compared to sorafenib, and is more capable of inhibiting liver cancer cells. 6. Conclusion In summary, UA has a good inhibitory effect on proliferation and promotes apoptosis of liver cancer cells, providing a new approach for the treatment and new drug research of liver cancer. Declarations Author contributions All authors were involved in the conceptualization and design of the study. Mei Shi provided experimental ideas. Meng Xu completed the first draft of the manuscript. Jinyu Wu, Zhimin Huang, Tingna Fu and Aitao Lin performed the material preparation, data collection and analysis. Guoliang Zhang was mainly responsible for reviewing the final manuscript. All authors participated in the editorial revision of the manuscript. All authors read and approved the final manuscript. Funding This work was supported by “2021 Anhui Province Major Difficult Diseases Collaborative Research Project of Traditional Chinese and Western Medicine” and “National Famous Traditional Chinese Medicine Seed Player Financial Cultivation Project in Anhui Province (Zhang Guoliang)”. Data availability The data generated in this study are available upon request to the corresponding author. Competing interests The authors declare no competing interests. Consent for publication All authors are aware of and have consented to publication. References Xu R, Su Q, Liao X et al (2026) A retrospective study of preoperative HBsAg levels predicting recurrence after curative resection in patients with HBV-associated hepatocellular carcinoma. Med (Baltim) 105(2):e47065 Wang P, Lin J, Su D (2025) Echinacoside as a Novel Ferroptosis Inducer in Hepatocellular Carcinoma: Mechanistic Insights from TP53/SLC7A11/GPX4 Pathway Modulation. Int J Mol Sci 27(1):411 Bokhtia RM, Gupta KB, Natalini A et al (2025) Biotin-Linked Ursolic Acid Conjugates as Selective Anticancer Agents and Target-Identification Tools for Cancer Therapy. Molecules 30(23):4588 Zhang S, Chen D, Zhang K et al (2025) ZIKV infection causes placental inflammation through activating PANoptosis. J Virol 99(12):e0175925 Chen Y, Wu L, Wang S et al (2025) The Anti-Tumor and Bortezomib-Sensitizing Effects of Apigenin in Multiple Myeloma. Curr Issues Mol Biol 47(9):717 Jung EJ, Kim HJ, Shin SC et al (2025) Tetraarsenic Hexoxide Enhanced the Anticancer Effects of Artemisia annua L. Polyphenols by Inducing Autophagic Cell Death and Apoptosis in Oxalplatin-Resistant HCT116 Colorectal Cancer Cells. Int J Mol Sci 26(16):7661 Xu M, Chen L, Wu J, Liu L, Shi M, Zhou H, Zhang G (2025) Mechanism of Hedyotis diffusa-Scutellaria barbata D. Don for treatment of primary liver cancer: analysis with network pharmacology, molecular docking and in vitro validation. Nan Fang Yi Ke Da Xue Xue Bao 45(1):80–89 English, Chinese Wang Y (2020) Fan Lulu Study on the Enhancement of Sorafenib's Proliferation Inhibition on Hepatocellular Carcinoma Cells by BET Protein Inhibitor JQ1 [J]. J Anhui Med Univ 55(08):1185–1188 Huang X, Sun Y, Tong H, Zhu J, Pan J, Wen P, He W (2025) Ursolic acid sensitizes bladder cancer to gemcitabine chemotherapy by concurrently targeting PI3K/AKT and JNK pathways. Transl Androl Urol 14(10):2902–2916 Liu T, Ma H, Shi W, Duan J, Wang Y, Zhang C et al (2017) Inhibition of STAT3 signaling pathway by ursolic acid suppresses growth of hepatocellular carcinoma. Int J Oncol 51(2):555–562 Yie Y, Zhao S, Tang Q, Zheng F, Wu J, Yang L et al (2015) Ursolic acid inhibited growth of hepatocellular carcinoma HepG2 cells through AMPKα-mediated reduction of DNA methyltransferase 1. Mol Cell Biochem 402(1–2):63–74 Fan YJ, Pan FZ, Cui ZG, Zheng HC (2024) The Antitumor and Sorafenib-resistant Reversal Effects of Ursolic Acid on Hepatocellular Carcinoma via Targeting ING5. Int J Biol Sci 20(11):4190–4208 Authors and Affiliations Meng Xu1 Jinyu Wu 2 ; Zhimin Huang 2 ; Mei Shi 3 ; Tingna Fu 1 ; Aitao Lin 2 ; Guoliang Zhang 3 Corresponding Author Guoliang Zhang [email protected] Wu J [email protected] . 