CN114470011A - Product, method and application for preventing and/or treating intestinal tract and liver inflammatory injury - Google Patents
Product, method and application for preventing and/or treating intestinal tract and liver inflammatory injury Download PDFInfo
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- CN114470011A CN114470011A CN202210072122.6A CN202210072122A CN114470011A CN 114470011 A CN114470011 A CN 114470011A CN 202210072122 A CN202210072122 A CN 202210072122A CN 114470011 A CN114470011 A CN 114470011A
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- inflammatory injury
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Abstract
The invention provides a product, a method and application for preventing and/or treating intestinal tract and liver inflammatory injury, wherein the product for preventing and/or treating intestinal tract and liver inflammatory injury is a black sorghum rice product, or a product containing black sorghum rice polysaccharide extracted from the black sorghum rice, and the product can have the effects of promoting intestinal tract movement and regulating intestinal tract health after being eaten for a long time. The sorghum Umi can regulate colon inflammation of mice by regulating the expression of cell factors and the conduction of related signal paths, and can prevent and regulate inflammatory injuries of intestinal tracts and livers by regulating the expression of the related factors and the signal paths in liver tissues through intestinal and hepatic axes. Has better application prospect in the aspects of preventing and treating inflammatory injuries of intestinal tracts and liver.
Description
Technical Field
The invention relates to the field of sorghum Umi application, in particular to a product, a method and application for preventing and/or treating intestinal tract and liver inflammatory injury.
Background
The health of the intestinal tract is in a mutual beneficial relationship with the host, and under healthy conditions, the cells in the intestinal tract are strictly controlled, for example, the balance between cytokines such as proinflammatory factors and anti-inflammatory factors is kept, and the structure of the intestinal microbiota is maintained. The direct connection between the diet and the intestinal tract exists, and the foods such as vegetables, fruits, whole grains and the like are rich in dietary fiber, polyphenol, folic acid, carotenoid and the like, and are very beneficial to the health of the intestinal tract. The gastrointestinal tract is a barrier that limits the interaction between the luminal contents of the intestinal flora and metabolic waste products, which are closely related and interacting with the liver in structure and function, known as the "gut-liver axis". At present, the incidence rate of intestinal inflammation and liver inflammation gradually rises, the disease course is long, and the risk of complicating colon cancer exists, so that more and more attention is paid to people.
Disclosure of Invention
The invention aims to provide a product, a method and application for preventing and/or treating intestinal tract and liver inflammatory injury.
In order to achieve the above objects, in a first aspect, the present invention provides a product for preventing and/or treating inflammatory injury of intestinal tract and liver, wherein the product is a product of black sorghum rice, or a product containing sorghum black rice polysaccharide powder extracted from the black sorghum rice.
According to one embodiment of the invention, the product is a food, a pharmaceutical, a nutraceutical or a food for special medical use.
In a second aspect, the present invention provides a method for preparing a product for preventing and/or treating inflammatory injury to the intestine and liver, comprising: the sorghum black rice is used as a raw material to prepare a product for preventing and/or treating intestinal tract and liver inflammatory injury.
According to an embodiment of the second aspect of the invention, the method comprises: drying the sorghum black rice, crushing and sieving to prepare the sorghum black rice whole powder.
According to an embodiment of the second aspect of the invention, the method comprises:
degreasing the whole sorghum black rice flour, refluxing in a water bath at 95 ℃, drying the filtered solid, desugarizing by 85% ethanol, drying the filtered solid, leaching in the water bath, and filtering to obtain a polysaccharide crude extract;
concentrating the polysaccharide crude extract, adding 4 times of volume of absolute ethyl alcohol, precipitating with ethanol overnight, and centrifuging to obtain crude polysaccharide;
washing the crude polysaccharide with anhydrous ethanol, acetone and diethyl ether, drying, re-dissolving the dried crude polysaccharide in water, removing protein, decolorizing with hydrogen peroxide at pH8.5, maintaining at 40 deg.C for 1 hr, concentrating, dialyzing for 48 hr, and vacuum freeze drying to obtain refined sorghum black rice polysaccharide powder.
In a third aspect, the invention provides an application of a sorghum Umi product in preventing and/or treating intestinal inflammatory injury.
