Target screening method of traditional Chinese medicine phellinus linteus and application of phellinus linteus in treatment of HPV positive tumors

Abstract

The invention discloses a screening method of action targets of traditional Chinese medicine phellinus linteus and application of the screening method to treatment of HPV positive tumors, and the screening method comprises the following steps: (1) screening potential action targets of phellinus linteus, respectively obtaining pathogenic targets of HPV infected cervical cancer by combining a GeneCards database, an OMIM database and a DisGeNET database, and taking intersection to obtain common targets; (2) obtaining a protein-protein interaction network of the intersection targets by using an STRING database, and constructing a PPI network between the intersection targets; (3) carrying out GO function enrichment and KEGG pathway enrichment analysis on the core target spot; and (4) carrying out molecular docking on the identified potential target spot of the phellinus linteus. The traditional Chinese medicine phellinus linteus ketone screened by the method disclosed by the invention can accurately target HPV virus, promote degradation of key oncoprotein E6 / E7 and play an anti-tumor role by regulating mitochondrial autophagy and reducing growth and metastasis of HPV-induced cervical cancer, and the medicine is identified to be low in cost and small in toxic and side effects and has relatively good treatment potential in HPV positive tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a traditional Chinese medicine phloretin target screening method and its application in treating HPV-positive tumors. BACKGROUND

[0002] As the fourth most common malignant tumor in women worldwide, the occurrence and development of cervical cancer are closely related to persistent infection of high-risk human papillomavirus (HPV). According to the epidemiological data of the World Health Organization in 2023, there are 528,000 new cases of HPV-positive cervical cancer worldwide each year, and China ranks second in the world in terms of disease burden. In 2024, the detection rate of high-risk HPV infection in China reached 63.0%, and 99.7% of cervical cancer cases were associated with HPV infection.

[0003] According to the "Guidelines for the Diagnosis and Treatment of Cervical Cancer (2021 Edition)", the current clinical treatment of cervical cancer has the following defects: radical surgery may lead to loss of fertility in patients and cause long-term complications such as pelvic adhesion and chronic pain, which seriously affect the quality of life; early cervical cancer patients still face the risk of recurrence and metastasis after routine radiotherapy combined with chemotherapy, therefore, revealing the molecular mechanism of HPV infection-induced cervical lesions and developing new drugs targeting HPV-positive tumors has important clinical significance for optimizing the prevention and treatment strategies for cervical cancer and improving patient outcomes. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a traditional Chinese medicine phloretin target screening method and its application in treating HPV-positive tumors. The phloretin screened by the method of the present application can precisely target and degrade HPV viral oncoproteins (E6 / E7), play a regulatory role in mitochondrial autophagy, inhibit the occurrence and development of HPV-induced cervical cancer, and improve the efficiency of anti-tumor treatment, while being low in cost and having fewer side effects.

[0005] The technical solution adopted by the present application is as follows: A traditional Chinese medicine phloretin target screening method, comprising the following steps: (1) Use the HERB database to screen the target points of potential compounds in phloretin, and combine the GeneCards database, the human Mendelian inheritance (OMIM) comprehensive database, and the DisGeNET database to obtain the pathogenic target points related to HPV infection of cervical cancer, and take the intersection to obtain the common potential therapeutic target points; (2) Use the STRING database to obtain the protein-protein interaction network of the intersection target points, and use the Cytoscape software to construct the PPI network between the intersection target points; (3) Perform GO function enrichment and KEGG pathway enrichment analysis on the core target points to analyze the main biological processes and signaling pathways involved; (4) Using AutoDock Vina software to perform molecular docking on the identified potential targets of Phellinus linteus flavones, determine the action of the Phellinus linteus flavone compounds and the target protein FASN, and analyze the binding energy, and the binding energy is less than -7.2 kcal / mol.

[0006] As a preferred embodiment of the present application, the main core target screened by the traditional Chinese medicine Phellinus linteus flavone target screening method described in the above scheme is FASN, SLC2A1, MIF, KLF5 and TOP1.

[0007] As a preferred embodiment of the present application, the core target KEGG analysis TOP20 signal pathways further include fatty acid metabolism, inflammatory response, PI3K-Akt signal pathway, human cytomegalovirus infection, chemical carcinogenesis-receptor activation, lipid and atherosclerosis, human papillomavirus infection, fluid shear stress and atherosclerosis, mitochondrial cell communication, human immunodeficiency virus type 1 infection, chemical carcinogenesis-active oxygen, small cell lung cancer, Kaposi's sarcoma-associated herpes virus infection, hypoxia-inducible factor 1 signal pathway, estrogen signal pathway, sphingolipid signal pathway, PD-L1 expression and PD-1 checkpoint pathway in cancer, chronic myeloid leukemia, central carbon metabolism in cancer, acute myeloid leukemia.

