Application of jellyfish snow rabbit in inhibiting ERK phosphorylation level and inhibiting MAPK pathway and evaluation method of jellyfish snow rabbit in inhibiting ERK phosphorylation level and MAPK pathway

Abstract

The invention discloses application of jellyfish and snow rabbits in inhibition of ERK phosphorylation level and MAPK pathway and an evaluation method of the jellyfish and snow rabbits, and relates to the technical field of biological medicines.The method comprises the following steps that 1, a treated jellyfish and snow rabbits breast cancer cell group and a control breast cancer cell group are established; and step 2, respectively dividing the two groups of samples in the step 1 into two parts, fixing one part in paraformaldehyde with the mass concentration of 10%, treating, embedding in paraffin for immunohistochemical analysis, and quickly freezing the other part for transcriptomics analysis and the like. According to the invention, UPLC-MS is adopted to determine the main components of SMM, the influence of SMM on breast cancer (BC) is evaluated through in-vivo experiments, in order to deeply clarify the mechanism, network pharmacology and transcriptomics analysis are integrated, finally, immunohistochemistry (IHC) is adopted to verify and explore the exact breast cancer resisting mechanism of SMM, and a scientific basis is provided for potential clinical application of SMM.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, specifically to the application of Saussurea medusa in inhibiting ERK phosphorylation level and inhibiting the MAPK pathway. Background Art

[0002] Breast cancer remains a major global health problem, seriously threatening women's health. According to statistics from the World Health Organization (WHO), breast cancer was the second most common cancer among women globally in 2022, accounting for 11.6% of all new cancer cases. Currently, the standard treatments for breast cancer include surgery, chemotherapy, radiotherapy, endocrine therapy, and molecular targeted therapy. Although these therapies have effectively reduced the mortality and recurrence rates, they often cause severe side effects, such as upper limb lymphedema, gastrointestinal disorders, endocrine disorders, and drug resistance, significantly affecting the quality of life of patients.

[0003] Traditional Chinese medicine (TCM) has been recognized for its multi-target therapeutic potential in cancer treatment and has shown relatively mild side effects and significant clinical efficacy in breast cancer management. For example, Rupixiao can exert anti-breast cancer effects by inducing cell cycle arrest, inhibiting MMP9, and EMT-mediated invasion. Similarly, Ruijian Sanjie Decoction promotes apoptosis of breast cancer cells by inhibiting anti-apoptotic proteins Bcl-2 and survivin, while activating caspase-3 / 7 and caspase-9. In addition, gossypol extracted from Arctium lappa shows anti-breast cancer potential by downregulating the expression of MMP-9 and uPA. Traditional Chinese medicine is increasingly regarded as a valuable resource for new cancer treatment drugs.

[0004] Saussurea medusa Maxim (SMM) is a traditional Tibetan medicinal plant that has long been used to treat arthritis, metabolic diseases, gynecological diseases, and various cancers. Its anti-cancer effects have been studied in esophageal cancer, liver cancer, and prostate cancer. However, the research on the anti-breast cancer properties of SMM is still very limited. Summary of the Invention

[0005] The purpose of the present invention is to make up for the deficiencies of the existing technology and provide the application of Saussurea medusa in inhibiting ERK phosphorylation level and inhibiting the MAPK pathway, providing a new path for the precise diagnosis and treatment of tumors.

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides an application of Saussurea medusa in inhibiting ERK phosphorylation level.

[0008] The present invention also provides an application of Saussurea medusa in inhibiting the MAPK pathway.

[0009] The present invention also provides a drug for inhibiting ERK phosphorylation levels, comprising at least Saussurea medusa.

[0010] The present invention also provides a drug for inhibiting the MAPK pathway, comprising at least Saussurea medusa.

[0011] The present invention also provides a drug for inhibiting tumor growth, comprising at least Saussurea medusa.

[0012] In one possible design, the tumor is selected from the group consisting of: esophageal cancer, liver cancer, prostate cancer, and breast cancer.

