Branched fatty acids and uses thereof

Abstract

The invention belongs to the field of nutrient substance research, particularly relates to branched-chain fatty acid and application thereof, and more particularly relates to application of branched-chain fatty acid in regulation and control of occurrence and development of colorectal cancer or providing nutritional support for patients with colorectal cancer. The research finds that the branched chain fatty acid can play a role in regulating the occurrence and development of the colorectal cancer. Experimental data show that the branched-chain fatty acid, especially the branched-chain fatty acid of which the branched chain is methyl, has the effect of inhibiting the growth of colorectal cancer cells, and also has the effect of regulating immune cells, so that tumor growth can be regulated through immunity. Therefore, the branched chain fatty acid can prevent and treat the colorectal cancer or provide nutritional support for patients with the colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the field of nutrient research, and specifically relates to branched-chain fatty acids and uses thereof, and more specifically to uses of branched-chain fatty acids in regulating the occurrence and development of colorectal cancer or providing nutritional support for colorectal cancer patients. Background Art

[0002] Colorectal cancer (including colon cancer and rectal cancer) is the third most common cancer in the world and the second leading cause of cancer-related death. The disease burden caused by it is serious and has become a major global public health problem. Previous studies have confirmed that colorectal cancer is a malignant tumor closely related to lifestyle, dietary factors and genetic factors. At present, the prevention and treatment of colorectal cancer is facing tremendous pressure. How to effectively prevent and control colorectal cancer based on the changing patterns of major risk factors, morbidity and mortality is an urgent problem to be solved. The three-level cancer prevention and control strategy is an important means of preventing and treating cancer, including zero-level prevention-preventing disease, primary prevention-preventing disease occurrence, secondary prevention-early detection, early diagnosis and early treatment, to tertiary prevention-preventing disease from developing in a serious or worsening direction. At the same time, the existing health strategy has also undergone great changes, from focusing on treating existing diseases to preventing diseases. This change provides more functional development opportunities for more nutrients.

[0003] Branched chain fatty acid (BCFA) refers to fatty acids with one or more branched alkyl groups or other functional groups on the alkyl chain of fatty acids. Branched alkyl groups located on the second carbon atom at the end of the alkyl group are called iso- or iso- fatty acids, and those located on the third carbon atom at the end of the alkyl group are called anteiso- or anteiso- fatty acids. Due to the unique branched structure of BCFA, it has good physical and chemical properties: low freezing point and pour point, good thermal stability and oxidative stability, good solubility, low foaming and good air and water permeability of the formed membrane. BCFA is widely present in nature, but the content is low. It is reported that a certain amount of BCFA with different structures exists in the milk and internal tissues of ruminants (such as cow and sheep milk and meat products), and in human milk. At the same time, BCFA is more abundant in sebum and the intestines of newborns. The BCFA spectrum of fetal hair and meconium shows that BCFAs are the main components of the gastrointestinal tract of normal and healthy full-term newborns, while the BCFAs in the intestines of premature infants are much less than those of normal and healthy babies.

[0004] Many studies have shown that BCFA has unique physiological regulatory functions, including improving the intestines. Current studies have found that BCFAs have the potential for preventing necrotizing enterocolitis. For example, Reference 1 discloses a method for preventing or treating a subject's gastrointestinal disease, wherein the gastrointestinal disease is necrotizing enterocolitis, and the method comprises administering one or more branched-chain fatty acids to the subject; Reference 2 discloses the use of a branched-chain fatty acid derived from goat milk in preventing or treating a gastrointestinal disease, wherein the gastrointestinal disease is inflammatory gastrointestinal disease or necrotizing enterocolitis, and the branched-chain fatty acid is prepared according to the following method: urea and ethanol are added to the fatty acid obtained from the total fat source of goat milk, and the mixture is refluxed and stirred to obtain a mixture, the mixture is crystallized under low temperature conditions, and the liquid portion is obtained after filtration, and after washing with sodium chloride, petroleum ether is added, stirred and filtered, and after stratification, the petroleum ether layer containing free fatty acids is dried with anhydrous sodium sulfate, and the petroleum ether is removed to obtain branched-chain fatty acids.

[0005] References:

[0006] Reference 1: CN101801200A;

[0007] Reference 2: CN115300530A. Summary of the invention

[0008] Problem that the invention aims to solve

[0009] There are already certain prevention and control strategies for colorectal cancer. For example, the three-level prevention and control strategy can effectively reduce the incidence of colorectal cancer. However, it is still difficult to fully implement the strategies of primary prevention, secondary early diagnosis and tertiary prevention of deterioration. The effective prevention and control of colorectal cancer and nutritional support for colorectal cancer patients are still issues to be solved.