According to an embodiment of the third aspect of the present invention, the product of the dark sorghum rice comprises dark sorghum rice whole flour or dark sorghum rice polysaccharides extracted from the dark sorghum rice.
According to an embodiment of the third aspect of the invention, the inflammatory bowel injury is DSS-induced inflammatory bowel injury.
In a fourth aspect, the invention provides an application of a sorghum Umi product in preventing and/or treating liver inflammatory injury.
According to an embodiment of the fourth aspect of the invention, the liver inflammatory injury is DSS-induced liver inflammatory injury.
The invention has the beneficial effects that:
the product for preventing and/or treating intestinal tract and liver inflammatory injury provided by the embodiment of the invention has the effects of promoting intestinal tract movement and regulating intestinal tract health after being eaten for a long time. The sorghum Umi can regulate colon inflammation of mice by inhibiting related inflammatory signal pathways and regulating the expression of cytokines, and can prevent and regulate inflammatory injuries of intestinal tracts and livers by regulating the expression of related factors and signal pathways in liver tissues through a bowel-hepatic axis. Has better application prospect in the aspects of preventing and treating inflammatory injuries of intestinal tracts and liver.
Drawings
FIG. 1 is a graph showing the effect of the SRPW and SRPS of the present invention on the regulation of colitis in mice. (A) Body weight change (B) mouse diet change (C) DAI score index (D) organ index (E) mouse colon histomorphometric change (HE, × 100). Indicates significant difference compared to Model group, p < 0.05;
FIG. 2 is a graph showing the effect of SRPW and SRPS of the present invention on the modulation of colonic inflammatory cytokines in mice with colitis. (A-D) expression of TNF-. alpha.IL-1. beta., iNOS, and IL-6mRNA was detected by the qRT-PCR method. (E-H) detecting protein expression amounts of key factors TNF-alpha, IL-6 and IL-10 by a Western blotting method;
FIG. 3 is a graph showing the expression effect of SRPW and SRPS of the present invention on the regulation of colon tissue-associated cytokines in colitis mice. (A-C) detecting the expression of CXCL-2, COX-2 and MUC-2 mRNAs by a qRT-PCR method;
FIG. 4 is a graph showing the effect of SRPW and SRPS of the present invention on the modulation of NF-. kappa.B signaling pathway in colon tissue of colitis mouse. (A-E) expression of NF-. kappa. B p65, MyD88, I.kappa.B.alpha., SIGIRR and TLR-4mRNA was detected by qRT-PCR method. (F-H) detecting protein expression amounts of key factors NF-kappa B p65, p-NF-kappa B p65 and I kappa B alpha by a Western blotting method;
FIG. 5 is a graph showing the effect of SRPW and SRPS of the present invention on the regulation of LPS expression in colon-blood-liver of colitis mice. (A-C) detecting the expression of LPS in colon, serum and liver, respectively, by ELISA;
FIG. 6 is a graph showing the effect of the SRPW and SRPS of the present invention on the regulation of mouse liver tissue cytokine caused by colitis. (A-E) expression of TNF- α, IL-1 β, iNOS, IL-6 and CXCL-2 mRNA was detected by the qRT-PCR method. (F-I) detecting protein expression amounts of key factors IL-6, TNF-alpha and IL-10 by a Western blotting method;
FIG. 7 is a graph showing the effect of SRPW and SRPS of the present invention on the regulation of NF-. kappa.B signaling pathway in liver tissue of colitis mice. (A-D) expression of NF-. kappa. B p65, MyD88, I.kappa.B.alpha., TLR-4 and SIGIRR mRNA was detected by qRT-PCR method. (E-G) detecting the protein expression amounts of key factors NF-kappa B p65, p-NF-kappa B p65 and I kappa B alpha by a Western blotting method.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
The materials, reagents and apparatus of the present invention are described below.
Kaoliang Wu millet, Jinmanwu agricultural science and technology development Co., Ltd.
SPF grade female BALB/c mice, 6-8 weeks old (18-20 g).
TRNzol, RIPA, PMSF, phosphatase inhibitor, 5xSDS loading buffer solution, TBS solution, Tris, Glycine, Tween20, BSA, PVDF membrane, dextran sodium sulfate, sulfasalazine enteric coated tablet, hematoxylin-eosin staining solution, SDS-PAGE gel preparation kit, LPSELISA kit, FastKing one-step genome-removing cDNA first-strand synthesis kit and SuperReal fluorescence quantitative kit.
