
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39300121
72536
10.1038/s41598-024-72536-3
Article
Oleanolic acid improves 5-fluorouracil-induced intestinal damage and inflammation by alleviating intestinal senescence
Bai Shi-rui 1
Zhao Bing-xiang 1
Zhao Qi 1
Ge Yu-chen 1
Li Man 1
Zhao Cheng-gang 2
Wu Xiao-jian 494179474@qq.com

2
Wang Xiao-bo wxb4320062@163.com

1
1 https://ror.org/02y7rck89 grid.440682.c 0000 0001 1866 919X School of Basic Medicine, Dali University, Dali, 671000 Yunnan China
2 https://ror.org/020rkr389 grid.452720.6 0000 0004 0415 7259 Microbiology Research Institute, Guangxi Academy of Agricultural Science, Nanning, 530007 Guangxi Province China
19 9 2024
19 9 2024
2024
14 2185222 5 2024
9 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
5-Fluorouracil (5-FU) is used as a standard first-line drug for colorectal cancer malignancy (CRC), but it brings a series of side effects such as severe diarrhea and intestinal damage. Our previous study found that a large number of senescent cells increased while 5-Fu induced intestinal damage, and anti-senescence drugs can alleviate its side effects of inflammatory damage. Oleanolic acid (OA) is a common pentacyclic triterpenoid mainly derived from food fungi and medicinal plants, and studies have shown that it mainly possesses hepatoprotective, enzyme-lowering, anti-inflammatory, and anti-tumor effects. But its role in senescence is still unclear. In the present study, we demonstrated for the first time that OA ameliorated 5-Fu-induced human umbilical vein endothelial cells (HUVECs) and human normal intestinal epithelial cells (NCM460) in a 5-Fu-induced cellular senescence model by decreasing the activity of SA-β-gal-positive cells, and the expression of senescence-associated proteins (p16), senescence-associated genes (p53 and p21), and senescence-associated secretory phenotypes (SASPs: IL-1β, IL-6, IL-8, IFN-γ and TNF-α). Meanwhile, in this study, in a BALB/c mouse model, we demonstrated that 5-FU induced intestinal inflammatory response and injury, which was also found to be closely related to the increase of senescent cells, and that OA treatment was effective in ameliorating these adverse phenomena. Furthermore, our in vivo and in vitro studies showed that OA could alleviate senescence by inhibiting mTOR. In colon cancer cell models, OA also enhanced the ability of 5-FU to kill HCT116 cells and SW480 cells. Overall, this study demonstrates for the first time the potential role of OA in counteracting the side effects of 5-FU chemotherapy, providing a new option for the treatment of colorectal cancer to progressively achieve the goal of high efficacy and low toxicity of chemotherapy.

Keywords

Oleanolic acid
5-Fluorouracil
Intestinal damage
mTOR
Foodborne fungus
Subject terms

Cancer
Cell biology
Diseases
Gastroenterology
Medical research
Pathogenesis
Guangxi Science and Technology ProgramGuiKe AB23075138 Zhao Cheng-gang Guangxi Special Crop Experimental StationTS202223 Wu Xiao-jian Special Project of Basic Scientific Research Business of Guangxi Academy of Agricultural SciencesGuinongke 2024YP080 Wang Xiao-bo issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

The treatment of advanced colorectal cancer (CRC) has been developing rapidly in recent years1. Although the introduction of new targeted drugs and immune drugs has brought more treatment options for patients, chemotherapy is still the cornerstone of advanced bowel cancer treatment2. As the core chemotherapeutic drug in the treatment of bowel cancer, fluorouracil has been recommended by authoritative domestic diagnostic and therapeutic guidelines and is widely used in various stages of bowel cancer treatment3–5. From the perspective of administration route, traditional 5-FU cannot be administered orally, and the continuous intravenous drip is not convenient for clinical use6. From the perspective of common adverse reactions, the adverse reactions of 5-FU mainly include gastrointestinal adverse reactions, hematologic toxicity, and so on5, and most of the patients will eventually suffer from abdominal pain, diarrhea, and other adverse reactions, and in severe cases, there will be blood in the stool. This seriously affects the continued development of current chemotherapeutic agents, so it is urgent to find a safe and effective drug to resist 5-FU chemotherapeutic injury.

Chemotherapy occupies an important position in the comprehensive treatment of CRC7, but the serious side effects of chemotherapeutic drugs, such as senescence and inflammatory damage, should not be ignored. It has been reported that the most important side effect of oxaliplatin is neurotoxicity8; irinotecan causes diarrhea in some patients9; and severe diarrhea and intestinal inflammation have been observed in patients treated with 5-FU chemotherapy10. On the other hand, we have learned that patients get the nutrients, such as sugars and lipids, needed for cancer cell growth from their diet. CRC is the most common cancer that originates in the intestine, and recent research suggests that CRC also directly transports and metabolizes dietary fructose and that ingestion of moderate amounts of fructose promotes CRC growth by enhancing the expression of hypoxia-responsive genes11. Thus, the relevant components of the diet also influence the progression of CRC to some extent. In our previous studies, we found that 5-FU induces normal cellular senescence and induces intestinal toxicity in clinical treatment12; artesunate inhibits irinotecan-induced intestinal damage and is also closely related to its intestinal senescence13. Chemotherapeutic agents almost always induce DNA damage, causing irreversible growth arrest along with cellular senescence. Based on our previous study, it is of great significance to find a food-based drug ingredient with natural composition, safety, and efficacy to resist 5-FU-induced normal cellular senescence and its induced intestinal toxicity in clinical treatments, which is of great importance to prevent the side effects of post-chemotherapy for CRC and thus improve the cure rate of CRC, and may be the most important means to prolong patients' survival time and improve their quality of life. It may be the most important means to prolong the survival time and improve the quality of life of patients14.