[0010] In addition, the current exploration of the physiological regulatory functions of branched-chain fatty acids has found that branched-chain fatty acids have the effect of preventing and treating inflammatory gastrointestinal diseases or necrotizing enterocolitis. However, there is still room for exploration of other physiological regulatory functions of branched-chain fatty acids.

[0011] Based on this, the purpose of the present invention is to provide the use of branched-chain fatty acids in regulating the occurrence and development of colorectal cancer or providing nutritional support for colorectal cancer patients.

[0012] Solutions for solving problems

[0013] Through long-term research by the inventors of the present invention, it is found that the above technical problems can be solved by the following technical solutions:

[0014] [1] Use of branched-chain fatty acids in (i) or (ii) as follows:

[0015] (i) preparing special medical purpose formula foods suitable for patients with intestinal cancer;

[0016] (ii) preparing a pharmaceutical composition for preventing and / or treating intestinal cancer.

[0017] [2]. The use according to [1], characterized in that the branched-chain fatty acid is a branched-chain fatty acid in which the branch is a methyl group.

[0018] [3]. The use according to [1] or [2], characterized in that the branched-chain fatty acid is iso-C15:0, anteiso-C15:0, iso-C17:0 or anteiso-C17:0.

[0019] [4]. The use according to any one of [1] to [3], characterized in that the intestinal cancer is colorectal cancer.

[0020] [5]. The use according to any one of [1] to [4], characterized in that the prevention and / or treatment of intestinal cancer includes inhibiting the activity of intestinal cancer cells, promoting the expression of IFN-β by macrophages, promoting the expression of IFN-β by dendritic cells, promoting the expression of antigen processing associated transporter by dendritic cells, promoting the expression of pro-inflammatory cytokines by dendritic cells, and inhibiting the expression of anti-inflammatory cytokines by dendritic cells.

[0021] [6]. The use according to [1] to [4] is characterized in that the prevention and / or treatment of intestinal cancer includes inhibiting the activity of intestinal cancer cells, promoting the expression of IFN-β by macrophages, promoting the expression of IFN-β by dendritic cells, promoting the expression of antigen processing associated transporter by dendritic cells, promoting the expression of pro-inflammatory cytokines by dendritic cells and inhibiting the expression of anti-inflammatory cytokines by dendritic cells.

[0022] [7]. The use according to [5] or [6], characterized in that the pro-inflammatory cytokines include any one or more of IL-1β and TNF-α.

[0023] [8] The use according to [5] or [6], characterized in that the pro-inflammatory cytokines include IL-1β and TNF-α.

[0024] [9]. The use according to [5] or [6], characterized in that the anti-inflammatory cytokine includes IL-10.

[0025] Effects of the Invention

[0026] The present invention has found that branched-chain fatty acids can play a role in regulating the occurrence and development of colorectal cancer. Experimental data show that branched-chain fatty acids, especially those with methyl groups, have the effect of inhibiting the growth of colorectal cancer cells. At the same time, they can also regulate immune cells, thereby regulating tumor growth through immunity. Therefore, branched-chain fatty acids can prevent and treat colorectal cancer, or provide nutritional support for colorectal cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Effect of BCFA in Example 1 on the viability of MC38, CT26 and HT29 cells.

[0028] Figure 2A : Lucia luciferase expression levels of RAW-Lucia ISG cells in Example 2 after being pretreated with BCFA for 24 hours and stimulated with ISD for 6 hours; among them, *P<0.05, **P<0.01, ***P<0.001.

[0029] Figure 2B : Lucia luciferase expression level of RAW-Lucia ISG cells in Example 2 after being pretreated with BCFA for 24 hours and stimulated with Poly(I:C) for 6 hours; wherein, ns is meaningless, *P<0.05, **P<0.01.

[0030] Figure 2C : Comparison results of the ability of anteiso-C15:0 to induce type I interferon with EPA, DHA and LA in Example 2; wherein, ns is meaningless, *P<0.05, **P<0.01.

[0031] Figure 2D : The expression level detection results of IFN-β gene and its downstream genes IP-10 and ISG15 mRNA after RAW-Lucia ISG cells were treated with anteiso-C15:0 and EPA for 24 hours in Example 2; ***P<0.001.

[0032] Figure 3A : In Example 3, the relative expression levels of IFN-β, IP-10 and ISG15 mRNA in DC cells were detected by real-time fluorescence quantitative PCR after anteiso-C15:0 treatment for 24 hours; among them, **P<0.01, ***P<0.001.

