CN112493483A - Use of chlorophyll-rich spinach extract for relieving intestinal inflammatory response and barrier dysfunction - Google Patents
Use of chlorophyll-rich spinach extract for relieving intestinal inflammatory response and barrier dysfunction Download PDFInfo
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- CN112493483A CN112493483A CN202011161218.7A CN202011161218A CN112493483A CN 112493483 A CN112493483 A CN 112493483A CN 202011161218 A CN202011161218 A CN 202011161218A CN 112493483 A CN112493483 A CN 112493483A
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Abstract
The present invention proposes the use of chlorophyll for the preparation of a food or pharmaceutical product, the chlorophyll being for use in the prevention or amelioration of an intestinal inflammatory response in a user whose intestinal barrier function is disturbed. Chlorophyll is suitable for use by a user having a dysfunction in the intestinal barrier, which can relieve the inflammatory response of the intestinal tract of the user. Therefore, a scientific research and clinical application foundation is laid for the chlorophyll in the aspect of preventing and treating intestinal inflammatory reaction.
Description
Technical Field
The invention relates to the field of food and medicine. In particular, the present invention relates to the use of a chlorophyll-rich spinach extract for alleviating intestinal inflammatory responses and barrier dysfunction.
Background
The intestine is the main medium between the human body and the environment. The intestinal tract is responsible not only for the absorption of essential dietary nutrients, but also for protecting the host from various ingested toxins and microorganisms. The intestinal barrier system is immune defenses by the mucus layer, intestinal epithelial cells and the lamina propria, all of which are susceptible to external factors (e.g., meal type, meal fat content, etc.).
Studies report that consumption of excess dietary fat can perturb barrier function to varying degrees, inducing inflammatory responses. Nowadays, there are more and more studies to prove that the effective alleviation of high fat-induced inflammatory conditions by food-derived active ingredients relies mainly on the intestinal barrier function, but the main mechanism of action is not yet clear.
High fat diets increase the abundance of sulfate-reducing bacteria, which dissociate the genotoxicity of taurocholic acid to produce H2S gas, which exacerbates the cycle of animal intestinal barrier degeneration. Some studies report that the content of sulfate-reducing bacteria, or specific species of sulfate-reducing bacteria, is increased in faeces in patients with impaired intestinal barrier. There are studies that suggest a mechanism by which sulfide-decomposing disulfide bonds disrupt intestinal barriers, and they have studied that increased sulfide production is a major cause of mucosal barrier disruption and colonic inflammation.
Chlorophyll is a pigment which is located on a plant thylakoid membrane and can perform photosynthesis, and is mainly divided into chlorophyll a and chlorophyll b, and chlorophyll c and chlorophyll d also exist in algae such as brown algae, diatom and the like. Such pigments are readily available in the daily diet. However, at present, there is still a need to study the influence and mechanism of chlorophyll on the high fat diet-induced intestinal inflammatory response and barrier dysfunction.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art.
The invention provides an application of chlorophyll in preparing food or medicines. According to an embodiment of the invention, the food or pharmaceutical product is for preventing or ameliorating an intestinal inflammatory response in a user, the user having a disturbed intestinal barrier function.
It has been reported that not all inflammatory responses of the intestine present a phenomenon of disturbed intestinal barrier function. The inventors of the present invention found that chlorophyll is suitable for use in users with a disturbed intestinal barrier function, which may alleviate the intestinal inflammatory response of the user. Therefore, the method lays the foundation of scientific research, dietary guidance and clinical application of chlorophyll in the aspect of preventing and treating intestinal inflammatory reaction.
According to an embodiment of the invention, the intestinal inflammatory response and intestinal barrier dysfunction are caused by a long-term high-fat meal intake. The inventors have found that chlorophyll is suitable for the inflammatory response of the gut and the dysfunction of the gut barrier caused by long-term intake of high fat diets. In particular, long-term high fat diets are due to long-term intake of high fat foods.