Mechanistic Target Of Rapamycin (mTOR) is a serine/threonine kinase that is an important regulator of cell growth and proliferation15,16. Studies have shown that mTOR is an important component of the oncogenic PI3K/AKT/mTOR signaling pathway and plays an important role in different basic cellular processes such as protein synthesis, cell proliferation and survival, and senescence17. Studies have found that dysregulation of mTOR signaling pathways is closely related to a variety of diseases, including cancer, intestinal inflammation, senescence, etc18. Overactivated mTOR signaling will directly or indirectly induce cancer, metabolism, and senescence-related diseases19. Inhibition of this state can effectively delay or treat diseases caused by excessive activation of mTOR and provide beneficial health effects under different pathological conditions20. Therefore, mTOR is a potential target that can treat a variety of diseases. There are many causes of cellular senescence, and it is not clear how to age so far, but we know that reactive oxygen species, DNA damage, protein homeostasis imbalance, and inflammation are all related to cellular senescence, and mTOR is involved in regulating these factors. It has been found that reducing mTORC1 activity can significantly prolong the lifespan of Caenorhabditis elegans, which has revealed the role of mTOR in the process of cellular senescence21.

OA is present in various edible mushrooms, and the extract components are safe and effective and have been approved for adjunctive therapy in patients with acute or chronic hepatitis22. It has been endowed with a wide range of biological activities with therapeutic potential due to its complex and multifactorial mechanisms. Evidence suggests that OA may be effective against dyslipidemia and diabetes mellitus by enhancing insulin response, maintaining beta cell function and survival, and preventing diabetic complications. In addition, OA is capable of antiviral, anticancer, anti-inflammatory, hepatoprotective, gastroprotective, hypolipidemic, and anti-atherosclerotic activities23. As a food-borne active ingredient, OA has been used in the treatment of a variety of diseases with remarkable effects, but its resistance to intestinal damage and cellular senescence induced by the chemotherapeutic drug 5-FU has been less studied, and its mechanism of action in anti-senescence has not yet been reported.

Overall, the present study aimed to explore in depth the beneficial effects of OA in a 5-FU-induced model of cellular senescence and intestinal injury in mice and to evaluate its anti-senescence effects. To further explore the effects of the active ingredients of food-borne OA on cancer growth and anticancer therapy in the prognostic process of CRC treatment and to provide new therapeutic options for cancer chemotherapy.

Results

OA alleviates HUVEC cells and NCM460 cells senescence caused by 5-FU

Our previous study showed that 5-FU can cause cellular senescence12, so we utilized this cell model to test whether OA (Fig. 1A) can resist senescence. As shown in Fig. 1B,C, different concentrations of OA could reduce the percentage of SA-β-Gal positive cells and restore the cell morphology. Combined with CCK8 experiments, we initially concluded that the optimal concentration of OA for anti-senescence in HUVEC cells was 10 μM (Fig. 1D). Subsequently, we selected normal human colonic epithelial cells-NCM460 to test whether OA had the same anti-senescence effect. Different concentrations of 5-FU (0.1–10 μM) were added to this cell to induce senescence, and according to the changes in the proportion of SA-β-Gal-positive cells, 3 μM of 5-FU was selected for the subsequent related experiments (Fig. 1E,F). Combining SA-β-Gal staining experiments with 72 h CCK8 experiments, it was likewise observed that cellular senescence caused by 5-FU was alleviated by different concentrations of OA treatments, and the optimal concentration of OA against NCM460 senescence was determined to be 10 μM (Fig. 1G–I)24. We further verified the anti-senescence effect of OA using Western blot experiments as well as QPCR experiments, and the results showed that the p16 protein expression level was slightly elevated on day 1 after 5-FU treatment, but there was no significant change compared with OA co-treatment. After the 3rd day of treatment, we found that the p16 protein level was significantly increased and OA treatment significantly reduced p16 expression (Fig. 1J,K); RT-qPCR assay at the same time point revealed that OA was able to reduce the expression of the P21 gene (Fig. 1L). Our results confirm that 5-FU treatment leads to significant cellular senescence, while OA treatment can effectively alleviate this phenomenon and resist the cellular senescence brought about by 5-FU.Fig. 1 OA alleviates HUVEC cells and NCM460 cells senescence caused by 5-FU. (A) The molecular formula of Oleanolic acid. (B, C) Representative images and statistical plots of the percentage of senescence-positive cells in HUVEC cells after treatment with 1 μM of 5-FU and different concentrations of OA (0–15 μM). Statistical graphs show the results of three replicated experiments. (D): HUVEC cells were treated with 1 μM of 5-FU and different concentrations of OA (0–15 μM). The CCK8 assay was used to determine cell viability. The statistics for the results of three replicate experiments are provided in the figure. (E) Representative photos of senescence-positive cells stained with SA-β-Gal in NCM460 cells treated with various concentrations of 5-FU (0.1–10 μM). (F): Statistical graph of the percentage of senescence-positive cells shown in Figure (E). The experiment was repeated three times. (G, H): Statistical graphs of senescence-positive cell images and their percentage of senescence-positive cells observed by SA-β-Gal staining in NCM460 cells after the addition of 5-FU (3 μM) to induce cellular senescence combined with different concentrations of OA (0–20 μM) treatment. The statistical graphs show the results of three replicated experiments. (I) Cell viability was detected in NCM460 cells after co-treatment with 5-FU (3 μM) as well as OA (0–20 μM) using the CCK8 assay, and the results of three replicate experiments are shown in the statistical graph. (J, K) Expression of p16 protein in cell models and statistical charts. (L) Expression of the p21 gene.

