Artemisia annua cell-derived exosome nano-vesicle and application of inclusion peroxidase A0A2U1N9S9 of exosome nano-vesicle in preparation of anti-colorectal cancer drugs

Abstract

The invention discloses an application of exosome nano-vesicles derived from artemisia annua cells and peroxidase A0A2U1N9S9 contained in the exosome nano-vesicles in preparation of anti-colorectal cancer drugs. According to the present invention, the anti-CRC activity analysis results show that the artemisia annua cell-derived exosome nano-vesicles can inhibit the growth of DLD-1 and HCT116 cell strains, and the active substance base of ACDENVs for inhibiting the growth of DLD-1 and HCT116 cell strains, namely peroxidase A0A2U1N9S9, is innovatively revealed for the first time based on the difference analysis of the proteomics technology; and a new research thought and a theoretical basis are provided for the research of anti-colorectal cancer drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to exosome nanovesicles derived from Artemisia annua cells and their contents.

[0002] Application of peroxidase A0A2U1N9S9 in the preparation of anti-colorectal cancer drugs. Background Technology

[0003] Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, with approximately 1.9 million new cases and over 900,000 deaths annually. It primarily occurs in the mucosal epithelial cells of the colon or rectum, typically originating from adenomatous polyps and gradually evolving into cancer over many years. Research indicates that the occurrence and development of CRC is a multi-step, multi-gene process. Key gene mutations include those in the RAS, p53, APC, and mismatch repair (MMR) genes. RAS gene mutations, such as K-Ras gene mutations, occur in approximately 50% of colorectal cancer patients, affecting cell proliferation and signal transduction; p53 gene mutations are present in approximately 75% of sporadic colorectal cancers, and the mutation frequency is correlated with tumor malignancy; APC gene mutations cause familial adenomatous polyposis, and APC gene loss is also common in patients without a family history; MMR genes, such as hMLH1 and hMSH2 gene mutations, lead to microsatellite instability and are closely associated with hereditary nonpolyposis colorectal cancer.

[0004] Artemisia annua L., also known as wormwood, is an annual herb belonging to the genus Artemisia in the family Asteraceae, native to China and Southeast Asia. It is the only natural source of the antimalarial drug artemisinin (ART) and also contains abundant flavonoids, volatile oils, and organic acids. In traditional Chinese medicine, Artemisia annua is used to clear heat and detoxify, cool the blood and stop bleeding, and eliminate dampness and jaundice. It can also be used to treat symptoms such as fever, headache, colds, and dysentery. Extracts of Artemisia annua, especially its main active ingredients, artemisinins (ARTs), such as dihydroartemisinin (DHA) and artesunate, have shown potential anticancer effects in recent years in CRC research. DHA can significantly reduce the survival rate of CRC cell lines (such as HCT116, SW480, and Caco-2) and inhibit cell growth in a dose-dependent manner. Meanwhile, DHA can arrest CRC cells in the G2 / M phase, preventing them from entering mitosis and thus inhibiting cell proliferation. The specific mechanisms include downregulating the expression of cell cycle proteins (such as CDK1, CCNB1, and PLK1), affecting cell cycle regulation. Studies have shown that DHA can also reduce the expression of cyclooxygenase-2 (COX-2) and decrease the production of prostaglandin E2 (PGE2), further inhibiting inflammatory responses and tumor growth. In conclusion, Artemisia annua extract and its main components, ARTs, demonstrate broad application potential in anti-CRC, particularly in selectively killing cancer cells, inhibiting angiogenesis, and reducing metastasis.

[0005] Exosomes (EXOs) are small membrane vesicles released into the extracellular environment after the fusion of intracellular multivesicular bodies (MVBs) with the cell membrane. Their diameter typically ranges from 30 to 150 nm. Studies have shown that EXOs contain abundant bioactive molecules, including proteins, lipids, nucleic acids, and small molecule compounds. They play important roles in intercellular communication, substance transport, immune regulation, disease biomarkers, drug delivery, and signal transduction, and are significant in various physiological and pathological processes. Plant-derived exosomes (PDEs) and animal-derived exosomes (ADEs) share similarities in structure, function, and applications, but also exhibit some significant differences. PDEs have significant advantages in safety, wide availability, natural functional molecules, drug delivery, and environmental friendliness, making them promising for applications in multiple fields. With further research and technological advancements, PDEs are expected to become important biomaterials with wide applications in pharmaceuticals, food, cosmetics, and other industries. However, there is currently very little research on the main active components of PDEs, and no literature reports have been found on the application of exosome nanovesicles derived from Artemisia annua cells and their contents, peroxidase A0A2U1N9S9, in the treatment of colorectal cancer. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a new application of exosome nanovesicles derived from Artemisia annua cells and their contents, peroxidase A0A2U1N9S9, in the preparation of anti-colorectal cancer drugs.