According to an embodiment of the present invention, the intestinal inflammation is associated with an up-regulation of gene expression of the pro-inflammatory factors IL-6, IL-1 β, MCP-1 and TNF- α in the colon of the user, and chlorophyll is used to down-regulate gene expression of the pro-inflammatory factors IL-6, IL-1 β, MCP-1 and TNF- α in the colon of the user. Researches show that the gene expression of proinflammatory factors IL-6, IL-1 beta, MCP-1 and TNF-alpha in colon of a user who takes a high-fat diet for a long time is up-regulated, and after the user takes chlorophyll, the gene expression of the proinflammatory factors IL-6, IL-1 beta, MCP-1 and TNF-alpha in the colon can be effectively down-regulated, so that intestinal inflammatory response is prevented or improved.
According to an embodiment of the invention, said intestinal inflammatory reaction is associated with an increase in the content of FITC-Dextran in the serum of the user, chlorophyll being used to lower the content of FITC-Dextran in the serum of said user. The research shows that the content of FITC-Dextran in the serum of users who take high-fat diet for a long time is increased, and the content of FITC-Dextran in the serum can be effectively reduced after the users take chlorophyll, so that the intestinal permeability is prevented or improved, and the intestinal inflammatory response is regulated.
According to an embodiment of the present invention, the intestinal inflammatory response is associated with down-regulation of the protein expression of ZO-1, Occludin, Claudin-1 and JAM-a in the intestinal tract of the user, and chlorophyll is used to up-regulate the protein expression of ZO-1, Occludin, Claudin-1 and JAM-a in the intestinal tract of the user. Researches show that the protein expression of ZO-1, Occludin, Claudin-1 and JAM-A in the intestinal tract of users who take high-fat diet for a long time is reduced, and after the users take chlorophyll, the protein expression of ZO-1, Occludin, Claudin-1 and JAM-A in the intestinal tract can be effectively up-regulated, so that the barrier function disorder of the intestinal tract can be prevented or improved, and the inflammatory response of the intestinal tract can be regulated.
According to an embodiment of the invention, the intestinal inflammatory response is associated with a down-regulation of protein expression of MUC2, KLF4 and TFF3 in the intestine of the user, and chlorophyll is used to up-regulate protein expression of MUC2, KLF4 and TFF3 in the intestine of the user. The research shows that the protein expression of MUC2, KLF4 and TFF3 in the intestinal tract of users who take high-fat diet for a long time is down-regulated, and the protein expression of MUC2, KLF4 and TFF3 in the intestinal tract can be effectively up-regulated after the users take chlorophyll, so that the intestinal barrier dysfunction is prevented or improved, and the intestinal inflammatory response is regulated.
According to an embodiment of the invention, the intestinal inflammatory response is highly correlated with an abundance of sulfate-reducing bacteria in the intestine of the user, and the chlorophyll is used for reducing the abundance of sulfate-reducing bacteria in the intestine of the user, and for providing H in the colon2The S content and the content of the trisulfide in the excrement are reduced. Researches show that some users who take high-fat diet for a long time have high abundance of the sulfate reducing bacteria in the intestinal tract, and after the users take chlorophyll, the abundance of the sulfate reducing bacteria in the intestinal tract can be effectively reduced, so that the functional disorder of the intestinal barrier can be prevented or improved, and the inflammatory reaction of the intestinal tract can be regulated.
According to an embodiment of the invention, the intestinal inflammatory response is associated with an increased lipopolysaccharide content in the serum of the user and the chlorophyll is used to lower the lipopolysaccharide content in the serum of the user. Researches show that the content of lipopolysaccharide in serum of some users who take high-fat diet for a long time is increased, and the content of lipopolysaccharide in intestinal tracts can be effectively reduced after the users take chlorophyll, so that intestinal inflammatory reaction is prevented or improved.
