Working solution with cell cycle regulation and cellular immune function and use method thereof

Abstract

The present application relates to the technical field of new use of compounds, and aims to provide a working solution with cell cycle regulation and cell immune function and a use method.The effective components of the working solution are butyric acid and palmitic acid, the rest is DMSO and ddH2O used as solvents, and cell culture medium used for providing cell nutrition and promoting cell growth and proliferation; wherein the mass ratio of butyric acid to the cell culture medium is 0.013:100, and the mass ratio of palmitic acid to the cell culture medium is 0.00064:100.The effect of the reagent for enhancing cell immunity and relieving cell cycle arrest when used for cell culture is very significant.The reagent components are all fatty acids, which are one of the components of body cell metabolites, and have no obvious toxic and side effects on body cells, so that the reagent for regulating cell cycle and enhancing cell immunity is safer, more convenient and has fewer side effects when used.

Description

Technical Field

[0001] This invention belongs to the field of new applications of compounds, and relates to a working solution and its method of use that has the function of regulating cell cycle and cellular immunity. Background Technology

[0002] Butyric acid, a short-chain fatty acid, not only provides energy to intestinal cells but also promotes their differentiation, proliferation, and maturation, enhances intestinal immunity, and possesses a wide range of pharmacological effects, including anti-inflammatory, antioxidant, antitumor, and metabolic regulatory activities. Studies have reported that butyric acid inhibits induced inflammatory responses in the mouse intestine. It can activate colonic immune cells such as macrophages, promote T lymphocyte differentiation, and ultimately suppress colonic inflammation. Butyric acid can also protect digestive gland cells from apoptosis by inhibiting the PERK-CHOP pathway of endoplasmic reticulum stress.

[0003] Palmitic acid, a long-chain saturated fatty acid, is often used as a feed additive. It can directly stimulate the host to produce an immune response, especially IgA antibodies, through multiple pathways such as promoting the production of pro-inflammatory cytokines IL-6 and TNF-α, or directly stimulate the host to produce IgA antibodies, thereby forming intestinal mucosal immunity and protecting the intestinal immune barrier function.

[0004] Current research on the application of butyric acid and palmitic acid is limited to the stimulatory or promoting effects of the two fatty acids on host cells to enhance the immune capacity of the host cells or the body. No research has been reported on the methods and effects of using butyric acid and palmitic acid in combination. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a new use for the combined use of butyric acid and palmitic acid, specifically to provide a working solution and method of use that has the function of regulating cell cycle and cellular immunity.

[0006] To solve the technical problem, the technical solution of the present invention is as follows:

[0007] A working solution is provided that has the functions of regulating cell cycle and cellular immunity. The effective components of the working solution are butyric acid and palmitic acid, with the balance being DMSO and ddH2O used as solvents, and cell culture medium used to provide cell nutrition and promote cell growth and proliferation. The mass ratio of butyric acid to cell culture medium is 0.013:100, and the mass ratio of palmitic acid to cell culture medium is 0.00064:100.

[0008] In this invention, the butyric acid has a CAS number of 107-92-6, a molecular formula of C4H8O2, and a molecular weight of 88.11; the palmitic acid has a CAS number of 57-10-3, and a molecular formula of C... 16 H32 O2 has a molecular weight of 256.424.

[0009] As a preferred embodiment of the present invention, the cell culture medium is any one of DMEM medium, 1640 medium, F12 medium or MEM medium; these are all mature commercial products and can be obtained commercially.

[0010] The active ingredients in cell culture media include amino acids, sugars, minerals, vitamins, oxidants, and antibiotics. For example, amino acids include glycine, tyrosine, alanine, histidine, phenylalanine, threonine, cysteine, valine, and glutamic acid. Sugars include glucose, galactose, cytose, polylactic acid, and polydextrose. Major minerals include calcium, phosphorus, magnesium, sodium, and potassium. Vitamins include vitamin B12, nicotinamide, acetylcholine, inositol, folic acid, vitamin E, and vitamin C. Oxidants include protease removers, glutathione, hydrogen peroxide, and indoletriol. Antibiotics include streptomycin, streptomycin B, and amphotericin B.