[0033] Figure 3B: In Example 3, real-time fluorescence quantitative PCR was used to detect the relative expression levels of Tapbp, Tap1, Tap2 and β2m mRNA in DC cells 24 hours after anteiso-C15:0 treatment; ***P<0.001.

[0034] Figure 3C : In Example 3, real-time fluorescence quantitative PCR was used to detect the relative expression levels of IL-1β, TNF-α and IL-10 mRNA in DC cells after anteiso-C15:0 treatment for 24 hours; ***P<0.001.

[0035] Figure 4A : The results of cell viability detection of T cells in Example 4 after treatment with anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C17:0 for 24 or 48 hours.

[0036] Figure 4B : Results of IFN-γ secretion level detection in the supernatant after DC cells were pretreated with anteiso-C15:0 and co-cultured with T cells in Example 4; ns is meaningless, ***P<0.001.

[0037] Figure 4C : In Example 4, flow cytometry was used to detect the expression of CD11c, CD80, CD86 and PD-L1 on the surface of DC cells.

[0038] Figure 4D : The expression results of CD11c, CD80, CD86 and PD-L1 on the surface of DC cells were quantitatively analyzed by flow cytometry in Example 4; ns is meaningless, *P<0.05, ***P<0.001. DETAILED DESCRIPTION

[0039] The following describes the embodiments of the present invention, but the present invention is not limited thereto. Various changes can be made within the scope of the present invention, and the embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0040] I. Definition of Terms

[0041] In the present invention, a numerical range expressed using “a value A to a value B”, “a value A - a value B”, “above a value A” or below a value A means a range including the endpoints A and B.

[0042] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0043] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0044] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0045] In the present invention, the term "about" is used to define the numerical range and parameters of the present invention are approximate values, and the specific relevant values ​​are presented as accurately as possible. Unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" usually means that the actual value is within ±5%, ±3%, ±1% or ±0.5% of a particular value or range.

[0046] In the present invention, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0047] In the present invention, for the convenience of expressing fatty acids, characters such as "C15:0", "iso-C15:0", "anteiso-C15:0" and the like are used to refer to different types of fatty acids, wherein "C15" indicates that the fatty acid contains 15 carbon atoms in total, "0" indicates that the fatty acid contains 0 unsaturated double bonds, "iso" indicates that the fatty acid contains a branched methyl group and the branched methyl group is located on the second carbon atom from the alkyl end of the fatty acid main chain, and "anteiso" indicates that the fatty acid contains a branched methyl group and the branched methyl group is located on the third carbon atom from the alkyl end of the fatty acid main chain.

[0048] In the present invention, the term "special medical purpose formula food" refers to a formula food specially processed and prepared to meet the special needs of people with specific disease states, such as colorectal cancer patients for nutrients or diets. This type of product must be consumed alone or in combination with other foods under the guidance of a doctor or clinical nutritionist. Special medical purpose formula foods can be used as a nutritional supplement to provide nutritional support for the treatment, rehabilitation and body function maintenance of people with specific disease states, such as colorectal cancer patients, but the special medical purpose formula foods described in the present invention are not intended to prevent and / or treat diseases such as colorectal cancer and other intestinal cancers.

[0049] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0050] II. Branched-chain fatty acids

[0051] The present invention does not particularly limit the source of branched fatty acids. For example, they can be separated and extracted from natural substances containing branched fatty acids, such as bacterial biofilms, animal sebum, mammary tissue, etc., or prepared by biological or chemical synthesis. At present, the common method in the art is to separate and extract branched fatty acids from lanolin. Free lanolin alcohol and free lanolin acid soap can be prepared by saponification reaction of lanolin, and then the branched fatty acids in lanolin are obtained through multiple steps such as alcohol soap separation process and preparation and extraction of fatty acids.

[0052] In some embodiments, the branched-chain fatty acid described herein is a single-branched-chain fatty acid.

[0053] In some embodiments, the branched-chain fatty acid described in the present invention is a branched-chain fatty acid whose branch chain is an alkyl group such as a methyl group; preferably, the branched-chain fatty acid is a branched-chain fatty acid whose branch chain is a methyl group.

[0054] In some specific embodiments, the branched-chain fatty acid described in the present invention includes any one of iso-C15:0, anteiso-C15:0, iso-C17:0 and anteiso-C17:0.

[0055] In some preferred embodiments, the branched-chain fatty acid described in the present invention is iso-C15:0, anteiso-C15:0, iso-C17:0 or anteiso-C17:0.

[0056] III. Uses of branched-chain fatty acids

[0057] The present invention provides the use of branched-chain fatty acids in the following (i) or (ii):

[0058] (i) preparing special medical purpose formula foods suitable for patients with intestinal cancer;

[0059] (ii) preparing a pharmaceutical composition for preventing and / or treating intestinal cancer.