According to an embodiment of the invention, the chlorophyll is derived from green fruits and vegetables, preferably spinach, lettuce, celery, rape, kiwi. The fruit and vegetable is rich in a large amount of chlorophyll, is convenient to obtain and has low cost.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a schematic representation of the relative expression changes of mRNA for inflammatory factors in the colon according to one embodiment of the present invention;
FIG. 2 shows a schematic diagram of the change in the concentration of FITC in serum according to one embodiment of the invention;
FIG. 3 shows an electron micrograph of a colon HE staining according to one embodiment of the invention;
FIG. 4 shows a schematic view of a colon mucosa thickness analysis according to an embodiment of the present invention;
FIG. 5 is a schematic diagram showing the analysis of the relative expression level of barrier proteins in the colon and intestine according to an embodiment of the present invention;
FIG. 6 shows an immunohistochemical electron micrograph of colon MUC2 according to one embodiment of the present invention;
FIG. 7A shows a schematic representation of the absolute abundance of the initial sulfate-reducing bacteria according to one embodiment of the present invention;
FIG. 7B shows a schematic diagram of a final (13 week) sulphate-reducing bacteria absolute abundance analysis according to one embodiment of the present invention;
FIG. 7C shows a colon H in accordance with one embodiment of the present invention2S content analysis schematic diagram;
FIG. 7D shows a schematic of analysis of trisulfide compounds in feces according to one embodiment of the present invention;
FIG. 8 shows a schematic diagram of an analysis of LPS concentration in serum according to an embodiment of the present invention.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Experimental methods
1. Chlorophyll extraction and content determination
Cleaning fresh spinach, draining, adding into a stirrer (Wiggens MB800, Germany), adding appropriate amount of 95% ethanol, stirring, and crushing. Taking 6 300mL centrifuge cups, adding 50g of spinach pomace and 150mL of ethanol according to the material-liquid ratio of 1:3, centrifuging for 10min at 4 ℃ under 8000g, taking supernatant, and continuously extracting and centrifuging the remaining spinach pomace until the spinach pomace in the centrifuge cups is grey white. Pouring the collected centrifugal supernatant into a rotary bottle, performing rotary evaporation at 36 ℃, pouring the centrifugal supernatant into a collecting bottle when the centrifugal supernatant is concentrated to the original volume of 1/4, and placing the collecting bottle in a refrigerator at 4 ℃ for refrigeration for 12 hours until the bottom is dark green precipitate and the top is yellow liquid. The lower dark green pellet was filtered at 4 ℃ using a Millipore membrane (0.22 μm) and stored at-20 ℃ until analysis and use (all experiments were performed protected from light).
Absorbing 1mL of chlorophyll extract by using a pipette, after diluting by 100 times or 1000 times in a gradient manner, respectively measuring the light absorption values at 645 nm and 663nm, taking 80% acetone as a control, and carrying out all experiments under the condition of keeping out of the sun. Calculating the chlorophyll content by using the Arnon formula:
chlorophyll a concentration (mg/L): ca＝12.71×A663-2.59×A645
Chlorophyll b concentration (mg/L): cb＝22.88×A645-4.67×A663
Total chlorophyll concentration (mg/L): c ═ Ca+Cb＝8.04×A663+20.29×A645
Equivalent dose conversion refers to the method of Huang-Ji-Han et al (2004), and the chlorophyll concentration of the target sample is 9 mg/mL.
2. Animal grouping and handling
In the experiment, C57BL/6N male mice with the age of 3 weeks are selected and bred in an SPF-level mouse room (room temperature 25 +/-2 ℃, relative humidity 45% -65%, day and night alternate light and shade for 12 h) of an experimental animal platform in western school district of Chinese agriculture university, after adaptive breeding for 7d, the mice are randomly divided into 4 groups, and each group is 12, and 4 mice are bred in one cage. The first group was control group (NC), diet control feed (10% kcal, D12450B; Research Diets), gavage saline; the second group was control + chlorophyll intervention group (NCCHL), diet control feed (10% kcal, D12450B; Research Diets), gavage chlorophyll (0.18mg/10g mouse body weight, dissolved in normal saline); the third group is high fat group (HFD), diet high fat diet (60% kcal, D12492; Research Diets), gavage normal saline; the fourth group was high fat + chlorophyll dried group (HFCHL), diet high fat diet (60% kcal, D12492; Research Diets), gavage chlorophyll (0.18mg/10g mouse body weight, dissolved in physiological saline). Feces were collected after 13 weeks of gavage, and after fasting for 6 hours, the mice were taken out of the cage, gently placed on a laboratory bench, and blood was collected by picking up the eyeballs of the mice with sterilized forceps. Mice were sacrificed by cervical dislocation, the colons of the mice were collected, weighed, immediately dosed with liquid nitrogen, and rapidly stored at-80 ℃ until the subsequent experiments.