[0011] The present invention also provides a method for preparing the aforementioned working solution, comprising the following steps:

[0012] (1) Palmitic acid was dissolved in DMSO at a ratio of 25.65 mg: 10 mL to obtain a palmitic acid mother liquor with a concentration of 10 mM; butyric acid was dissolved in ddH2O at a ratio of 0.881 g: 10 mL to obtain a butyric acid mother liquor with a concentration of 1 M.

[0013] (2) Take palmitic acid stock solution and butyric acid stock solution according to the ratio of stock solution to cell culture medium of 2.5 μL / mL and 1.5 μL / mL respectively; then add them to the cell culture medium in sequence and mix well to prepare working solution; the percentage content of butyric acid and palmitic acid in working solution is 0.013% and 0.00064% respectively.

[0014] The present invention further provides a method for using the aforementioned working solution, which is used for cell culture. When using the solution, the working concentrations of butyric acid and palmitic acid are 1.5 mM and 25 μM, respectively, meaning that there are 0.013 g of butyric acid and 0.00064 g of palmitic acid per 100 g (mL) of cell culture medium.

[0015] As a preferred embodiment of the present invention, the working fluid is used in any of the following methods:

[0016] (1) Pretreatment: For ongoing cell culture experiments, discard the original cell culture medium in the well plate; add 2 mL of working solution to pretreatment the cells, then discard the working solution and wash twice with Hank's solution, and then perform subsequent operations on the cells according to the experimental protocol;

[0017] (2) Simultaneous processing: For ongoing cell culture experiments, discard the original cell culture medium in the well plate and wash twice with Hank's solution; while performing subsequent operations on the cells according to the experimental protocol, use 2 mL of working solution for cell culture.

[0018] (3) Post-processing: After processing the cells according to the experimental protocol, wash them twice with Hank's solution and add 2 mL of working solution for cell culture.

[0019] Description of the invention principle:

[0020] The combined reagents provided by this invention can regulate cell cycle and cellular immune function. Butyric acid can directly enhance cellular innate immune function by affecting the upregulation of the key interferon-stimulating factor ISG15, while palmitic acid can alleviate cell cycle arrest and promote cell growth and replication by regulating proteins such as Cyclin A2.

[0021] According to literature reports, butyric acid can induce immature dendritic cells and promote the differentiation of T lymphocytes, enabling them to exert their unique cellular immune function. Palmitic acid can enhance the specific immune function of immune cells by increasing the expression levels of inflammatory responses such as interleukin and specific antibodies, thereby improving their ability to resist pathogen invasion.

[0022] This invention uses epithelial cells and other cells with weak inflammatory responses and lacking specialized immune function as research subjects. In-depth research revealed that butyrate can activate epithelial cell interferon-related pathways through interferon-stimulating factor ISG15, directly enhancing cellular innate immune function and improving the epithelial cell immune barrier. When palmitic acid is used concurrently, the transcription and expression levels of ISG15, an innate immune molecule in the interferon pathway, are significantly higher than when palmitic acid is absent, indicating that palmitic acid has a synergistic promoting effect on the direct enhancement of cellular innate immune function produced by butyrate. Palmitic acid can alleviate cell cycle arrest caused by butyrate by regulating proteins such as Cyclin A2, promoting cell growth and replication. Furthermore, when butyrate is used in conjunction, the expression level of Cyclin A2 is upregulated more significantly than when palmitic acid is used alone, suggesting that butyrate has a synergistic promoting effect on the cell cycle arrest and cell growth and replication-promoting effects produced by palmitic acid.

[0023] Based on existing results and literature reports, butyric acid inhibits inflammation and apoptosis, and can alleviate the inflammatory response and cell damage caused by palmitic acid, thereby regulating cell state. Palmitic acid can regulate the cell cycle and promote cell growth, thereby increasing cell number. This invention combines butyric acid and palmitic acid in a specific ratio, which can keep cells in a good growth state. The two work synergistically to achieve optimal regulation of cell cycle and cellular immune function.