Petroleum ether, absolute ethyl alcohol, acetone, ether, 30% hydrogen peroxide, formaldehyde, xylene, isopropanol, methanol and the like are analytically pure.
The instrument comprises: the device comprises a multifunctional crusher, an electric heating blowing drying box, an electronic balance, a constant-temperature water bath, a freeze dryer, a quick-freezing centrifuge, a freeze grinding instrument, a vortex mixer, a rotary evaporator, an acidimeter, a mouse IVC system, a biological tissue embedding machine, a paraffin slicer, a magnetic stirrer, a PCR instrument, an electrophoresis tank, a transfer printing tank, an enzyme labeling instrument and a-80 ℃ ultra-low temperature refrigerator.
The product for preventing and/or treating inflammatory lesions of the intestine and liver according to the present invention will be described below.
The product for preventing and/or treating intestinal tract and liver inflammatory injury according to the embodiment of the invention can be a product of the sorghum black rice, or a product containing sorghum black rice polysaccharide extracted from the sorghum black rice, and the like.
The preparation method of the product for preventing and/or treating intestinal tract and liver inflammatory injury can be obtained by the following method.
Taking the preparation of the whole sorghum black rice flour as an example, the whole sorghum black rice is dried, crushed and sieved by adopting a 100-mesh sieve, for example, to prepare the whole black rice flour (SRPW) for standby.
Taking preparation of sorghum black rice polysaccharide powder as an example, weighing the whole sorghum black rice powder, degreasing with petroleum ether for three times (1:4), refluxing in water bath at 95 ℃, drying the filtered solid, desugaring with 85% ethanol, drying the filtered solid, adding distilled water according to the ratio of 1:10, leaching in water bath at 85 ℃ for 2h, filtering to obtain a polysaccharide crude extract, extracting for three times, and combining the filtrates. Concentrating the crude extractive solution, adding 4 times volume of anhydrous alcohol, precipitating with ethanol overnight, centrifuging to obtain crude polysaccharide, washing with anhydrous alcohol, acetone and diethyl ether for 2 times, and drying at 60 deg.C. Adding water to the dried crude polysaccharide for redissolving, removing protein for 3 times by Sevag method, adding hydrogen peroxide for decolorization under the condition of pH8.5, keeping the temperature at 40 ℃ for 1h, concentrating, dialyzing for 48h, and freeze-drying in vacuum to obtain refined sorghum black rice polysaccharide powder (SRPS) for later use.
The following description will discuss the use of the sorghum Umi product for preventing and/or treating inflammatory injury of intestinal tract and liver with reference to the specific examples.
1. Test grouping
SPF-grade female BALB/c mice were selected and acclimatized in the IVC system for 1 week, and then randomly divided into five groups of 12 mice each. Respectively a Normal group (Normal group), a Model group (Model group), a Positive control group (Positive group), a sorghum black rice whole flour group (SRPW group) and a sorghum black rice polysaccharide group (SRPS group). Normal saline for intragastric administration of Normal group and Model group, 200mg/kg sulfasalazine enteric-coated tablet for intragastric administration of Positive group, 500mg/kg whole sorghum black rice powder for intragastric administration of SPRW group, and 400mg/kg sorghum black rice polysaccharide for intragastric administration of SRPS group.
2. Model induction of inflammatory injury
Except for the Normal group, five groups of mice all received DSS via drinking water. Distilled water is drunk on 0-7 days, 2.5% DSS is drunk on 8-10 days, and 5% DSS is introduced on 11-16 days to perform inflammatory injury model induction.
3. General physical observation
Daily food intake, water intake, weight change, mental state and hair color quality of each group of mice were recorded, and stool character and hematochezia condition of the mice were observed. The Disease Activity Index (DAI) was calculated according to the scoring criteria of Table 1-1, and the formula is shown in (1-1).
TABLE 1-1 DAI scoring criteria
4. Histological morphology observation
Taking mouse colon tissue with proper size, washing with running water for 24h, dehydrating with gradient ethanol, clearing with xylene, soaking in molten paraffin, and embedding with paraffin. The embedded paraffin blocks were sliced to a thickness of 5 μm. Hematoxylin-eosin (HE) staining followed by microscopic observation.