[0060] For branched chain fatty acids, as described above in Section II.

[0061] The present invention has found that a variety of branched-chain fatty acids have the effect of directly inhibiting the growth of intestinal cancer cells, and at the same time, they also have the effect of regulating immune cells, such as regulating the activity of immune cells, regulating the ability of immune cells to release cytokines (such as type I interferon, IL-1β, TNF-α and IL-10, etc.), and regulating the antigen presentation of immune cells (such as increasing the relative expression level of mRNA of antigen presentation-related genes Tapbp, Tap1, Tap2 and β2m in DC cells, etc.). In addition, it is known that branched-chain fatty acids are the main components of trace fatty acids in human milk fat, and are important nutrients in the intestines of infants and young children before birth, and play an important role in maintaining the normal physiological functions of organisms. Therefore, branched-chain fatty acids can be used as nutrients to prepare special medical formula foods suitable for intestinal cancer patients, providing nutritional support for intestinal cancer patients. At the same time, branched-chain fatty acids can also be used to prepare pharmaceutical compositions for the prevention and / or treatment of intestinal cancer.

[0062] In some embodiments, the intestinal cancer described in the present invention includes at least one of colorectal cancer, duodenal cancer, jejunal cancer and ileal cancer; preferably colorectal cancer.

[0063] The present invention does not impose any particular limitation on other ingredients in the special medical purpose formula food except branched-chain fatty acids, as long as they comply with the requirements of relevant laws and regulations.

[0064] The special medical formula food described in the present invention can be a complete nutritional formula food, a specific complete nutritional formula food or an incomplete nutritional formula food (mainly fat / fatty acid components); wherein, a complete nutritional formula food refers to a special medical formula food that can be used as a single nutritional source to meet the nutritional needs of a target population, such as intestinal cancer patients, and a specific complete nutritional formula food refers to a special medical formula food that can be used as a single nutritional source to meet the nutritional needs of a target population, such as intestinal cancer patients, under specific diseases or medical conditions, such as intestinal cancer; an incomplete nutritional formula food refers to a special medical formula food that can meet part of the nutritional needs of a target population, such as intestinal cancer patients, and is not suitable as a single nutritional source.

[0065] Example

[0066] The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out under normal conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used are all commercially available conventional products.

[0067] Example 1: BCFA inhibits the growth of colorectal cancer cells

[0068] 1 Experimental methods

[0069] 1.1 Cell lines and cell culture

[0070] Mouse colon cancer cells MC38, CT26 and human colon cancer cells HT29 were ordered from the American type culture collection (ATCC). According to the culture conditions recommended by ATCC, the above cells were cultured in DMEM, RPMI-1640 or McCoy's medium supplemented with 10% v / v fetal bovine serum, 100 IU / mL penicillin and streptomycin. All cells were cultured in a cell culture incubator at 37°C and 5% v / v CO2, and no mycoplasma contamination was detected.

[0071] 1.2 Cell viability assay

[0072] Cell viability was detected by Cell Counting Kit-8 (CCK-8). HT29, CT26, and MC38 cells in the logarithmic growth phase were inoculated into 96-well plates with a cell suspension (8000 cells / well) at a volume of 200 mL / well and cultured for 12 hours until the cells were completely attached. The treatment group, solvent control group, and blank control group were treated with different doses of BCFA, BSA solvent (PBS containing BSA), and culture medium, respectively. BCFA monomers (iso-C15:0, iso-C17:0, anteiso-C15:0, and anteiso-C17:0) were dissolved in PBS containing 0.22 g BSA. Cells were treated with different concentrations of BCFA monomers, and 4 replicate wells were set up in each group and incubated in a 37°C, 5% v / v CO2 incubator for 48 hours. After the culture was completed, 10 μL of CCK-8 reagent was added to each well. After incubation for 4 h, the absorbance (OD) value at 450 nm was measured using a Biotek Synergy H1 microplate reader. Cell viability (%) = [(OD value of the treatment group - OD value of the blank control) / (OD value of the solvent control - OD value of the blank control)] × 100. The above method was used to evaluate the effect of BCFA on the proliferation of CT26, MC38 and HT29 cells. The experiment was repeated three times.