1 Graduate School of Guangxi University of Traditional Chinese Medicine, No.13 Wuhe Avenue, Qingxiu District, 530000 Nanning, Guangxi, China 2 The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine, No. 89 – 9 Dongge Road, 530000 Nanning, Guangxi, China 3 The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, NO. 117 Meishan Road, 230031 Hefei, Anhui, China Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 18 Mar, 2026 Read the published version in Cytotechnology → Version 1 posted Editorial decision: Revision requested 26 Feb, 2026 Reviews received at journal 25 Feb, 2026 Reviewers agreed at journal 16 Feb, 2026 Reviewers invited by journal 16 Feb, 2026 Editor assigned by journal 17 Jan, 2026 Submission checks completed at journal 17 Jan, 2026 First submitted to journal 16 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8619071","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":593358373,"identity":"dd2c69eb-25b6-4194-8ca2-4ab43220e738","order_by":0,"name":"朦 徐","email":"","orcid":"","institution":"广西中医药大学","correspondingAuthor":false,"prefix":"","firstName":"朦","middleName":"","lastName":"徐","suffix":""},{"id":593358374,"identity":"c3f15e4a-7240-424c-9101-83873968e574","order_by":1,"name":"金玉 吴","email":"","orcid":"","institution":"广西中医药大学","correspondingAuthor":false,"prefix":"","firstName":"金玉","middleName":"","lastName":"吴","suffix":""},{"id":593358375,"identity":"6b207b32-9e3a-49d7-8786-e83059689d84","order_by":2,"name":"志敏 黄","email":"","orcid":"","institution":"广西中医药大学","correspondingAuthor":false,"prefix":"","firstName":"志敏","middleName":"","lastName":"黄","suffix":""},{"id":593358376,"identity":"913ca2eb-37e6-4bc8-9d18-d8328aa81e1a","order_by":3,"name":"爱桃 林","email":"","orcid":"","institution":"广西中医药大学","correspondingAuthor":false,"prefix":"","firstName":"爱桃","middleName":"","lastName":"林","suffix":""},{"id":593358377,"identity":"bf0b1846-0cce-47be-b112-dfd05a48e7ea","order_by":4,"name":"庭娜 付","email":"","orcid":"","institution":"广西中医药大学","correspondingAuthor":false,"prefix":"","firstName":"庭娜","middleName":"","lastName":"付","suffix":""},{"id":593358378,"identity":"2a938554-0725-4fbd-8b43-84d66a0bc911","order_by":5,"name":"美 施","email":"","orcid":"","institution":"安徽中医药大学","correspondingAuthor":false,"prefix":"","firstName":"美","middleName":"","lastName":"施","suffix":""},{"id":593358379,"identity":"ab32c1cc-7c53-4065-84cf-750592978f7f","order_by":6,"name":"国梁 张","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYDACCSjNxwwiKyTkGJiJ1cIGVnnGwpgELSCCsaUisYGQDvnZzc8efvl1WI6NnfnZw68NEunz23kPfmCosYnGpYVxzjFzY9m+w8ZszGxAxg6J3A2H+ZIlGI6l5eKyjlkiwUxasudwYhszA5BxBqiFmcdAgrHhME4tbBLp36Ba2IGMNol0+WYe4x/4tPBI5JhJfvgB0sJjJvmxTSKB4TCPGV5bJCRyyqQZG9KBfuEpk2Y4I2G4AajFIgGPX+RnpG+T/PHHWo6f/ziQUVEnL99/xvjGhxobnFrAQcDbBmXwwIQS8CgHAcYff2AMAipHwSgYBaNgZAIAy/5Q08pFdxoAAAAASUVORK5CYII=","orcid":"","institution":"安徽中医药大学","correspondingAuthor":true,"prefix":"","firstName":"国梁","middleName":"","lastName":"张","suffix":""}],"badges":[],"createdAt":"2026-01-16 13:12:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8619071/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8619071/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10616-026-00926-8","type":"published","date":"2026-03-18T15:59:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":102997510,"identity":"52b6f7b0-9afe-48e4-a4ad-e27fa92c0388","added_by":"auto","created_at":"2026-02-19 12:33:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":103727,"visible":true,"origin":"","legend":"\u003cp\u003eCCK8 experiment\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8619071/v1/243a87efa7b4661b9144bbb3.png"},{"id":102997511,"identity":"590d6e66-88d5-4e5a-a688-f2d0d2f22c93","added_by":"auto","created_at":"2026-02-19 12:33:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22882,"visible":true,"origin":"","legend":"\u003cp\u003eCCK8 experiment(1 is LO-2, 2 is HepG2, 3 is HepG2+UA(40 µM, cultured for 24 hours), 4 is HepG2+80 µM z-vad fmk apoptosis inhibitor, 5 is HepG2+sorafenib (10 µmol/L, cultured for 24 hours), 6 is HepG2+UA(40 µM, cultured for 24 hours) +80 µM z-vad fmk apoptosis inhibitor, 7 is HepG2+sorafenib (10 µmol/L, cultured for 24 hours)+80 µM z-vad fmk apoptosis inhibitor)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8619071/v1/b2594feae3b8ab94255782aa.png"},{"id":103056443,"identity":"b825ab03-2c49-404e-8702-719c251643fa","added_by":"auto","created_at":"2026-02-20 09:10:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":215293,"visible":true,"origin":"","legend":"\u003cp\u003eHepG2 cell apoptosis rate (A is LO-2, B is HepG2, C is HepG2+UA(40 µM, cultured for 24 hours), D is HepG2+80 µM z-vad-fmk apoptosis inhibitor, E is HepG2+sorafenib (10 µmol/L, cultured for 24 hours), F is HepG2+UA(40 µM, cultured for 24 hours) +80 µM z-vad-fmk apoptosis inhibitor, G is HepG2+sorafenib (10 µmol/L, cultured for 24 hours)+80 µM z-vad-fmk apoptosis inhibitor)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8619071/v1/419f83e6bc6a19af7d5f0b7e.png"},{"id":103050031,"identity":"1e272b54-a369-4174-bbf4-8b90e392395f","added_by":"auto","created_at":"2026-02-20 07:47:46","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132268,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative