Device for treating or ameliorating tumor and age-related diseases and application and method thereof

Abstract

The invention provides a device for treating or improving tumors and age-related diseases and application and a method thereof.The hyperbaric oxygen device comprises a hyperbaric oxygen cabin and a cabin control console, the pressure of the cabin control console is set to be 1.5-3.0 ATA, the oxygen concentration is larger than or equal to 92%, and the time is 90-180 min; experiments prove that the level of SASP factors is effectively reduced through hyperbaric oxygen therapy, and a new treatment direction is provided for tumors and age-related diseases; and the hyperbaric oxygen therapy can reverse the side effect of SASP level increase caused by the anticancer drug, and a new idea is provided for reducing the side effect of the anticancer drug.

Description

Technical Field

[0001] The present invention relates to a device for treating or improving tumors and age-related diseases and an application and method thereof, belonging to the technical field of biomedicine. Background Art

[0002] The paper "Progress in Research on Cellular Senescence-Associated Secretory Phenotype Factors and Their Molecular Regulatory Mechanisms" disclosed that cellular senescence is a state of permanent growth arrest in which proliferating cells are separated from the cell cycle. One of its notable characteristics is the secretion of a large number of bioactive substances, namely, cellular senescence-associated secretory phenotype (SASP) factors. The secretion of SASP factors can be roughly divided into three stages: rapid paracrine phase of DNA damage, early SASP, and mature SASP. The molecular regulatory mechanism of SASP factors is complex, involving DNA damage response, p38 mitogen-activated protein kinase (MAPK) signaling pathway, nuclear factor κB (NF-κB) and CCAAT / enhancer binding protein β (C / EBPβ) activation, SASP gene epigenetic changes, gene post-transcriptional regulation and autophagy. SASP factors can change the occurrence of various pathological states caused by the cell microenvironment, and have become drug targets for regulating aging effects, providing a new treatment direction for tumors and age-related diseases.

[0003] In addition, SASP has diverse functions and plays an important role in many diseases, especially age-related diseases, such as cardiovascular disease, atherosclerosis, diabetes, osteoarthritis, tumors, etc. It is related to DNA damage and mitochondrial dysfunction, and is a phenotypic direction with research potential and value.

[0004] The types, characteristics and regulatory effects of SASP in several common malignant tumors such as liver cancer, breast cancer, prostate cancer, etc. The senescence-associated secretory phenotype (SASP) plays a leading role in promoting the malignant transformation of normal tissue cells, the invasion and metastasis of tumor cells. In addition, SASP can also accelerate cell senescence while activating related regulatory pathways to promote the immune system to eliminate senescent cells.

[0005] The mainstream view is that cell senescence is one of the basic measures for the body to prevent cell malignancy. However, SASP can promote inflammation and cell proliferation by changing the cell microenvironment, thereby promoting tumor occurrence and development, indicating that cell senescence is an obstacle to tumor progression; but if cell senescence is not cleared in time, it will enhance tumor occurrence through SASP. SASP may be an important marker for poor tumor prognosis.

[0006] In order to regulate SASP, the academic community has explored a variety of interventions, including exercise, dietary restrictions, stress reduction, and some pharmacological means, including but not limited to: coenzyme Q10, vitamin C, resveratrol, rapamycin, etc. Although these methods have shown certain effects, most of them lack specificity and are difficult for the general population to implement continuously. For example, lifestyle changes (including exercise and dietary restrictions) require a high level of personal commitment and may not be sufficient to achieve the desired results. Similarly, the research and development and approval process of small molecule drugs is long and costly, and they face important issues of dosage control and side effect management, which limits their scope of application and can only be circulated in the non-medical market. Summary of the invention

[0007] The present invention provides an apparatus for treating or improving tumors and age-related diseases, and its application and method. The present invention proves through experiments that effective hyperbaric oxygen therapy reduces the level of SASP factor, providing a new treatment direction for tumors and age-related diseases; and hyperbaric oxygen therapy can reverse the side effect of increased SASP level caused by anticancer drugs, providing a new idea for reducing the side effects of anticancer drugs.

[0008] To achieve this object, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides the use of a hyperbaric oxygen device in the preparation of a device for treating or improving tumors and age-related diseases, characterized in that the pressure of the hyperbaric oxygen device is set to 1.5-3.0 ATA, and the oxygen concentration is set to ≥92%.