3. Intestinal wall permeability detection
After 11 weeks of rearing, FITC-labeled dextran was dissolved in PBS at a concentration of 100mg/ml, after fasting for 4 hours, 150. mu.l of FITC dextran solution was orally administered to each mouse, blood was collected after 4 hours, serum was separated by centrifugation at 1000rpm at 4 ℃ for 20min, and FITC fluorescence intensity in serum was measured using a multifunctional fluorescence microplate reader at an excitation wavelength of 495nm and an emission wavelength of 535 nm.
4. HE staining of colon tissue in mice
And (3) cutting a part of colon tissues, fixing the cut part of colon tissues in 4% paraformaldehyde for 48 hours, then embedding paraffin, slicing, performing hematoxylin-eosin staining experiments, performing microscopic examination, and collecting and analyzing images. The specific experimental steps are as follows: (1) and (4) dehydrating. Sequentially soaking the tissue mass with alcohol of increasing concentration: 85% ethanol (30min), 95% ethanol (30min), 100% ethanol (1 h); then, soaking the tissue block by dimethylbenzene until the tissue block is completely transparent; (2) and (5) wax dipping. Placing the transparent tissue block into melted paraffin, placing the tissue block into a paraffin dissolving box, and keeping the temperature for 2 hours until the paraffin is completely immersed into the tissue block; (3) and (4) embedding. Pouring paraffin embedded tissue blocks into an embedding box, and cooling and solidifying the paraffin into blocks; (4) slicing, spreading and baking. The wax block was fixed on a microtome and cut into 5 μm slices. The cut thin slices were flattened in water at 42 ℃ and attached to a glass slide, and dried overnight in a 37 ℃ incubator. Then baking the slices at 60 ℃ for 1 h; (5) dewaxing and rehydration. Removing paraffin from the slices by using dimethylbenzene, and then sequentially soaking in alcohol with gradually decreased concentration: 100% ethanol (5min), 95% ethanol (5min), 70% ethanol (5min), 50% ethanol (5min), and distilled water; (6) and (5) HE staining. Staining the slices in hematoxylin water solution for 2min, washing with tap water for 2 times (each time for 2 min), and observing the cell nucleus to be blue under microscope; soaking the slices in 1% hydrochloric acid for differentiation for 5s to remove excessive staining agent, and washing the slices with tap water for 2 times, each time for 2 min; then placing the slices in eosin staining agent for 2min, and washing with tap water for 2 times, each for 2 min; (7) and (4) dehydrating. Soaking the slices with the following reagents in sequence: 70% alcohol (5s), 95% alcohol (1min), 100% alcohol (5min), xylene (10 min); (8) and (6) sealing the sheet. And (4) dripping gum on the transparent slice, covering a cover glass, sealing, airing and observing.
5. Immunohistochemistry (expression of mouse Colon goblet cell Muc 2)
Placing the paraffin section of colon tissue in a 70 deg.C oven, baking for 2h, dewaxing and rehydrating, washing with PBS buffer solution with pH of 7.4 for 3 times, 3min each time, adding a certain amount of citrate buffer solution with pH of 6.0 into a microwave box, heating with microwave to boiling, placing the dewaxed and hydrated tissue section on a high temperature resistant plastic section frame, placing in the boiled buffer solution, treating with middle-grade microwave for 10min, taking out the microwave box, cooling with flowing tap waterHowever, the slides were removed from the buffer, rinsed twice with distilled water, then 2 times with PBS buffer, 3min each time, 1 drop of 3% H was added to each section2O2Incubation for 10min at room temperature to block endogenous peroxidase activity, 3 washes in PBS buffer for 3min each, PBS solution was removed, 1 drop of the corresponding primary anti-MUC 2 antibody (1:500) was added to each section and incubated overnight at 4 ℃, 3 washes in PBS buffer for 3min each, PBS solution was removed, 1 drop of peroxidase-conjugated secondary antibody was added to each section, incubation for 30min at room temperature, 3 washes in PBS buffer for 5min each, PBS solution was removed, 1 drop of freshly prepared DAB solution (diaminobenzidine) was added to each section, and observation and photographing were performed under an optical microscope.
