Application of dihydromyricetin in preparation of inflammatory factor inhibitor for regulating immune homeostasis

Abstract

The invention belongs to the technical field of biology, and particularly discloses application of dihydromyricetin in preparation of an inflammatory factor inhibitor for regulating immune homeostasis, the inhibitor comprises a medicine for reducing the content of inflammatory factors in cells and / or the content of cyclin, and specifically comprises dihydromyricetin; the inflammatory factors comprise Il8, Il1-alpha, Il1-beta and Il6. According to the application, by establishing a H2O2 induced human skin fibroblast premature senility model and a H2O2 induced mouse fibroblast premature senility model, the dihydromyricetin is found to be used as an inhibitor, so that overexpression of inflammatory factors including Il8, Il1-alpha, Il1-beta and Il6 can be effectively reduced, expression of cyclin P16 is also reduced, and the inflammatory factors such as inflammatory factors including Il8, Il1-alpha, Il1-beta and Il6 can be effectively inhibited. The anti-aging and anti-inflammatory effects are effectively achieved in mammals, and the immune homeostasis is maintained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the field of molecular biology, and in particular to the application of an inflammatory factor overexpression inhibitor in regulating immune homeostasis. BACKGROUND

[0002] In recent years, with the in-depth study of immunology, the imbalance of immune homeostasis caused by inflammatory factors can cause various diseases, such as autoimmune diseases, chronic inflammation, allergic reactions, etc. The maintenance of immune homeostasis depends on the dynamic balance of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6. However, when inflammatory factors are overexpressed, it can lead to systemic immune disorders, causing tissue damage and disease progression. Some scholars have proposed that inflammation is one of the main causes of immune aging, and avoiding the overexpression of inflammatory factors is the key to restoring the vitality of the immune system and preventing or reducing the occurrence of various diseases.

[0003] Currently, commonly used broad-spectrum anti-inflammatory drugs such as glucocorticoids can inhibit inflammatory reactions, but long-term use can easily cause immune suppression, metabolic abnormalities, and other side effects. Biological agents targeting specific inflammatory factors such as anti-TNF-α monoclonal antibodies, although they improve efficacy, still have differences in response rates and infection risks. Therefore, the development of new types of selective and safe inflammatory factor expression inhibitors to restore immune homeostasis through precise regulation of key signaling pathways has become a hot research topic.

[0004] Currently, maca polysaccharides are used to prepare immunomodulatory drugs or anti-inflammatory drugs. For example, patent CN105367677A introduces a preparation method of maca polysaccharides and its immunomodulatory activity, which can be used for cancer prevention and treatment, anti-aging, and antioxidant applications. However, the use of maca polysaccharides can increase the expression of pro-inflammatory factors TNF-alpha 、 IL-1 beta 、 IL-6 , which can easily promote the occurrence of inflammation. At the same time, the use of maca polysaccharides has a certain dose dependence, making the pharmacological effect of maca polysaccharides increase with the increase of the dose. If the dose is not appropriate, it may overactivate the immune system, leading to overexpression of inflammatory factors and systemic inflammation. Therefore, the use of maca polysaccharides requires different dose designs for different individuals and precise control of the dose. Some use microorganisms to inhibit inflammation, such as patent CN119506125A which uses Lactobacillus crispatus ProSci-38 to reduce the expression levels of inflammatory factors IL-2 、 IL-10 、 IL-12 、 TNF-alpha , but this bacterial agent is applied to specific parts of the human body, with a small range of action.

[0005] Therefore, there is an urgent need for an inhibitor that can effectively inhibit the overexpression of inflammatory factors to regulate immune homeostasis, and the inhibitor needs to be simple to operate and have a wide range of effects while in action. SUMMARY

[0006] The present application aims to overcome the shortcomings of the prior art and provide the application of dihydromyricetin in the preparation of an inflammatory factor inhibitor for regulating immune homeostasis, and the application value of dihydromyricetin (DHM) in regulating immune homeostasis.

[0007] The application of an inflammatory factor overexpression inhibitor in regulating immune homeostasis, wherein the inhibitor comprises a drug that reduces the content of inflammatory factors and / or the content of cell cycle proteins in cells.