[0024] Compared with the prior art, the technical effects of the present invention are:

[0025] 1. The reagent described in this invention is highly effective in enhancing cellular immunity and alleviating cell cycle arrest. Experiments have shown that it can significantly increase the expression levels of the innate immune marker ISG15 and the cell cycle marker Cyclin A2 in IPEC-J2 cells. When applied to cell infection virus assays, the reagent exhibits antiviral activity, can alleviate cell cycle arrest caused by viral infection, significantly reduce intracellular viral copy number, and inhibit viral protein expression.

[0026] 2. Unlike current drugs such as hormones, antibiotics, and synthetic chemicals used for cellular immune function and cell cycle regulation, the reagent components butyric acid and palmitic acid are both fatty acids, which are components of the body's cellular metabolic products. They have no obvious toxic side effects on the body's cells and are safer, more convenient, and have fewer side effects when used as reagents to regulate the cell cycle and enhance cellular immunity. Attached Figure Description

[0027] Figure 1 The structural formulas of butyric acid and palmitic acid, the components of the reagent described in this invention, are shown below.

[0028] Figure 2 The results of the cytotoxicity tests on the reagent components are as follows: 0–3 mM butyric acid had no effect on cell survival, and 0–50 μM palmitic acid had no effect on cell survival.

[0029] Figure 3The effects of different combinations of the reagent components on cellular immune function and cell cycle were investigated. 1 was the untreated control (butyric acid to palmitic acid mass ratio 0%:0%), 2 was palmitic acid treatment (butyric acid to palmitic acid mass ratio 0%:0.00128%, working concentration of palmitic acid 50 μM), 3 was treatment with a combination of butyric acid and palmitic acid (butyric acid to palmitic acid mass ratio 0.0065%:0.00096%, working concentrations of butyric acid and palmitic acid were 0.75 mM and 37.5 μM, respectively), and 4 was treatment with a combination of butyric acid and palmitic acid (butyric acid...). Butyric acid and palmitic acid were used in the following treatment groups: 1) Butyric acid and palmitic acid were used in a 0.013%:0.00064% mass ratio (working concentrations of butyric acid and palmitic acid were 1.5 mM and 25 μM, respectively); 2) Butyric acid and palmitic acid were used in combination (butyric acid and palmitic acid mass ratio was 0.195%:0.00032% mass ratio (working concentrations of butyric acid and palmitic acid were 2.25 mM and 12.5 μM, respectively); 3) Butyric acid was used in a 0.026%:0% mass ratio (working concentration of butyric acid was 3 mM). The expression levels of the innate immune molecule ISG15 and the cell cycle-related factor Cyclin A2 were measured in each group of cells. When butyric acid and palmitic acid are used in combination, the transcriptional level of ISG15 and the expression levels of ISG15 and Cyclin A2 are significantly increased. When the mass ratio of butyric acid to palmitic acid is 0.013%:0.00064%, the expression levels of ISG15 and Cyclin A2 are the highest, and the regulatory effect on cellular immune function and cell cycle is the best.

[0030] Figure 4 This describes the application of the reagents in different combinations in cell virus infection assays. 1 is an untreated control (butyric acid to palmitic acid mass ratio 0%:0%), 2 is palmitic acid treatment (butyric acid to palmitic acid mass ratio 0%:0.00128%, working concentration of palmitic acid 50 μM), 3 is a treatment using a combination of butyric acid and palmitic acid (butyric acid to palmitic acid mass ratio 0.0065%:0.00096%, working concentrations of butyric acid and palmitic acid are 0.75 mM and 37.5 μM, respectively), and 4 is a treatment using a combination of butyric acid and palmitic acid (butyric acid... Butyric acid and palmitic acid were used in the following treatment groups: 1) Butyric acid and palmitic acid were used in a ratio of 0.013% to 0.00064% (working concentrations of butyric acid and palmitic acid were 1.5 mM and 25 μM, respectively); 2) Butyric acid and palmitic acid were used in combination (butyric acid and palmitic acid were used in a ratio of 0.195% to 0.00032% (working concentrations of butyric acid and palmitic acid were 2.25 mM and 12.5 μM, respectively); 3) Butyric acid was used in combination (butyric acid and palmitic acid were used in a ratio of 0.026% to 0% (working concentration of butyric acid was 3 mM). After cell infection with the virus in each group, the transcription and expression levels of PEDV N protein were measured. The combined use of butyric acid and palmitic acid significantly inhibited intracellular viral replication through regulation of cellular immune function and the cell cycle.