expression levels of proteins in each group of HepG2 (A is LO-2, B is HepG2, C is HepG2+UA(40 µM, cultured for 24 hours) , D is HepG2+80 µM z-vad-fmk apoptosis inhibitor, E is HepG2+sorafenib (10 µmol/L, cultured for 24 hours), F is HepG2+UA(40 µM, cultured for 24 hours) +80 µM z-vad-fmk apoptosis inhibitor, G is HepG2+sorafenib (10 µmol/L, cultured for 24 hours)+80 µM z-vad-fmk apoptosis inhibitor)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8619071/v1/c09cbfa2ddf48fa1a9ab5cee.jpeg"},{"id":102997514,"identity":"e3b2db83-3401-4a11-9e6d-c653b7cab3ff","added_by":"auto","created_at":"2026-02-19 12:33:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95958,"visible":true,"origin":"","legend":"\u003cp\u003eCCK8 experiment\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8619071/v1/3c420c9a3d8b9f9316e7e57d.png"},{"id":102997513,"identity":"76861f32-1e0e-4af3-b246-0636624b2391","added_by":"auto","created_at":"2026-02-19 12:33:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":232979,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial membrane potential detection (JC-1) (LO-2, HepG2, HepG2+UA(40 µM, cultured for 24 hours), HepG2+optimal concentration of ROS inhibitor NAC, HepG2+sorafenib (10 µmol/L, cultured for 24 hours), HepG2+UA(40 µM, cultured for 24 hours) +optimal concentration of ROS inhibitor NAC, HepG2+sorafenib (10 µmol/L, cultured for 24 hours)+optimal concentration of ROS inhibitor NAC)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8619071/v1/3509304a8b88d881ccdd7e68.png"},{"id":102997516,"identity":"2b3d6ced-52eb-4b60-9474-ef0c1625a01b","added_by":"auto","created_at":"2026-02-19 12:33:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":71308,"visible":true,"origin":"","legend":"\u003cp\u003eROS flow cytometry analysis (A for LO-2, B for HepG2, C for HepG2+UA(40 µM, cultured for 24 hours), D for HepG2+optimal concentration of ROS inhibitor NAC, E for HepG2+sorafenib (10 µmol/L, cultured for 24 hours), F for HepG2+UA(40 µM, cultured for 24 hours) +optimal concentration of ROS inhibitor NAC, G for HepG2+sorafenib (10 µmol/L, cultured for 24 hours)+optimal concentration of ROS inhibitor NAC)\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8619071/v1/a303dbc8b722846cf1e4f352.png"},{"id":105223414,"identity":"d6be7858-580c-4daf-8eaa-332a0be554e0","added_by":"auto","created_at":"2026-03-23 16:06:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1295660,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8619071/v1/8fed5e66-0e35-49b2-9b57-ace296eb87ae.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Study on the effect of Ursolic acid on liver cancer cells using z-vad-fmk and ROS inhibitors","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePrimary liver cancer (PLC) is malignant tumor with a mortality rate and incidence rate that are almost identical. Its malignancy is extremely high, and PLC ranks third as a cause of death among malignant tumors worldwide(Xu R et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). At present, the treatment for PLC mainly includes liver resection, radiotherapy and chemotherapy, radiofrequency ablation, immunotherapy, use of molecular targeted drugs, and liver transplantation, and their efficacy is not significant(Wang P et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This also indicates the importance and urgency of continuing to research PLC treatment methods.\u003c/p\u003e \u003cp\u003eUrsolic acid (UA) belongs to the pentacyclic triterpenoid class and is widely present in various plants. It has attracted attention to its anti-cancer effects in various experimental studies, including cell apoptosis, tumor growth inhibition, and autophagy. The existence of this effect indicates its potential to inhibit tumor cell proliferation and promote apoptosis. However, due to its inherent limitations such as rapid systemic clearance, poor water solubility, and low oral bioavailability, UA is subject to various restrictions in clinical use. Therefore, this has also become a new direction and necessity for our research. Enable it to break through its own limitations and fully utilize it in the treatment of liver cancer(Bokhtia RM et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor z-vad fmk, which is an inhibitor of cell apoptosis, it was found in Zhang Shuqing's study of ZIKV infection that the combination of z-vad fmk with other substances can greatly increase the inhibition of ZIKV induced cell death(Zhang S et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e); When Ye Chen studied APG, he learned that APG