[0008] Specifically, the method for reducing the content of inflammatory factors in cells comprises inhibiting the expression of SASP-related genes. Specifically, the SASP comprises at least one of IL-8, IL-1 alpha , IL-1 beta and IL-6 . Specifically, the method for reducing the content of inflammatory factors in cells is to inhibit the synthesis of SASP-related genes, and the SASP-related genes comprise at least one of IL-8, IL-1 alpha and IL-1 beta . And / or, the method for reducing the content of inflammatory factors in cells is to inhibit the translation of SASP-related genes, and the SASP-related genes comprise IL-6 . Specifically, the method for reducing the content of cell cycle proteins in cells comprises inhibiting the expression of cell cycle protein-related genes. Specifically, the cell cycle protein comprises P16. Preferably, the model for regulating immune homeostasis used in the present application comprises an H2O2-induced human skin fibroblast premature aging model and an H2O2-induced mouse fibroblast premature aging model. Specifically, the commonly used premature aging models mainly include UV ultraviolet irradiation, adriamycin, and hydrogen peroxide. Specifically, the inflammation also includes the expression of cell cycle protein P16. Specifically, the H2O2-induced human skin fibroblast (HFF-1) premature aging model is established by using 50-200 μM H2O2 to treat human skin fibroblasts for 1-1.5 h and then performing SA-β-gal staining. Preferably, the H2O2 induces the human skin fibroblast (NIH-3T3) premature aging model, and the SA-β-gal staining is performed after the human skin fibroblasts are treated with 100 μM H2O2 for 1 h, so that the cell positive rate is high and the cell survival rate is high; Specifically, the H2O2 induces the NIH-3T3 cell premature aging model: SA-β-gal staining is performed after the mouse fibroblasts are treated with 400 μM H2O2 for 1-1.5 h; Preferably, the H2O2 induces the NIH-3T3 cell premature aging model, and the SA-β-gal staining is performed after the mouse fibroblasts are treated with 400 μM H2O2 for 1 h, so that the SA-β-gal positive cell rate can be significantly improved; Specifically, the DHM is an inhibitor with a concentration of 50 μM, and the treatment time is 48 h; Preferably, the inhibitor regulates the expression of inflammatory factors through MAP3K7 the pathway; Specifically, the inhibitor regulates the expression of inflammatory factors through MAP3K7 the pathway. Il1-β and Il8 .

[0009] Specifically, the inhibitor reduces the expression of SASP.

[0010] Specifically, the expression inhibitor of the inflammatory factor has an anti-inflammatory and anti-aging effect in mammals.

[0011] Mechanism of the present application: the present application inhibits the expression of SASP (IL-8, IL-1β, IL-6) and P16 by dihydro myricetin, and regulates the MAP3K7 (TAK1) pathway, thereby effectively delaying cell aging and improving immune homeostasis, and having a significant anti-inflammatory, anti-aging and immune regulation effect.

[0012] The beneficial effects of the present application are: (1) The present application adds DHM, which can significantly reduce the expression of SASP-related pro-inflammatory factors (IL-8, IL-1β, IL-6), effectively regulate the balance of the immune system, and relieve chronic inflammatory state. IL-8, IL-1 alpha, IL-1 beta, IL-6

[0013] (2) The present application adds DHM to inhibit the expression of cell cycle proteins, reduce cell cycle arrest, maintain cell proliferation ability, and delay aging process. P16

[0014] (3) The present application adds DHM to reduce SA-β-gal positive cells, improve the condition of skin and tissue aging, and can be applied to the development of drugs for anti-skin aging and organ aging.

[0015] ​​(4) The inhibitor DHM developed in the present application can regulate the expression of inflammatory factors MAP3K7 and Il1-β and Il8 through the PI3K / Akt pathway.