[0073] 2 Experimental results

[0074] Mouse colon cancer cells (CT26, MC38) and human colon cancer cells (HT29) were treated for 48 hours, and the CCK-8 study detected the effects of four common BCFAs (iso-C15:0, iso-C17:0, anteiso-C15:0 and anteiso-C17:0) in ruminants on the viability of colorectal cancer cells. Different concentrations (0-50 μM) of iso-C15:0, iso-C17:0, anteiso-C15:0 and anteiso-C17:0 were used to detect the effects of BCFA on the viability of colon cancer CT26, MC38 and HT29 cells. Figure 1 As shown in the figure, the inhibitory effect of anteiso-C17:0 on the viability of CT26, MC38 and HT29 cells was dose-dependent. After anteiso-C17:0 (0-50 μM) treated cells for 48 hours, the cell viability decreased by about 5%-50%. Anteiso-C15:0 had a more obvious inhibitory effect on CT26 and HT29 cells, with an inhibition rate of about 8%-50%, and the effect of inhibiting the viability of MC38 cells was not obvious. In contrast, after treatment with iso-C15:0 and iso-C17:0, only the number of CT26 cells was observed to decrease in a dose-dependent manner. iso-C15:0 and iso-C17:0 had no obvious effect on the viability of MC38 and HT29 cells.

[0075] Example 2: BCFA promotes the expression of type I interferon (IFN-β) induced by Poly(I:C) and ISD

[0076] 1 Experimental methods

[0077] 1.1Raw-Lucia ISG fluorescence reporter system

[0078] RAW-Lucia TM ISG was purchased from InvivoGen, USA. The cell line was constructed by stably integrating the Lucia luciferase reporter gene induced by interferon regulatory factor (IRF) from the mouse RAW 264.7 macrophage cell line. TM ISG cells can be used to monitor IRF activation by measuring Lucia luciferase activity. TM4Lucia / Gaussia (a Lucia and Gaussia luciferase detection reagent) can easily monitor the level of IRF-induced Lucia luciferase in the cell culture supernatant. Therefore, the level of luciferin reflects the ability of macrophages to secrete type I interferon (IFN-β). The cells were cultured in RPMI-1640 medium containing 10% v / v fetal bovine serum, 100 IU / mL penicillin and streptomycin, 1% v / v essential amino acids, 1% v / v L-glutamine and 0.1% v / v 2-mercaptoethanol at 37°C and 5% v / v CO2 in a cell culture incubator.

[0079] Treatment of RAW-Lucia using BCFA monomers and other substances TM ISG cells, by detecting the expression level of luciferase, to explore whether BCFA can promote the secretion of IFN-β by macrophages. RAW-Lucia were planted in a 96-well plate at a density of about 10,000 cells / well. TM After the ISG cells were fully attached, they were treated with 50 μM iso-C15:0, iso-C17:0, anteiso-C15:0 and anteiso-C17:0, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), linoleic acid (LA) and 5 μg / mL polyinosinic acid (Poly(I:C)), 1 μg / mL interferon-stimulated DNA (ISD) alone or in combination with RAW-Lucia TM ISG cells, take 30 μL of cell culture supernatant and mix with 50 μL QUANTI-Luc in dark place. TM Mix well in a 96-well white plate and immediately detect the luciferase level using a Biotek Synergy H1 microplate reader. TM ISG cells were not treated with any substance. Luciferase activity (compared to control) = (value of treatment group - blank background value) / (value of control group - blank background value).

[0080] 1.2 Cell transfection experiment

[0081] Add the cells to be transfected to OPTI-MEM medium at a concentration of 30μL / mL, then add 1μg / mL ISD or 5μg / mL Poly(I:C) and mix well. Add 2μL Lipofection 2000 to 30μL OPTI-MEM medium, mix well, let stand for 5 minutes, then add to ISD or Poly(I:C), let stand for 20 minutes, and replace with normal medium after 6 hours.

[0082] 1.3 Real-time fluorescence quantitative PCR

[0083] First, cells were lysed with Trizol lysis buffer, and then cellular RNA was extracted using the Direct-zol-96 MagBead RNA Kit (ZYMO-Research). TM RNA was converted to cDNA using RT reagen Kit. SYBR Premix Ex Taq TM II for real-time quantitative PCR reaction, and detected by CFX96 Touch Real-Time PCR Detection System. The specificity of amplification was determined by melting curve. Primer β-Actin was used as an internal reference. The relative expression change was calculated by 2-ΔΔCt method. The PCR primer sequences used in the present invention are shown in Table 2 below.