OA attenuates the inflammatory expression of NCM460 cells caused by 5-FU

The damage caused by 5-FU chemotherapy to the gastrointestinal tract is also not negligible. Our previous study confirmed that 5-FU treatment causes inflammation of intestinal cells, so we hypothesized that the side effects of 5-FU chemotherapy might also be related to the chemical inflammation of the gastrointestinal mucosa produced during its treatment. To confirm this hypothesis, we continued to conduct subsequent studies with NCM460 cells. First, we examined the expression levels of inflammation-related proteins p-p65, p-p38 and related total proteins in 5-FU and OA + 5-FU co-treated cells using Western blot experiments. Based on the results of the previous experiments, we chose to treat the cells with 5-FU at a concentration of 3 µM and OA at a concentration of 10 µM, and examined the protein samples of the cells on days 1 and 3. It was found that the expression of cellular inflammation-related proteins p-p65 and p-p38 increased after 5-FU treatment, whereas OA treatment was able to significantly reduce the level of inflammatory proteins, which effectively alleviated the phenomenon of cellular inflammation caused by 5-FU in a time-dependent manner (Fig. 2A–C). Similarly, we also performed RT-qPCR of inflammation-related genes (IL-1, IL-6, IL-8, IFN-γ, TNF-α) on cells at the same time point, and OA also significantly reduced the expression of inflammatory factors (Fig. 2D–H).Fig. 2 OA attenuates the inflammatory expression of NCM460 cells caused by 5-FU. (A) In NCM460 cells, Western blot was used to detect the expression of inflammation-related proteins p-p65 and p-p38 after treatments with 5-FU (3 μM) and OA (10 μM). (B, C) Statistical graphs of p-p65 and p-p38 protein expression, the graphs show the results of three experimental replicates. (D, F) Expression of inflammation-related factors (IL-1, IL-6, IL-8, IFN-γ, TNF-α) in NCM460 cells model and statistical graphs. The experiment was repeated three times.

OA alleviates the effects of intestinal damage brought on by 5-Fu in the BALB/c mice model

Based on our previous results in in vitro experiments, we next established a diarrhea model with BALB/c mice to evaluate the efficacy and safety of OA in mitigating intestinal damage caused by 5-Fu. Our experiments were divided into three groups: the control group, the 5-FU treatment group, and the OA combination therapy group (Fig. 3A). Compared with the control group, the body weight of mice in the 40 mg/kg 5-Fu treatment group decreased from day 4 and was lower than that in the control group, while the body weight of mice in the 5-FU treatment group was significantly increased compared with the 5-FU treatment group (Fig. 3B)25. As shown in Fig. 3C–D, the intake and water intake of the 5-FU treatment group were significantly reduced, and OA treatment improved this phenomenon, which confirmed that OA can alleviate the intestinal toxicity caused by 5-FU. We also monitored the diarrhea of mice in all groups and found that OA significantly improved the diarrhea caused by 5-FU (Fig. 3E). After the experiment, we dissected the ileal and colon tissues of mice for follow-up-related studies. We found that the colon length of mice after 5-FU treatment was significantly shorter than that of the control group, and the phenomenon of colon surface inflammation was obvious, while OA restored these phenomena (Fig. 3F). Subsequently, we froze sections of some tissues and analyzed them histologically using HE staining. The results showed that after 5-FU treatment, the crypts of colon tissue rupture and tissue vacuoles appeared in mice, the depth of the crypts increased, and there was an obvious accumulation of inflammatory factors. Notably, after OA combination therapy, intestinal damage was significantly reduced and the structural integrity of the intestine was effectively restored (Fig. 3G–H). Subsequently, we measured the expression levels of inflammation-associated proteins p-p38 and p-p65 in intestinal tissue, and the expression of these proteins was upregulated by 5-FU treatment, and the combination therapy with OA was effective in reducing these expressions (Fig. 3I–J)26,27. At the same time, RT-qPCR was used to detect the pro-inflammatory factors IL-1β, IL6, IL-8, IFN-γ and TNF-α, and the results showed that OA treatment in mouse colon tissues also effectively reduced the expression of these inflammatory factors (Fig. 3K–L)28. The results of our in vitro and in vivo experiments collectively elucidate the significant protective effect of OA against 5-FU-induced intestinal injury.Fig. 3 OA alleviates the effects of intestinal damage brought on by 5-Fu in the BALB/c mice model. (A) Diagram of animal experimental program in BALB/c mouse model. (B–E) Body weight records, food and water intake records, diarrhea scoring index records (scoring criteria as shown in Table 1) (F) Representative images of colon length in experimental mice. (G, H) Summary images of H&E staining results of frozen sections of ileum and colon of experimental mice. (I, J) Expression of inflammation-related proteins p-p65 and p-p38 detected by Western blot experiment in colon tissues of experimental mice. The experiment was repeated three times and analyzed for statistical significance. (K) RT-qPCR assay to detect the expression of pro-inflammatory factors IL-1β, IL6 IL-8, IFN-γ and TNF-α in colon tissues of experimental mice.