[0016] (5) The inhibitor DHM developed in the present application has anti-aging and anti-inflammatory effects in mammals, and provides a new treatment strategy for studying autoimmune diseases, chronic inflammatory diseases and age-related inflammatory disorders. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 SA-β-gal staining bright field images of Example 1 and Comparative Example 1 (Figure A is the staining bright field image of HFF-1 without H2O2 induction; Figure B is the staining bright field image of HFF-1 with 100 μM H2O2 induction); Figure 2 mRNA expression levels of markers P16, Il1-β , Il6 , Il8 in Comparative Example 1 and Comparative Example 2 detected by qRT-PCR (wherein the gray rectangular bar represents no H2O2 induction; the blue rectangular bar represents H2O2 induction p <0.05,*** p <0.001); Figure 3 Cell SA-β-gal staining images of Comparative Example 1, Comparative Example 3 and Example 1 (Con Comparative Example 1 without H2O2 induction; H2O2+DMSO Comparative Example 3 with H2O2 induction but only adding DMSO for control; H2O2+DHM Example 1 with H2O2 induction and adding DHM, wherein blue represents SA-β-gal positive); Figure 4 Cell SA-β-gal staining results of Comparative Example 1, Comparative Example 3 and Example 1; Figure 5 mRNA expression levels of markers P16, Il1-β , Il6 , Il8 in Comparative Example 2 and Example 1 detected by qRT-PCR (wherein the blue rectangular bar represents H2O2 induction without adding other substances; the orange bar represents H2O2 induction with DHM added p <0.05,*** p <0.001); Figure 6 Pl6 and IL6 protein expression in cells of Comparative Examples 1-3 and Example 1 determined by western blot; Figure 7To determine the protein cyclin in Comparative Examples 1-3 and Example 1 P16 and pro-inflammatory cytokines IL6 The protein expression level is a measure of Figure 6 Quantification of protein expression levels (Figure A shows cell cycle proteins) P16 The expression levels of pro-inflammatory cytokines were measured; Figure B shows the expression levels of pro-inflammatory cytokines. IL6 Expression level determination; ns: no significant change, * p <0.05,** p <0.01, use Student's test to analyze the significance of the difference); Figure 8 For the detection of MAP3K7 gene interference efficiency in Example 2; Figure 9 Example 2: Detection of MAP3K7 gene expression level after adding DHM; Figure 10 The expression levels of pro-inflammatory cytokines were detected after adding DHM in Example 2 (Figure A is shown). Il1-β Expression level determination; Figure B shows Il6 Expression level determination; Figure C shows Il8 The expression levels of various genes were determined, including the detection of mRNA expression levels of each gene in cells using qRT-PCR. (ns: no significant change, *p<0.05, **p<0.01, ***p<0.001=3; all gene quantifications used the internal reference gene gpdh for relative quantification). Figure 11 The results of SA-B-gal staining in NIH 3T3 cells in Comparative Example 4 and Example 3 are shown (top left: before H2O2 induction, bottom left: after induction, bottom right: after H2O2 induction and DHM application; blue indicates SA-B-gal positivity). Figure 12 Quantitative statistics of SA-β-gal positive cells in NIH 3T3 cells for comparative examples 4 and 5 (where ns: indicates no significant change, ***) p <0.001,** p <0.01); Figure 13 For comparative examples 4 and 5, the mRNA expression level of the aging marker SASP was detected by qRT-PCR (where ns: indicates no significant change, ***). p <0.001,** p <0.01); Figure 14 Western blot was used to determine the protein expression levels of p16 and IL6 in non-senescent cells for comparative examples 4-6 and example 3. Figure 15For the quantitative statistics of SA-β-gal positive cells in NIH 3T3 cells in Comparative Examples 5-6 and Example 3 (where ns: indicates no significant change, ***) p <0.001,** p <0.01); Figure 16 For comparative examples 5 and 6, the mRNA expression level of the aging marker SASP was detected by qRT-PCR (where ns: indicates no significant change, ***). p <0.001,** p <0.01); Figure 17 Comparative Examples 4-6 and Example 3 are... Figure 14 Quantification of p16 protein expression (where ns: indicates no significant change, ***) p <0.001,** p <0.01); Figure 18 Comparative Examples 4-6 and Example 3 are... Figure 14 Quantification of IL6 protein expression (where ns: indicates no significant change, ***) p <0.001,** p <0.01). Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0019] The experimental materials, reagents, and instruments required for this application are listed below.

