Application of composition in preparation of product for improving spleen deficiency type intestinal mucosal barrier damage

Abstract

The invention relates to application of a composition in preparation of a product for improving spleen deficiency type intestinal mucosal barrier damage. Tests prove that the composition not only can improve intestinal epithelial cell apoptosis of mice with spleen deficiency, but also can improve an intercellular tight connection structure of the mice with spleen deficiency, and finally improves spleen deficiency type intestinal mucosal barrier damage. Therefore, the composition has the effect of improving the spleen deficiency type intestinal mucosal barrier damage and can be used for preparing products for improving the spleen deficiency type intestinal mucosal barrier damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine compositions, and relates to the application of a composition in the preparation of a product for improving the damaged intestinal mucosal barrier of the spleen-deficiency type. Background Art

[0002] The intestine is the main organ for absorbing nutrients and water. At the same time, it also constitutes an important barrier against harmful substances and pathogens in the external environment. The normal intestinal mucosal barrier is mainly composed of a mechanical barrier, a chemical barrier, a biological barrier, and an immune barrier. Among them, the mechanical barrier composed of intestinal epithelial cells and their tight junctions is the most important. It is not only the key for the intestinal mucosa to resist the invasion of pathogens or harmful substances in the external environment into the body, but also the structural basis for maintaining the selective permeability of the intestinal epithelium and its barrier function. There are many reasons for the damage of the intestinal mucosal barrier, such as infectious factors (bacterial infection or viral infection), immune factors (autoimmune diseases or immune deficiency), physical factors (trauma or surgery), chronic disease factors (diabetes or chronic liver disease), stress factors (psychological stress or physiological stress), etc. Different causes of intestinal mucosal barrier damage require different intervention measures.

[0003] Feng Shibin et al. from Anhui Agricultural University published a literature titled "Effect of Jiawei Sijunzi Decoction on the Expression of Tight Junction Proteins and ERK / MAPK Pathway in the Small Intestine of Spleen-Deficiency Dogs" in the Journal of Yunnan Agricultural University. The literature mentioned that spleen deficiency can cause damage to the mechanical barrier of the intestinal mucosa. At present, for the development of products to improve the damaged intestinal mucosal barrier, most are based on intestinal mucosal barrier damage models induced by chemical induction (such as dextran sulfate sodium or benzalkonium chloride), drug induction (5-fluorouracil), physical injury induction, chronic stress induction, etc., to further develop products for improving intestinal mucosal damage. However, there is no report on these products for the damaged intestinal mucosal barrier of the spleen-deficiency type.

[0004] In view of this, developing a product that can effectively improve the damaged intestinal mucosal barrier of the spleen-deficiency type has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] To solve the problems existing in the prior art, the present invention provides the application of a composition in the preparation of a product for improving the damaged intestinal mucosal barrier of the spleen-deficiency type. The technical solution adopted by the present invention is as follows:

[0006] The application of a composition in the preparation of a product for improving the damaged intestinal mucosal barrier of the spleen-deficiency type, wherein the composition comprises the following raw materials: 33.7 wt% of pumpkin peel, 20.8 wt% of pumpkin pedicel, 12.9 wt% of pumpkin vein, 7.9 wt% of Poria cocos, 9.0 wt% of Tremella fuciformis, and 15.7 wt% of xylooligosaccharide.

[0007] In a preferred embodiment of the present invention, the improvement of the damaged intestinal mucosal barrier of spleen deficiency type is the improvement of the damaged intestinal mechanical barrier of spleen deficiency type.

[0008] In a preferred embodiment of the present invention, the improvement of the intestinal mucosal barrier of spleen deficiency type includes the improvement of intestinal epithelial cell apoptosis in spleen-deficient mice.

[0009] In a preferred embodiment of the present invention, the improvement of the intestinal mucosal barrier of spleen deficiency type includes the improvement of the intercellular tight junction structure in spleen-deficient mice.