Table 1 Diarrhea scoring criteria.

0score	No diarrhea, normal stools	
1score	Mild diarrhea, soft stools	
2score	Moderate diarrhea with thin, unformed stools	
3score	Severe diarrhea, watery stools	

In the BALB/c mouse model, OA was able to alleviate intestinal senescence caused by 5-Fu

Our previous research has confirmed that 5-FU causes intestinal senescence, producing a cascade of intestinal inflammation. So we evaluated the mitigating effect of OA on intestinal senescence in a mouse model. After freezing sections of ileum and colon tissue from experimental mice, staining with the SA-β-gal kit revealed that 5-FU resulted in blue staining of the colon and ileum of mice, while OA combination therapy significantly improved intestinal senescence with little blue deposition in tissue sections (Fig. 4A,B). In addition, the Western blot results of animal tissues showed that the expression level of p16 protein under the action of 5-FU was significantly higher than that in the control group, while these indicators were down-regulated in the OA combination therapy group (Fig. 4C,D). At the same time, OA co-treatment in animal models effectively reduced the RNA expression of the senescence-related genes p53 and p21 (Fig. 4E,F). Based on these results, our study confirms the significant alleviating effect of OA on intestinal senescence in vivo, which further demonstrates the potential properties of OA in anti-senescence.Fig. 4 In the BALB/c mouse model, OA was able to alleviate intestinal senescence caused by 5-Fu. (A, B) Frozen sections of mouse ileum and colon tissues were stained with SA-β-gal showing the results as shown. (C, D) Western blot experiment to detect the expression of the senescence-related protein p16 in mouse colon tissue. The results of three replicate experiments were statistically significant. (E, F) RNA expression levels of p53 and p21 in the colon tissues of experimental mice. The results of three replicate experiments were statistically significant.

In vivo and in vitro models, OA resists 5-FU-induced senescence by inhibiting mTOR

The mTOR signaling pathway is important in senescence-related diseases, and there is growing evidence that it strongly affects longevity and senescence. Based on this, in order to further confirm the relevant mechanism of OA anti-senescence, we separately measured the expression of phosphorylated mTOR proteins in in vitro and in vivo experiments. Based on previous literature research, we know that OA plays a larger role in a short period of time. So we performed Western blot detection on NCM460 cells after cell collection at 4/6/8 h. It was found that 5-FU activated the expression of phosphorylated mTOR while OA inhibited its expression and was time-dependent (Fig. 5A–C). An mTOR activation experiment was subsequently designed to verify this. MHY1485, a potent, cell-permeable mTOR agonist, was selected based on references. In the NCM460 cell senescence model, MHY1485 (3 μM) was added in combination, and through SA-β-Gal staining, we found that OA effectively inhibited mTOR, while its inhibitory effect was weakened by adding MHY1485 (Fig. 5G,H). We simultaneously phosphorylated mTOR in colonic tissue samples (Fig. 5D–F). Consistent results at the cellular level were obtained. The above results show that OA in vivo and in vitro resists 5-FU-induced senescence by inhibiting mTOR.Fig. 5 In vivo and in vitro models, OA resists 5-FU-induced senescence by inhibiting mTOR. (A) Western blot detection of p-mTOR, p-AMPK activation in cell samples at the 4th, 6th, and 8th hour after treatment with 5-FU (3 μM) and OA (10 μM) added to NCM460 cells. (B, C) Three replicates were performed with statistically significant results. (D) Western blot detection of p-mTOR expression in mouse colon tissue. (E, F) Statistical graphs of p-mTOR protein expression, and the experiments were repeated three times and were statistically significant. (G) Images of SA-β-Gal staining of cells treated with 5-FU (3 μM), OA (10 μM), and MHY (3 μM) were added in the NCM460 cells model. (H) Statistical graph of the percentage of senescence-positive cells in Figure (G). Three replicated experiments were statistically significant.