6. Absolute quantification of sulfate-reducing bacteria
(1) Construction of plasmid standard product by specific flora primer
And (3) verifying the specificity of a primer (dsrA gene) by agarose electrophoresis aiming at a target flora query primer, cutting and recovering the primer to obtain a purified DNA fragment, connecting the purified DNA fragment with a PMD-19T carrier, transferring the purified DNA fragment into an escherichia coli competent cell, activating at 37 ℃, carrying out amplification culture in an LB liquid culture medium, successfully transferring the competent cell of the specific flora primer into the competent cell for 12 hours at 37 ℃, reserving 1ml of bacterial liquid for preservation, sending 500ul of bacterial liquid to a sequencing company for sequencing, centrifuging the remained bacterial liquid, collecting bacteria, extracting plasmids and obtaining plasmids. The sequencing result is compared with the strain sequence in the NCBI database, and the strain with 100 percent of identity is the same strain, and can be used as a plasmid standard substance for subsequent experiments.
(2) Method for determining change of number of sulfate reducing bacteria in mouse excrement by absolute quantitative qPCR (quantitative qPCR)
Diluting the sulfate reducing bacteria plasmid standard substance at intervals of 10 times, determining the number of floras by a qPCR method with sample DNA, drawing a standard curve graph by taking the logarithm of the standard substance copy number as an abscissa and the cycle number (Cq) as an ordinate according to the obtained result, keeping the amplification efficiency at 90-110% to be good, and R2>0.99 is available. Substituting the Cq value of the sample into a formula for calculation to finally obtain the copy number of the sulfate reducing bacteria flora.
(3) In vitro disulfide bond cracking experiment and determination of hydrogen sulfide and trithiogen compounds in feces
Sodium sulfide (Na) was determined using DTNB as a disulfide model compound2S), N-acetylcysteine (NAC), Glutathione (GSH) and the potency of cysteine to cleave S-S bonds. The methylene blue method was used to quantify hydrogen sulfide and trisulfide compounds.
7. Gene expression assay
The total RNA of each tissue is extracted by referring to a Trizol Reagent total RNA extraction kit of Beijing Tiangen Biochemical technology Co., Ltd, and real-time fluorescent quantitative PCR detection is carried out after cDNA synthesis.
8. Tissue protein immunoblotting
Extracting tissue protein, determining protein concentration, performing SDS-PAGE gel electrophoresis, performing membrane transfer and sealing, incubating antibody, and performing ECL development.
Example 1 Effect of chlorophyll on Gene expression of high fat diet-induced intestinal proinflammatory factors
As shown in figure 1, compared with the NC group and NCCHL group mice, the expression levels of proinflammatory factors IL-6, IL-1 beta, MCP-1 and TNF-alpha genes in colon of HFD group mice are obviously increased, and the dietary supplement of spinach extract rich in chlorophyll can obviously reduce the expression of the proinflammatory factors IL-6, IL-1 beta, MCP-1 and TNF-alpha genes in colon.
Example 2 chlorophyll effects on high-fat diet-induced intestinal permeability
High fat diets enhance intestinal permeability by directly stimulating proinflammatory signal secretion, or indirectly by increasing intestinal barrier-disrupting cytokines (IL1 β, IL6, IFN γ, and TNF α), which will increase the incidence and mortality of inflammation-related diseases, impairing the integrity of the intestinal barrier. The intestinal permeability test (fig. 2) was performed three weeks before the mice became necrotized, and after injecting fluorescein isothiocyanate-labeled Dextran (FITC-Dextran, a fluorescent tracer) into the intestinal lumen of the mice, the content of FITC-Dextran in the serum was measured, and it was found that the content of FITC in the serum of HFD group was significantly increased and the content of FITC in HFCHL group was significantly decreased compared to HFD group, indicating that the intestinal permeability was significantly improved.
