Medicine capable of reversing abnormal lipid metabolism of NK (Natural Killer) cells and application

Abstract

The invention provides a medicine capable of reversing abnormal lipid metabolism of NK (Natural Killer) cells and application of the medicine. According to the invention, it is found that lipid accumulation can induce in-vivo and in-vitro immune cell dysfunction and oxidative stress phenomena, and it is found that selenium, selenocystine (SeCys2), selenomethionine (SeMet) and nano-selenium (SeNPs) in different forms are selected for intervention that the selected selenium medicine, especially nano-selenium, can reverse fatty acid-induced NK92 cell lipid accumulation and activity decline. Further experimental exploration finds that the nano-selenium can reverse fatty acid induced NK92 cell mitochondrial function and nutrition metabolism disorder.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a drug capable of reversing abnormal lipid metabolism of NK cells and its application. Background Art

[0002] As an emerging systemic cancer treatment strategy, tumor immunotherapy has attracted much attention. Among them, natural killer cells (NK cells) have shown wide application potential in innate and acquired immunity due to their unique advantages of being non-MHC restricted and able to directly kill tumor cells without prior sensitization. NK cells accurately kill target cells by releasing lytic granules containing pore-forming perforins and granzymes, making them an important research direction for tumor immunotherapy.

[0003] However, dyslipidemia, a characteristic of the metabolic system, is closely related to the morbidity and mortality of diabetes, obesity, cardiovascular disease, and cancer. Metabolic disorders not only affect human health, but also seriously interfere with the function of anti-tumor immune cells. In particular, abnormal changes in fatty acid uptake and metabolism not only contribute to the growth and metastasis of cancer cells, but also provide a breeding ground for the occurrence of tumors by weakening anti-tumor immune responses. Abnormal lipid metabolism has become a key factor in leading to the functional failure of immune cells such as NK cells and weakening the anti-tumor effect. In obese individuals or mice, lipid accumulation destroys the normal function of cells, causing NK cells to fall into metabolic and functional paralysis, thereby accelerating the development of tumors.

[0004] At present, the research and development of drugs for abnormal lipid metabolism of NK cells is still insufficient. The existing single small molecule inhibitors have problems such as easy metabolism, weak accumulation ability in tumor sites, and low bioavailability, making it difficult to achieve efficient and accurate lipid metabolism regulation. At the same time, long-term single medication is also prone to cause side effects such as immune resistance and immunotoxicity. Therefore, actively exploring or developing new drugs that can inhibit abnormal lipid metabolism of NK cells and enhance tumor immunity has become an important frontier topic that needs to be overcome in the field of tumor immunotherapy. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a drug or preparation that can inhibit abnormal lipid metabolism of NK cells to improve tumor immunity.

[0006] Selenium is an essential trace element in organisms and plays an important role in a series of biological functions. It also acts as a cofactor of the antioxidant enzyme glutathione peroxidase (GSH-Px) and binds to selenoproteins involved in antioxidant defense. It participates in various biological processes in the form of selenoproteins and maintains the redox balance of immune cells. It can promote the proliferation and differentiation of various immune cells and activate immune cells to participate in immune regulation. At the same time, selenium can participate in lipid metabolism by regulating the levels of total cholesterol and triglycerides in the body through selenoproteins. Therefore, it is of great research significance to explore whether selenium can regulate the abnormal lipid metabolism of NK cells and enhance their immune activity. However, there are few studies on whether selenium can regulate the abnormal lipid metabolism of NK cells and enhance their anti-tumor immune activity. Screening highly efficient and low-toxic selenium drugs to achieve the effect of inhibiting lipid accumulation-induced abnormal cell metabolism and regulating the body's immune system will have important clinical application value.

[0007] Therefore, the present application provides a drug capable of reversing abnormal lipid metabolism in NK cells, wherein the drug comprises a selenium-containing preparation.

[0008] Specifically, the selenium-containing preparation is one or more combinations of SeCys2, SeMet or nano-selenium preparations.

[0009] Specifically, the nano-selenium preparation is nano-selenium that is synthesized by modifying with a modifier and can stabilize naked nano-selenium.

[0010] Specifically, the modifier includes one or more combinations of folic acid, functionalized polysaccharides, polymers or amino acids.

[0011] Specifically, the functionalized polysaccharide includes, but is not limited to, one or more natural or synthetic polysaccharides such as dextran, chitosan, fungal polysaccharides (such as lentinan, ganoderma polysaccharide, etc.), plant polysaccharides (such as wolfberry polysaccharide, ginkgo polysaccharide, etc.); the polymer includes PEG (polyethylene glycol), PEI (polyethyleneimine), PAMAM (dendritic polymer, ethylenediamine core), etc.; the amino acid includes cysteine, glycine, etc.