OA enhanced the antitumor activity of 5-FU against HCT116 cells as well as SW480 cells

To further evaluate OA for the suitability of combination 5-FU for CRC We assessed whether the combination with OA affects the anti-tumor effect of 5-FU. We first used the CCK8 method to treat HCT116 cells and SW480 cells separately with different concentrations of 5-FU and OA for 72H, and then measured the viability of cells under each condition (Fig. 6A,E). We found that different concentrations of 5-FU could effectively inhibit cell viability and kill tumor cells in a concentration-dependent manner, and OA also showed a killing effect on tumor cells at a certain concentration. Subsequently, we selected 5-FU at concentrations of 5 μM and 7 μM in combination with OA at a concentration of 10 μM for HCT116 cells and SW480 cells, respectively, and through cell viability assays, we found that the number of tumor cells surviving under these conditions became smaller. This indicates that OA can enhance the anti-tumor activity of 5-FU against HCT116 cells and SW480 cells and also suggests the potential anti-tumor effect of OA. Subsequently, we performed cell cloning experiments on these two tumor cells and stained the tumor cells with different concentrations of 5-FU combined with OA for crystal violet. As shown in the figure (Fig. 6C,D,G,H), 5 μM/7 μM 5-FU has had an impact on colony formation, and OA combined treatment further reduces the formation of cancer cell colonies. The results showed that OA could enhance the killing effect of 5-FU on CRC cells.Fig. 6 OA enhanced the antitumor activity of 5-FU against HCT116 cells as well as SW480 cells. (A) Cell viability of HCT116 cells under each condition was detected by the CCK8 method after treating HCT116 cells individually with different concentrations of 5-FU (1–15 μM) and OA (1–25 μM) for 72H, respectively. Three replicates were performed with statistically significant results. (B) For HCT116 cells treated with different concentrations of 5-FU (3/5/7/10 μM) and OA (10 μM) alone and in combination for 72 h of the two, cell viability was determined using the CCK8 assay. Three replicates were performed with statistically significant results. (C, D) Representative images of crystal violet staining experiments and statistical graphs of the percentage of clonal cells in HCT116 cells treated with 5-FU (5/7 μM) and 0A (10 μM). Three replicates were performed with statistically significant results. (E–H) The same experiment was performed in SW480 cells and the results are shown. All experiments were repeated three times and were statistically significant.

Discussion

In this study, we first demonstrated that 5-FU effectively induced senescence in HUVECs and NCM460 cell lines by establishing a cellular senescence model, and we discovered the potential properties of OA in anti-senescence. Furthermore, we found that 5-FU induced cellular senescence through activation of the mTOR signaling pathway, whereas OA treatment significantly downregulated the expression of phosphorylated mTOR, which counteracted the pro-senescence effect of 5-FU during chemotherapy. Meanwhile, we found that 5-FU inhibited tumor cell growth in HCT116 and SW480 cells, and the combination of 5-FU and OA enhanced its anti-tumor effect. 5-Fu-induced intestinal injury caused intestinal inflammation and the accumulation of intestinal epithelial senescent cells, and intestinal inflammation-related factors and senescence-related genes were significantly increased. The anti-senescence drug OA in this study could alleviate 5-Fu-induced intestinal injury and senescence by reducing the expression of these inflammatory factors and SASP. We also found significant anti-senescence effects of OA in our in vitro experiments. Our study provides new solutions and ideas for the limited use of 5-FU chemotherapy in clinical treatment, provides a theoretical basis and new treatment options for the clinical alleviation of more chemotherapy-induced side effects, and provides an experimental basis for the development of novel anti-senescence drugs.

Based on current research on commonly used chemotherapies for CRC, we have learned that dietary interventions during chemotherapy can reduce specific nutrients required for cancer metabolism and also enhance its efficacy by depriving cancer of escaping nutrients and signals29. For example, food-related compounds can limit the ability of cancer cells to repair DNA damage, thereby enhancing the efficacy of chemotherapy. In the current study, the food compound OA showed significant anti-inflammatory effects in several diseases. These results provide an additional theoretical basis for the treatment of major clinical diseases with OA. In the present study, OA significantly reversed the inflammatory manifestations such as diarrhea induced by 5-FU treatment in vivo, as well as intestinal damage in terms of the colon and ileum. Thus, the problem of serious side effects caused by the use of 5-FU in clinical chemotherapy can be more easily solved, and a new protocol strategy and theoretical basis for the subsequent cancer treatment can be provided.

So how does OA attenuate 5-FU-induced senescence? Our study found that 5-FU-induced cellular senescence and intestinal damage are closely linked to the mTOR signaling pathway. mTOR is considered a key regulator of cell growth, proliferation, survival, and autophagy30, interacting with multiple proteins to form distinct complexes that regulate fundamental processes in the cell. Dysregulation of its activity is associated with a wide range of diseases and disorders, and activation of mTOR is detrimental to a long and healthy life. While we found that OA was able to attenuate 5-FU-induced cellular senescence and intestinal damage by inhibiting mTOR activation, we simultaneously added mTOR activators at the cellular level to further validate this phenomenon and obtained results consistent with the previous ones. We demonstrated that this could be a potential mechanism related to anti-senescence in OA, and we will continue to explore this phenomenon in future studies.