[0013] This invention also provides a method for verifying the inhibitory effect of Saussurea medusa on ERK phosphorylation level and MAPK pathway in breast cancer cells, comprising the following steps:

[0014] Step 1: Establish the breast cancer cell group after treatment with Saussurea medusa and the control breast cancer cell group;

[0015] Step 2: Divide the two groups of samples from Step 1 into two parts. One part is fixed in 10% paraformaldehyde and then paraffin-embedded for immunohistochemical analysis. The other part is quick-frozen for transcriptomics analysis.

[0016] Step 3: Statistically analyze the experimental results of Step 2, and combine them with anti-breast cancer target screening, network pharmacology analysis of Saussurea medusa in treating breast cancer, and transcriptomic analysis of Saussurea medusa's anti-breast cancer effect to verify the anti-breast cancer mechanism of Saussurea medusa.

[0017] In one possible design, step 3 includes at least the following steps: extracting Saussurea medusa, identifying compounds in the Saussurea medusa extract, and screening the Saussurea medusa extract for genes; obtaining breast cancer-related genes from the GeneCards database, and performing KEGG pathway and GO enrichment analysis using Bioconductor in R language; fixing the tumor tissue from step 2 in 4% paraformaldehyde for 24 hours, rinsing with running water, embedding in paraffin, blocking 5μm thick tissue sections with goat serum for 30 minutes, and then incubating with primary antibody; the next day, adding secondary antibody and DAB chromogenic agent, counterstaining with hematoxylin, scoring the immunohistochemical signal according to staining intensity and positive tissue area, performing quantitative image analysis using Image-Pro Plus 6.0 software, and performing data analysis using SPSS 28.0 and GraphPad Prism 10.

[0018] In one possible design, step 1 includes at least the following: 4–5 week old female BALB / c mice are housed in groups under standard laboratory conditions at 20°C with a 12-hour light / dark cycle, with free access to food and water. The mice are anesthetized with isoflurane / oxygen masks, and the hair on the lower abdomen is removed with depilatory cream. A single subcutaneous injection of 1×10⁻⁶ ppm of [a specific substance] is administered into the inguinal mammary fat pad. 6 Phosphate-buffered saline (PBS) was injected into 4T1 breast cancer cells. Tumor formation was assessed on day 7 post-injection, and body weight and tumor volume were measured every two days thereafter. After 7 days, tumor-bearing mice were randomly divided into two groups. The control group was maintained on a standard diet, while the experimental group was fed 6g of Saussurea medusa extract per kilogram of body weight by gavage daily for four weeks.

[0019] The present invention also provides a method for inhibiting tumor growth, the method comprising administering Saussurea medusa to a subject in need of such treatment.

[0020] Compared with existing technologies, the application of this siRNA targeting LOC124906852 in tumors has the following beneficial effects:

[0021] Network pharmacology, as a systematic and comprehensive research method, is increasingly popular in the study of TCM mechanisms due to its alignment with the holistic principles of traditional medicine. Visualization techniques can elucidate the interactions between bioactive compounds and disease targets, deepening our understanding of TCM treatment mechanisms. Meanwhile, transcriptomics, with its high-throughput characteristics, has become a powerful tool for molecular-level research. Network pharmacology provides theoretical predictions, while transcriptomics focuses on tissue-specific gene expression; the integration of the two forms a powerful strategy for elucidating drug mechanisms.

[0022] This invention uses UPLC-MS to determine the main components of SMM and evaluates its effect on breast cancer (BC) through in vivo experiments. To further elucidate its mechanism, this invention integrates network pharmacology and transcriptomics analysis. Finally, immunohistochemistry (IHC) is used to verify and explore the exact anti-breast cancer mechanism of SMM, providing a scientific basis for its potential clinical application. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0024] Figure 1 This is an overlapped extract ion chromatogram of the identified SMM compounds;

[0025] Figure 2A schematic diagram illustrating the relationship between drug component targets and 148 potential therapeutic targets related to breast cancer genes analyzed by Wayne.