[0084] Table 2 Real-time fluorescence quantitative PCR primer sequences

[0085]

[0086] 2 Experimental results

[0087] In order to explore the effect of BCFA on immune cells, RAW-Lucia ISG cells were first pretreated with iso-C15:0, iso-C17:0, anteiso-C15:0 and anteiso-C17:0 for 24 hours, then stimulated with ISD and Poly(I:C) for 6 hours, and finally the RAW-Lucia ISG fluorescence reporter system was used to detect the effect of different BCFA alone or in combination with ISD and Poly(I:C) on the production of type I interferon by immune cells. Figure 2A and 2B As shown in the results, iso-C15:0, iso-C17:0, anteiso-C15:0 and anteiso-C17:0 can induce the production of type I interferon when used alone, among which anteiso-C15:0 has the most obvious effect. When used in combination with ISD and Poly(I:C), the three BCFAs can significantly enhance the production of type I interferon in immune cells, and the effect is significantly better than that of the single treatment group (P<0.05). The above results show that BCFA can promote the production of type I interferon by cells induced by exogenous nucleic acids, and type I interferon signaling is the key to the initiation of T cell antigen-specific response, suggesting that BCFA can participate in cellular immune regulation.

[0088] The present invention found that anteiso-C15:0, whether used alone or in combination with ISD and Poly(I:C), can strongly induce immune cells to produce type I interferon. Considering the extensive research on EPA, DHA and LA in anti-cancer, the present invention compared the ability of anteiso-C15:0 to induce type I interferon with EPA, DHA and LA. Figure 2C As shown, RAW-Lucia ISG cells were treated with 50 μM anteiso-C15:0 and EPA, DHA and LA for 24 hours. The results showed that compared with EPA, DHA and LA, anteiso-C15:0 could significantly enhance the production of type I interferon (P<0.05). In order to further verify the effect of anteiso-C15:0 on mRNA levels, real-time fluorescence quantitative PCR was used to detect the effects of anteiso-C15:0 and EPA on the mRNA expression levels of interferon (IFN-β) gene and its downstream genes interferon-induced protein-10 (IP-10) and interferon-stimulated gene 15 (ISG15) in RAW-Lucia ISG cells. The results showed that compared with EPA, anteiso-C15:0 could significantly increase the relative mRNA expression levels of IFN-β, IP-10 and ISG15 in immune cells (P<0.05) ( Figure 2D ). Therefore, it is shown that BCFA can specifically induce the expression of type I interferon mRNA level compared with other fatty acids (EPA, DHA and LA).

[0089] Example 3: Effect of BCFA on intracellular cytokines of mouse bone marrow-derived dendritic cells

[0090] 1 Experimental methods

[0091] 1.1 Experimental animals

[0092] Female C57BL / 6 wild-type mice were purchased from Shanghai Experimental Animal Center. OT-1TCR transgenic mice were purchased from Shanghai Model Organism Center. All transgenic mice were of C57BL / 6 background, and both male and female mice of the same litter were used in this study. All mice were 7 to 9 weeks old when used. Mice were kept in the SPF animal room of Shanghai Jiao Tong University School of Medicine, and all animal experimental procedures were reviewed and approved by the Animal Ethics Committee of Shanghai Jiao Tong University.

[0093] 1.2 Isolation of mouse bone marrow-derived dendritic cells (BMDC)

[0094] The mice were killed by cervical dislocation, the skin was removed, the femoral tissue was cleaned, and the tibia of the mouse hind legs was taken with forceps and scissors. The bone marrow was repeatedly rinsed from both ends of the tibia into a 50 mL centrifuge tube with sterile PBS. Centrifuge at 1500 rpm for 5 minutes. The cell suspension was washed twice with RPMI1640 medium containing L-glutamine, 10% v / v fetal bovine serum (FBS), 50 μM β-mercaptoethanol, and 1% v / v Pen-Strep, centrifuged at 1500 rpm for 5 minutes, filtered with a 40 μM cell sieve, and the cells were counted. Approximately 200×10 4 The cells were cultured in RPMI 1640 medium (containing L-glutamine, 10% v / v FBS, 50 μM β-thiol, 1% v / v Pen-Strep) containing 20 ng / mL GM-CSF, and cultured in a 37°C, 5% v / v CO2 incubator for 5 days, and supplemented with 10 mL BMDC medium on the 3rd day.

[0095] 1.3DC cell treatment

[0096] BMDC cells were treated with 50 μM anteiso-C15:0 for 24 hours, while BMDC cells without any treatment were used as a control group. Then, BMDC cells in different treatment groups were incubated with OVA protein (100 μg / 200 μL) for 1 hour.

[0097] 1.4 Real-time fluorescence quantitative PCR

[0098] As described in Example 2.