5. ELISA method
According to the kit determination method, the content of LPS in serum, liver and colon tissues is determined.
6. qRT-PCR method
6.1 TRNzol method for extracting tissue RNA
Placing appropriate amount of colon or liver tissue into EP tube, adding 1mL TRNzol (lysate) for lysis, grinding with cryo-grinder, standing at room temperature for 5min, and rotating at 12000rpm for 10min at 4 deg.C. The supernatant was aspirated, 1/5 volumes of chloroform were added, and the solution was shaken well for 15s using a vortex mixer. After standing at room temperature for 3min, the mixture was centrifuged at 4 ℃ for 15min at 12000 rpm. The liquid in the EP tube is divided into three layers: the lowest layer is a pink transparent organic phase, the middle layer is white, and the upper layer is a colorless aqueous phase. The aqueous phase was transferred to an enzyme-deactivated EP tube and its volume was recorded. Adding isopropanol with the same volume into an EP tube, uniformly mixing, and standing for 10min at room temperature. Centrifugation was carried out at 12000rpm at 4 ℃ for 10min to remove the supernatant. Add 1mL of the RNase-free ddH2O75% ethanol, wash the precipitate 3 times. Centrifuging at 4 deg.C and 10000rpm for 5min, pouring out liquid, air drying at room temperature, adding appropriate amount of ddH without RNase2O, fully blowing and uniformly mixing by using a liquid transfer gun to dissolve the RNA in the ddH2O, and finally storing at-80 ℃ in a sealed manner.
6.2 cDNA Synthesis
Inserting an EP tube into an ice box to accurately absorb reagents according to the table 1-2 to prepare a reaction solution, uniformly mixing the reagents, and putting the mixture into a PCR instrument for reaction, wherein the reaction conditions of the instrument are as follows: the reaction is carried out for 15min at the temperature of 42 ℃ and for 3min at the temperature of 95 ℃.
TABLE 1-2 genomic DNA removal reaction procedure
6.3 real-time fluorescent quantitative PCR analysis
PCR reaction solutions were accurately prepared on an ice bench as shown in tables 1-3. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 30s, the instrument was then set to 40 cycles with cycling conditions of 95 ℃ for 3s and 60 ℃ for 30s, and melting curve conditions of 95 ℃ for 15s, 60 ℃ for 15s, and 95 ℃ for 15 s. mRNA levels were normalized to GADPH mRNA by 2-ΔΔCtThe method performs a result calculation.
TABLE 1-3 PCR reaction solution preparation System
TABLE 1-4 primer sequences
7. Western blotting method
7.1 Total protein extraction from tissues
An appropriate amount of tissue was placed in a 1.5mL EP tube, 400 μ L of RIPA lysate (RIPA: PMSF: phosphatase inhibitor: 100: 1: 1) was added according to the amount of tissue, and after grinding for 30 seconds with a cryo-grinder, the tube was shaken for 30 seconds on a vortex mixer every 10min for 30 min. Centrifuging at 12000rpm at 4 deg.C for 10min, and collecting supernatant to obtain total protein product. The operations were all performed on ice.
7.2 determination of protein concentration
(1) Preparation of protein standards: and adding 0.8mL of protein standard preparation solution into a protein standard tube (total 20mg of BSA), and carrying out ultrasonic dissolution to obtain a 25mg/mL BSA standard solution. Long-term storage at-20 deg.C. Taking a proper amount of protein standard solution, and diluting to a final concentration of 0.5 mg/mL.
(2) The operation procedure is as follows: a. according to the quantity of the samples, adding the reagent A and the reagent B according to the ratio of 50:1 to prepare working solution, and fully and uniformly mixing. b. And (3) sucking the standard solution prepared in the step (1) into a 96-well plate according to the sequence of 0, 1, 2, 4, 8, 12, 16 and 20 mu L, and adding a standard diluent to make up the volume of the standard solution to 20 mu L, wherein the concentrations of the standard in the wells are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/mL in sequence. Adding a tissue protein sample into the sample hole, and arranging a plurality of holes at the same time. 200 mu L of BCA working solution is added into each hole, and the mixture is placed at 37 ℃ for 20-30 min. Measuring OD value with enzyme-labeling instrument at 562nm, drawing standard curve to calculate total protein concentration, diluting with lysate, and adding 5xSDS protein loading buffer for storage.