[0026] Figure 3 A schematic diagram of network pharmacology analysis for SMM in the treatment of breast cancer;

[0027] Figure 4 This serves as a control for the effect of SMM on tumor growth in vivo.

[0028] Figure 5 A schematic diagram of differentially expressed genes in the transcriptome of SMM's anti-breast cancer mechanism;

[0029] Figure 6 A schematic diagram of transcriptomic enrichment analysis of the SMM anti-breast cancer mechanism;

[0030] Figure 7 This is a schematic diagram showing the mI HC images of tumors in the SMM treatment group and the control group. Detailed Implementation

[0031] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0032] In at least one embodiment, the present invention provides the application of Saussurea medusa in inhibiting ERK phosphorylation levels.

[0033] In at least one embodiment, the present invention also provides the application of Saussurea medusa in inhibiting the MAPK pathway.

[0034] In at least one embodiment, the present invention also provides a drug for inhibiting ERK phosphorylation levels, comprising at least Saussurea medusa.

[0035] In at least one embodiment, the present invention also provides a drug for inhibiting the MAPK pathway, comprising at least Saussurea medusa.

[0036] In at least one embodiment, the present invention also provides a drug for inhibiting tumor growth, comprising at least Saussurea medusa.

[0037] The tumors mentioned are selected from the group consisting of: esophageal cancer, liver cancer, prostate cancer, and breast cancer.

[0038] In at least one embodiment, the present invention also provides a method for inhibiting tumor growth, the method comprising administering Saussurea medusa to a subject in need of such treatment.

[0039] In at least one embodiment, the present invention also provides a method for verifying the inhibitory effect of Saussurea medusa on ERK phosphorylation level and MAPK pathway in breast cancer cells, comprising the following steps:

[0040] Step 1: Establish the breast cancer cell group after treatment with Saussurea medusa and the control breast cancer cell group;

[0041] Step 2: Divide the two groups of samples from Step 1 into two parts. One part is fixed in 10% paraformaldehyde and then paraffin-embedded for immunohistochemical analysis. The other part is quick-frozen for transcriptomics analysis.

[0042] Step 3: Statistically analyze the experimental results of Step 2, and combine them with anti-breast cancer target screening, network pharmacology analysis of Saussurea medusa in treating breast cancer, and transcriptomic analysis of Saussurea medusa's anti-breast cancer effect to verify the anti-breast cancer mechanism of Saussurea medusa.

[0043] Step 3 includes at least the following steps: extracting Saussurea medusa, identifying compounds in the Saussurea medusa extract, and screening the Saussurea medusa extract for genes; obtaining breast cancer-related genes from the GeneCards database, and performing KEGG pathway and GO enrichment analysis using Bioconductor in R language; fixing the tumor tissue from Step 2 in 4% paraformaldehyde for 24 hours, rinsing with running water, embedding in paraffin, blocking 5μm thick tissue sections with goat serum for 30 minutes, and then incubating with primary antibody; the next day, adding secondary antibody and DAB chromogenic agent, counterstaining with hematoxylin, scoring the immunohistochemical signal according to staining intensity and positive tissue area, performing quantitative image analysis using Image-Pro Plus 6.0 software, and performing data analysis using SPSS 28.0 and GraphPad Prism 10.

[0044] Step 1 includes at least the following: 4–5 week old female BALB / c mice are housed in groups under standard laboratory conditions of 20°C and a 12-hour light / dark cycle, with free access to food and water. The mice are anesthetized with isoflurane / oxygen masks, and the hair on the lower abdomen is removed with depilatory cream. A single subcutaneous injection of 1×10⁻⁶ ppm of BALB / c into the mammary fat pad in the groin is administered. 6 Phosphate-buffered saline (PBS) was injected into 4T1 breast cancer cells. Tumor formation was assessed on day 7 post-injection, and body weight and tumor volume were measured every two days thereafter. After 7 days, tumor-bearing mice were randomly divided into two groups. The control group maintained a standard diet, while the experimental group was given Saussurea medusa extract by gavage at a dose of 6 g / kg daily for four weeks.