[0099] 2 Experimental results

[0100] DC is an important professional antigen presenting cell in the body. It can participate in tumor immune response and play a key role in activating anti-tumor T cell response. Therefore, the present invention explores the immunomodulatory effect of anteiso-C15:0 on DC cells. First, the real-time fluorescence quantitative PCR method was used to detect the effect of anteiso-C15:0 on the expression levels of genes related to the type I interferon signaling pathway (IFN-β, IP-10 and ISG15) in DC cells. The results showed that ( Figure 3A ), when DC cells were treated with anteiso-C15:0 for 24 hours, the relative expression levels of IFN-β, IP-10 and ISG15 mRNA in the cells were significantly increased (P<0.05).

[0101] Usually, DC cells are activated after antigen recognition and migrate to the draining lymph nodes, while presenting the processed antigens on the major histocompatibility (MHC) proteins to T cells. Therefore, the present invention further explored the expression of antigen presentation-related genes (Tapbp, Tap1, Tap2 and β2m) in DC cells under the intervention of anteiso-C15:0. These genes encode antigen processing-related transport proteins (TAPs), which are involved in the presentation of MHC-I class antigens and the intracellular transport of endogenous peptides. The results are as follows Figure 3B As shown in the results, it was found that compared with the control group, the anteiso-C15:0 treatment group could significantly increase the relative expression levels of Tapbp, Tap1, Tap2 and β2m mRNA in DC cells (P<0.05). This indicates that anteiso-C15:0 can stimulate the expression of antigen processing transporter genes, which may promote the presentation of MHC-I class antigens in the tumor microenvironment. MHC-I class antigen presentation is a prerequisite for cytotoxic T cells to recognize and kill tumor cells.

[0102] Usually after recognizing the pathogen pattern, mature DC cells will start the antigen presentation mechanism and highly express MHC-II class, co-stimulatory molecules and cytokines that promote inflammatory response. Immature DC cells have strong antigen capture ability, but the expression level of MHC-II class molecules is low, and they can produce cytokines that inhibit inflammatory response (such as IL-10). In order to evaluate the effect of anteiso-C15:0 on genes related to inflammatory response (IL-1β, TNF-α and IL-10) in DC cells, the present invention detected the expression levels of these genes in DC cells after treatment. The experimental results show that ( Figure 3C ), the anteiso-C15:0 treatment group could significantly increase the expression of IL-1β and TNF-α in DC cells (P<0.05), and reduce the expression of IL-10 (P<0.05).

[0103] Example 4: Effects of BCFA-treated DCs on T lymphocyte proliferation and IFN-γ secretion

[0104] 1 Experimental methods

[0105] 1.1 Isolation of mouse bone marrow-derived dendritic cells (BMDC)

[0106] Same as described in Example 3.

[0107] 1.2 Isolation of mouse CD8+ T cells

[0108] Under sterile conditions, spleen tissues of 8-week-old OT-1 mice were obtained and the spleen was ground with PBS containing 2% v / v FBS. The cell suspension was filtered through a 70 μm cell sieve and centrifuged at 1500 rpm for 10 minutes to obtain a cell pellet. The supernatant was then discarded, and red blood cell lysis buffer was added. The cells were lysed on ice for 3-5 minutes and centrifuged at 1500 rpm for 10 minutes to obtain spleen cells without red blood cells. The culture medium was added according to the cell amount to resuspend the cells and the cells were separated according to EasySep. TM Mouse CD8+T Cell Isolation Kit Instructions Magnetic beads were used to separate CD8+T cells from spleen tissue. The extracted CD8+T cells were resuspended in RPMI 1640 medium containing 10 ng / mL IL-2, and the cells were counted. The cells were then seeded in 96-well plates (10×10 4 / well) were cultured.

[0109] 1.3 T cell treatment and viability assay

[0110] T cells were treated with 50 μM anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C17:0 for 24 or 48 hours, respectively, and the T cell viability was detected using the Cell Counting Kit-8 (CCK-8) method.

[0111] 1.4 T cell and DC co-culture experiment

[0112] BMDC cells were treated with 50 μM anteiso-C15:0, Poly(I:C) and ISD for 24 h, respectively. BMDC cells in different treatment groups were incubated with OVA protein (100 μg / 200 μL) for 1 h, and then BMDC cells were co-cultured with T cells at a ratio of 5:1 for 48 h.

[0113] 1.5 Flow cytometry

[0114] Cell surface protein staining: The collected cells were first blocked with 100 μL Fc receptor blocker (anti-mouse CD16 / 32) prepared at a ratio of 1:100 and incubated at room temperature for 15 minutes. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and stain with Fixable Viability Stain 780 prepared at a ratio of 1:1000 for 10 minutes to mark and distinguish between live and dead cells. Wash with PBS three times, add the corresponding cell surface protein antibody and stain for 30 minutes in the dark. After staining, resuspend the cells with PBS and analyze them using the BD FACS Aria II flow cytometer. The acquired data was analyzed using FlowJo software.