7.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
(1) Preparing glue: the optimum separation gel concentration is selected according to the molecular weight of the target protein, and the mixture is prepared according to the instruction of an SDS-PAGE gel kit. The optimum separation ranges for the different concentrations of separation gel are shown in tables 1-5.
TABLE 1-5 optimum separation Range for separation gels of different concentrations
(2) SDS-PAGE electrophoresis: before sample adding, boiling and denaturing the sample in boiling water bath for 5min, determining a proper sample adding volume, then adding the sample, starting electrophoresis at a constant voltage of 80V, and changing the voltage to 120V when the protein strip moves to a position 1-2 cm below the concentrated gel until the protein leaves the separation gel and enters the electrophoresis solution.
(3) Film transfer: transferring the film by adopting a semi-dry transfer method: soaking a filter paper sheet and the PVDF membrane which is activated by methanol in a wet transfer liquid, taking down the separation gel, clamping the gel and the PVDF membrane in the middle by using filter paper (the gel is above the PVDF membrane) to form a sandwich structure, paying attention to remove bubbles, and setting the conditions of an instrument to be transfer membrane current of 70 mA/membrane 1 h/membrane (<120KD) or 2.5 h/membrane (>120 KD).
(4) Blocking and hybridizing: after membrane transfer is finished, selecting a band range according to the indication of a pre-stained marker, cutting off a target protein band, blocking for 2h at 4 ℃ by using 5% BSA, and washing for 5 times by using TBS-T solution, wherein each time is 5 min; incubating primary antibody of the target protein, standing overnight at 4 ℃, and cleaning the strip with prepared TBS-T solution after the end of the next day of primary antibody hybridization; the secondary antibody was incubated at 4 ℃ for 2h to complete the washing of the strip with TBS-T solution.
(5) Color development: the color developing solution is divided into solution A and solution B, and when the color developing solution is used, the solution A and the solution B are mixed in equal volume to prepare working solution. And (4) putting the cleaned strip in the step (4) into an instrument, uniformly adding working solution into the surface of the strip, selecting exposure time for developing, and then storing the picture.
8. Data results and analysis
The test data is analyzed and charted by Excel software, and the Image J software is used for carrying out quantitative analysis on the test picture result. The test data are statistically analyzed by SPSS22.0 software, and significance comparison is carried out by One-way ANOVA, and the statistical significance is achieved when p is less than 0.05.
Second, result and analysis
1. Macro-regulation effect of sorghum black rice on colitis mice
Observing the living state of the mice every day, wherein the weight change of each group of mice is shown as A in figure 1, and on 8-10 days, the Normal group of mice has good mental state, liveness and mobility and agility in action; model group mice are listened, lazy and sluggish; the conditions of the mice in the SRPW and SRPS groups are similar to those in the normal group. After 11 days, four groups of mice, except Normal, showed signs of listlessness, laziness, and bradykinesia. Day 16 body weights, Normal and SRPW groups differed significantly from Model group (p <0.05), indicating that DSS had an effect on mouse body weight in this experiment. Meanwhile, in the first 7 days, particularly in the SRPW group, the food consumption is on the rise, probably because the sorghum and black rice whole flour has the effects of promoting intestinal peristalsis and relaxing bowel. However, the food consumption tended to decrease in the four groups except the Normal group, beginning on day 10. As shown in B of fig. 1).
The DAI score index is used for scoring according to body weight change, stool characteristics and hematochezia conditions, and is widely applied to evaluating the molding degree of the colitis of the mice. The Model panel score was significantly different from the Normal panel by panels indicated as C in fig. 1, indicating successful molding. And the Positive, SRPW and SRPS cohorts all had significant differences from the Model cohort (p < 0.05). As shown by D in fig. 1, the Model group renal and spleen indices were significantly higher than the Normal group, and the thymus index was significantly lower than the Normal group (p < 0.05). Through the regulation of the sorghum Umi, organ indexes of the SRPW group and the SRPS group are in opposite trends, and are remarkably different from those of the Model group (p < 0.05). As shown in E in figure 1, observed under a 100-fold microscope, the colon and intestinal lumen textures of Normal mice are clear, mucous membranes are complete and continuous, and glands are regularly and clearly arranged; the colon and intestinal cavity texture of the Model group is fuzzy, the mucous membrane is incomplete, the intestinal cavity structure is damaged, and inflammatory cell infiltration is obvious; the colon mucosa of the Positive group, the SRPW group and the SRPS group is relatively complete, the gland arrangement is relatively regular, the structure is clear, the inflammatory cell infiltration is reduced, and no obvious ulcer occurs, wherein the SRPW group and the SRPS group are relatively close to the Normal group.