[0045] The verification method provided by this invention includes the following steps:

[0046] 1. SMM preparation

[0047] The Saussurea medusa (SMM) was purchased from Tibet Yunmi Co., Ltd., and the original identification was completed by Dr. Sun XX of the Department of Pharmacy of XX University. The dried SMM was soaked in 95% ethanol at a material-to-liquid ratio of 1:10 (1g SMM to 10ml ethanol) for 48 hours. After filtration, the ethanol in the filtrate was removed by rotary evaporation, and the remaining solution was further concentrated by reflux extraction.

[0048] 2. SMM compound identification

[0049] SMM extract was analyzed using high-performance liquid chromatography-mass spectrometry (HPLC-MS) with an Agilent 1260 HPLC system tandem with a Triple Quad 6470 mass spectrometer. Chromatographic separation was performed, and mass spectrometry detection was performed using an electrospray ionization (ESI) source with positive / negative polarity switching mode and a scan range of m / z 100–1500. Chemical components were identified using Compound Discoverer 3.0 software in conjunction with the mzCloud and Thermo Scientific mzVault spectral libraries.

[0050] 3. Screening of target genes from SMM extracts

[0051] SMM compound structure data were obtained from the PubChem database, and their SMILES expressions were input into the Swiss TargetPrediction platform to filter known and predicted human targets with a probability threshold >0.1.

[0052] 4. Identification of potential breast cancer target genes

[0053] Using "breast cancer" as the keyword, we obtained breast cancer-related genes from the GeneCards database, standardized the genes and defined the species ("human") using UniProtKB in the UniProt database, and set a scoring threshold of >10.

[0054] 5. Pathway and functional enrichment analysis

[0055] To investigate the mechanism of SMM in treating breast cancer, we used Bioconductor in R (v3.6.1) to perform KEGG pathway and GO enrichment analysis. The pathway and target gene assessment adopted a corrected P value ≤ 0.01, and the top 20 GO items and KEGG pathways with the highest enrichment were analyzed.

[0056] 6. Cell Culture

[0057] The mouse breast cancer cell line 4T1 was kindly provided by Dr. Ma Xueman's research group. The cells were cultured in Duchenne modified Eagle medium (DMEM; Procel 1) with the addition of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Procel 1). The culture conditions were 1% oxygen, 94% nitrogen, and 5% carbon dioxide.

[0058] 7. Animal models and tumor induction

[0059] All animal experiments were approved by the Ethics Committee of the Medical College of Qinghai University. Female BALB / c mice (4–5 weeks old) were purchased from Xi'an Huisheng Biotechnology Co., Ltd., and housed in groups (8 mice / cage) under standard laboratory conditions (20℃, 12-hour light / dark cycle) with free access to food and water. Tumor induction: Mice were anesthetized with isoflurane / oxygen masks (2–3% isoflurane, 1L / min), and hair was removed from the lower abdomen with depilatory cream. A single subcutaneous injection of 1×10⁻⁶ ppm of isoflurane was administered into the fat pad of the mammary gland in the groin. 6 Four T1 breast cancer cells were injected with phosphate-buffered saline (PBS). Tumor formation was assessed on day 7 post-injection, and body weight and tumor volume were measured every two days thereafter.

[0060] Seven days later, tumor-bearing mice were randomly divided into two groups. The control group maintained a standard diet, while the experimental group was given SMM extract by gavage daily (at a ratio of 6g of Saussurea medusa extract per kilogram of body weight per day) for four weeks. After treatment, the mice were euthanized under CO2 anesthesia, and the tumors were removed. Tumor samples from both the control and experimental groups were divided into two parts: one part was fixed in 10% paraformaldehyde, processed, and then embedded in paraffin for IHC; the other part was quick-frozen for transcriptomics analysis.

[0061] 8. RNA extraction and sequencing were performed in both the control and experimental groups.