[0007] The technical solution adopted in this invention is as follows:

[0008] On the one hand, this invention provides the application of peroxidase A0A2U1N9S9 in the preparation of anti-colorectal cancer drugs,

[0009] The amino acid sequence of the peroxidase A0A2U1N9S9 is shown in SEQ ID NO.1, and its coding sequence is shown in SEQ ID NO.2.

[0010] This invention analyzes the heterologous expression and bioactivity of peroxidase A0A2U1N9S9 in Escherichia coli. The results show that peroxidase A0A2U1N9S9 inhibits the growth of colorectal cancer cell lines HCT116 and DLD-1 by inducing apoptosis.

[0011] On the other hand, the present invention also provides the application of exosome nanovesicles derived from Artemisia annua cells containing the above-mentioned peroxidase A0A2U1N9S9 in the preparation of anti-colorectal cancer drugs, wherein the exosome nanovesicles derived from Artemisia annua cells have an inhibitory effect on the growth of DLD-1 and HCT116 cell lines.

[0012] As a preferred embodiment of the present invention, the method for preparing the exosome nanovesicles derived from Artemisia annua cells includes:

[0013] The following steps are required:

[0014] (1) Culture Artemisia annua cells, collect the culture medium, and centrifuge at 4℃ and 8000-10000 g for 15-20 min to remove cell debris;

[0015] (2) The clarified culture medium was concentrated using a hollow fiber dialysis tube, and the concentrate was collected;

[0016] (3) Add 0.971 M / L sucrose solution to the lower layer of the ultra-high speed centrifuge tube, then add the concentrate to the ultra-high speed centrifuge tube, centrifuge at 4℃ and 100000-150000 g for 80-100 min, and collect the sucrose layer.

[0017] (4) Add PBS buffer to the sucrose layer and centrifuge at 4℃ and 2000-4000 g to concentrate the extract, thus obtaining Artemisia annua.

[0018] Cell-derived exosome nanovesicles.

[0019] In a preferred embodiment of the present invention, the average particle size of the exosome nanovesicles derived from Artemisia annua cells is [missing information].

[0020] 113.2±17.8 nm (mean±SD).

[0021] In a preferred embodiment of the present invention, the exosome nanovesicles derived from Artemisia annua cells are spherical with a centrally concave depression.

[0022] The average potential was -22.8 ± 1.3 mV (mean ± SD).

[0023] Compared with the prior art, the present invention has the following superior effects:

[0024] This invention provides a novel application of exosomal nanovesicles derived from Artemisia annua cells and their internal component, peroxidase A0A2U1N9S9, in the preparation of anti-colorectal cancer drugs. Anti-CRC activity analysis showed that exosomal nanovesicles derived from Artemisia annua cells inhibited the growth of DLD-1 and HCT116 cell lines. Furthermore, based on differential proteomics analysis, this invention innovatively reveals for the first time the active substance underlying the inhibition of DLD-1 and HCT116 cell line growth by ACDENVs—peroxidase A0A2U1N9S9—providing new research ideas and theoretical basis for anti-colorectal cancer drug research. Attached Figure Description

[0025] Figure 1 shows the standard curve for protein quantification of ACDENVs using the BCA method in Example 1.

[0026] Figure 2 shows the detection results of ACDENVs particle size in Example 2.

[0027] Figure 3 shows the detection results of the ACDENVs potential in Example 2.

[0028] Figure 4 shows the results of transmission electron microscopy detection of ACDENVs in Example 2.

[0029] Figure 5 shows the effect of ACDENVs on the survival rate of DLD-1 and HCT116 cell lines in Example 3. A: Change in the survival rate of HCT116 cell line after treatment with the positive control drug cisplatin; B: Change in the survival rate of DLD-1 cell line after treatment with the positive control drug cisplatin; C: Change in the survival rate of HCT116 cell line after treatment with different concentrations of ACDENVs; D: Change in the survival rate of DLD-1 cell line after treatment with different concentrations of ACDENVs; n=3.