has anti-tumor properties, and z-vad-fmk can significantly weaken the effect of APG and accelerate the occurrence of tumors(Chen Y et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Currently, numerous studies have shown that ROS at toxic levels play a role in promoting apoptosis and killing cancer cells in anti-cancer targets. NAC is often used as an antioxidant to inhibit ROS and prevent cancer cell death; but if the concentration of NAC is too low, it can also induce excessive oxidative stress and ROS production, thereby inducing cancer cell death (Jung EJ et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, this article starts with z-vad-fmk and ROS inhibitors to investigate the inhibitory effect of UA on proliferation and promotion of apoptosis in liver cancer cells, providing a new possibility for new drug research. According to the preliminary experiments conducted by the research group, the optimal concentration and time of UA is 40 \u0026micro; M UA for 24 hours(Xu M et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e"},{"header":"2. Experimental materials:","content":"\u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e1.1 Cells: LO-2(normal human hepatocellular), HepG2(human hepatic carcinoma cell lines).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e1.2 Drugs and Reagents: UA, z-vad-fmk apoptosis inhibitor, sorafenib (10\u0026micro;mol/L, cultured for 24h)(Yu W et al. 2020), ROS inhibitor NACare purchased from Anhui Zhongkang Biological Technology Co., Ltd., CCK8 assay kit (Lot No.: BJ05203090), Caspase-8 are purchased from BIOSS, β-Actin (Lot No.: 19C10509) is purchased from Zsbio, Caspase-3 (Lot No.: GR3241586-6), Caspase-9 (Lot No.: GR305416-13), Caspase-7 (Lot No.: 7).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e"},{"header":"3. Experimental protocol","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Comparison of the effects of UA and sorafenib on cells by adding z-vad-fmk apoptosis inhibitor:\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Groups are: HepG2, HepG2\u0026thinsp;+\u0026thinsp;0, 20, 40, 80\u0026micro;M z-vad-fmk apoptosis inhibitor.\u003c/h2\u003e \u003cp\u003eCCK8 experiment: HepG2 cells added with UA: the pancreatin digests the logarithmic-phase cells, which are processed and put in the incubator for being cultured (monolayer cells fully cover the bottom of the pore (96-pore flat plate)) over night under conditions of 5%CO2 and 37℃. It is then observed under an inverted microscope. HepG2 are divided into 5 groups. Each group is added with: 0\u0026micro;M, 20\u0026micro;M, 40\u0026micro;M, and 80\u0026micro;M z-vad-fmk apoptosis inhibitor respectively, and cultured for 12h, 24h, 48h and 72h. After that, each pore is added with 10ul CCK8 and continued the culture for another 4 hours. The absorbance of each pore is measured at OD450nm of the enzyme-linked immunosorbent assay. At the same time, blank pores (substratum, CCK8) are set as well. (Cell viability = (experimental pores - blank pores)/ (control pores - blank pores)).\u003c/p\u003e \u003cp\u003e3.1.2 Groups are: LO-2, HepG2, HepG2\u0026thinsp;+\u0026thinsp;UA (40\u0026micro;mol/L, cultured for 24h), HepG2\u0026thinsp;+\u0026thinsp;optimal concentration of z-vad-fmk apoptosis inhibitor, HepG2\u0026thinsp;+\u0026thinsp;sorafenib (10\u0026micro;mol/L, cultured for 24h), HepG2\u0026thinsp;+\u0026thinsp;UA (40\u0026micro;mol/L, cultured for 24h)\u0026thinsp;+\u0026thinsp;optimal concentration of z-vad-fmk apoptosis inhibitor, HepG2\u0026thinsp;+\u0026thinsp;sorafenib (10\u0026micro;mol/L, cultured for 24h)\u0026thinsp;+\u0026thinsp;optimal concentration of z-vad-fmk apoptosis inhibitor.\u003c/p\u003e \u003cp\u003eCCK8 experiment: Cells are LO-2 and HepG2. The pancreatin digests the two groups of logarithmic-phase cells, which are processed and put in incubator for being cultured (monolayer cells fully cover the bottom of the pores (96-pore flat plate)), overnight under conditions 5%CO2 and 37℃, and observed under an inverted microscope. HepG2 cells are divided into 6 groups as mentioned above. Each pore is added with 10ul CCK8 and continued the culture for another 4 hours. The absorbance of each pore is measured at OD450nm of the enzyme-linked immunosorbent assay. At the same time, blank pore (substratum, CCK8) are set. (Cell viability = (experimental pores - blank pores)/ (control pores - blank pores)).\u003c/p\u003e \u003cp\u003eCell apoptosis: Cells are centrifuged and collected, and then performed with centrifugal washing by cold PBS. A total of 1\u0026ndash;10*10^5 cells are collected and diluted with double diluted water to take 500ul 1*Binding Buffer suspended cells. Then 5ulAnnexin V-FITC and 10ulPI are added as well for to conduct cytometry analysis.