[0062] Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. [The following text appears to be unrelated and possibly a separate instruction:] ...using... Spectrophotometer (IMPLEN, CA, USA)

[0063] RNA purity, concentration, and integrity were assessed using the RNA assay kit (Life Technologies, CA, USA) and the RNA Nano 6000 assay kit (Agilent Technologies, CA, USA). RNA sequencing was performed on the Illumina HiSeq2500 platform (Jiangsu Sanshu Biotechnology Co., Ltd.).

[0064] 9. Immunohistochemistry (IHC)

[0065] Tumor tissue was fixed in 4% paraformaldehyde for 24 hours, rinsed with running water, embedded in paraffin, and 5 μm thick tissue sections were blocked with goat serum for 30 minutes, followed by incubation with primary antibody (4°C overnight). The next day, secondary antibody and DAB chromogenic agent were added, and hematoxylin was used for counterstaining. Immunohistochemical signal scores were based on staining intensity and positive tissue area. Quantitative image analysis was performed using Image-Pro Plus 6.0 software. Primary antibodies ERK (#9102), p-ERK (#9106), JNK (#9252), p-JNK (#9251), p38 (#9212), and p-p38 (#9216) were purchased from Cell Signaling Technology (Danvers, MA, USA).

[0066] 10. Statistical Analysis

[0067] Data analysis was performed using SPSS 28.0 and GraphPad Prism 10. Results are expressed as mean ± standard deviation. Student's t-test was used for comparisons between groups, and the significance level was set at P < 0.05.

[0068] result

[0069] 1. Chemical composition of SMM extract

[0070] HPLC-MS analysis identified 25 compounds in the SMM extract, including quercetin, isoquercetin, kaempferol-7-glucoside, eleutheroside, rutin, chlorogenic acid, scopolamine, honeysuckle glycoside, luteolin-8-C-glucoside, 6-methoxyluteolin, apigenin-7-glucoside, digitalis flavonoids, caffeic acid, osthol, xanthine ethyl ester, ethyl gallate, quercetin, (+)-syringin, DL-3-phenyllactic acid, arctigenin, lycoside, syringin, apigenin, and arctiin. Detailed chemical spectra of the 25 components are shown in Table 1. Overlap extraction ion chromatograms (EIC) of the identified compounds are shown in [Table 1]. Figure 1 .

[0071] Table 1: Detailed chemical spectra of 25 components

[0072]

[0073] 2. Screening of anti-breast cancer targets

[0074] Venn diagram analysis matched drug targets with breast cancer-related genes, identifying 218 potential therapeutic targets (see...). Figure 2 These targets are derived from 25 SMM active ingredients (a total of 355 targets) obtained from STITCH and Swiss Target Prediction, and 4188 breast cancer-related genes from GeneCards.

[0075] 3. Network pharmacology analysis of SMM in the treatment of breast cancer

[0076] To investigate the biological functions of these 218 targets, GO and KEGG enrichment analyses were performed, yielding 1568 biological processes (BP), 49 cellular components (CC), 136 molecular functions (MF), and 178 KEGG pathways (see [link to analysis]). Figure 3 ).

[0077] GO analysis revealed that the target is located in the membrane raft, microdomain, and transferase complex, and is involved in peptide responses, peptidyl serine modification / phosphorylation, and oxidative stress responses. KEGG analysis revealed key pathways including PI3K-Akt, MAPK, Ras signaling pathways, microRNAs in focal adhesion, endocrine resistance, and cancer, as well as pathways in lipid metabolism, chemical carcinogenesis, and viral infection.

[0078] 4. The effect of SMM on tumor growth in vivo

[0079] To evaluate the in vivo anti-breast cancer potential of SMM, an in situ tumor model was established. For example... Figure 4 As shown, SMM treatment significantly inhibited tumor growth. The tumor weight in the treatment group was significantly lower than that in the control group (P<0.05), indicating that it has a potent anti-tumor effect.