[0030] Figure 6 shows the SDS-PAGE analysis results of total protein from Artemisia annua cells and ACDENVs in Example 4. Protein markers of 10-180 kDa were selected. A: Total protein from Artemisia annua cells; B1 and B2 are both total protein from ACDENVs.

[0031] Figure 7 is a Venn diagram showing the mass spectrometry identification results of Artemisia annua cells and total protein of ACDENVs in Example 5.

[0032] Figure 8 shows the KEEG analysis results of 62 common proteins in Example 5.

[0033] Figure 9 shows the recombinant plasmid map of heterologous expression of A0A2U1N9S9 in Escherichia coli in Example 6.

[0034] Figure 10 shows the effect of A0A2U1N9S9 on the survival rate of DLD-1 and HCT116 cell lines in Example 6. A: Changes in the survival rate of HCT116 cell line after treatment with different concentrations of A0A2U1N9S9; B: Changes in the survival rate of DLD-1 cell line after treatment with different concentrations of A0A2U1N9S9.

[0035] Figure 11 shows the flow cytometry detection of apoptosis in HCT116 and DLD-1 cells by peroxidase A0A2U1N9S9 in Example 6, n=3; ** indicates comparison with the control group (nc), P < 0.01; A: Detection of apoptosis in HCT116 cell line by A0A2U1N9S9; B: Statistical results of apoptosis in HCT116 cell line by A0A2U1N9S9; C: Detection of apoptosis in DLD-1 cell line by A0A2U1N9S9; D: Statistical results of apoptosis in DLD-1 cell line by A0A2U1N9S9. Detailed Implementation

[0036] The present invention will be further illustrated below through specific embodiments. The following embodiments are specific implementations of the present invention, but the implementation of the present invention is not limited to the following embodiments.

[0037] Example 1: Extraction of exosome nanovesicles (ACDENVs) derived from Artemisia annua cells

[0038] Artemisia annua cells were suspended and cultured at a ratio of 5 g / 100 mL of MS liquid medium for 14 days at 120 rpm and 25°C. After culture, the culture medium was collected and centrifuged at 4°C and 9000 g for 20 min to completely remove cell debris. The clear culture medium was stored at 4°C or used immediately for downstream experiments. The culture medium was concentrated to 200 mL using hollow fiber dialysis tubes, and the concentrate was collected and aliquoted into Beckman ultracentrifuge tubes. 7.00 mL of 0.971 M / L sucrose solution was slowly added to the bottom layer of each tube, and the mixture was centrifuged at 4°C and 150000 g for 90 min. The sucrose layer (approximately 6.00 mL) was collected, and 18.00 mL of PBS buffer was added. The mixture was concentrated to 2.00 mL using a 30 KD ultracentrifuge tube at 4°C and 3000 g, yielding ACDENVs. Quantification was performed using a BCA protein quantification kit.

[0039] like Figure 1 The results showed that the standard curve for protein quantification of ACDENVs using the BCA method was y = 0.6432x - 0.096, R0. 2=0.9998. The calculated protein concentration of the extracted ACDENVs was 15.58 mg / mL. The extracted ACDENVs were stored at -80℃ for later use.

[0040] Example 2: Identification of physical properties and structural morphology characterization of ACDENVs

[0041] ACDENVs were diluted with ultrapure water to a final concentration of 100 µg / mL, and their particle size and potential were measured using nanoflow cytometry. Simultaneously, an appropriate amount of the 100 µg / mL ACDENVs solution was added dropwise to a 300-mesh carbon-supported copper mesh for adsorption for 90 s, followed by negative staining with 5% phosphotungstic acid for 1 min. The sample was then air-dried overnight at room temperature before being examined by transmission electron microscopy.

[0042] The results showed that the average particle size of ACDENVs was 113.2 nm. Figure 2 It carries a weak negative charge, with an average potential of -22.8mV. Figure 3 ).like Figure 2 , Figure 3 As shown, the particle size and potential detection results of ACDENVs both show single symmetrical sharp peaks, indicating that the extracted ACDENVs have high purity. Transmission electron microscopy revealed that ACDENVs exhibit a hollow, concave spherical shape. Figure 4 The above physical morphological characteristics are consistent with those of previously reported exosomes (EXOs), indicating that high-purity ACDENVs were extracted by sucrose density gradient centrifugation.

[0043] Example 3: Activity analysis of ACDENVs against DLD-1 and HCT116 cell lines

[0044] The DLD-1 and HCT116 cell lines used in this study were obtained from the Shanghai National Certified Cell Bank. These cell lines were cultured in DMEM or RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and passaged in a 37°C carbon dioxide incubator.