[0008] The present application also provides a use of the traditional Chinese medicine Phellinus linteus flavone in the treatment of HPV-positive tumors as described in the above scheme, comprising the following steps: (1) Take the liquid nitrogen-frozen tumor cells for resuscitation and subculture, and when the cells enter the logarithmic growth phase and the density reaches 80%-90%, use trypsin to digest and collect the cells, count and prepare into a cell suspension; (2) Select 6-week-old female BALB / c-nude mice with a body weight of 16-18 g, and subcutaneously inoculate HPV-positive tumor cells in the right front limb armpit, with an inoculation amount of 5×10 6 cells and an inoculation volume of 0.1 mL to establish a subcutaneous xenograft model; (3) Intraperitoneally inject the mice with Phellinus linteus flavones, once every 2 days, for continuous intervention for 2 weeks; during the period, weigh the mice every 2 days, and measure the longest diameter and shortest diameter of the tumor body using a vernier caliper, and record the tumor volume change; (4) Calculate the tumor volume according to the formula V (mm 3 ) = 1 / 2 × length × width 2 , and draw a tumor growth curve and tumor weight.

[0009] As a preferred embodiment of the present application, the concentration of the Phellinus linteus flavones is 15 mg / kg. Significant anti-tumor effect is observed in the HPV-positive tumor in vivo model, and lower toxicity is shown.

[0010] As a preferred embodiment of the present application, after step (3), the subcutaneous tumor tissue is taken out for electron microscope observation, and tissue sections are prepared for HE and IHC staining.

[0011] As a preferred embodiment of the present application, after step (3), the internal organs of the mouse are dissected, and the heart, liver, spleen, lung and kidney are stripped for paraffin fixation, tissue sectioning and staining to evaluate the safety of the drug in vivo.

[0012] Compared with the prior art, the present application has the following beneficial effects: 1. By the traditional Chinese medicine Sanghunag flavone target screening method of the present application, traditional Chinese medicine Sanghunag flavone with potential therapeutic targets can be screened, the main core targets are FASN, SLC2A1, MIF, KLF5 and TOP1, which can precisely target cervical cancer HPV to degrade HPV viral oncoproteins (E6 / E7).

[0013] 2. The present application finds that the Sanghunag flavone screened by the present application can inhibit the expression of viral oncoproteins E6 and E7 when acting on HPV positive cells, reduce the expression of target protein FASN, and induce changes in mitochondrial autophagy marker proteins. Traditional drugs usually have a single target on tumor cells, which is easy to cause drug resistance. However, the Sanghunag flavone in the present application can regulate mitochondrial autophagy to reduce the drug resistance of tumor cells and improve the efficiency of anti-tumor.

[0014] 3. In the present application, the molecular docking binding energy is required to be less than -7.2 kcal / mol. It is found that when the value is lower than this value, the interaction between the ligand and the receptor molecule is stronger, and the complex formed is more stable.

[0015] 4. Sanghunag flavone extraction has mature technology and low cost. Compared with traditional drugs (cisplatin and paclitaxel) with high toxicity and side effects, Sanghunag flavone is more mild and has smaller toxicity and side effects. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 (A) Schematic diagram of the source of Sanghunag flavone; (B) HPLC identification of the structural chemical composition of Sanghunag flavone.

[0017] Figure 2 (A) Establishment of drug concentration evaluation of Sanghunag flavone on HPV infected cells; (B) Morphological changes of HPV infected cells treated with Sanghunag flavone; (C) EDU experiment to detect the inhibitory effect of Sanghunag flavone on the proliferation of HPV positive cells; (D) Transwell experiment to detect the inhibitory effect of Sanghunag flavone on the migration and invasion of HPV positive cells; (E) Flow cytometry to detect the early apoptosis effect of Sanghunag flavone on HPV positive cells.

[0018] Figure 3(A) Construction of an in vivo mouse model of HPV-positive cervical cancer and a treatment pattern of mulberry sanghuang ketone; (BC) Photographs of tumor growth in tumor-bearing mice before and after intraperitoneal injection of 5 mg / kg and 15 mg / kg mulberry sanghuang ketone; (D) Tumor volume in tumor-bearing mice before and after mulberry sanghuang ketone treatment; (E) Tumor weight before and after mulberry sanghuang ketone administration; (F) Pathological analysis of internal organs in mice before and after mulberry sanghuang ketone administration.