[0031] Figure 5 This describes the application of the reagent in cell virus infection assays using different treatment methods. Lane 1 contains cell samples uninfected with PEDV, lane 2 contains PEDV-infected samples, and lane 3 contains PEDV-infected samples treated with the reagent; A represents pretreatment before virus infection, B represents simultaneous treatment during virus infection, and C represents post-virus infection treatment. All three treatment methods can regulate cellular immune function and cell cycle to inhibit intracellular viral replication.

[0032] Figure 6 This study illustrates the application of the reagent treatment in a cell virus infection assay. Cells treated with a combination of butyric acid and palmitic acid (butyric acid to palmitic acid mass ratio of 0.013%:0.00064%, working concentrations of butyric acid and palmitic acid of 1.5 mM and 25 μM, respectively) were infected with PEDV, and the expression of viral proteins, cell cycle, and related marker molecules was detected. 1 represents the cell group not infected with PEDV, 2 represents the cell group treated with the reagent combination, 3 represents the cell group infected with PEDV, and 4 represents the cell group infected with PEDV after using the reagent combination. A shows the changes in cell cycle in each group; B shows the expression of cell cycle-related proteins such as Cyclin A2 and PEDV N protein. Detailed Implementation

[0033] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0034] Example 1

[0035] IPEC-J2 cells were cultured in 96-well plates until the cell density reached 60%. 0, 1, 1.5, 2, 2.5, and 3 mM butyric acid, and 1, 5, 10, 20, and 50 μM palmitic acid were added to each well, respectively. A negative control was also included. Each group had eight replicate wells. After incubation at 37°C for 12 h, 10 μL of CCK-8 (5 mg / mL) was added to each well, and the cells were incubated at 37°C in the dark for 2 h. The absorbance at 450 nm was measured for each well. Cell viability (%) = OD 450 nm (Control group) / OD 450 nm (treatment group) × 100.

[0036] Experimental results:

[0037] The results of the cell viability assay are attached. Figure 2 As shown, butyric acid, a reagent component, has no significant effect on cell survival in the concentration range of 0–5 mM, and palmitic acid, a reagent component, has no significant effect on cell survival in the concentration range of 0–50 μM.

[0038] Example 2

[0039] (1) Cell treatment: IPEC-J2 cells were cultured in 6-well plates until the cell density reached 70-80%. The culture medium was discarded, and the cells were washed twice with Hank's solution. Cells were then treated according to the following methods: no control (butyric acid to palmitic acid mass ratio 0%:0%), palmitic acid treatment (butyric acid to palmitic acid mass ratio 0%:0.00128%, working concentration of palmitic acid 50 μM), combined butyric acid and palmitic acid treatment (butyric acid to palmitic acid mass ratio 0.0065%:0.00096%, working concentrations of butyric acid and palmitic acid 0.75 mM and 37.5 μM, respectively), and combined butyric acid and palmitic acid treatment (butyric acid to palmitic acid mass ratio 0.013%:0.00064%). Six groups were formed by pretreatment with butyric acid and palmitic acid at working concentrations of 1.5 mM and 25 μM, respectively; combined treatment with butyric acid and palmitic acid at a mass ratio of 0.195%:0.00032% and working concentrations of 2.25 mM and 12.5 μM, respectively; and butyric acid treatment at a mass ratio of 0.026%:0% and working concentration of 3 mM. Each group was pretreated with 0 μL, 0 μL, 1.5 μL, 3 μL, 4.5 μL, and 6 μL of 1 M butyric acid stock solution and 0 μL, 10 μL, 7.5 μL, 5 μL, 2.5 μL, and 0 μL of 10 mM palmitic acid stock solution, respectively.