[0020] 1. Test materials (1) The human skin fibroblast cell line HFF-1 was purchased from Zhejiang Meisen Cell Technology Co., Ltd., catalog number: CTCC-001-0346; (2) The mouse embryonic fibroblast cell line NIH-3T3 was obtained from the Genetics and Evolution Laboratory of Sichuan Agricultural University.

[0021] 2. Main reagents for the experiment Table 1 Main Reagents 3. Main experimental instruments Table 2 Main Instruments Example 1 Cell Culture (1) Cell recovery: Sterilize consumables in advance and preheat the medium. After quickly thawing the cells, centrifuge to remove the cryopreservation solution, resuspend and inoculate in a T25 bottle, mix crosswise, and place in a 37°C 5% CO2 incubator; (2) Cell passage: After observing 90% confluence, sterilize the consumables and preheat the reagents, rinse with PBS, trypsinize for 1 min, stop by adding 3 times the culture medium when the cells round up under a microscope, inoculate at a ratio of 1:3, mix, and continue to culture; (3) Cell cryopreservation: Sterilize the consumables and preheat the reagents, resuspend in 10% DMSO cryopreservation solution after digestion and centrifugation, and store after gradient cooling (4°C→-20°C→-80°C→liquid nitrogen); Example 2 H2O2-induced premature aging model of human skin fibroblasts (1) Cell counting: Sterilize the consumables, prepare a HFF-1 cell suspension, take 10 μL and drop into a counting plate, count the cells in the four corners under a microscope, and calculate the cell concentration per milliliter according to the average cell count × 10 4 .

[0022] (2) Hydrogen peroxide-induced HFF-1 cell aging: Sterilize the consumables and count the plates, wash with PBS after 24 h, add 100 μM H2O2 and incubate for 1 h, change the medium and culture for 3 days to 80% of the aging area.

[0023] (3) DAPI staining procedure: Dilute DAPI (1:1000) with PBS, fix HFF-1 cells for 15 min, wash with PBS 3 times, incubate DAPI in the dark for 10 min after washing with PBS, and examine under a fluorescence microscope after washing with PBS. The fixed sample can be stored at 4°C in the dark.

[0024] (4) SA-β-gal staining steps: Fix for 15 min after washing with PBS, wash with PBS 3 times, add the staining working solution and incubate overnight at 37°C (seal the membrane to prevent evaporation), and add 1 mL of PBS for temporary storage at 4°C or seal the sample for long-term storage after microscopic examination.

[0025] Table 3 β-Gal Staining Working Solution Example 3 Addition of DHM to H2O2-treated human skin fibroblasts DHM was dissolved in DMSO and added to 50 μM DHM-treated HFF-1 cells for 48 h to determine Il8, Il1-alpha, Il1-β and Il6 expression, as well as the expression of cell cycle protein P16. Other treatments are as follows: Table 4 H2O2-treated human skin fibroblasts Experimental Example 1 Determination of expression level of HFF-1 cell related genes (1) Reverse transcription ①Removal of residual genomic DNA, prepare the following mixture in RNase Free centrifuge tube, mix well and incubate at 42°C for 2 min; Table 5 De-genome system ②Preparation of reverse transcription reaction system Table 6 Reverse transcription reaction system ③PCR reaction program Table 7 Reverse transcription PCR program Note: Dilute cDNA after reverse transcription and mark well, store in -20°C refrigerator.

[0026] (2) qRT-PCR determination Use Hieff ® qPCR SYBR Green Master Mix (No Rox) kit, set three replicate holes in 96-well plate with 20 μL system per hole (2 μL cDNA, 10 μL Master Mix, 0.8 μL forward and reverse primers, and ddH2O), run standard qPCR program.

[0027] (3) Protein extraction ①Prepare lysis solution containing protease / phosphatase inhibitor (add 1 μL inhibitor per 100 μL) and 4xLoading Buffer mixture; ②Aspirate 6-well plate medium and wash with PBS, add 150 μL mixture per well, scrape the cells and transfer to EP tube, boil in water bath for 5 min, aliquot and store at -80°C.