[0010] In a preferred embodiment of the present invention, the improvement of the intestinal mucosal barrier of spleen deficiency type includes a significant increase in the expression levels of intestinal tight junction proteins Claudin-1, Occludin, and ZO-1.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] The composition of the present invention has been proven by experiments to be able to improve both the intestinal epithelial cell apoptosis in spleen-deficient mice and the intercellular tight junction structure in spleen-deficient mice, and ultimately improve the damaged intestinal mucosal barrier of spleen deficiency type; at the same time, it can improve symptoms such as reduced food intake, listlessness, and weight loss caused by spleen deficiency. Therefore, the composition of the present invention has the effect of improving the damaged intestinal mucosal barrier of spleen deficiency type and can be used to prepare products for improving the damaged intestinal mucosal barrier of spleen deficiency type. Brief Description of the Drawings

[0013] Figure 1 It is a schematic diagram of Tunel staining of the colon tissues of the test animals in each group of the present invention;

[0014] Figure 2 It is a schematic diagram of immunohistochemical staining of the colon tissues of the test animals in each group of the present invention;

[0015] Figure 3 It is a schematic diagram of the expression levels of three tight junction proteins in the colon tissues of the test animals in each group of the present invention;

[0016] Figure 4 It is a schematic diagram of the statistical analysis of the hair condition scores of the test animals in each group of the present invention;

[0017] Figure 5 It is a schematic diagram of the statistical analysis of the average daily food intake of the test animals in each group of the present invention in the 3rd week;

[0018] Figure 6 It is a schematic diagram of the statistical analysis of the weight changes of the test animals in each group of the present invention before and after the experiment;

[0019] Figure 7 It is a schematic diagram of the statistical analysis of the autonomous activity evaluation of the test animals in each group of the present invention;

[0020] Figure 8 Photographs of the behavioral states of the test animals in each group of the present invention;

[0021] In the figure, CON is the blank group, MOD is the model group, and JTTC is the composition group. Specific embodiments

[0022] The present invention will be specifically described below in conjunction with specific drawings and embodiments. It is necessary to point out here that the following embodiments are only for further illustration of the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention still fall within the protection scope of the present invention.

[0023] The raw materials used in the embodiments are all conventional commercially available raw materials. Among them, the manufacturer of the rhubarb medicinal material is Bozhou Baichuan Pharmaceutical Co., Ltd.

[0024] In the present invention, the specific weight ratio of the composition is as follows: pumpkin peel 33.7 wt%, pumpkin pedicel 20.8 wt%, pumpkin fiber 12.9 wt%, Poria cocos 7.9 wt%, Tremella fuciformis 9.0 wt%, and xylooligosaccharide 15.7 wt%. Its preparation method is as follows: select pumpkin peel, pumpkin pedicel, pumpkin fiber, Poria cocos, and Tremella fuciformis raw materials according to the weight ratio, pick and remove impurities, and then soak them in water; after soaking, start decocting, decocting 3 times, each time for 30 min, combine the decoctions of the 3 times, and filter; start concentrating until it becomes a thick paste; then carry out low-temperature drying to make the moisture in the mixture ≤8% and the ash content ≤5%; after testing and meeting the requirements, ultrafinely pulverize it with a jet mill to finally pulverize it to 500 mesh; after pulverization, fully mix and blend it with xylooligosaccharide to obtain the product. It should be noted that the composition of the present invention is basically the same as the composition in the Chinese invention patent "Capsule formula and preparation method for treating constipation mainly made of pumpkin" (publication number: CN1533805A), the difference is only that aloe is not added, and the other components and the corresponding preparation methods are exactly the same, and it is actually the reduced formula Tiantian Capsule (JTTC).

[0025] The instruments used in the embodiments are all conventional commercially available instruments.

[0026] Example 1

[0027] 1. Preparation of the test sample

[0028] 1.1 Decoction of rhubarb: Take 250 g of raw rhubarb, add 2000 ml of water, soak for 0.5 h, decoct over low heat for 15 min, cool naturally, filter with 4 layers of gauze, combine the decoctions, and concentrate to 250 ml using a rotary evaporator, equivalent to 1 g / ml of raw medicine.

[0029] 1.2 Test sample of the composition: Weigh 1 g of the composition and dissolve it in 10 mL of physiological saline to obtain a test sample of the complex with a composition concentration of 0.1 g / mL.

[0030] 2. Animal grouping, model establishment and administration

[0031] 2.1 Test animals and animal grouping

[0032] The test animals were selected as mice of the KM strain (SPF level). All test animals were of clean grade, with the certificate number SCXK(Jing)2021 - 0011, a total of 30. All test animals were fed in a rack - type cage, 5 in each cage, and male and female were caged separately. The room temperature was maintained at about 24 °C, the relative humidity was 40 - 60%, and the light cycle was 12 h. During the whole test process, the test animals were given ordinary feed and free drinking water. The mice were fed adaptively for 1 week, weighed, and randomly divided into 3 groups according to their body weight. Then, model establishment and administration were carried out. The method of model establishment referred to "The influence of Guiqi Baizhu Decoction on the expression of immune factors and aquaporins in spleen - deficient mice" (published by Gansu University of Chinese Medicine in Lishizhen Medicine and Materia Medica).