[0040] (1) Sample collection: For samples used to detect cell and protein expression, discard the culture supernatant, add RIPA lysis buffer to lyse and harvest, and store at -80℃. For samples used to detect transcription, discard the culture supernatant, add Trizol lysis buffer to lyse and harvest, and store at -80℃.

[0041] (2) Lysated samples for detecting cellular protein expression were prepared for SDS-PAGE by adding protein loading buffer. 20 μL of protein sample was added to each well, and the mixture was kept at a constant voltage of 120 V for 1 h. The protein was then transferred to a PVDF membrane under constant current conditions of 0.26 A for 45 min. The membrane was blocked with 5% skim milk at room temperature for 1 h, and washed three times with TBST for 10 min each time. The PVDF membrane was cut to size according to the protein molecular weight and incubated overnight at 4 °C with 1:500 diluted ISG15 antibody, Cyclin A2 antibody, and cell GAPDH antibody, respectively. The next day, the membrane was washed three times with TBST, incubated with HRP-labeled secondary antibody (1:1000 diluted) at 37 °C for 1.5 h, and washed three times with TBST. ECL substrate was added, and imaging analysis was performed using a chemiluminescence imaging system.

[0042] (3) Total RNA was extracted from cell lysate samples used for transcriptional level detection using an RNA extraction kit and reverse transcribed. The reverse transcription system was as follows: 8 μL of RNase-free water, 4 μL of 4x gDNA wiper mix, and 4 μL of RNA were mixed and reacted at 42°C for 2 min. Then, 4 μL of 5x HiScript IT qRT Super Mix 11 was added, mixed, and briefly centrifuged. The reaction was carried out at 37°C for 15 min and terminated at 85°C for 5 s. Using ISG15 specific primers and cDNA as a template, amplification was performed. The amplification system and conditions were as follows: 10 μL of SYBR, 7.2 μL of RNase-free water, 0.4 μL each of forward and reverse primers, and 2 μL of cDNA. Cycles were performed at 95°C for 10 min, 95°C for 15 s, 58°C for 50 s, and 72°C for 2 s, for a total of 40 cycles. The melting curve ranged from 65°C to 95°C.

[0043] Experimental results: The expression levels of cellular innate immune molecule ISG15 and cell cycle-related factor Cyclin A2 were detected in each group of cells. The results are shown in the attached figure. Figure 3 As shown, when butyric acid and palmitic acid are used in combination, the transcriptional level of ISG15 and the expression levels of ISG15 and Cyclin A2 are significantly increased. When butyric acid and palmitic acid are used in combination at a mass ratio of 0.013%:0.00064%, the expression levels of ISG15 and Cyclin A2 are the highest, and the regulatory effect on cellular immune function and cell cycle is the best.