[0019] Figure 4 (A) Intersection of targets related to mulberry sanghuang ketone, HPV infection, and cervical cancer yielded 58 shared targets; (B) FASN, SLC2A1, MIF, KLF5, and TOP1 were identified as the main core targets of mulberry sanghuang ketone; (C) GO enrichment analysis of core targets; (D) KEGG analysis of core targets revealed the TOP20 signaling pathways.

[0020] Figure 5 (A) Molecular docking analysis of lindane and target protein FASN; (B) Effect of different concentrations of lindane on HPV-positive cells; (C) Transmission electron microscopy detection of lindane-induced formation of mitochondrial autophagy vesicles in HPV-positive cells; (D) In ​​vivo histopathological analysis of lindane in HPV-positive tumors and detection of target protein FASN, viral oncoproteins E6 and E7, and tumor proliferation markers. Detailed Implementation

[0021] Typical embodiments embodying the features and advantages of the present invention will be specifically described in the following description. It should be understood that the present invention can have various variations in different embodiments without departing from the scope of the present invention, and the descriptions and illustrations herein are for illustrative purposes only and not intended to limit the present invention.

[0022] This embodiment discloses a method for screening targets of the traditional Chinese medicine Sanghuang ketone, including the following steps: (1) In order to identify the important chemical components of mulberry twig extract, its active ingredient was identified as morinone by UPLC-Orbitrap-Exploris-120-MS. Figure 1 AB).

[0023] (2) Using the HERB database, 125 potential compounds in linalool were screened for targets. Combined with the GeneCards database, the Human Mendelian Inheritance (OMIM) database, and the DisGeNET database, 119 pathogenic targets of HPV infection and 1730 potential targets of cervical cancer were obtained. The intersection was taken to obtain 58 common potential therapeutic targets related to HPV-positive cervical cancer. Figure 4 A) Using the STRING database, the protein-protein interaction network (PPI) of the intersecting targets was obtained, and the PPI network between the intersecting targets was constructed using Cytoscape software; (3) GO and KEGG enrichment analysis of the targets of Sanghualactone (SAL) (Fig. Figure 4 C-D) showed that the compound significantly affected multiple biological processes and signaling pathways closely related to tumor occurrence and development.

[0024] In terms of biological processes, Sanghualactone mainly regulated the response of cells to external stimuli and oxidative stress, inflammatory response, and protein metabolism, etc.; cell components were concentrated in the perinuclear region, membrane raft, and receptor complex; and molecular functions were related to kinase binding, chromatin binding, and protein dimerization, etc.

[0025] KEGG pathway analysis further found that Sanghualactone was significantly enriched in the top 20 pathways, including metabolism, lipid, viral infection, PI3K-Akt signaling pathway, HIF-1 signaling pathway, PD-1 / PD-L1 immune checkpoint, etc.

[0026] In summary, Sanghualactone may play an anti-tumor role through regulating immune inflammatory response, tumor metabolic reprogramming, virus carcinogenic process, and immune checkpoint molecules, etc., and has potential therapeutic value in HPV-related tumors.

[0027] (4) The main core targets of the screened Sanghualactone were FASN, SLC2A1, MIF, KLF5, and TOP1 (Fig. Figure 4 B). Molecular docking was performed on the identified potential targets of Sanghualactone using AutoDock Vina software, and the results showed that the target protein FASN (light purple) and the Sanghualactone compound (red) formed 3 hydrogen bonds and 1 π-π interaction, with a binding energy score of -8.781 kcal / mol (Fig. Figure 5 A).

[0028] (5) HPV virus-encoded genes E6 and E7 genes are most important for cell growth, which mainly regulate cell cycle, promote viral genome amplification, and late gene expression. We found that under the treatment of Sanghualactone, the expression of viral E6 and E7 cancer proteins was reduced, and the protein level of FASN was significantly down-regulated, accompanied by significant changes in autophagy key proteins PINK, Parkin1, P62, and LC3BII (Fig. Figure 5 B). Transmission electron microscopy (TEM) was also used to observe the process of autophagy flow, and the results showed that Sanghualactone, as a natural inhibitor of FASN, played an important role in regulating mitochondrial autophagy in HPV-positive cells (Fig. Figure 5 C-D).