[0010] Further preferably, the pressure of the high-pressure oxygen device is set to 2.0 ATA, and the oxygen concentration is set to ≥92%.

[0011] Preferably, the age-related diseases refer to age-related diseases resulting in a significant increase in the levels of various SASPs, including but not limited to cardiovascular disease, atherosclerosis, diabetes, and osteoarthritis.

[0012] Preferably, the use time of the hyperbaric oxygen device is set to 90-180 minutes each time; further preferably, the use time of the hyperbaric oxygen device is set to 90 minutes each time.

[0013] Preferably, the high-pressure oxygen device is used at a frequency of not less than 6 times per week, and the interval between two adjacent high-pressure oxygen treatments is not less than 24 hours.

[0014] The second aspect of the present invention provides the use of a hyperbaric oxygen device in improving the side effects of anticancer drugs, wherein the pressure of the hyperbaric oxygen device is set to 1.5-3.0 ATA, and the oxygen concentration is set to ≥92%; further preferably, the pressure of the hyperbaric oxygen device is set to 2.0 ATA, and the oxygen concentration is set to ≥92%.

[0015] Preferably, the use time of the hyperbaric oxygen device is set to 90-180 minutes each time; further preferably, the use time of the hyperbaric oxygen device is set to 90 minutes each time.

[0016] Preferably, the high-pressure oxygen device is used at a frequency of not less than 6 times per week, and the interval between two adjacent high-pressure oxygen treatments is not less than 24 hours.

[0017] Preferably, the anticancer drug includes but is not limited to Bleomycin.

[0018] Preferably, the side effects of the anticancer drug include increased protein levels of Mmp3 and IL6 caused by the anticancer drug.

[0019] The third aspect of the present invention provides a hyperbaric oxygen device for treating or improving tumors and age-related diseases, comprising a hyperbaric oxygen chamber and a chamber console, wherein the chamber console is set to a pressure of 1.5-3.0 ATA, an oxygen concentration of ≥92%, and a time of 90-180 min; further preferably, the pressure of the hyperbaric oxygen device is set to 2.0 ATA, and the oxygen concentration is set to ≥92%.

[0020] A fourth aspect of the present invention provides a method for reducing SASP levels for non-therapeutic purposes, comprising the following steps:

[0021] S1. Place the test object in a hyperbaric oxygen chamber;

[0022] S2. Use high-purity oxygen with an oxygen purity of ≥99.99% to continuously ventilate the hyperbaric oxygen chamber until the replacement oxygen concentration in the hyperbaric oxygen chamber reaches ≥92%;

[0023] S3. Treatment is carried out for 90-180 minutes at a pressure of 1.5-3.0ATA. During this period, the chamber is flushed with pure oxygen for 5-10 seconds every 150-300 seconds to ensure that the oxygen content in the chamber is stable.

[0024] Preferably, the method further comprises the following steps: repeating steps S1-S3 at least 6 times per week, with the interval between two adjacent steps being at least 24 hours.

[0025] Further preferably, the pressure of the high-pressure oxygen device is set to 2.0 ATA, and the oxygen concentration is set to ≥92%.

[0026] Preferably, reducing the level of SASP comprises reducing the secretion of at least one SASP factor, wherein the SASP factors include IL1β, IL4, IL6, IL10, CCL1, CCL2, CCL3, CCL4, CCL7, CCL8, CCL9, CCL11, CCL22, CXCL1, CXCL2, CXCL3, CXCL9, CXCL10, CXCL11, CX3CL3, TNFα, GM-CSF, IL13, Timp1, Adamsts4, Mmp3, Mmp8, Mmp9, and Mmp13.

[0027] Preferably, the test object in step S1 includes experimental animals, humans or cells.

[0028] In the present invention, reducing the SASP level for non-therapeutic purposes includes but is not limited to preparing an immortalized SASP cell model, which will provide great convenience for high-throughput screening of SASP inhibitors and exploring the SASP mechanism, etc., and has broad application prospects in the field of biomedical research.

[0029] In the present invention, reducing the SASP level for non-therapeutic purposes includes reducing the increase in the SASP level caused by anticancer drugs.