[0028] (4) Western blot operation Electrophoresis: prepare SDS-PAGE gel, concentrate the gel to Marker separation at 80 V, then adjust to 100 V; Membrane transfer: wet transfer method (280 mA constant current) to transfer protein to methanol activated PVDF membrane (order: sponge-filter paper-gel-membrane-filter paper-sponge), ice bath operation; Blocking and antibody incubation: block with 5% skim milk powder for 2 h, primary antibody (diluted with BSA) for 1 h at room temperature or overnight at 4°C, secondary antibody (diluted with 5% skim milk powder) for 1 h after TBST washing; Development and analysis: ECL reagent development, Image J gray scale analysis, GraphPad Prism 8 statistical difference.

[0029] (1) Result analysis Example 1 and Comparative Examples 1-3 are to verify the effect of the inhibitor in inhibiting inflammatory factors, using H2O2 induced human skin fibroblast (HFF-1) premature aging model for testing. The staining results can be seen: in the H2O2 treatment group, with the increase of the concentration, the number of SA-β-gal positive cells increases ( Figure 1 ). The calculation results of the positive rate of senescent cell staining (%) show that the average value of SA-β-gal staining positive rate is 66.61% after H2O2 100 μM treatment for 1 h, and the number of viable cells is high. H2O2 100 μM treatment for 1 h can significantly up-regulate the mRNA levels of pro-inflammatory cytokines Il1-β , Il6 , Il8 and cell cycle proteins P16 ( Figure 2 ), using 50 μM of DHM for 48 h for testing, the results show that DHM significantly reduces the cell SA-β-gal staining positive rate in the HFF-1 cell premature aging model ( Figure 3 and Figure 4 ), and significantly down-regulates the mRNA levels of cell pro-inflammatory cytokines Il1-β , Il8 and cell cycle proteins P16 ( Figure 5 ), the protein expression levels of related proteins cell cycle proteins P16 and pro-inflammatory cytokines IL6 are detected, the results show that: DHM down-regulates the expression of cell cycle protein P16, but the results of pro-inflammatory cytokines IL6 are not significantly down-regulated ( Figure 6 and Figure 7 ). It is proved that DHM has anti-inflammatory and anti-aging activity in human skin fibroblast cell line.

[0030] Experimental Example 2 Verification of the conservation of DHM action pathway This experimental example has the same cell culture steps as Example 1, and the test steps are the same as Experimental Example 1, in order to verify the conservation of the DHM action pathway, the human homologous gene tak1 is silenced in HFF-1 cells MAP3K7 .

[0031] The results show that siRNA significantly silences the expression of the gene ( Figure 8 ). At the same time, there is no significant change in the expression of the RNAi group after adding DHM ( MAP3K7 Figure 9 ​Results of cytokine cytokine detection showed that in HFF-1 cells, silencing... MAP3K7 Adding DHM after expression did not alter pro-inflammatory cytokines. Il1-β , Il8 Synthesis of related genes ( Figure 10 A and Figure 10 C). And silence. MAP3K7 Post-DHM treatment can significantly downregulate pro-inflammatory cytokines. Il-6 The expression ( Figure 10 B). This DHM is... Il1-β , Il8 Expression regulation depends on MAP3K7 way.

[0032] Example 4: H2O2-induced premature aging model of mouse fibroblasts The cell culture process is the same as in Example 1.

[0033] (1) Cell counting: UV-sterilized consumables were used to prepare NIH-3T3 cell suspension by digestion. 10 μL of the suspension was added to a counting chamber, and the number of cells in the four corner squares was examined under a microscope. The cell count was calculated by multiplying the average cell count by 10. 4 Calculate the cell concentration per milliliter.

[0034] (2) Hydrogen peroxide-induced senescence of NIH-3T3 cells: sterilized consumables were counted and plated. After 24 h, the cells were washed with PBS, incubated with 100 μM H2O2 for 1 h, and cultured for 3 days until 80% of the senescent area was reached.

[0035] (3) DAPI staining procedure: Dilute DAPI (1:1000) with PBS, fix NIH-3T3 cells for 15 min, wash 3 times with PBS, incubate with DAPI for 10 min in the dark, wash with PBS and examine under a fluorescence microscope. After fixation, the sample can be stored at 4℃ in the dark.