In summary, our results indicate that food-grade OA attenuated 5-FU-induced intestinal injury while alleviating intestinal inflammation and intestinal senescence. These results demonstrate the development of OA as a food-grade bacterial functional agent for ameliorating and preventing intestinal damage induced by cancer chemotherapy, effectively protecting the patient's intestinal tract, and enhancing the efficacy of cancer chemotherapy. It also provides a theoretical basis for dietary intervention to improve cancer therapy and prognosis. At present, there are still some limitations in our research on OA and alleviating intestinal injury during 5-FU chemotherapy. In fact, the functional roles of OA are complex and diverse, and they play different roles in different diseases. We will conduct relevant studies in the future to further explore the mechanism of the anti-senescence role of OA during chemotherapy.

Materials and methods

Instruments and materials

Tianjin Jinyao Pharmaceutical Co. provided the medication 5-fluorouracil (H12020959). Solarbio (Beijing, China) provided L-glutamine(G0200), penicillin–streptomycin liquid(P1400), while Selleck (Shanghai, China) provided Oleanolic acid(S2334) and MHY1485(S7811). The Cell Counting Kit-8 (Cat. No: PF00004) was sold by Sevier Biotechnology (Wuhan, China). Cell Signaling Technology (Danvers, MA) provided the p-p38 (Thr180/Tyr182), p38 (ET1602-26), p65(ET1603-12) phospho-mTOR (Ser2448) (Cat#5536), and p-p65 (S536) antibodies, while Abcam provided the anti-GAPDH antibody (Cat#ab181602). p16 (SR34-02) purchased from HUABIO (Hangzhou, China). Peroxidase-conjugated Affinipure goat anti-mouse IgG (H + L) (Cat#SA00001) and peroxidase-conjugated Affinipure goat anti-rabbit IgG (H + L) (GB23303) were available from Solarbio (Beijing, China).

Cell processing and culture

In Roswell Park Memorial Institute 1640 medium (RPMI-1640, PMI150110, Procell Wuhan, China), supplemented with 10% fetal bovine serum (FBS, C04001-050, xpbiomed), 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 2 mM/mL L-glutamine. HUVECs cells were brought from Shanghai Institutes for Biological Science of the Chinese Academy of Science and NCM460 cells were purchased from the Hunan Feng-hui Biotechnology Co., Ltd. The Dulbecco's Modified Eagle Medium (DMEM, PMI150210, Procell, Wuhan, China) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 g/mL streptomycin, and 2 mM/mL L-glutamine was used to cultivate the human colon cancer cell lines SW480 and HCT116. These cells were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Duchenne modified Eagle's medium (DMEM) was used to cultivate the cells. Cells were grown in an incubator at 37 °C with 5% stabilized CO2. Before each experiment, stock solutions of OA and 5-FU, storing them at − 20 °C. In this investigation, the maximum Dimethyl sulfoxide (DMSO, D8371, Solarbio, Beijing) concentration was 0.1% (all controls had a DMSO concentration of 0.1%).

We established a cellular senescence model by 5-FU induction of senescence in both HUVEC cells and NCM460 cells lines. Briefly, 5-FU (1 μM/3 μM) was given to the entire medium to treat and culture the cells when they had grown to 60–70% fusion, and changes in cell shape could be seen under a microscope.

Test for cell viability

Cell viability was assessed with the CCK8 technique. In a 96-well cell culture dish, we cultured four cell lines—HUVEC, NCM460, HCT116, and SW480—using appropriate whole media for 1000 cells and 100μL of medium per well, respectively. Cell viability was measured at 450 nm for 72 h using a cellular microtiter plate reader device after the cells had been cultured for 3 days with various dosage combination treatments. To this end, 10 μL of the CCK8 reagent and 90 μL of the working reagent of the corresponding medium were added to each well of the 96-well culture plate. This was done for 2 h at 37 °C. (Three independent replicates were carried out in each of the three replicate experimental wells that were set up for each concentration.) Different cells received individual or drug-combination treatments. The tests used OA (1–25 μM) and 5-FU (1–15 μM). The average and standard deviation from three separate experiments represent the results.

Western blot

Protein concentrations in NCM460 cells and animal tissue samples were found using the Western blotting method. Using RIPA buffer, total protein was extracted from tissues or cells. The BCA method was used to calculate the protein concentration (Cat#PC0020, Solarbio, Beijing, China). The lysate was added to 2.5xSDS loading buffer after boiling for 10 min. The proteins were loaded with the same quantity of protein and electrophoresed using 8–12% SDS–PAGE, depending on the molecular weight. Primary antibodies were treated with proteins overnight at 4 °C after being transferred to a PVDF membrane (Cat#1620177, BIO-RAD, USA). After being cleaned with tris buffered saline (TBS) containing 0.1% Tween-20, the strips were then incubated for an hour at room temperature on a shaker with the appropriate secondary antibody given to 5% skim milk at a concentration of 1:10,000. The PVDF membrane was cut horizontally to detect proteins of different molecular weights. Using a fusion-independent imsenescence system (Germany) and the Western Blot Plus Blot Reagent Kit (Bio-Rad, Hercules, CA), strip images were recorded using electrochemiluminescence detection. Band intensities were measured using ImageJ analysis software, all original WB images were shown in supplementary information.