[0030] Compared with the prior art, the beneficial effects and significant progress of the technical solution of the present invention are: the device for treating or improving tumors and age-related diseases provided by the present invention can effectively reduce the systemic SASP level, including reducing the SASP factors IL1β, IL4, IL6, IL10, CCL1, CCL2, CCL3, CCL4, CCL7, CCL8, CCL9, CCL11, CCL22, CXCL1, CXCL2, CXCL3, CXCL9, CXCL10, CXCL11, CX3CL3, TNFα, GM-CSF, IL13, Timp1, Adamsts4, Mmp3, Mmp8, Mmp9, Mmp13. The present invention provides a new treatment direction for tumors and age-related diseases; and hyperbaric oxygen therapy can reverse the side effects of increased SASP levels caused by anticancer drugs, providing a new idea for reducing the side effects of anticancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments of the present invention are briefly introduced below.

[0032] Figure 1 It is the experimental flow chart of Example 2;

[0033] Figure 2 is a statistical graph of serum SASP factors of three groups of mice in Example 2;

[0034] Figure 3 is a graph of the gene set enrichment analysis (GSEA) results of the SASP of Example 3;

[0035] Figure 4 This is a result diagram of the gene expression level of each solid tissue organ in Example 3;

[0036] Figure 5 is the immunoblotting result diagram of Example 4;

[0037] Figure 6 This is a graph showing the in vitro experimental results of Example 4 at different pressures;

[0038] Figure 7 This is a diagram showing the results of the high pressure and high oxygen combined screening experiment of Example 4. DETAILED DESCRIPTION

[0039] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0040] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0041] In order to more fully understand the present invention, the professional terms in the present invention are explained below.

[0042] The complex of bleomycin and iron is embedded in DNA, causing single-strand and double-strand breaks in DNA. It does not cause RNA chain breaks. The first step of action is that the dithiazole ring of this product is embedded between the GC base pairs of DNA, and at the same time, the positive charge of the terminal tripeptide amino acid reacts with the phosphate group of DNA to unwind it. The second step of action is that the complex of this product and iron leads to the generation of superoxide or hydroxyl free radicals, causing DNA chain breaks. It is ineffective when taken orally and requires intramuscular or intravenous injection. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer published a preliminary list of carcinogens for reference. Etoposide combined with cisplatin and bleomycin is on the list of Class 1 carcinogens, and bleomycins are on the list of Class 2B carcinogens.

[0043] Gene Set Enrichment Analysis (GSEA) is an enrichment method proposed by the Broad Institute. It also provides the corresponding analysis software GSEA and a gene set database. GSEA analyzes whether all genes in a gene set are enriched at the top or bottom of the sorted list. If they are enriched at the top, it can be said that, overall, the gene set is up-regulated. Conversely, if they are enriched at the bottom, they are down-regulated.

[0044] Example 1

[0045] Experimental methods

[0046] 1.1. Construction of natural aging model in mice

[0047] Unbred C57BL / 6J male mice were housed in an SPF animal room until they were 20 months old.

[0048] 1.2. Hyperbaric oxygen treatment

[0049] Starting from the age of 20 months, the naturally aging model mice were given hyperbaric oxygen therapy 6 times a week for 4 weeks, with an interval of ≥24 hours between two hyperbaric oxygen treatments. Hyperbaric oxygen therapy was carried out in an animal experimental chamber purchased from Shandong Weifang Huaxin Oxygen Industry Co., Ltd. The treatment plan includes: placing C57BL / 6J mice in a closed ventilation cage and placing them in the chamber; using high-purity oxygen (Shanghai Chloro Min Gas Co., Ltd., oxygen purity ≥99.99%) to continuously ventilate the animal experimental chamber until the oxygen concentration in the chamber is replaced to ≥92%, which takes 20-30 minutes; setting automatic parameters and starting the program: the pressure increase and pressure reduction time are 10 minutes respectively, and in

[0050] HBO treatment was performed for 90 minutes at a pressure of 2.0 ATA, with the chamber flushed with pure oxygen for 5-10 seconds every 150-300 seconds to ensure a stable oxygen content in the chamber.

[0051] Example 2

[0052] Experimental methods

[0053] 2.1 Experimental Grouping

[0054] like Figure 1 As shown, three groups were set up in this embodiment: one group was a young control group (YNG), which was 2-month-old C57BL / 6J male mice; the other two groups were naturally aged 20-month-old C57BL / 6J aged male mice, which were given different treatment schemes respectively. The OLD+HBO group was naturally aged mice treated with hyperbaric oxygen, and the hyperbaric oxygen treatment was completed according to the method in step 1.2 of Example 1. The OLD group was naturally aged mice receiving sham treatment (SHAM), which were placed in the same ventilation cage and put into the animal experimental chamber for the same time, but no ventilation treatment was performed.