Example 3 Effect of chlorophyll on the integrity of the intestinal Barrier in high fat diet mice
The intestinal barrier is a complex defense system consisting of intestinal epithelial cells, intestinal mucus layers, an intestinal mucosal immune system and microorganisms colonized by the intestinal epithelium. Changes in the colon were observed for each group of mice using HE staining (fig. 3), and mucosal thickness was calculated (fig. 4). The results show that compared with the control group (NC), the high-fat group (HFD) mice have certain damage to the intestinal tract structure, the colon mucosa is thinned, and the high-fat supplement of the spinach extract (HFCHL) rich in chlorophyll can obviously increase the thickness of the intestinal tract mucus layer, so that the intestinal tract flora is prevented from directly contacting with the intestinal epithelial cells, and the intestinal tract mild inflammation is induced. In addition, there was a certain increase in mucosal thickness between the chlorophyll-rich spinach extract group (NCCHL) and the control group (NC) on the control diet gavage, indicating that the chlorophyll-rich spinach extract can significantly protect the integrity of the intestinal barrier.
The intestinal physical barrier is a direct histological barrier between the host and the external environment, mainly consists of intestinal epithelial cells and tight connection among the cells, and is an important barrier for preventing the invasion of pathogenic bacteria by the host. The intestinal tight junction structure is composed of a plurality of protein complexes, including a cytoplasmic protein ZOs family, transmembrane proteins Occludin, Claudins, and a connection adhesion molecule JAM family. Further determining the expression of physical barrier related proteins in the intestinal tissues of four groups of mice, as shown in FIG. 5, HFD can significantly reduce the protein expression of ZO-1, Occludin, Claudin-1 and JAM-A in the intestinal tracts compared with NC, indicating that HFD can seriously damage the physical barriers of mice, and the relative expression amounts of ZO-1, Occludin, Claudin-1 and JAM-A proteins in the colon tissues of HFCHL mice are significantly increased compared with the HFD group.
The intestinal chemical barrier is mainly composed of the intestinal mucus layer, which is composed of a highly glycosylated polymeric network of mucins secreted by goblet cells (MUC proteins are the major mucins). The network is interconnected by disulfide bonds, which are produced by goblet cells in the intestinal epithelium. The pore size of this hydrated gel-like structure in the inner mucus layer does not allow bacterial penetration. In the outer mucus layer, the mucus network is looser due to the activity of proteases, thus allowing bacteria to invade. As shown in FIG. 5, compared with the NC group, HFD can significantly reduce the protein expression of MUC2, KLF4 and TFF3, and supplement of spinach extract rich in chlorophyll can significantly increase the protein expression of MUC2, KLF4 and TFF3, and has a certain repair function on the chemical barrier of the intestinal tract.
Furthermore, as shown in fig. 6, immunohistochemistry results further showed that the NCCHL group mice had significantly increased mucin expression from colon tissue MUC2 compared to the NC group. Notably, the HFD group significantly down-regulated the expression level of MUC2, and MIC2 protein staining of goblet cells in the colon was significantly enhanced in the HFCHL group mice, and therefore, chlorophyll-rich spinach extract might improve impaired mucus barrier in high-fat diet mice, mainly by increasing the expression of MUC 2.