[0045] DLD-1 and HCT116 cell lines were seeded into 96-well plates (2 × 10⁶ DLD-1 cells per well). 5 2.2 × 10⁶ HCT116 cells per well 5 Cells were then treated with ACDENVs for 48 hours, and the positive control drug cisplatin was also used for 48 hours. Cell viability was assessed by the MTT assay, and absorbance was measured at a wavelength of 570 nm.

[0046] The results showed that the positive control drug cisplatin inhibited the growth of HCT116 cell line by an IC50. 50The IC50 value was 0.76 ± 0.14 µg / mL, which inhibited the growth of DLD-1 cell line. 50 It was 1.22 ± 0.18 µg / mL ( Figure 5 (A, B). Within the detection concentration range of 15.625-500 µg / mL, ACDENVs inhibited the growth of both DLD-1 and HCT116 cell lines. Figure 5 (C, D), its IC50 inhibitory effect on the growth of HCT116 cell line 50 The IC50 value was 442.512 ± 26.22 µg / mL, which inhibited the growth of DLD-1 cell line. 50 The concentration was 261.068 ± 13.11 µg / mL, highlighting the potential of ACDENVs as a novel anti-CRC drug.

[0047] Example 4: Extraction and SDS-PAGE analysis of total protein from Artemisia annua cells and ACDENVs

[0048] An appropriate amount of Artemisia annua cells were placed in a mortar and rapidly ground with liquid nitrogen. The ground cell powder was then placed in a centrifuge tube, and protein lysis buffer was added at a mass-to-volume ratio of 1:5. Lysis was performed at 4℃ and 80 g for 2 h, followed by centrifugation at 4℃ and 12000 g for 20 min. The supernatant was collected as the total protein extract from Artemisia annua cells. Total ACDENVs protein was extracted using an exosome protein extraction kit (BBE). The total protein from Artemisia annua cells and ACDENVs was quantified using a BCA protein quantification kit. A 12% separating gel was prepared, and an equal amount of total Artemisia annua cell protein was simultaneously analyzed by SDS-PAGE. After electrophoresis, silver staining was used for color development.

[0049] The results showed that, according to SDS-PAGE and silver staining, the total protein of ACDENVs exhibited a clear enrichment band at around 35 kDa. Figure 6 This indicates that ACDENVs exhibit protein enrichment in Artemisia annua.

[0050] Example 5: Mass spectrometry identification and analysis of total proteins from Artemisia annua cells and ACDENVs

[0051] Based on the results of Example 4, compared to the total protein in Artemisia annua cells, the total protein in ACDENVs showed protein enrichment, suggesting that it may be the main component responsible for the biological activity of ACDENVs. Based on this, proteomic identification was performed on the total proteins of Artemisia annua cells and ACDENVs, and enriched proteins were screened using differential expression protein analysis. Both components were then subjected to proteomic analysis.

[0052] Depend on Figure 7It was found that 125 proteins were identified in the total protein of ACDENVs and 1189 proteins were identified in the total protein of Artemisia annua cells. Differential analysis of proteomics data revealed that 62 proteins were shared by Artemisia annua cells and the total protein of ACDENVs.

[0053] Studies have shown that redox imbalance is one of the key characteristics of cancer. The core function of peroxidase is to catalyze the decomposition of reactive oxygen species such as hydrogen peroxide, regulating the intracellular redox balance. For example, myeloperoxidase can produce oxidants such as hypochlorous acid, damaging the extracellular matrix and promoting tumor invasion and metastasis. Inflammatory cells in the body produce large amounts of H2O2; sufficient peroxidase activity can remove excess H2O2, reducing its oxidative damage to the DNA of normal intestinal epithelial cells, thereby playing a role in preventing carcinogenesis. All of the above research results indicate that the application of peroxidase in the treatment of colorectal cancer is a promising anti-cancer strategy. KEEG analysis was performed on 62 proteins found in Artemisia annua cells and total ACDENVs (Acinetobacter acetamipridae). Figure 8 It was found that three peroxidases involved in phenylpropane biosynthesis—A0A2U1N9S9, A0A2U1PUY6, and A0A2U1MXR2—are highly expressed in ACDENVs. Since the amino acid sequences encoding A0A2U1PUY6 and A0A2U1MXR2 have not yet been successfully heterologously expressed, this invention first performs heterologous expression and biological activity analysis on A0A2U1N9S9.