[0055] 2.2. Blood collection from mice

[0056] After the last hyperbaric oxygen treatment, the aged mice continued to be raised in the animal room for 1-2 weeks, and then the mice were bled and killed in accordance with relevant ethical requirements. The specific operation was as follows: after proper anesthesia, the mice were fixed with one hand, the eyeballs were clamped with elbow forceps and quickly removed, about 300-500μl of the mouse blood was collected with a 1.5ml EP tube, gauze was used to stop bleeding, and then the mouse limbs were quickly fixed, and tissues and organs were collected after perfusion.

[0057] 2.3. Serum sample acquisition and testing

[0058] The collected blood was placed vertically in the dark at room temperature for 30-45 minutes to allow the blood to coagulate and stratify on its own. It was then centrifuged at 2000 rpm for 15 minutes at 4°C. The upper light yellow serum was aspirated with a pipette and dispensed into 1.5 ml cryovials, which were then transferred to a -80 degree refrigerator for storage. Finally, the protein levels of various SASPs were detected using a mouse luminex liquid phase chip.

[0059] The experimental results are as follows Figure 2 As shown, compared with young mice, the levels of various SASPs in the serum of old mice were significantly increased, while they were significantly decreased after hyperbaric oxygen treatment, including IL1β, IL4, IL6, IL10, CCL1, CCL2, CCL3, CCL4, CCL7, CXCL10, CXCL11, CX3CL3, TNFα, and GM-CSF.

[0060] Example 3

[0061] The experimental grouping and experimental methods of this example are the same as those of Example 2. After perfusion and dissection 1-2 weeks after the end of hyperbaric oxygen therapy, appropriate amounts of skin, liver, heart, brain, lung, and kidney tissues are collected, packaged into cryopreservation tubes, plunged into liquid nitrogen for quick freezing, and then stored in a -80 degree refrigerator. Subsequently, transcriptome sequencing (bulk RNA-seq) of each solid tissue organ and corresponding bioinformatics analysis are performed.

[0062] The results of SASP gene set enrichment analysis (GSEA) are as follows Figure 3 As shown in the figure, the overall SASP levels of aged solid tissues and organs, except the heart, have been significantly reversed to varying degrees, with the skin having the most obvious effect. This may be because the skin has the fastest metabolic renewal rate, making it most affected by the SASP related to cell aging and most easily alleviated, while heart cells hardly regenerate, so the changes are the smallest.

[0063] The gene expression levels of each solid tissue and organ were also analyzed separately. Figure 4As shown in the figure, typical SASP factors (including IL6, IL13, Cxcl1, Cxcl2, Cxcl3, Cxcl9, Ccl2, Ccl8, Ccl9, Ccl11, Ccl22, Timp1, Adamsts4, Mmp3, Mmp8, Mmp9, and Mmp13) showed a downward trend in different tissues and organs, that is, the Log2 FoldChange values ​​were all negative, which was represented by the blue system ( Figure 4 a). Cdkn1a, a gene closely related to SASP and also a marker gene for cell senescence, showed obvious upregulation after aging and downregulation after hyperbaric oxygen treatment in solid tissues and organs ( Figure 4 b).

[0064] Example 4

[0065] In order to further verify the application scope of the effect of hyperbaric oxygen in reducing SASP, this example conducted an in vitro experiment.

[0066] Immunoblotting

[0067] A human prostate stromal cell, called PSC27, was induced by the small molecule drug Bleomycin, and treated with or without hyperbaric oxygen. The method of hyperbaric oxygen treatment was consistent with the single treatment method of mice in step 1.2 of Example 1. The results of immunoblotting are shown in Figure 5 As shown in Figure 2, Bleomycin induced an increase in the protein levels of typical SASP factors, including Mmp3 and IL6, and this trend was reversed by HBO treatment (Supplementary Figure 2). Figure 5 a). In order to analyze the overall transcriptome characteristics, RNA sequencing was performed on normal cells, Bleomycin-induced cells, and cells treated with hyperbaric oxygen after induction. GSEA analysis of the sequencing results showed that compared with normal cells, induced cells had obvious upregulation of SASP production, while hyperbaric oxygen treatment significantly reversed this characteristic (see Appendix Figure 5 b).