2. Regulation effect of sorghum black rice on colon inflammation mouse intestinal tract
2.1 Regulation of intestinal tissue cytokines in mice with colitis
Under healthy conditions, there is a tight control between gut cells and gut immune system cells to ensure tolerance to gut microbiota and thus a balance between proinflammatory and anti-inflammatory factors and other cytokines. The major players in the transition between acute and chronic inflammation include IL-1. beta. and IL-6, etc., while TNF-. alpha.secretion may further promote the expression of other factors and activation of signaling pathways. As shown in FIG. 2, the expression of TNF-. alpha.IL-1. beta., iNOS, and IL-6mRNA in the Normal group was significantly different from that in the Model group (p <0.05), in which the TNF-. alpha.and IL-1. beta. mRNA expression levels were significantly decreased in the Positive, SRPW, and SRPS groups (p <0.05), the iNOSmRNA expression level was significantly decreased in the Positive and SRPW groups (p <0.05), and the IL-6mRNA expression level was significantly decreased in the Positive and SRPS groups (p <0.05), as shown by A to D in FIG. 2. Meanwhile, protein expressions of TNF-alpha, IL-6 and IL-10 show that, as shown in the E to H in figure 2, protein expressions of a Normal group and a Model group are all significantly different (p <0.05), and protein expressions of TNF-alpha and IL-6 are significantly reduced by a Positive group, an SRPW group and an SRPS group (p <0.05), and protein expression of IL-10 is significantly improved (p <0.05), which is consistent with the trend of the Normal group and the mRNA expression regulation result.
Chemokines are small cytokines secreted from cells, and have a function of chemotactic targeting immune cells when a human body defends and removes foreign substances such as invading pathogens. The intestinal mucosa is the largest interface of the body contacting with the external environment, and has the barrier functions of selectively permeating and absorbing nutrient substances and defending the invasion of microorganisms and inflammatory factors in the intestinal tract. The balance of intestinal mucosal factor expression also plays a crucial role. As can be seen from FIG. 3, the CXCL-2, COX-2 and MUC-2mRNA expression in the Normal group is significantly different from that in the Model group (p <0.05), while the Positive, SRPW and SRPS groups also inhibit the CXCL-2, COX-2 and MUC-2mRNA expression, and are significantly different from that in the Model group (p <0.05), which tends to be at the level of the Normal group. In conclusion, the sorghum Umbelliferae is shown to regulate the generation of inflammation by regulating and inhibiting the gene and protein expression of inflammatory factors.
2.2 Regulation of NF- κ B Signal pathway in intestinal tissue of Mesorghums Umbelliferae mice with colitis
In recent years, studies of signal pathways in the intestinal tract associated with IBD have mainly focused on nuclear factor- κ B (NF- κ B), mitogen-activated protein kinase (MAPK), peroxisome proliferator-activated receptors (PPARs), and the like. NF- κ B is a family of transcription factor proteins and is an important factor in transcriptional regulation, including five subunits, Rel, p65, RelB, p50 and p 52. All proinflammatory cytokine genes comprise NF-kB binding sites, the expression of the proinflammatory cytokine genes is increased along with the production and secretion capacity of inflammatory factors such as IL-6, TNF-alpha, IL-1 and the like, and the proinflammatory cytokine genes are used as a core effect for controlling the secretion of the proinflammatory cytokines and directly involved in the occurrence of mucosal tissue damage. As shown in FIG. 4, the mRNA expression of NF-. kappa. B p65, MyD88, I.kappa.B.alpha.and TLR-4 in the Normal group were all significantly different from that in the Model group (p <0.05) as shown by A to E in FIG. 4, indicating that the NF-. kappa.B signaling pathway was activated. Wherein the Positive and SRPS groups significantly down-regulated the expression of NF- κ B p65 mRNA (p < 0.05); the SRPW group significantly down-regulated the expression of MyD88 mRNA (p < 0.05); the Positive, SRPW, and SRPS groups all significantly down-regulated expression of I κ B α mRNA (p <0.05) and significantly up-regulated expression of SIGIRR mRNA (p < 0.05).