[0012] Use of selenium-containing preparations in any of the above-mentioned medicines.

[0013] The improvements of the present application bring the following advantages: The present invention has found through screening research that, compared with other forms of selenium, nano-selenium and its preparations have better reversal of lipid accumulation-induced NK cell dysfunction and enhance its anti-tumor killing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Lipid accumulation can induce immune cell dysfunction and oxidative stress in vivo and in vitro.

[0015] in( Figure 1-A) is 4T1 breast cancer mouse ( Figure 1 -A) and LLC lung cancer mice / ( Figure 1 -B) Lipid accumulation in CD8+T cells, CD4+T cells, and NK1.1 cells in spleen and tumors in vivo; ( Figure 1 -C, D) The proportion of CD8+T cells, CD4+T cells and NK1.1 cells in the blood, spleen and tumor of LLC lung cancer mice; ( Figure 1 -E) is the lipid accumulation in NK92 cells after NK92 cells were induced by different contents of breast cancer (MDA-MB-231) cell culture supernatant (TCM) for 24 hours; ( Figure 1 -F) Lipid accumulation in NK92 cells after treatment with different concentrations of palmitic acid and oleic acid alone or in combination; ( Figure 1 -G) is a fluorescence photograph of lipid accumulation in NK92 cells after Bodipy493 / 503 staining after NK92 cells were treated with palmitic acid and oleic acid at different concentrations alone or in combination for 24 hours; ( Figure 1 -H) is the killing effect of different contents of breast cancer (MDA-MB-231) cell culture supernatant (TCM) on MDA-MB-231 cells after treating NK92 cells for 24 hours; ( Figure 1 -I) is the survival rate of NK92 cells after 24h treatment with palmitic acid and oleic acid at different concentrations alone or in combination; ( Figure 1 -J) is the secretion of cytokine IFN-γ by NK92 cells after NK92 cells were treated with different concentrations of palmitic acid and oleic acid alone or in combination for 24 hours.

[0016] Figure 2 This is an in vitro experimental study on the reversal of fatty acid-induced lipid accumulation and decreased activity of NK92 cells by selenium.

[0017] in( Figure 2 -A) is the inhibition of lipid accumulation of NK92 cells induced by oleic acid by SeNPs, SeMet and SeCys2; ( Figure 2 -B) is the inhibition of lipid accumulation of NK92 cells induced by oleic acid and palmitic acid by SeNPs, SeMet and SeCys2; ( Figure 2 -C) is the inhibition of lipid accumulation of NK92 cells induced by tumor culture medium supernatant (TCM) by SeNPs, SeMet and SeCys2; ( Figure 2 -D) is a fluorescence photo of lipid accumulation in NK92 cells induced by oleic acid after treatment with SeNPs, SeMet and SeCys2 for 24 hours, after Bodipy493 / 503 staining; ( Figure 2-E) After NK92 cells induced by oleic acid and palmitic acid were treated with SeNPs, SeMet and SeCys2 for 24 hours, the expression of CD36 on the surface of NK92 cells was detected by flow cytometry; ( Figure 2 -F) is the killing effect of SeNPs, SeMet and SeCys2 on NK92 cells induced by palmitic acid after 24 hours of treatment; when the effector-target ratio is 1:2 ( Figure 2 -G) and effector target are 3:4 ( Figure 2 -H), the killing effect of SeNPs on tumor cells after NK92 cells were treated with palmitic acid and oleic acid alone or in combination for 24 hours; ( Figure 2 -I, J) After SeNPs treated NK92 cells induced by palmitic acid and oleic acid alone or in combination for 24 hours, they secreted TNF-α and IFN-γ cytokines when killing tumor cells.

[0018] Figure 3 Selenium drugs reverse fatty acid-induced mitochondrial dysfunction in NK92 cells.