[0115] 1.6 Enzyme-linked immunosorbent assay (ELISA)

[0116] The supernatant of T cells and DC cells after 48 hours of co-culture was used for cytokine determination. Cell treatment and supernatant collection: The IFN-γ content in the supernatant of the cell culture medium was detected by Mouse IFN gamma ELISA Kit, High Sensitivity (Serum, Plasma, TCM) from PBL Assay Science, USA. The specific experimental steps were carried out according to the instructions provided by the kit.

[0117] 2 Experimental results

[0118] Dendritic cells are professional antigen-presenting cells that have the unique ability to cross-present exogenous antigens to MHC molecules and activate antigen-specific cytotoxic T cells. The present invention isolated T cells from OT-1 mice and co-cultured them with BCFA-treated DC cells to evaluate the effect of BCFA on T cell activation. In order to evaluate the effect of BCFA on the proliferation capacity of T cells, we used the CCK8 method to detect the cell viability of T cells within 24 or 48 hours after treatment with anteiso-C15:0, anteiso-C17:0, iso-C15:0 and iso-C17:0. The results showed that in all treatment groups, the proliferation of T cells was promoted, and the cell viability exceeded 100% ( Figure 4A ). This suggests that BCFA does not inhibit the activity of T cells.

[0119] Next, we explored the interaction between T cells and DC after co-culture. We first treated DC cells with 50μM anteiso-C15:0, ISD, and Poly (I:C) for 24 hours, then used OVA as an antigen to stimulate DC cells, and co-cultured DC cells and T cells at a ratio of 5:1. After 48 hours of co-culture, flow cytometry was used to detect the activation state and function of DC cells; at the same time, the supernatant was collected and the level of IFN-γ secreted by T cells was detected by Elisa. The Elisa results showed that ( Figure 4B ), after T cells were activated by BCFA-treated DC cells, the amount of IFN-γ released was significantly higher than that in the OVA-treated group (P<0.05), while the level of IFN-γ was comparable to that in the ISD group and the Poly(I:C) group (P>0.05). IFN-γ is an important cytokine secreted by helper T cells (TH1), which can promote the differentiation of T cells into cytotoxic T lymphocytes (CTL). Flow cytometry results showed that ( Figure 4C and 4D ), after anteiso-C15:0 pretreatment and co-culture with T cells for 48 hours, the proportion of DC (CD11c+IA / IE+) mature in the BCFA-treated group was significantly higher than that in the blank control group (P<0.05), and slightly lower than that in the ISD and Poly(I:C)-treated groups (P<0.05). In addition, compared with the blank control group, the expression of costimulatory molecules CD80 and CD86 and PD-L1 on the surface of DC cells in the BCFA-treated group was significantly increased (P<0.05). Therefore, BCFA may promote the activation and maturation of DC cells, enhance the antigen presentation function of DC cells, and further promote the production of IFN-γ by T cells.

Claims

1. Use of branched-chain fatty acids in the preparation of special medical formula foods suitable for patients with intestinal cancer.

2. The use according to claim 1, characterized in that The branched-chain fatty acid is a branched-chain fatty acid whose branch chain is a methyl group.

3. The use according to claim 1 or 2, characterized in that The branched-chain fatty acid is iso-C15:0, anteiso-C15:0, iso-C17:0 or anteiso-C17:

0.

4. The use according to any one of claims 1 to 3, characterized in that The intestinal cancer is colorectal cancer.

5. Use of branched-chain fatty acids in the preparation of a pharmaceutical composition for preventing and / or treating intestinal cancer.

6. The use according to claim 5, characterized in that The branched-chain fatty acid is a branched-chain fatty acid whose branch chain is a methyl group.

7. The use according to claim 5 or 6, characterized in that The branched-chain fatty acid is iso-C15:0, anteiso-C15:0, iso-C17:0 or anteiso-C17:

0.

8. The use according to any one of claims 5 to 7, characterized in that The intestinal cancer is colorectal cancer.

9. The use according to any one of claims 5 to 8, characterized in that The prevention and / or treatment of intestinal cancer includes inhibiting the vitality of intestinal cancer cells, promoting the expression of IFN-β by macrophages, promoting the expression of IFN-β by dendritic cells, promoting the expression of antigen processing-related transporters by dendritic cells, promoting the expression of pro-inflammatory cytokines by dendritic cells, and inhibiting any one or more of the expression of anti-inflammatory cytokines by dendritic cells.

10. The use according to claim 9, characterized in that The pro-inflammatory cytokines include any one or more of IL-1β and TNF-α, and the anti-inflammatory cytokines include IL-10.

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