As shown by F to H in FIG. 4, the protein expression levels of NF-. kappa. B p65 and I.kappa.B.alpha.in the Normal group and the Model group were significantly different (p <0.05), the phosphorylation expression of NF-. kappa. B p65 protein in the Positive group and the SRPS group was significantly reduced (p <0.05), and the protein expression level of I.kappa.B.alpha.in the Positive group and the SRPW group was significantly reduced (p < 0.05). Consistent with the trend of results described above. The sorghum Umbellata is shown to be capable of reducing the expression of inflammatory factors and regulating colonic inflammation by inhibiting the activation of NF-kB signal channels.
3. Sorghum black rice regulating effect on colon enteritis mouse liver based on intestine-liver axis
The direct connection between the diet and the intestinal tract exists, and the foods such as vegetables, fruits, whole grains and the like are rich in dietary fiber, polyphenol, folic acid, carotenoid and the like, and are very beneficial to the health of the intestinal tract. The gastrointestinal tract is a barrier that limits the interaction between the luminal contents of the intestinal flora and metabolic waste products, which are closely related and interacting with the liver in structure and function, known as the "gut-liver axis". LPS is an endotoxin, and is hardly released from cell walls, and when bacteria die or the like, it is released by dissolving, destroying cells, and exerts its toxicity by acting on animal cells or the like. As shown in FIG. 5, the expression of LPS in Normal group was significantly different from that in Model group (p <0.05) in colon, serum and liver as shown in A to C in FIG. 5, indicating that the inflammatory factors induced by DSS entered the liver through colon-blood and damaged the liver. While the Positive group, the SRPW group and the SRPS group all obviously reduce the expression of LPS (p is less than 0.05), which indicates that the sorghum Umbelliferae can play a certain role in regulating the inflammation of the liver through the intestine-liver axis.
3.1 Regulation of colitis mouse liver by cell factor of Kaoliang black rice
Referring to FIG. 6, as shown by A to D in FIG. 6, the expression of TNF-. alpha.IL-1. beta. and iNOS mRNAs in the Normal group was significantly different from that in the Model group (p < 0.05). The Positive group, the SRPW group and the SRPS group all significantly down-regulate the expression of the three genes (p is less than 0.05), and the Positive group and the SRPS group down-regulate the expression of IL-6mRNA, but have no significant difference. In liver tissues, the Normal and SRPW groups also significantly down-regulated CXCL-2 mRNA expression (p <0.05) (fig. 6E). From the protein expression results, as shown in FIGS. 6F to I, the protein expression of IL-6, TNF-. alpha.and IL-10 in the Normal group were all significantly different from that in the Model group (p < 0.05). The protein expression of IL-6 and TNF-alpha in the Positive group, SRPW group and SRPS group is significantly different from that in the Model group (p <0.05), and tends to the normal group. The expression trend of the IL-10 protein in the SRPW group is opposite to that in the Model group, and the expression trend is remarkably different (p < 0.05). The sorghum Umbellatus can regulate the expression of cell factors through the intestine-liver axis so as to regulate the inflammatory injury of the liver.
3.2 Regulation of colitis mouse liver by NF-kB Signal pathway of Umi sorghum
Referring to FIG. 7, from A to D in FIG. 7, it can be seen that the expression of mRNA of NF-. kappa. B p65, MyD88 and TLR-4 in the Normal group was significantly different from that in the Model group (p <0.05), indicating that NF-. kappa.B signaling pathway was activated. The Positive group, the SRPW group and the SRPS group all significantly down-regulate the expression of NF-kappa B p65, MyD88 and TLR-4mRNA (p is less than 0.05), and tend to normal level. Meanwhile, the SRPS group significantly improves the expression of SIGIRR mRNA (p < 0.05). As can be seen from E to G in FIG. 7, the expression of NF- κ B p65 protein in the Model group was significantly different from that in the other four groups (p <0.05), while the expression level of Iκ B α was significantly different from that in the Normal group, the Positive group and the SRPS group (p <0.05), which is substantially consistent with the gene expression results. The sorghum Umbelliferae shows that the NF-kB signal channel can be adjusted by the intestinal-hepatic axis of the Umbelliferae, so that the expression of related cell factors is inhibited, and the aim of adjusting liver injury is fulfilled.