\u003c/p\u003e \u003cp\u003eWestern blot experiment: LO-2 and HepG2 cells are inoculated in 6-pore plates, and HepG2 cells are divided into 6 groups as mentioned above. Then, 600\u0026micro;l RIPA cell lysate is added for lysis and extraction of total tissue protein. SDS-PAGE gel preparation is performed according to the TRAKRA product catalog, concentrate gel (2ml) 10% and separate it (10ml). 5X SDS-PAGE protein sample loading buffer is added to the collected protein samples by 1:4 proportion. The filter paper and PVDF membrane are immersed in the membrane rotating buffer for 5min, and then performed with constant flow trarsmembrane through the membrane rotating device. After that, the protein membrane is rinsed in Western washing solution for 5 min, and then added with Western blocking solution (5% skim milk powder) for 2 hours. Then the filter paper and PVDF membrane are immersed in the membrane transfer buffer for 5min, and performed with the constant flow trarsmembrane through the membrane transfer device. After finishing the membrane transfer, the protein membrane is rinsed in Western washing solution for 5 min, and then put in Western blocking solution(5% skim milk powder) for 2 hours. After that, the samples are diluted with primary antibody diluent at 4℃ overnight in an appropriate proportion(see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and reacted with the secondary antibody(1:2000 dilution) marked by horseradish peroxidase for 1.2h. Finally the proteins are detected by ECL luminescence kit according to relevant instructions.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimary antibody dilution ratios\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDilution Ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecies And Genus\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaspase-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1: 2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaspase-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1: 2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaspase-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1: 1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaspase-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1: 2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e3.2 Comparison of the effects of UA and sorafenib on cells through the addition of ROS inhibitor NAC:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Groups are: HepG2, HepG2\u0026thinsp;+\u0026thinsp;0, 2.5, 5.0, 7.5\u0026micro;M ROS inhibitor NAC.\u003c/h2\u003e \u003cp\u003eCCK8 experiment: The process is the same as that of CCK8 experiment in 2.1.\u003c/p\u003e \u003cp\u003e3.2.2 Groups are: LO-2, HepG2, HepG2\u0026thinsp;+\u0026thinsp;UA(40\u0026micro;mol/L, cultured for 24h), HepG2\u0026thinsp;+\u0026thinsp;optimal concentration of ROS inhibitor NAC, HepG2\u0026thinsp;+\u0026thinsp;sorafenib (10\u0026micro;mol/L, cultured for 24h), HepG2\u0026thinsp;+\u0026thinsp;UA(40\u0026micro;mol/L, cultured for 24h)\u0026thinsp;+\u0026thinsp;optimal concentration of ROS inhibitor NAC, HepG2\u0026thinsp;+\u0026thinsp;sorafenib(10\u0026micro;mol/L, cultured for 24h)\u0026thinsp;+\u0026thinsp;optimal concentration of ROS inhibitor NAC.\u003c/p\u003e \u003cp\u003eMitochondrial membrane potential detection(JC-1): Cells of each group are collected, and washed twice with PBS. Part of the cells taken to make them resuspended in 0.5ml substratum by 500ul JC-1 staining working solution. The cells are cultured in incubator with conditions of 37℃ and 5% CO2 for 20min, and then collected based on centrifugation at 600g for 4min. After that, the JC-1 staining buffer solution is taken to wash cells twice, and then proper amount of JC-1 staining buffer is used again to make the cells resuspended for conducting flow cytometry.