[0054] Example 6: Heterologous expression of peroxidase A0A2U1N9S9 in Escherichia coli and its activity analysis on DLD-1 and HCT116 cell lines

[0055] The amino acid and base sequences encoding A0A2U1N9S9 are shown in SEQ ID NO. 1-2. Homologous recombination was used to construct the base sequence encoding A0A2U1N9S9 between the NdeI and XhoI restriction sites of plasmid pET-28b(+)-MBP. Positive plasmids were selected and transformed into *E. coli* DH5α competent cells. A0A2U1N9S9 was heterologously expressed in 1 L shake flasks. Total protein was extracted, purified by nickel column chromatography, and the heterologously expressed A0A2U1N9S9 was used for downstream activity experiments. The activity analysis method for A0A2U1N9S9 in DLD-1 and HCT116 cell lines was the same as in Example 3. For flow cytometry analysis, HCT116 and DLD-1 cells were treated with A0A2U1N9S9 at concentrations of 25-100 µg / mL for 48 h, respectively. Cells were harvested and stained with Annexin V-FITC / 7-AAD. The apoptosis rate was quantified by flow cytometry.

[0056] The results showed that the recombinant plasmid map of A0A2U1N9S9 heterologously expressed in E. coli was as follows: Figure 9 As shown, the bioactivity of A0A2U1N9S9 cells was analyzed after heterologous expression and purification. It was found that A0A2U1N9S9 significantly inhibited the growth of DLD-1 and HCT116 cell lines. Figure 10 Its IC50 inhibitory effect on the growth of HCT116 cell line 50 The IC50 value was 66.08 ± 3.25 μg / mL, which inhibited the growth of DLD-1 cell line. 50 The concentration was 64.03 ± 15.31 μg / mL, while the IC50 of ACDENVs inhibiting the growth of HCT116 and DLD-1 cell lines was 64.03 ± 15.31 μg / mL. 50 The values ​​were 442.512±26.22 µg / mL and 261.068±13.11 µg / mL, respectively. Figure 5 The inhibition effect of A0A2U1N9S9 is better than that of ACDENVs.

[0057] The above results indicate that A0A2U1N9S9 possesses anti-CRC activity, specifically manifested in its ability to inhibit the growth of CRC cell lines HCT116 and DLD-1. Furthermore, it demonstrates that peroxidase A0A2U1N9S9 is the pharmacodynamic basis for the anti-CRC activity of ACDENVs. Flow cytometry analysis (…) Figure 11 It was found that peroxidase A0A2U1N9S9 inhibits the growth of HCT116 and DLD-1 cells by inducing apoptosis.

Claims

1. The application of peroxidase A0A2U1N9S9 in the preparation of anti-colorectal cancer drugs, wherein the amino acid sequence of peroxidase A0A2U1N9S9 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The coding sequence of the peroxidase A0A2U1N9S9 is shown in SEQ ID NO.

2.

3. The application according to claim 1 or 2, characterized in that, The peroxidase A0A2U1N9S9 inhibits the growth of colorectal cancer cell lines HCT116 and DLD-1 by inducing apoptosis.

4. Application of Artemisia annua cell-derived exosome nanovesicles containing peroxidase A0A2U1N9S9 in the preparation of anti-colorectal cancer drugs.

5. The application according to claim 4, characterized in that, The method for preparing the exosome nanovesicles derived from Artemisia annua cells includes the following steps: (1) Culture Artemisia annua cells, collect the culture medium, and centrifuge at 4℃ and 8000-10000 g for 10-15 min to remove cell debris; (2) The clarified culture medium was concentrated using a hollow fiber dialysis tube, and the concentrate was collected; (3) Add 0.971 M / L sucrose solution to the lower layer of the ultra-high speed centrifuge tube, then add the concentrate to the ultra-high speed centrifuge tube, centrifuge at 4℃ and 100000-150000 g for 80-100 min, and collect the sucrose layer. (4) Add PBS buffer to the sucrose layer and centrifuge at 4℃ and 2000-4000 g to concentrate the extract, thus obtaining Artemisia annua. Cell-derived exosome nanovesicles.

6. The application according to claim 4, characterized in that, The exosome nanovesicles derived from Artemisia annua cells The average particle size is 113.2 ± 17.8 nm.

7. The application according to claim 4, characterized in that, The exosome nanovesicles derived from Artemisia annua cells are hollow, concave spherical shapes with an average potential of -22.8 ± 1.3 mV.

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