[0036] (4) SA-β-gal staining procedure: after washing with PBS, fix for 15 min, wash with PBS 3 times, add staining working solution and incubate overnight at 37℃ (seal with film to prevent evaporation), after microscopic examination, add 1 mL PBS and store temporarily at 4℃ or seal for long-term preservation.

[0037] Example 5: Mouse fibroblasts treated with DHM and H2O2 DHM was dissolved in DMSO, and 400 μM DHM was added to senescent NIH-3T3 cells for 48 h. SASP was then measured. Il8, Il1-alpha, Il1-beta and Il6 The expression levels of [specific proteins] and cyclin P16 were analyzed. Other treatments are shown in the table below: Table 8. Mouse fibroblasts treated with H2O2 Experimental Example 3 The determination step was the same as in Example 1, and the specific results are analyzed as follows: Example 5 and Comparative Examples 4-6 were to verify the effect of the inhibitor on the inhibition of inflammatory factors, and a mouse fibroblast premature aging model was tested using H2O2induction. The use of H2O2at a concentration of 400 μM significantly increased the SA-β-gal positive cell rate (as shown in Figure 11 and Figure 12 ), showing that the H2O2-induced mouse fibroblast premature aging model was successfully established, which significantly up-regulated the mRNA levels of pro-inflammatory cytokines Il1-α , Il6 , the protein levels of senescence-associated proteins cyclin P16 and pro-inflammatory cytokine IL6 (as shown in Figure 13 ), and when the model was added with 50 μM DHM for 48 h, the SA-β-gal positive cell rate was significantly reduced (as shown in Figure 14 ), the mRNA levels of pro-inflammatory cytokines Figure 15 , Il1-α , Il6 , Il8 were significantly down-regulated (as shown in Figure 16 ), the protein expression levels of cyclin P16 and pro-inflammatory cytokine IL6 were significantly down-regulated (as shown in Figure 17 and Figure 18 ), and the decrease in these indicators showed that DHM could reduce the expression of SASP and play an anti-aging and anti-inflammatory role in NIH-3T3.

[0038] The above only describes preferred embodiments of the present application, and it should be understood that the present application is not limited to the forms disclosed herein, and should not be considered as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concepts described herein, by the above teachings or related art or knowledge. Any modification and change made by those skilled in the art without departing from the spirit and scope of the present application shall be within the protection scope of the claims of the present application.

Claims

1. Use of dihydromyricetin in the preparation of an inflammatory factor inhibitor for modulating immune homeostasis, characterized in that: The inhibitors include drugs that reduce the amount of inflammatory factors and / or the amount of cell cycle proteins in cells.

2. Use according to claim 1, characterized in that: The method of reducing the amount of inflammatory factors in cells includes inhibiting the expression of SASP-related genes.

3. Use according to claim 2, characterized in that: The SASP includes at least one of IL-8, IL-1α , IL-1β and IL-6 .

4. Use according to claim 3, characterized in that: The method of reducing the amount of inflammatory factors in the cell is when synthesis of SASP-related genes is inhibited, the SASP-related genes include IL-8, IL-1α and IL-1β at least one of; and / or, the method of reducing the amount of inflammatory factors in the cell is when translation of SASP-related genes is inhibited, the SASP-related genes include IL-6 .

5. The use according to claim 1, characterized in that: The method of reducing the amount of cell cycle proteins in cells includes inhibiting the expression of cell cycle protein-related genes.

6. Use according to claim 5, characterized in that: The cell cycle proteins include P16.

7. Use according to claim 1, characterized in that: The inhibitors modulate the expression of inflammatory factors by MAP3K7 pathway regulation.

8. Use according to claim 7, characterized in that: The inhibitors modulate the inflammatory response by MAP3K7 Inflammatory factors modulated by the pathways include Il1-β and Il8 .

9. The use according to claim 1, characterized in that: The application of dihydromyricetin in mammalian anti-inflammatory and anti-aging drugs.

10. The use according to claim 1, characterized in that: The application of dihydromyricetin in drugs for regulating immune homeostasis.

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