\u003c/p\u003e \u003cp\u003eROS: Cells: HepG2 cells are divided into 5 groups, which are HepG2, 0, 20, 40, and 80\u0026micro;M UA. The cells in each of the above groups are collected to regulate the cell concentration to (1\u0026ndash;10)x10^6/ml. Then they are add with 10umol/L DCFH-DA, cultured for 20min under 37℃ condition, and mixed every 3-5min to make it fully act. Meanwhile the non-stained cells are set as control. After that, the cells are washed twice with PBS to conduct flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Comparison of the effects of UA and sorafenib on cells by adding z-vad-fmk apoptosis inhibitor:\u003c/h2\u003e \u003cp\u003eIt can be known by CCK8 experiment that: the cell viability enhances gradually with the concentration increase of z-vad-fmk apoptosis inhibitor, and becomes basically stable when the concentration reaches 80\u0026micro;M (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The lowest inhibition effect of cell viability appears when the cells are cultured for 24h \u003cem\u003e(P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). UA (40\u0026micro;M, cultured for 24h) has a greater inhibitory effect on the viability of liver cancer cells than that of the sorafenib (10\u0026micro;mol/L, cultured for 24h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). From cell apoptosis experiment, it can be observed that the intracellular fluorescence intensity of Group C is significantly greater than that of Group E under the fluorescence microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). From Western blot experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), it can be observed that when UA is added to HepG2(40\u0026micro;M, cultured for 24h), the relative protein expression in HepG2 is almost the same as that of the status of adding sorafenib to HepG2(10\u0026micro;mol/L, cultured for 24h) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e4.2 Comparison of the effects of UA and sorafenib on cells through the addition of ROS inhibitor NAC:\u003c/p\u003e \u003cp\u003eAccording to the results of CCK8 experiment, the cell viability enhances gradually with the concentration increase of ROS inhibitor NAC. And the inhibition effect of drug on cell viability becomes basically stable it reaches 7.5\u0026micro;M (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The lowest inhibition effect on cell viability appears when the cells are cultured for 48h (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). It can be observed from mitochondrial membrane potential detection (JC-1) experiment that, under the fluorescence microscope, the intracellular fluorescence intensity of Group C is significantly weaker than that of Group E (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). It can be known from ROS experiment that: When the UA(40 \u0026micro;M, cultured for 24 hours) is added to HepG2, the DCF fluorescence is lower than that of adding sorafenib (10\u0026micro;mol/L, cultured for 24h) to HepG2. This means that, for HepG2, the reactive oxygen species at the time when adding UA(40 \u0026micro;M, cultured for 24 hours) is lower than that adding sorafenib (10\u0026micro;mol/L,cultured for 24 hours). And this further proves that UA has a stronger effect on HepG2 than sorafenib (10\u0026micro;mol/L, cultured for 24h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5. Discussion","content":"\u003cp\u003eResearch has shown that UA is present in many medicinal plants and has low toxicity. It has a wide range of pharmacological effects, including regulating immunity, protecting the liver, anti-tumor effects, etc. It can promote cell apoptosis, reverse tumor drug resistance, inhibit tumor cell proliferation, and increase the efficacy of chemotherapy in cancer treatment(Huang X et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). For liver cancer, studies by Liu T and Yee Y(Liu T et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e;Yie Y et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) have shown that UA can inhibit the growth of liver cancer cells through the Stat3 pathway or by reducing AMPK α - mediated DNA methyltransferase 1. In the Yin Jie Fan8 (Fan YJ et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) study, it was shown that UA can significantly reduce liver enzyme indicators in liver cancer mouse models, inhibit the invasion and migration of liver cancer cells, and also reduce the resistance of liver cancer cells to sorafenib.\u003c/p\u003e \u003cp\u003eThis study mainly demonstrated the inhibitory effect of UA on proliferation and promoting apoptosis of liver cancer cells by adding z-vad-fmk inhibitor and ROS inhibitor NAC, and using sorafenib as a positive control group. After adding the inhibitor, it can be seen that UA has a greater effect on liver cancer cells compared to sorafenib, and is more capable of inhibiting liver cancer cells.\u003c/p\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003eIn summary, UA has a good inhibitory effect on proliferation and promotes apoptosis of liver cancer cells, providing a new approach for the treatment and new drug research of liver cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors were involved in the conceptualization and design of the study.