[0080] 5. Transcriptomic validation of the anti-breast cancer mechanism of SMM

[0081] Transcriptomic analysis further validated the network pharmacology results, identifying 75 differentially expressed genes (DEGs) between the control and model groups (49 upregulated and 26 downregulated, see below). Figure 5 GO and KEGG enrichment analysis of DEGs confirmed their association with 2220 BP, 69 CC, 177 MF, and 173 KEGG pathways (see [link to article]). Figure 6 GO analysis highlighted cellular components such as contractile fibers, sarcomeres, and myofibrils. SMM targets are primarily involved in biological processes such as skeletal muscle tissue development, muscle tissue development, and myocyte differentiation. Functionally, these targets are associated with cellular activities such as ubiquitin-like protein transferase activity, ubiquitin-protein ligase activity, and ubiquitin-protein transferase activity. KEGG pathway enrichment analysis further showed that these targets were significantly enriched in pathways such as the oxytocin signaling pathway, fluid shear stress and atherosclerosis, hypertrophic cardiomyopathy, osteoclast differentiation, focal adhesion, and the MAPK signaling pathway.

[0082] 6. SMM exerts its anti-breast cancer effect through the MAPK signaling pathway. Network pharmacology and transcriptomics analysis showed that SMM mainly exerts its anti-cancer effect through the MAPK signaling pathway. IHC confirmed the involvement of key pathway proteins (ERK, JNK, p38, p-ERK, p-JNK, and p-p38). Compared with the model group, p-ERK expression was significantly reduced in breast cancer tissues of the SMM treatment group, while p-JNK and p-p38 levels did not show significant changes (see [link to article]). Figure 7 These findings suggest that SMM inhibits breast cancer progression by regulating ERK activation in the MAPK pathway.

[0083] Traditional Chinese medicine (TCM) has long been a valuable resource for drug development, utilizing the synergistic effects of multiple components to combat complex diseases such as cancer. SMM, a high-altitude herb, has traditionally been considered to possess potential anti-tumor properties. Preliminary studies have shown that SMM extract can inhibit cancer cell proliferation; however, its precise molecular mechanism against breast cancer, particularly its involvement in the MAPK signaling pathway, remains largely unexplored.

[0084] This invention identified the active compounds in SMM and predicted their anti-breast cancer potential using network pharmacology, which was subsequently validated in vivo. Transcriptomic and immunohistochemical analyses revealed that SMM exerts its therapeutic effect by inhibiting the MAPK signaling pathway, particularly the ERK axis.

[0085] In this invention, HPLC-MS analysis confirmed that the main components of SMM are flavonoids, with 12 of the 25 identified compounds belonging to this class, including quercetin, isoquercetin, kaempferol-7-glucoside, rutin, luteolin-8-C-glucoside, apigenin-7-glucoside, digitalis flavonoids, zeulatin, 6-methoxyluteolin, quercetin, and apigenin. Flavonoids, as a class of polyphenolic secondary metabolites, have become promising candidates for breast cancer treatment due to their multi-target activity and good safety profile. In vivo experiments using an orthotopic mouse model in this invention showed that SMM treatment significantly reduced tumor weight, suggesting that its anti-tumor effect is mediated by these flavonoids.

[0086] The integration of network pharmacology and transcriptomics in tumor research enables the systematic identification of therapeutic targets and mechanistic pathways. This invention utilizes this integration strategy to reveal that SMM primarily exerts its anti-breast cancer effect by regulating the MAPK signaling pathway. The MAPK pathway plays a central role in tumorigenesis and progression, comprising three main branches: ERK, JNK, and p38. The ERK pathway, the most extensively studied branch, is mainly associated with cell proliferation, differentiation, and survival. JNK signaling can promote tumor cell apoptosis or tumor growth and metastasis under specific conditions. The p38 pathway primarily regulates inflammation and stress responses, playing diverse roles in tumor progression by influencing cell cycle regulation, apoptosis, and the inflammatory cascade.