[0033] Table 1 Test animals and animal grouping

[0034] Group Test animals Gender Body weight Number of animals Blank group KM mice Half male and half female (20±2)g 10 Model group KM mice Half male and half female (20±2)g 10 Composition group KM mice Half male and half female (20±2)g 10

[0035] 2.2 Test animal model establishment and administration

[0036] Table 2 Test animal model establishment and administration

[0037]

[0038]

[0039] 3 Detection of test animals

[0040] 3.1 Detection of routine physiological state indicators of test animals in each group

[0041] During the experiment, the body weight of the animals was recorded daily. During the 3rd week, the hair condition, behavior state, food intake and body weight of the test animals in each group were recorded. Among them, in the evaluation of the behavior state of the test animals in each group (i.e., the evaluation of the spontaneous activity of the animals), the test animals in each group were put into the spontaneous activity recorder in turn every day and the number of activities within the unit time was counted.

[0042] The animal hair was scored according to the "Expert Consensus on Traditional Chinese Medicine Diagnosis and Treatment of Spleen Deficiency Syndrome (2023)" and "Methodology of Traditional Chinese Medicine Experimental Animal Models", and the appearance signs of spleen-deficiency mice were scored. The scoring criteria were as follows: ① Hair condition: smooth, shiny, no yellowing, no piloerection, scored 0 points; messy, dull, slightly yellowing, no piloerection, scored 1 point; messy, dirty, dry, yellowing, no piloerection, scored 2 points; messy, dirty, dry, yellowing, piloerection, scored 3 points.

[0043] 3.2 Detection of intestinal mucosal barrier in each group of test animals

[0044] 3.2.1 Sample collection and storage

[0045] After 12 weeks, the test animals in each group were sacrificed. After taking blood from the abdominal aorta, the colon parts of each mouse in each group of test animals were immediately cut. Three mice were randomly selected from each group, and a section about 0.5 cm long was cut at the proximal colon (connected to the cecum), rinsed and immersed in 10% formalin for staining. The remaining colon tissues were labeled and placed in a plastic bag for storage at -80 °C for subsequent detection.

[0046] 3.2.2 TUNEL staining

[0047] a) Fixation and dehydration: After fixing the colon tissue with 4% paraformaldehyde as the fixative for 24 h, transfer it to an automatic tissue dehydrator for dehydration operation;

[0048] b) Embedding: Embed the dehydrated colon tissue with paraffin to form tissue paraffin blocks;

[0049] c) Sectioning: Cut the paraffin block embedding the colon tissue into tissue sections with a thickness of 4 μm;

[0050] d) Dewaxing: Place the tissue sections in xylene solution, dewax for 10 min, then replace with fresh xylene solution and continue dewaxing for 10 min;

[0051] e) Hydration: Hydrate the tissue sections with ethanol solutions of different concentrations. The specific sequence is absolute ethanol for 5 min → 95% ethanol for 5 min → 80% ethanol for 5 min → 75% ethanol for 5 min → distilled water for 2 min;

[0052] f) Rinse twice with PBS and treat the tissue sections with Proteinase K working solution at 20 μg / mL for 15 - 30 min;

[0053] g) Add PBS containing 2% hydrogen peroxide, react at room temperature for 5 min, and rinse twice with PBS;

[0054] h) Prepare the TUNNEL reaction mixture. For the treatment group, mix 50 μl of TdT with 450 μl of fluorescein-labeled dUTP solution; for the negative control group, add only 50 μl of fluorescein-labeled dUTP solution. For the positive control group, first add 100 μl of DNase 1 and react for 15 - 25 × 10 min. The subsequent steps are the same as those for the treatment group;

[0055] i) After the glass slides are dried, carefully suck off the excess liquid around the sections with filter paper, add 50 μl of the TUNNEL reaction mixture (for the negative control group, add only 50 μl of fluorescein-labeled dUTP solution to the specimen), cover with a coverslip or sealing film, and react in a dark and humid box at 37 °C for 1 h;