Real-time QPCR

The 800 L TRIzol reagent (CW0580, Cwbio, China) was used to treat animal tissue samples and cultured cells in 6-well plates. Using an RT kit, total RNA was obtained, extracted, and reverse-transcribed into cDNA for real-time fluorescence quantitative PCR (Cat: G3337-50, Servicebio). The SYBR Green qPCR Master Mix (G3326-1, Servicebio) operating instructions was followed, and three duplicates of the real-time fluorescence quantitative PCR amplification were run. Using 18S ribosomal RNA sequences as a control, the comparative CT method was used to determine the relative amount of cDNA. In Table 2, gene-specific mouse and human PCR primers are listed. (All primers were obtained from Tsingke Biotech Co., Ltd.).Table 2 Primer sequences for quantitative RT-qPCR.

Primer names	Primer sequences	
h-18S	F-5′-TTGACGGAAGGGCACCACCAG-3′

R-5′-GCACCACCACCCACGGAATCG-3′

	
h-IL-6	F-5′-ACTCACCTCTTCAGAACGAATTG-3′

R-5′-CCATCTTTGGAAGGTTCAGGTTG-3′

	
h-P21	F-5′-GTGGCCTTGTCGCTGTCTT-3′

R-5′-GCGCTTGGAGTGATAGAAATCTG-3′

	
h-TNF-α	F-5′-CAAGATGATCTGAGTGTGAGGGT-3′

R-5′-GCAATACGGACTTGCTCACAGA-3′

	
h-IL-1β	F-5′-ATGATGGCTTATTACAGTGGCAA-3′

R-5′-GTCGGAGATTCGTAGCTGGA-3′

	
h-IL-8	F-5′-TTTTGCCAAGGAGTGCTAAAGA-3′

R-5′-AACCCTCTGCACCCAGTTTTC-3′

	
h-IFN-γ	F-5′-TCGGTAACTGACTTGAATGTCCA-3′

R-5′-TCGCTTCCCTGTTTTAGCTGC-3′

	
m-P21	F-5′-CCTGGTGATGTCCGACCTG-3′

R-5′-CCATGAGCGCATCGCAATC-3′

	
m-P53	F-5′-CCCCTGTCATCTTTTGTCCCT-3′

R-5′-AGCTGGCAGAATAGCTTATTGAG-3′

	
m-IL-1β	F-5′-GAAATGCCACCTTTTGACAGTG-3′

R-5′-TGGATGCTCTCATCAGGACAG-3′

	
m-IL-6	F-5′-CTGCAAGAGACTTCCATCCAG-3′

R-5′-AGTGGTATAGACAGGTCTGTTGG-3′

	
m-IL-8	F-5′-TCGAGACCATTTACTGCAACAG-3′

R-5′-CATTGCCGGTGGAAATTACTT-3′

	
m-IFN-γ	F-5′-ATGAACGCTACACACTGCATC-3′

R-5′-CCATCCTTTTGCCAGTTCCTC-3′

	
m-TNF-α	F-5′-CAGGCGGTGCCTATGTCTC-3′

R-5′-CGATCACCCCGAAGTTCAGTAG-3′

	

Cell clone formation experiment

Add different concentrations of 5-FU, OA or a combination of the two in the experimental group and continue to culture until 14 days or until the number of cells in the majority of individual clones was greater than 50, during which the liquid was changed every 3 days and the cell status was observed; after the completion of the cloning, the clones were washed once with PBS, and each well was fixed by adding 1 mL of 4% paraformaldehyde for 30–60 min, and washed once with PBS; and then the clones were stained with 0.1% crystal violet working solution for 10 min. Staining was performed for 10 min. Each monoclonal colony in each well was counted and evaluated for statistical significance using ImageJ software (version 1.8.0.172).

Model for cell senescence

Model for 5-FU-induced senescence of HUVEC cells: After HUVEC cells were grown in 24-well plates to a fusion level of 70–80%, 5-FU at a concentration of 1 μM was added for co-treatment. The cells were then cultured for 4 days at 37 °C in an incubator with 5% carbon dioxide before being stained with SA-β-Gal. The 5-FU treatment alone and the OA treatment were equivalent. Co-treatment involved adding OA (1–25 μM) and 5-FU (1 μM) to the culture media, then treating the cells continuously for 4 days. Staining with SA-β-Gal was done.

Model for 5-FU-induced NCM460 cells senescence: NCM460 cells were cultivated in 24-well plates until each well's area reached 65–75%. In an incubator set to 37 °C, 5% CO2, the cells were then continuously exposed to 3 μM 5-FU. SA-β-Gal staining was carried out till day 4. The 5-FU-alone treatment for OA was equivalent. The cells were continuously treated for 4 days with OA (1–25 μM) and 5-FU (3 μM) added to the culture medium for co-treatment. Staining with SA-β-Gal was done.