[0029] The present embodiment discloses a kind of Chinese medicine Sanghualactone in vitro inhibits tumor effect, comprising the following steps: (1) Functional experiments were conducted in HPV-positive cervical cancer cells. The CCK-8 assay was used to detect the effect of different concentrations of linalool (0-100 μM) on cell viability, reflecting the inhibitory effect of the drug on the proliferation of HPV-positive cervical cancer cells, and determining the appropriate drug concentration. At the same time, the morphology of HPV-positive cells under different drug concentrations was monitored. Figure 2 AB).

[0030] (2) The EdU assay directly reflects the inhibitory effect of linalool on cell proliferation by detecting cellular DNA synthesis activity, and is used to evaluate the direct effect of the drug on tumor cell replication activity. Figure 2 C).

[0031] (3) The Transwell assay was used to evaluate the inhibitory effect of lindrine on cell migration and invasion, which can simulate the metastasis process of tumor cells in vivo. The results showed that lindrine treatment reduced the migration and invasion levels of HPV-positive cells. Figure 2 D). (4) Flow cytometry detected changes in mitochondrial membrane potential, and the results showed that mulberry lintein induced apoptosis in HPV-positive cells, revealing that mulberry lintein induces apoptosis through the mitochondrial pathway. Figure 2 E).

[0032] The above experiments comprehensively verified the antitumor activity of lindane from multiple perspectives, including proliferation, migration, apoptosis, and mitochondrial membrane potential. In particular, the observation of mitochondrial function and autophagy strongly supports its in vitro antitumor efficacy.

[0033] This invention also provides an application of the traditional Chinese medicine Sanghuang ketone as described above in the in vivo treatment of HPV-positive tumors, including the following steps: (1) Take the tumor cells frozen in liquid nitrogen, revive and passage them. When the cells enter the logarithmic growth phase and the density reaches 80%~90%, digest them with trypsin and collect the cells. After counting, prepare a cell suspension. (2) Six-week-old female BALB / c-nude mice, weighing 16-18g, were randomly divided into a control group (A: saline), a low-dose control group (B: 5 mg / kg linalool), and a high-dose control group (C: 15 mg / kg linalool). The mice were then administered 5 × 10⁻⁶ mg / kg of linalool. 6 HPV-positive tumor cells were injected into the right forelimb axilla of mice at a volume of 0.1 mL per mouse to establish a subcutaneous tumor model. Figure 3 A); (3) After inoculation for 6-7 days, tumors can be observed to grow, and anti-tumor drugs are detected: a control group (injection of the same amount of normal saline), a low-dose drug group (intra-abdominal injection of 0.1 mL of chamonix flavone at 5 mg / kg, every 2 days), and a high-dose drug group (intra-abdominal injection of 0.1 mL of chamonix flavone at 15 mg / kg, every 2 days), with an intervention time of 2 weeks. The weight is measured every 2 days, and the longest diameter (length) and the shortest diameter (width) of the tumor are measured using a vernier caliper, and the change in tumor volume is recorded Figure 3 B-C). (4) The tumor volume is calculated according to the formula V (mm 3 )=1 / 2 x length x width 2 , and the tumor growth curve and the tumor weight are plotted with the growth weeks as the horizontal coordinate and the tumor volume as the vertical coordinate, so as to evaluate the treatment effect of the drug chamonix flavone Figure 3 D-E).

[0034] After step (3), the subcutaneous tumor tissue is taken, electron microscopy is observed, and tissue sections are prepared for HE and IHC staining.

[0035] After step (3), the internal organs of the mouse are dissected, the heart, liver, spleen, lung and kidney are stripped and paraffin-fixed, tissue sections are prepared and stained, and the safety of the drug in the body is evaluated.

[0036] In combination with the HE staining of the internal organs of the mouse, the safety of the drug in the body is evaluated. The results show that the high-dose chamonix flavone (15 mg / kg) of the application does not observe adverse reactions while inhibiting tumor growth, which shows that it has great potential in the treatment of HPV-positive tumors Figure 3 F).

[0037] In the research of the application, the in-vivo anti-tumor effect and safety of the traditional Chinese medicine chamonix flavone obtained through screening are systematically evaluated by constructing an HPV-positive cervical cancer (CC) nude mouse transplanted tumor model. As shown in the accompanying Figure 3 After the tumor-bearing mice are intervened by intra-abdominal injection of the low-dose chamonix flavone group (5 mg / kg) and the high-dose chamonix flavone group (15 mg / kg) every other day for two weeks, compared with the control group, each drug administration group, especially the high-dose group of the application, shows a significant tumor growth inhibition effect Figure 3 B-C), and the tumor volume and weight are significantly reduced Figure 3 D). In addition, pathological observation is performed on the main organs (including the heart, spleen, lung and kidney) Figure 3 F), and no obvious tissue damage or pathological structure change is observed, which preliminarily indicates that the traditional Chinese medicine chamonix flavone has good in-vivo safety within the dosage range.