[0068] Stress screening experiment

[0069] This example uses different pressures (1.5ATA, 2.0ATA, 2.5ATA, 3.0ATA) to perform hyperbaric oxygen treatment in vitro. Figure 6 As shown, different pressures can reduce the typical SASP expression level of this cell model, including IL1α, IL1β, CXCL1, CXCL3, GM-CSF, MMP1 and IL6, among which the effect of 2.0ATA limiting SASP is relatively the most significant.

[0070] Hyperbaric and hyperoxia combined screening experiment

[0071] The cell model was further treated with 2.0 ATA of high pressure oxygen, and 2.0 ATA of high pressure and high oxygen under normal pressure to explore the effects of high pressure and high oxygen separately. Figure 7 As shown in the figure, high pressure and high oxygen alone also have a certain effect on reducing SASP, especially high pressure, but the effect is not as good as high pressure oxygen. This shows that high pressure and high oxygen may play a synergistic role in the process of high pressure oxygen limiting the generation of SASP.

[0072] The applicant declares that, in the description of the above specification:

[0073] The descriptions of the terms "this embodiment", "an embodiment of the present invention", "as shown in...", "further", "a further improved technical sub-scheme", etc., mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily for the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without causing any contradiction.

[0074] Finally, it should be noted that:

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them.

[0076] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. The non-essential improvements, adjustments or replacements made by those skilled in the art based on the contents of this specification are all within the scope of protection required by the present invention.

Claims

1. Use of a hyperbaric oxygen device in the preparation of a device for treating or improving tumors and age-related diseases, characterized in that: The pressure of the high-pressure oxygen device is set to 1.5-3.0 ATA, and the oxygen concentration is set to ≥92%.

2. The use according to claim 1, characterized in that The age-related diseases include cardiovascular disease, atherosclerosis, diabetes, and osteoarthritis.

3. Application of a hyperbaric oxygen device in improving the side effects of anticancer drugs, characterized in that: The pressure of the high-pressure oxygen device is set to 1.5-3.0 ATA, and the oxygen concentration is set to ≥92%.

4. The use according to claim 1 or 3, characterized in that: The use time of the hyperbaric oxygen device is set to 90-180 minutes each time.

5. The use according to claim 1 or 3, characterized in that: The frequency of use of the hyperbaric oxygen device is no less than 6 times per week, and the interval between two adjacent hyperbaric oxygen treatments is no less than 24 hours.

6. A hyperbaric oxygen device for treating or improving tumors and age-related diseases, characterized in that: It comprises a hyperbaric oxygen chamber and a chamber console, wherein the chamber console is set to have a pressure of 1.5-3.0 ATA, an oxygen concentration of ≥92%, and a time of 90-180 min.

7. A method for reducing SASP levels for non-therapeutic purposes, characterized in that: The following steps are involved: S1. Place the test object in the hyperbaric oxygen chamber; S2. Use high-purity oxygen with an oxygen purity of ≥99.99% to continuously ventilate the hyperbaric oxygen chamber until the replacement oxygen concentration in the hyperbaric oxygen chamber reaches ≥92%; S3. Treatment is carried out for 90-180 minutes at a pressure of 1.5-3.0 ATA. During this period, the chamber is flushed with pure oxygen for 5-10 seconds every 150-300 seconds to ensure that the oxygen content in the chamber is stable.

8. The method for reducing SASP levels for non-therapeutic purposes according to claim 7, characterized in that: The following steps are also included: Repeat steps S1-S3 at least 6 times a week, with a gap of at least 24 hours between two consecutive times.

9. The method for reducing SASP levels for non-therapeutic purposes according to claim 7, characterized in that: Lowering the level of SASP includes reducing the secretion of at least one SASP factor, wherein the SASP factors include IL1β, IL4, IL6, IL10, CCL1, CCL2, CCL3, CCL4, CCL7, CCL8, CCL9, CCL11, CCL22, CXCL1, CXCL2, CXCL3, CXCL9, CXCL10, CXCL11, CX3CL3, TNFα, GM-CSF, IL13, Timp1, Adamsts4, Mmp3, Mmp8, Mmp9, and Mmp13.

10. The method for reducing SASP levels for non-therapeutic purposes according to claim 7, characterized in that: The test object in step S1 includes experimental animals, humans or cells.

Images