SA-β-GAL staining

Senescence-associated β-galactosidase (SA-β-Gal) activity was detected in HUVECs and NCM460 cells by using the SA-β-Gal staining kit (Cat# G1580, Sola bio, Beijing, China). 24-well plates were injected with the cells. After cells were exposed to various doses of OA, 5-FU, or a combination of the two for a predetermined amount of time, senescence-associated staining was carried out. The cells were rinsed once with TBS and the original media was discarded. Following the addition of the fixative solution, the cells were kept at room temperature for 15 min. Then comes, three rounds of three-minute TBS washes in ice-cold water were performed on the cells. The cells were finally imaged using an Olympus IX71 light microscope from Shanghai, China, and the blue-green coloration seen beneath the microscope helped identify the cells as senescent cells. To calculate the proportion of SA-β-gal positive cells to total cells in each group, 1000 cells were counted in seven randomly selected fields. The mean and standard deviation of three replicates are used to express the results. In order to evaluate intestinal tissue senescence, animal colonic and ileal tissues were cut into 5-μm-thick frozen slices after being treated with OCT embedding solution. The tissue sections underwent a single TBS wash after being brought back to room temperature, then 1 mL of section fixation solution is covered with tissue section surface and fixed at room temperature for at least 25 min. The pieces were then gently cleaned using TBS three times for a total of five minutes. The excess liquid was absorbed using filter paper to completely cover the tissue sections in stained working solution before the sections were overnight incubated at 37° C. Microscopic examination and photo documentation were done following a 48-h staining period.

Animal experiment

Male BALB/c mice that were 8 weeks old were utilized to create the intestinal damage model .We selected male BALB/C mice to establish the model of 5-fu-induced intestinal senescence in mice because the male mice had higher adult rate, better stability and reproducibility than the female mice. According to Dali University's Institutional Committee for Laboratory Animals standards, approval was acquired to perform this animal experimentation program (Number: 2022-P2-21, Dali, Yunnan, China). The experimental mice were kept in housing with the proper levels of humidity and temperature, and food and water were freely available to them. Twenty-four mice were randomly assigned to one of three groups—no treatment control, 5-FU monotherapy, or OA plus 5-FU combination therapy—each of which contained six mice. Without providing any treatment, the control group was monitored and recorded. In the administration group, 5-FU (40 mg/kg) was injected intraperitoneally a total of five times (every other day), While in the group receiving combination therapy, 5-FU (40 mg/kg) and OA (5 mg/kg) were administered simultaneously, injections every other day for a total of 5 injections. The experimental mice were observed and recorded on day 10 and sacrificed after cervical dislocation. Retain its ileal and colon tissues for subsequent experiments. Some tissues were stained with hematoxylin and eosin(H&E), and the kit was purchased from Solabron, Beijing (Cat# G1120) and senescence-related staining and examined for histopathology and morphometry; other tissues were left untreated and utilized for RNA and Western blot detection later.

H&E staining experiment

For the colon and ileum tissues obtained from animal experiments, the tissue samples were first treated with OCT embedding medium before being snap-frozen and subjected to frozen sectioning. Then fix the 5 μM sections with acetone for 15 min, followed by hematoxylin/eosin (H&E) staining, and seal the sections with neutral gum for preservation. The histopathologic changes were then observed under a microscope and documented using photographs. The nucleus’ chromatin and the cytoplasm's ribosomes appeared purple-blue when stained with hematoxylin; the extracellular matrix turned red when stained with eosin.

Analysis of experimental data

All experimental data in this research design was statistically analyzed using GraphPad Prism software (version 5.0). To compare the means of the two groups, the Student's t-test was performed, and ANOVA was utilized for multiple comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, P ≤ 0.05 were deemed statistically significant, whilst NS was deemed not statistically significant.

Ethics approval and consent to participate

This study and included experimental procedures were approved by the Dali University's Institutional Committee for Laboratory Animals standards, approval was acquired to perform this animal experimentation program (Number: 2022-P2-21, Dali, Yunnan, China). All animal housing and experiments were conducted in strict accordance with the institutional Guidelines for Care and Use of Laboratory Animals. Moreover, this study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org/).

Supplementary Information

Supplementary Figures.

Abbreviations

5-FU 5-Fluorouracil

OA Oleanolic acid

mTOR Mechanistic target of rapamycin

DMSO Dimethyl sulfoxide

HUVEC Human umbilical vein endothelial cell

NCM460 Normal intestinal epithelial cells

HCT116; SW480 Human colon cancer cells

H&E Hematoxylin-eosin

CCK8 Cell counting kit-8

IL1-β Interleukin-1β

IL-6 Interleukin-6

IL-8 Interleukin-8

TNF-α Tumor necrosis factor-alpha

CTRL Control

D Day

H Hour

MHY MHY1485

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72536-3.

Acknowledgements

This work was financially supported by the Guangxi Science and Technology Program (GuiKe AB23075138); Special Project of Basic Scientific Research Business of Guangxi Academy of Agricultural Sciences (Guinongke 2024YP080); Guangxi Special Crop Experimental Station (TS202223).

Author contributions

Shi-rui Bai: Conceptualization, Formal analysis, Writing—Original Draft, Validation. Bing-xiang Zhao: Data Curation. Qi Zhao: Software, Data Curation. Yu-chen Ge: Validation. Man Li: Investigation. Cheng-gang Zhao: Supervision. Xiao-bo Wang: Writing—Review & Editing, Project administration. Xiao-jian Wu: Funding acquisition. All authors reviewed and approved the final version of the manuscript.

Data availability

Data used to support this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Shi-rui Bai, Bing-xiang Zhao, and Qi Zhao.
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