[0038] In summary, the traditional Chinese medicine obtained by screening according to the application can effectively inhibit the growth of HPV-positive cervical cancer in vivo, and does not cause obvious systemic toxicity, and has the potential to be further developed as an anti-tumor candidate drug.

[0039] The above embodiments are only preferred embodiments of the present application, and cannot be used to limit the scope of protection of the present application, and any non-substantial changes and substitutions made by those skilled in the art on the basis of the present application are within the scope of protection of the present application.

Claims

1. A traditional Chinese medicine Sanghuang flavone target screening method, characterized in that, Includes the following steps: (1) Use the HERB database to screen potential targets of compounds in linalool, and combine the GeneCards database, the Human Mendelian Inheritance (OMIM) database and the DisGeNET database to obtain pathogenic targets related to HPV infection and cervical cancer, and take the intersection to obtain common potential therapeutic targets. (2) Using the STRING database, the protein-protein interaction network of the intersection targets was obtained, and the PPI network between the intersection targets was constructed using Cytoscape software; (3) Perform GO function enrichment and KEGG pathway enrichment analysis on the core targets to analyze the main biological processes and signaling pathways involved; (4) The potential targets of mulberry senna identified were molecularly docked using AutoDock Vina software to determine the interaction between mulberry senna compounds and the target protein FASN, and the binding energy was analyzed. The binding energy was less than -7.2 kcal / mol.

2. The method according to claim 1, characterized in that, The main core targets screened using the traditional Chinese medicine mulberry sanghuang ketone target screening method as described in claim 1 are FASN, SLC2A1, MIF, KLF5 and TOP1.

3. The method according to claim 1, characterized in that, It also includes the KEGG analysis of the top 20 signaling pathways for core targets: fatty acid metabolism, inflammatory response, PI3K-Akt signaling pathway, human cytomegalovirus infection, chemical carcinogenesis-receptor activation, lipids and atherosclerosis, human papillomavirus infection, fluid shear stress and atherosclerosis, mitochondrial cell communication, human immunodeficiency virus type 1 infection, chemical carcinogenesis-reactive oxygen species, small cell lung cancer, Kaposi's sarcoma-associated herpesvirus infection, hypoxia-inducible factor 1 signaling pathway, estrogen signaling pathway, sphingolipid signaling pathway, PD-L1 expression and PD-1 checkpoint pathway in cancer, chronic myeloid leukemia, central carbon metabolism in cancer, and acute myeloid leukemia.

4. The use of the traditional Chinese medicine of the flavonoids of Phellinus baumii in the treatment of HPV-positive tumors, characterized in that, Includes the following steps: (1) Take the tumor cells frozen in liquid nitrogen, revive and passage them. When the cells enter the logarithmic growth phase and the density reaches 80%~90%, digest them with trypsin and collect the cells. After counting, prepare a cell suspension. (2) 6-week-old female BALB / c-nude mice, weighing 16-18 g, were inoculated with HPV-positive tumor cells under the right axillary subcutaneous of the front limbs, with 5 x 10 6 cells per mouse and 0.1 mL of inoculum to establish a subcutaneous xenograft model; (3) Intraperitoneal injection of mulberry ketone into mice was administered once every 2 days for 2 weeks. During this period, the weight of the mice was measured every 2 days, and the longest and shortest diameters of the tumor were measured using calipers to record the changes in tumor volume. (4) Calculate the tumor volume according to the formula V (mm 3 ) = 1 / 2 x length x width 2 Draw the tumor growth curve and tumor weight.

5. The application of the traditional Chinese medicine Sanghuangone in treating HPV-positive tumors according to claim 4, characterized in that, The concentration of the linalool is 15 mg / kg.

6. The application of the traditional Chinese medicine Sanghuangone in treating HPV-positive tumors according to claim 4, characterized in that: After step (3), the experiment ended and subcutaneous tumor tissue was taken for electron microscopy observation. Tissue sections were prepared for HE and IHC staining.

7. The use of the traditional Chinese medicine of the flavonoids of the Phellinus baumii according to any one of claims 4-6 for the treatment of HPV-positive tumors, characterized in that, After step (3), the internal organs of the mice were dissected, and the heart, liver, spleen, lungs and kidneys were dissected for paraffin fixation, tissue sectioning and staining to assess the safety of the drug in vivo.

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