[0044] Example 3

[0045] (1) Cell treatment: IPEC-J2 cells were cultured in 6-well plates until the cell density reached 70-80%. The culture medium was discarded, and the cells were washed twice with Hank's solution. Cells were then treated according to the following methods: no control (butyric acid to palmitic acid mass ratio 0%:0%), palmitic acid treatment (butyric acid to palmitic acid mass ratio 0%:0.00128%, working concentration of palmitic acid 50 μM), combined butyric acid and palmitic acid treatment (butyric acid to palmitic acid mass ratio 0.0065%:0.00096%, working concentrations of butyric acid and palmitic acid 0.75 mM and 37.5 μM, respectively), and combined butyric acid and palmitic acid treatment (butyric acid to palmitic acid mass ratio 0.013%:0.00064%). Six groups were formed by pretreatment with butyric acid and palmitic acid at working concentrations of 1.5 mM and 25 μM, respectively; combined butyric acid and palmitic acid treatment (butyric acid to palmitic acid mass ratio of 0.195%:0.00032%, working concentrations of butyric acid and palmitic acid of 2.25 mM and 12.5 μM, respectively); and butyric acid treatment (butyric acid to palmitic acid mass ratio of 0.026%:0%, working concentration of butyric acid of 3 mM). Each group was pretreated with 0 μL, 0 μL, 1.5 μL, 3 μL, 4.5 μL, and 6 μL of 1 M butyric acid stock solution and 0 μL, 10 μL, 7.5 μL, 5 μL, 2.5 μL, and 0 μL of 10 mM palmitic acid stock solution, respectively. PEDV infection was then performed at an MOI of 1.

[0046] (2) Sample collection: For samples used to detect cell and protein expression, discard the culture supernatant, add RIPA lysis buffer to lyse and harvest, and store at -80℃. For samples used to detect transcription, discard the culture supernatant, add Trizol lysis buffer to lyse and harvest, and store at -80℃.

[0047] (2) Lysated samples for detecting cellular protein expression were prepared for SDS-PAGE by adding protein loading buffer. 20 μL of protein sample was added to each well, and the mixture was kept at a constant voltage of 120 V for 1 h. The protein was then transferred to a PVDF membrane under constant current conditions of 0.26 A for 45 min. The membrane was blocked with 5% skim milk at room temperature for 1 h, and washed three times with TBST for 10 min each time. The PVDF membrane was cut to size according to the protein molecular weight and incubated overnight at 4 °C with 1:500 diluted PEDV N protein antibody and cellular GAPDH antibody, respectively. The next day, the membrane was washed three times with TBST, incubated with HRP-labeled secondary antibody (1:1000 diluted) at 37 °C for 1.5 h, and washed three times with TBST. ECL substrate was added, and imaging analysis was performed using a chemiluminescence imaging system.

[0048] (3) Total RNA was extracted from cell lysate samples used for transcriptional level detection using an RNA extraction kit and reverse transcribed. The reverse transcription system was as follows: 8 μL of RNase-free water, 4 μL of 4x gDNA wiper mix, and 4 μL of RNA were mixed and reacted at 42°C for 2 min. Then, 4 μL of 5x HiScript IT qRT Super Mix 11 was added, mixed, and briefly centrifuged. The reaction was carried out at 37°C for 15 min and terminated at 85°C for 5 s. Amplification was performed using 5'UTR-specific primers with cDNA as a template. The amplification system and conditions were as follows: 10 μL of SYBR, 7.2 μL of RNase-free water, 0.4 μL each of forward and reverse primers, and 2 μL of cDNA. Cycles were performed at 95°C for 10 min, 95°C for 15 s, 58°C for 50 s, and 72°C for 2 s, for a total of 40 cycles. The melting curve ranged from 65°C to 95°C.

[0049] Experimental results: Changes in viral copy number and PEDV N protein expression levels were detected in each group of cells after PEDV infection. The results are attached. Figure 4 As shown, when butyric acid and palmitic acid are used in combination, both the PEDV viral copy number and the expression level of N protein decrease to some extent.