Example 4 Effect of chlorophyll on the Absolute abundance of sulfate-reducing bacteria in the gut of high-fat diet mice
Sulfide production by sulfate-reducing bacteria reduces disulfide bonds in the mucus network in the gut, thereby disrupting the polymeric MUC2 network. As a result, the mucus layer becomes less viscous and more permeable, causing toxic compounds and bacteria present in the intestinal lumen to contact the surface of epithelial cells, thereby causing inflammation. As shown in fig. 7, by measuring the absolute abundance of the initial sulfate-reducing bacteria (fig. 7A) and the final (after 13 weeks) sulfate-reducing bacteria (fig. 7B), no significant difference was found in the absolute quantification of the initial sulfate-reducing bacteria. Supplementation with 13 weeks of high-fat diet significantly increased the absolute abundance of sulfate-reducing bacteria, while supplementation with chlorophyll-rich spinach extract decreased the abundance of sulfate-reducing bacteria in the intestinal tract of high-fat diet mice. Sulfide production by sulfate-reducing bacteria may reduce disulfide bonds in the mucus network, thereby cleaving the polymeric MUC2 network, thereby further determining H in the colon2S (FIG. 7C) and trisulfide in stool (FIG. 7D), supplementation of chlorophyll-rich spinach extract was found to reduce H in the colon2S and the level of trisulphide in the feces indicate that chlorophyll-rich spinach extract prevents sulphide formation by down-regulating the abundance of sulphate-reducing bacteria, avoiding damage to the mucus barrier.
In addition, studies indicate that high-fat and high-sugar diet can increase the abundance of Desulfovibrio (Desulfovibrio) in sulfate-reducing bacteria, can produce excessive LPS (lipopolysaccharide), further induces intestinal leakage, causes the LPS to permeate intestinal epithelial cells, enter a blood circulation system and induce metabolic endotoxemia. Therefore, the present study measured the LPS levels in the blood of mice. As shown in fig. 8, the serum LPS levels in HFD mice were significantly increased compared to NC group, indicating that high fat diet caused "intestinal leakage" in mice, while the serum LPS levels in HFCHL group were significantly lower than HFD, indicating that chlorophyll-rich spinach extract decreased intestinal permeability, thereby preventing the intestinal barrier function from being impaired and alleviating intestinal inflammation.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (9)
1. Use of chlorophyll in the preparation of a food or pharmaceutical product for preventing or ameliorating an inflammatory response of the gut in a user having a disturbed gut barrier function.
2. Use according to claim 1, wherein the intestinal inflammatory response and intestinal barrier dysfunction are caused by long-term high fat meal intake.
3. Use according to claim 1, wherein the intestinal inflammatory response is associated with an up-regulation of the gene expression of the pro-inflammatory factors IL-6, IL-1 β, MCP-1 and TNF- α in the colon of the user, and chlorophyll is used to down-regulate the gene expression of the pro-inflammatory factors IL-6, IL-1 β, MCP-1 and TNF- α in the colon of the user.
4. Use according to claim 1, characterized in that said intestinal inflammatory reaction is associated with an increase in the FITC-Dextran content in the serum of the user, chlorophyll being used to lower the FITC-Dextran content in the serum of said user.
5. Use according to claim 1, wherein the intestinal inflammatory response is associated with down-regulation of the protein expression of ZO-1, Occludin, Claudin-1 and JAM-A in the intestinal tract of a user and chlorophyll is used to up-regulate the protein expression of ZO-1, Occludin, Claudin-1 and JAM-A in the intestinal tract of the user.
6. The use according to claim 1, wherein the intestinal inflammatory response is associated with a down-regulation of the protein expression of MUC2, KLF4 and TFF3 in the intestine of a user, and chlorophyll is used to up-regulate the protein expression of MUC2, KLF4 and TFF3 in the intestine of said user.
7. The use according to claim 1, wherein the intestinal inflammatory response is associated with a high absolute abundance of sulfate-reducing bacteria in the intestine of the user, and chlorophyll is used to reduce the absolute abundance of sulfate-reducing bacteria in the intestine of the user and to provide H in the colon2The S content and the content of the trisulfide in the excrement are reduced.
8. Use according to claim 1, wherein the intestinal inflammatory response is associated with an elevated lipopolysaccharide content in the serum of a user and chlorophyll is used to lower the lipopolysaccharide content in the serum of the user.
9. Use according to claim 1, characterized in that chlorophyll is derived from green vegetables and fruits, preferably spinach, lettuce, celery, rape, kiwi.
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