[0019] in( Figure 3 -A) is a flow cytometric graph of changes in mitochondrial membrane potential in NK92 cells treated with SeNPs, SeMet and SeCys2 after induction with palmitic acid, oleic acid combined with palmitic acid and tumor culture medium supernatant (TCM) for 24 h; ( Figure 3 -B) is the fluorescence photo of mitochondrial mass in NK92 cells treated with SeNPs, SeMet and SeCys2 and induced by palmitic acid for 24 hours; ( Figure 3 -C) is the percentage of mitochondrial membrane potential dissipation in NK92 cells treated with SeNPs, SeMet and SeCys2 after palmitic acid induction for 24 hours; ( Figure 3 -D) is the fluorescence intensity of mitochondrial mass in NK92 cells treated with SeNPs, SeMet and SeCys2 after palmitic acid induction for 24 hours; ( Figure 3 -E) is the intracellular NADPH / NADP+ ratio and NADPH content after SeNPs treated palmitic acid-induced NK92 cells for 24 hours; ( Figure 3 -F, G) are the mitochondrial superoxide production in NK92 cells induced by palmitic acid and oleic acid after treatment with SeNPs, SeMet and SeCys2 for 24 hours; ( Figure 3 -H) is the status of mitochondrial respiratory chain complex I / IV / V in palmitic acid-induced NK92 cells treated with SeNPs, SeMet and SeCys2 for 24 hours; ( Figure 3 -I) is the kinetics of total ROS production in palmitic acid-induced NK92 cells treated with SeNPs, SeMet and SeCys2 for 24 hours.

[0020] Figure 4 Selenium reverses fatty acid-induced metabolic dysfunction in NK92 cells.

[0021] in( Figure 4 -A) is the flow cytometry graph of SeNPs, SeMet and SeCys2 reversing the inhibition of glucose uptake by NK92 cells by palmitic acid, oleic acid and their combination. Figure 4 -B) Glucose intake ratio; Figure 4 -C) SeNPs reversed the inhibition of ATP production by NK92 cells induced by palmitic acid combined with oleic acid. DETAILED DESCRIPTION

[0022] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0023] Selenium is an essential trace element for the human body. It plays an important role in anti-inflammatory, anti-oxidation and maintaining immune homeostasis. The development of drugs based on selenium that can regulate lipid metabolism disorders and enhance the activity of immune cells has important clinical application prospects. The present invention found that nano-selenium can inhibit the decline in immune activity caused by abnormal lipid metabolism of NK cells. The present invention provides an important basis for the design of drugs or lead compounds based on selenium to improve the effect of NK cell tumor immunotherapy; the present invention organically combines selenium with strategies based on NK cell therapy to achieve effective inhibition of tumor malignant progression.

[0024] The present invention found that lipid accumulation can induce immune cell dysfunction and oxidative stress in vivo and in vitro. After intervention with different forms of selenium, selenocystine (SeCys2), selenomethionine (SeMet) and nano-selenium (SeNPs), it was found that selenium can reverse the lipid accumulation and activity decline of NK92 cells induced by fatty acids. Further experimental exploration found that selenium can reverse the mitochondrial function and nutritional metabolism disorders of NK92 cells induced by fatty acids.

[0025] Experiment 1: Lipid accumulation can induce immune cell dysfunction and oxidative stress in vivo and in vitro.

[0026] Experimental content: First, the spleens and tumors of 4T1 breast cancer mice and LLC lung cancer mice were collected, and the lipid accumulation in CD4+T cells, CD8+T cells and NK1.1+ cells in the tissues was detected by flow cytometry.

[0027] Result analysis: Please refer to Figure 1. Since there are highly accumulated lipids in the microenvironment of many tumors, and these lipids play an important immunosuppressive role on many immune cells in the microenvironment. So we first explored the lipid accumulation of immune cells in the spleen and tumors in mice with different tumors. The results showed that the lipid accumulation in CD8+T cells and NK1.1 cells in tumors of 4T1 breast cancer mice and LLC lung cancer mice was higher than that in the spleen. At the same time, the content of CD8+T cells and NK1.1 cells in the blood and spleen of LLC lung cancer mice was higher than that in tumor tissues. Next, after inducing NK92 cells with the supernatant of the culture medium (TCM) of MDA-MB-231 cells for 24 hours, we found that NK92 cells also had obvious lipid accumulation, and with the increase of the proportion of TCM, the lipid accumulation in NK92 cells was more significant, showing a certain dose-dependency. Oleic acid (OA) can induce lipid accumulation in NK92 cells to different degrees, and when oleic acid (200μM) and palmitic acid (100μM) are used in combination, they can also significantly induce intracellular lipid accumulation. Different concentrations of MDA-MB-231 cell culture supernatant (TCM) induced NK92 cells, which inhibited the killing effect of NK92 cells on MDA-MB-231 cells. Different concentrations of palmitic acid and oleic acid alone or in combination had a certain killing effect on NK92 cells after 24 hours of treatment, and significantly reduced the ability of cells to secrete IFN-γ. These results indicate that tumor-infiltrated NK cells have lipid accumulation, and lipid accumulation can induce immune cell dysfunction and oxidative stress in vivo and in vitro.

[0028] Experiment 2: In vitro experimental study on the reversal of fatty acid-induced lipid accumulation and decreased activity of NK92 cells by selenium.