[0050] Example 4

[0051] IPEC-J2 cells were cultured in 24-well plates. When the cells reached 80% confluence, they were treated with three different methods: (A) Pretreatment before viral infection: The cell culture medium was discarded, and 3 μL of 1M butyric acid stock solution and 5 μL of 10mM palmitic acid stock solution were added to the culture medium. The cells were treated with a combination of butyric acid and palmitic acid (butyric acid to palmitic acid mass ratio of 0.013%:0.00064%, working concentrations of butyric acid and palmitic acid were 1.5mM and 25μM, respectively). The cells were cultured at 37°C for 4 h, and the culture medium containing the aforementioned... (A) For the reagent-containing culture medium, wash twice with Hank's solution, add PEDV virus solution to the culture medium at an MOI=1 infection ratio, incubate at 37℃ for 4 hours, discard the virus-containing culture medium, wash twice with Hank's solution, and then add culture medium containing 5% trypsin and continue culturing for 20 hours; (B) Simultaneous treatment during virus infection: discard the cell culture medium, wash twice with Hank's solution, add PEDV virus solution to the culture medium at an MOI=1 infection ratio, incubate at 37℃ for 4 hours, discard the virus-containing culture medium, wash twice with Hank's solution, add PEDV virus solution to the culture medium at an MOI=1 infection ratio, incubate at 37℃ for 4 hours, discard the virus-containing culture medium, and then add culture medium containing 5% trypsin and continue culturing for 20 hours; After washing twice with s solution, add 3 μL of 1M butyric acid stock solution and 5 μL of 10mM palmitic acid stock solution to the culture medium. Treat the cells with the combination of butyric acid and palmitic acid (butyric acid to palmitic acid mass ratio of 0.013%:0.00064%, working concentrations of butyric acid and palmitic acid are 1.5mM and 25μM, respectively) and continue culturing with 5% trypsin for 20 h; (C) Post-viral infection treatment: Discard the cell culture medium, add 3 μL of 1M butyric acid stock solution and 5 μL of 10mM palmitic acid stock solution to the culture medium, and treat the cells with the combination of butyric acid and palmitic acid. Cells (butyrate and palmitic acid mass ratio of 0.013%:0.00064%, working concentrations of butyrate and palmitic acid of 1.5 mM and 25 μM, respectively) were cultured at 37°C for 4 h. The culture medium containing the reagents was discarded, and the cells were washed twice with Hank's solution. PEDV virus solution was added to the culture medium at an MOI of 1, and the cells were incubated at 37°C for 4 h. The virus-containing culture medium was discarded, and the cells were washed twice with Hank's solution. The cells were then cultured again in medium containing 5% trypsin for 4 h, followed by the addition of the reagents and a further 16 h of culture. Cells uninfected with the virus served as the negative control group, and cells infected with the virus but not treated with the reagents served as the positive control group. The expression of PEDV N protein and the cellular reference protein β-actin were detected.

[0052] Experimental results:

[0053] The effects of different treatments on viral protein expression are shown in the attached figure. Figure 5 As shown, all three treatments can effectively inhibit the expression of PEDV N protein.

[0054] Example 5

[0055] (1) Cell treatment: IPEC-J2 cells were cultured in 6-well plates until the cell density reached 70-80%. The culture medium was discarded, and the cells were washed twice with Hank's solution. Then, 3 μL of 1M butyric acid stock solution and 5 μL of 10mM palmitic acid stock solution were added to the culture medium. The cells were treated with a combination of butyric acid and palmitic acid (butyric acid to palmitic acid mass ratio of 0.013%:0.00064%, working concentrations of butyric acid and palmitic acid of 1.5mM and 25μM, respectively). An untreated group was set up as a control. PEDV infection was performed at an MOI of 1.

[0056] (2) Sample collection: For samples used to detect cell and protein expression, discard the culture supernatant, add RIPA lysis buffer, lyse and harvest, and store at -80℃. For samples used to detect copy number, discard the culture supernatant, add Trizol lysis buffer, lyse and harvest, and store at -80℃.

[0057] (3) Lysated samples used for cell and protein expression detection were prepared for SDS-PAGE electrophoresis by adding protein loading buffer. 20 μL of protein sample was added to each well, and the mixture was kept at a constant voltage of 120 V for 1 h. The protein was then transferred to a PVDF membrane under constant current conditions of 0.26 A for 45 min. The membrane was blocked with 5% skim milk powder at room temperature for 1 h, and washed three times with TBST for 10 min each time. The PVDF membrane was cut to size according to the protein molecular weight and incubated overnight at 4℃ with 1:1000 diluted PEDV N and β-actin monoclonal antibodies, respectively. The next day, the membrane was washed three times with TBST, incubated with HRP-labeled secondary antibody (1:1000 diluted) at 37℃ for 1.5 h, and washed three times with TBST. ECL substrate was added, and imaging analysis was performed using a chemiluminescence imaging system.