\u0026nbsp;Mei Shi\u0026nbsp;provided experimental ideas. Meng Xu completed the first draft of the manuscript.\u0026nbsp;Jinyu Wu, Zhimin Huang, Tingna Fu and Aitao Lin\u0026nbsp;performed the material preparation, data collection and analysis. Guoliang Zhang was mainly responsible for reviewing the final manuscript. All authors participated in the editorial revision of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by “2021 Anhui Province Major Difficult Diseases Collaborative Research Project of Traditional Chinese and Western Medicine” and “National Famous Traditional Chinese Medicine Seed Player Financial Cultivation Project in Anhui Province (Zhang Guoliang)”.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data generated in this study are available upon request to the corresponding author.\u003c/p\u003e\n\n\u003cp\u003eCompeting interests The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors are aware of and have consented to publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXu R, Su Q, Liao X et al (2026) A retrospective study of preoperative HBsAg levels predicting recurrence after curative resection in patients with HBV-associated hepatocellular carcinoma. Med (Baltim) 105(2):e47065\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang P, Lin J, Su D (2025) Echinacoside as a Novel Ferroptosis Inducer in Hepatocellular Carcinoma: Mechanistic Insights from TP53/SLC7A11/GPX4 Pathway Modulation. Int J Mol Sci 27(1):411\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBokhtia RM, Gupta KB, Natalini A et al (2025) Biotin-Linked Ursolic Acid Conjugates as Selective Anticancer Agents and Target-Identification Tools for Cancer Therapy. Molecules 30(23):4588\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Chen D, Zhang K et al (2025) ZIKV infection causes placental inflammation through activating PANoptosis. J Virol 99(12):e0175925\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Wu L, Wang S et al (2025) The Anti-Tumor and Bortezomib-Sensitizing Effects of Apigenin in Multiple Myeloma. Curr Issues Mol Biol 47(9):717\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung EJ, Kim HJ, Shin SC et al (2025) Tetraarsenic Hexoxide Enhanced the Anticancer Effects of Artemisia annua L. Polyphenols by Inducing Autophagic Cell Death and Apoptosis in Oxalplatin-Resistant HCT116 Colorectal Cancer Cells. Int J Mol Sci 26(16):7661\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu M, Chen L, Wu J, Liu L, Shi M, Zhou H, Zhang G (2025) Mechanism of Hedyotis diffusa-Scutellaria barbata D. Don for treatment of primary liver cancer: analysis with network pharmacology, molecular docking and in vitro validation. Nan Fang Yi Ke Da Xue Xue Bao 45(1):80\u0026ndash;89 English, Chinese\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y (2020) Fan Lulu Study on the Enhancement of Sorafenib's Proliferation Inhibition on Hepatocellular Carcinoma Cells by BET Protein Inhibitor JQ1 [J]. J Anhui Med Univ 55(08):1185\u0026ndash;1188\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang X, Sun Y, Tong H, Zhu J, Pan J, Wen P, He W (2025) Ursolic acid sensitizes bladder cancer to gemcitabine chemotherapy by concurrently targeting PI3K/AKT and JNK pathways. Transl Androl Urol 14(10):2902\u0026ndash;2916\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu T, Ma H, Shi W, Duan J, Wang Y, Zhang C et al (2017) Inhibition of STAT3 signaling pathway by ursolic acid suppresses growth of hepatocellular carcinoma. Int J Oncol 51(2):555\u0026ndash;562\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYie Y, Zhao S, Tang Q, Zheng F, Wu J, Yang L et al (2015) Ursolic acid inhibited growth of hepatocellular carcinoma HepG2 cells through AMPKα-mediated reduction of DNA methyltransferase 1. Mol Cell Biochem 402(1\u0026ndash;2):63\u0026ndash;74\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan YJ, Pan FZ, Cui ZG, Zheng HC (2024) The Antitumor and Sorafenib-resistant Reversal Effects of Ursolic Acid on Hepatocellular Carcinoma via Targeting ING5. Int J Biol Sci 20(11):4190\u0026ndash;4208\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAuthors and Affiliations\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng Xu1 Jinyu Wu\u003csup\u003e2\u003c/sup\u003e; Zhimin Huang\u003csup\u003e2\u003c/sup\u003e; Mei Shi\u003csup\u003e3\u003c/sup\u003e; Tingna Fu\u003csup\u003e1\u003c/sup\u003e; Aitao Lin\u003csup\u003e2\u003c/sup\u003e; Guoliang Zhang\u003csup\u003e3\u003c/sup\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorresponding Author\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuoliang Zhang [email protected]\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu J [email protected]. \u003csup\u003e1\u003c/sup\u003eGraduate School of Guangxi University of Traditional Chinese Medicine, No.13 Wuhe Avenue, Qingxiu District, 530000 Nanning, Guangxi, China \u003csup\u003e2\u003c/sup\u003eThe First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine, No. 89\u0026thinsp;\u0026ndash;\u0026thinsp;9 Dongge Road, 530000 Nanning, Guangxi, China \u003csup\u003e3\u003c/sup\u003eThe First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, NO. 117 Meishan Road, 230031 Hefei, Anhui, China\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cytotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cyto","sideBox":"Learn more about [Cytotechnology](http://link.springer.com/journal/10616)","snPcode":"10616","submissionUrl":"https://submission.nature.com/new-submission/10616/3","title":"Cytotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ursolic acid, Hepatocellular carcinoma cells, Cell proliferation, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-8619071/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8619071/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To investigate the effect of Ursolic acid (UA) on liver cancer cells through cell experiments, in order to inhibit cell proliferation and induce apoptosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod:\u003c/strong\u003e By adding z-vad-fmk apoptosis inhibitor and ROS inhibitor NAC, CCK8, cell apoptosis, Western blot experiments, mitochondrial membrane potential detection, and ROS flow cytometry were conducted. Compared with the positive drug sorafenib, the inhibitory effect of UA on liver cancer cells and its promotion of apoptosis were studied. The results of CCK8 confirmed that when UA was at its optimal concentration, its inhibitory effect on liver cancer cell viability was greater than that of sorafenib; Through cell apoptosis experiments, it can be observed that under fluorescence microscopy, the fluorescence intensity in the UA+HepG2 group is significantly stronger than that in the sorafenib+HepG2 group; Western blot experiments showed that the relative expression levels of proteins in HepG2 were almost equal between the HepG2+UA and sorafenib+HepG2 groups; through ROS experiments, it was found that when the reactive oxygen species in the HepG2+UA group were lower than those in the sorafenib+HepG2 group, it indicated that the former had a stronger effect of UA on HepG2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e UA has a significant inhibitory effect on proliferation and promotes apoptosis in liver cancer cells.\u003c/p\u003e","manuscriptTitle":"Study on the effect of Ursolic acid on liver cancer cells using z-vad-fmk and ROS inhibitors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-19 12:33:02","doi":"10.21203/rs.3.rs-8619071/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-26T13:38:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T11:29:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207452950744820839822837119804593128388","date":"2026-02-16T16:33:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-16T13:00:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-17T14:04:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-17T14:03:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cytotechnology","date":"2026-01-16T12:37:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cytotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cyto","sideBox":"Learn more about [Cytotechnology](http://link.springer.com/journal/10616)","snPcode":"10616","submissionUrl":"https://submission.nature.com/new-submission/10616/3","title":"Cytotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"21ba003c-ecdf-4412-a828-fad2955727a3","owner":[],"postedDate":"February 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:02:45+00:00","versionOfRecord":{"articleIdentity":"rs-8619071","link":"https://doi.org/10.1007/s10616-026-00926-8","journal":{"identity":"cytotechnology","isVorOnly":false,"title":"Cytotechnology"},"publishedOn":"2026-03-18 15:59:17","publishedOnDateReadable":"March 18th, 2026"},"versionCreatedAt":"2026-02-19 12:33:02","video":"","vorDoi":"10.1007/s10616-026-00926-8","vorDoiUrl":"https://doi.org/10.1007/s10616-026-00926-8","workflowStages":[]},"version":"v1","identity":"rs-8619071","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8619071","identity":"rs-8619071","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}