[0029] Experimental content: Please refer to Figure 2 NK92 cells were induced with fatty acids and different tumor cell culture medium supernatants (TCM) for different time periods to detect intracellular lipid accumulation, cell apoptosis ratio, mitochondrial membrane potential dissipation, mitochondrial superoxide production, total cellular ROS production, and IFN-γ and TNF-α expression in cells.

[0030] Results: Three selenium drugs, Lentinan nanoselenium (SeNPs), selenomethionine (SeMet), and selenocystine (SeCys2), were selected for subsequent experimental exploration. When NK92 cells were treated with SeCys2, SeMet, and SeNPs after lipid accumulation, we found that the addition of selenium could significantly inhibit the lipid accumulation of NK92 cells induced by oleic acid, oleic acid combined with palmitic acid, and different levels of tumor supernatant (TCM). Fluorescence photos of lipid accumulation in NK92 cells after Bodipy493 / 503 staining also significantly reflected the reversal effect of selenium drugs on lipid accumulation. CD36 is a transmembrane protein involved in fatty acid uptake and metabolism, and plays a vital role in lipid homeostasis. Elevated CD36 can lead to excessive cellular fatty acid uptake. Treatment with selenium drugs can reduce the content of CD36 in NK92 cells induced by oleic acid combined with palmitic acid. Selenium drugs can enhance the killing effect of NK92 cells induced by palmitic acid and oleic acid alone or in combination on tumor cells (MCF-7), and increase the secretion of TNF-α and IFN-γ cytokines. These results suggest that selenium can reverse fatty acid-induced lipid accumulation and decreased antitumor activity in NK92 cells.

[0031] Experiment 3: Selenium reverses fatty acid-induced mitochondrial dysfunction in NK92 cells.

[0032] Experimental content: After determining that lipid accumulation induces NK92 cell dysfunction, different concentrations of different forms of selenium were administered for intervention to detect changes in the above indicators.

[0033] Result analysis: Please refer to Figure 3 . The mitochondrial network coordinates cellular metabolic processes and also plays a key role as a signaling hub and potential biomarker in disease progression. When NK92 cells were treated with SeCys2, SeMet, and SeNPs after lipid accumulation, we found that the addition of selenium could significantly inhibit the dissipation of mitochondrial membrane potential of NK92 cells induced by palmitic acid, oleic acid combined with palmitic acid, and tumor supernatant, as shown by flow cytometry and fluorescence photography. Mitochondrial mass was stained with Mito Tracker Green, and the results showed that selenium drugs could improve the mitochondrial mass of NK92 cells inhibited by palmitic acid. Nano-selenium could increase the NADPH / NADP+ ratio and NADPH content in palmitic acid-induced NK92 cells. SeNPs, SeMet, and SeCys2 could inhibit the production of mitochondrial superoxide in NK92 cells induced by palmitic acid and oleic acid, increase the content of mitochondrial respiratory chain complex I / IV / V, and reduce the total ROS production. These results indicate that selenium can reverse fatty acid-induced mitochondrial dysfunction in NK92 cells.

[0034] Experiment 4: Selenium reverses fatty acid-induced metabolic dysfunction in NK92 cells.

[0035] Experimental content: In order to evaluate the regulatory effect of selenium on lipid-accumulating NK cell metabolism, the effects of different forms of selenium intervention on glucose uptake and ATP production in fatty acid-induced NK92 cells were first detected.

[0036] Result analysis: Please refer to Figure 4 Flow cytometry showed that SeNPs, SeMet and SeCys2 could increase the glucose uptake of NK92 cells induced by palmitic acid and oleic acid; SeNPs could increase the ATP production of NK92 cells induced by palmitic acid and oleic acid.

[0037] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A drug capable of reversing abnormal lipid metabolism in NK cells, characterized in that: The medicine includes a selenium-containing preparation.

2. The drug according to claim 1, characterized in that The selenium-containing preparation is one or more combinations of SeCys2, SeMet or nano-selenium preparations.

3. The drug according to claim 2, characterized in that The nano-selenium preparation is nano-selenium that is synthesized by modifying with a modifier and can stabilize naked nano-selenium.

4. The drug according to claim 3, characterized in that The modifications include one or more combinations of functionalized polysaccharides, polymers or amino acids.

5. The drug according to claim 4, characterized in that The modifier includes one or more combinations of dextran, chitosan, fungal polysaccharide, plant polysaccharide, PEG, PEI, PAMAM, folic acid, cysteine, and glycine.

6. Use of a selenium-containing preparation in the medicine according to any one of claims 1 to 5.

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