[0058] For cell cycle analysis, each tube of cell suspension was incubated with propidium iodide staining solution at 37°C in the dark for 30 minutes. After staining, flow cytometry was used to detect red fluorescence at an excitation wavelength of 488 nm, while simultaneously detecting light scattering to analyze cellular DNA content.

[0059] Experimental results:

[0060] Cell cycle analysis results of cells treated with a combination of butyric acid and palmitic acid (butyric acid to palmitic acid mass ratio of 0.013%:0.00064%, working concentrations of butyric acid and palmitic acid of 1.5 mM and 25 μM, respectively) after viral infection are shown in the attached figure. Figure 6 As shown in Figure A, the expression of cellular and viral proteins is as follows (see attached figure). Figure 6 As shown in B: After PEDV infection, the virus-induced S-phase arrest in the treatment group was significantly alleviated, the expression of the cell cycle-related Cyclin A2 protein was significantly upregulated, and the level of viral N protein decreased.

Claims

1. A working solution with functions of regulating cell cycle and cellular immunity, characterized in that, The active ingredients of this working solution are butyric acid and palmitic acid, with the remainder being DMSO and ddH2O used as solvents, and cell culture medium used to provide cell nutrition and promote cell growth and proliferation. Palmitic acid plays a synergistic role in enhancing the innate immune function of cells directly produced by butyric acid, and alleviates cell cycle arrest caused by butyric acid by regulating CyclinA2 protein, thereby promoting cell growth and replication. The mass ratio of butyric acid to cell culture medium is 0.013:100, and the mass ratio of palmitic acid to cell culture medium is 0.00064:

100.

2. The working fluid according to claim 1, characterized in that, The butyric acid has CAS number 107-92-6, molecular formula C4H8O2, and molecular weight 88.11; the palmitic acid has CAS number 57-10-3, molecular formula C... 16 H 32 O2 has a molecular weight of 256.

424.

3. The working fluid according to claim 1, characterized in that, The cell culture medium is any one of DMEM, 1640, F12 or MEM medium, and its active ingredients include amino acids, sugars, minerals, vitamins, oxidants and antibiotics.

4. The method for preparing the working solution with cell cycle and cellular immune function as described in claim 1, characterized in that, Includes the following steps: (1) Palmitic acid was dissolved in DMSO at a ratio of 25.65 mg: 10 mL to obtain a palmitic acid mother liquor with a concentration of 10 mM; butyric acid was dissolved in ddH2O at a ratio of 0.881 g: 10 mL to obtain a butyric acid mother liquor with a concentration of 1 M. (2) Take palmitic acid stock solution and butyric acid stock solution according to the ratio of stock solution to cell culture medium of 2.5 μL / mL and 1.5 μL / mL respectively; then add them to the cell culture medium in sequence and mix well to prepare working solution; the percentage content of butyric acid and palmitic acid in working solution is 0.013% and 0.00064% respectively.

5. The method of using the working solution with cell cycle and cellular immune function as described in claim 1, characterized in that, This working solution is used for cell culture.

6. The method according to claim 5, characterized in that, Use the working fluid according to any of the following methods: (1) Pretreatment: For ongoing cell culture experiments, discard the original cell culture medium in the well plate; add 2 mL of working solution to pretreat the cells, then discard the working solution and wash twice with Hank's solution, and then perform subsequent operations on the cells according to the experimental protocol; (2) Simultaneous processing: For ongoing cell culture experiments, discard the original cell culture medium in the well plate and wash twice with Hank's solution; while performing subsequent cell operations according to the experimental protocol, use 2 mL of working solution for cell culture. (3) Post-processing: After manipulating the cells according to the experimental protocol, wash them twice with Hank's solution and add 2 mL of working solution for cell culture.

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