Therefore, according to the product for preventing and/or treating intestinal tract and liver inflammatory injury provided by the embodiment of the invention, the sorghum black rice whole powder and the polysaccharide are used for intervening DSS-induced colitis mice, the sorghum black rice promotes appetite, the food consumption of the mice can be increased, and the effects of promoting intestinal tract movement and regulating intestinal tract health can be realized after long-term eating of the product. The sorghum Umi can regulate colon inflammation of mice by regulating the expression of cell factors and inhibiting the activation of signal channels, and regulates the expression of related factors and signal channels in liver tissues through a bowel-liver axis, which indicates that the sorghum Umi can prevent and regulate inflammatory injuries of intestines and livers.
Therefore, the scope of the present invention should not be limited to the disclosure of the embodiments, but includes various alternatives and modifications without departing from the scope of the present invention, which is defined by the claims of the present patent application.
Claims (10)
1. A product for preventing and/or treating intestinal tract and liver inflammatory injury, wherein the product is a product of Usnea sorghum, or a product containing Usnea sorghum polysaccharide extracted from Usnea sorghum.
2. The product for preventing and/or treating inflammatory injury of the intestine and liver according to claim 1, wherein the product is a food, a pharmaceutical or a health product.
3. A method for preparing a product for preventing and/or treating intestinal and liver inflammatory injury, which is characterized by comprising the following steps:
the sorghum black rice is used as a raw material to prepare a product for preventing and/or treating intestinal tract and liver inflammatory injury.
4. The method for preparing a product for preventing and/or treating inflammatory injury of the intestine and liver according to claim 3, which comprises: drying the sorghum black rice, crushing and sieving to prepare the sorghum black rice whole powder.
5. The method for preparing a product for preventing and/or treating inflammatory injury of the intestine and liver according to claim 4, which comprises:
degreasing the whole sorghum black rice flour, refluxing in a water bath at 95 ℃, filtering, and drying the solid to obtain sorghum black rice degreased powder;
desugarizing the sorghum black rice defatted powder by 85% ethanol, filtering, drying the solid, leaching in water bath, and filtering to obtain a polysaccharide crude extract;
concentrating the polysaccharide crude extract, adding 4 times of volume of absolute ethyl alcohol, precipitating with ethanol overnight, and centrifuging to obtain crude polysaccharide;
washing the crude polysaccharide with anhydrous ethanol, acetone and diethyl ether, drying, re-dissolving the dried crude polysaccharide in water, removing protein, decolorizing with hydrogen peroxide at pH8.5, maintaining at 40 deg.C for 1 hr, concentrating, dialyzing for 48 hr, and vacuum freeze drying to obtain refined sorghum black rice polysaccharide powder.
6. An application of sorghum Umi product in preventing and/or treating intestinal inflammatory injury is provided.
7. The use of the kaoliang ugli nakaoliang nakai comprises whole kaoliang nakaoliang powder or kaoliang polysaccharide extracted from said kaoliang nakaoliang.
8. The sorghum Umi preparation for use in the prevention and/or treatment of inflammatory injury in the intestinal tract according to claim 6, wherein the inflammatory injury in the intestinal tract is Dextran Sodium Sulfate (DSS) -induced inflammatory injury in the intestinal tract.
9. An application of sorghum Umi product in preventing and/or treating liver inflammatory injury is provided.
10. The sorghum Umi preparation according to claim 9, for use in the prevention and/or treatment of liver inflammatory injury, wherein the liver inflammatory injury is DSS-induced liver inflammatory injury.
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Cited By (1)
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| CN113768028A (en) * | 2021-09-14 | 2021-12-10 | 辽宁省农业科学院 | Method for separating protein from ustilago sorghum and application of method |
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| CN113768028A (en) * | 2021-09-14 | 2021-12-10 | 辽宁省农业科学院 | Method for separating protein from ustilago sorghum and application of method |
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