[0087] To further validate the network pharmacology and transcriptomics results, this invention used immunofluorescence to assess the expression of key MAPK-related proteins (p-ERK, p-p38, and p-JNK). The results showed that the expression levels of p-p38 and p-JNK remained unchanged after SMM treatment, while the phosphorylation level of ERK (p-ERK) was significantly reduced. This finding is consistent with the well-established role of the MEK-ERK pathway in tumor cell proliferation and survival. ERK is a key target for cancer therapy, and inhibitors of this pathway have shown significant clinical efficacy. The FDA-approved BRAF / MEK inhibitor combination encorafenib / binimetinib has demonstrated good efficacy in melanoma treatment, further highlighting the therapeutic potential of ERK inhibition. In summary, the results of this invention indicate that inhibition of the ERK axis is the main mechanism of SMM's antitumor activity.

[0088] This invention integrates UPLC-MS, network pharmacology, and transcriptomics analyses to explore the therapeutic potential of SMM in breast cancer. The results indicate that SMM (particularly its flavonoid components) may inhibit breast cancer growth by targeting the ERK axis in the MAPK signaling pathway. These results highlight SMM as a promising natural therapy and provide new insights into the role of herbal compounds in cancer treatment.

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Use of jellyfish Aequorea coerulescens in inhibiting the phosphorylation level of ERK.

2. Use of jellyfish Aequorea coerulescens in inhibiting the MAPK pathway.

3. A medicament for inhibiting the phosphorylation level of ERK, characterized by, At least comprising jellyfish Aequorea coerulescens.

4. A medicament for inhibiting the MAPK pathway, characterized by, At least comprising jellyfish Aequorea coerulescens.

5. A medicament for inhibiting tumor growth, characterized by comprising the compound of claim 1. At least comprising jellyfish Aequorea coerulescens.

6. The medicament for inhibiting tumor growth according to claim 5, wherein Wherein the tumor is selected from the group consisting of esophageal cancer, liver cancer, prostate cancer, breast cancer.

7. A method for verifying the inhibitory effect of Aequorea victoria green fluorescent protein and Rana sylvatica on the phosphorylation level of ERK and the MAPK pathway of breast cancer cells, characterized by, The method comprises the following steps: Step 1, establishing a group of breast cancer cells treated with jellyfish Aequorea coerulescens and a control group of breast cancer cells; Step 2, dividing the two groups of samples in step 1 into two parts, one of which is fixed in 10% polyformaldehyde and paraffin-embedded for immunohistochemical analysis after treatment, and the other is quick-frozen for transcriptomic analysis; Step 3, statistically analyzing the test results of step 2, and verifying the mechanism of jellyfish Aequorea coerulescens against breast cancer by combining anti-breast cancer target screening, network pharmacology analysis of jellyfish Aequorea coerulescens for treating breast cancer, and transcriptomic analysis of jellyfish Aequorea coerulescens against breast cancer.

8. The authentication method of claim 7, wherein, Step 3 at least comprises the following steps: extracting jellyfish Aequorea coerulescens, identifying the compounds of jellyfish Aequorea coerulescens extract, and performing gene screening on the jellyfish Aequorea coerulescens extract; obtaining breast cancer-related genes from the GeneCards database and performing KEGG pathway and GO enrichment analysis using R language Bioconductor; the tumor tissue in step 2 is fixed in 4% polyformaldehyde for 24 hours, washed with running water, paraffin-embedded, 5 μm thick tissue sections are blocked with goat serum for 30 minutes, then incubated with primary antibody; the next day, secondary antibody and DAB developer are added, hematoxylin is re-stained, the immunohistochemical signal is scored according to the staining intensity and positive tissue area, quantitative image analysis is performed using Image-Pro Plus 6.0 software, and data analysis is performed using SPSS 28.0 and GraphPad Prism 10.

9. The authentication method according to claim 7 or 8, characterized in that, Step 1 includes at least 4-5 weeks old female BALB / c mice are group-housed under standard laboratory conditions at 20°C, 12 hours light / dark cycle, free access to food and water, anesthetized with isoflurane / oxygen mask, hair removal cream to remove the lower abdominal hair, a single subcutaneous injection of 1 x 10 6 6g jellyfish rabbit extract per kg body weight per day for four weeks.

10. A method of inhibiting tumor growth, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-9. The method comprises administering jellyfish Aequorea coerulescens to a subject in need thereof.

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