[0056] j) Add to the washing and termination reaction buffer preheated to 37 °C, incubate at 37 °C for 30 min, and rinse 3 times with PBS;

[0057] k) Add 50 μl of DIG-POD to the specimen on the glass slide, cover with a coverslip or sealing film, and react in a dark and humid box at 37 °C for 30 min;

[0058] l) Rinse 3 times with PBS; add 50 - 100 μl of DAB substrate to the tissue and react for 15 - 25 × 10 min;

[0059] m) Rinse 3 times with PBS; after photographing, counterstain with hematoxylin or methyl green, immediately rinse with tap water after a few seconds. Dehydrate with gradient ethanol, clear with xylene, and mount with neutral gum;

[0060] n) Place the mounted slide under a fluorescence microscope, observe the apoptosis of mouse colonic epithelial cells, and photograph and record.

[0061] 3.2.3 Immunohistochemical staining

[0062] a) Fixation and dehydration: Use 4% paraformaldehyde as the fixative to fix the colonic tissue for 24 h, and then transfer it to an automatic tissue dehydrator for dehydration;

[0063] b) Embedding: Embed the dehydrated colonic tissue with paraffin to form a tissue paraffin block;

[0064] c) Sectioning: Cut the paraffin block containing the embedded colonic tissue into tissue sections with a thickness of 4 μm;

[0065] d) Deparaffinization: Place the tissue sections in xylene solution, deparaffinize for 10 min, then replace with fresh xylene solution and continue deparaffinizing for 10 min;

[0066] e) Hydration: Hydrate the tissue sections with ethanol solutions of different concentrations. The specific sequence is absolute ethanol for 5 min → 95% ethanol for 5 min → 80% ethanol for 5 min → 75% ethanol for 5 min → distilled water for 2 min;

[0067] f) Antigen repair: Place the processed tissue sections into a container filled with EDTA or citrate buffer solution, transfer it to a microwave oven, and boil for 10 - 15 min;

[0068] g) Take out the tissue sections, cool them to room temperature, rinse the sections with PBS solution for 3 min × 3 times, then add an endogenous peroxidase blocker (3% H2O2 solution) to the sections and incubate at room temperature for 10 min;

[0069] h) Serum blocking: Wash with PBS solution for 5 min × 3 times, discard the PBS, place the sections on slides, add 100 μl of goat serum working solution to each section, and incubate at room temperature for 10 - 15 min;

[0070] i) Primary antibody incubation: After removing the serum, add 100 μl of primary antibody solution (Claudin - 1: 1:1000; Occludin: 1:1000; Zo - 1: 1:1000) to the corresponding sections, put them into an incubation box, and incubate overnight at 4°C.

[0071] j) Secondary antibody incubation: Wash with PBS solution for 5 min × 3 times, add 100 μl of HRP - labeled goat anti - rabbit secondary antibody solution to the corresponding sections, and incubate at room temperature for 60 min;

[0072] k) Immunohistochemical staining: Wash the tissue sections with PBS solution for 5 min × 3 times, add 100 μl of fresh DAB solution to each section, incubate at room temperature for 1 - 2 min, then rinse the sections with tap water for 1 - 2 min, counterstain with hematoxylin solution for 1 min, soak in tap water for 15 - 30 min, dehydrate, and mount with neutral resin;

[0073] l) Place the sections under an optical or inverted fluorescence microscope for observation, and take photos of different fields of view.

[0074] 4 Result analysis

[0075] 4.1 Detection of intestinal mucosal barrier indexes in the colon of test animals in each group

[0076] 4.1.1 Detection results of apoptosis in colon tissues of test animals in each group

[0077] Figure 1 It is a schematic diagram of Tunel staining of the colon tissue of test animals in each group. It is the terminal - deoxynucleotidyl - transferase - mediated dUTP nick - end labeling method, which is a common method for detecting DNA fragmentation (apoptosis). As Figure 1 can be seen, compared with the blank group, the apoptosis of colon epithelial cells in the model group increased significantly (specifically, it can be determined through Figure 1Compared with the model group, the colon epithelial cell apoptosis in the combination group was significantly reduced (specifically, Figure 1 (reflected by the green fluorescence intensity).

[0078] In summary, the composition can significantly reduce the expression of apoptosis-related proteins and inhibit apoptosis of colon epithelial cells, ultimately significantly improving intestinal epithelial cell apoptosis in spleen-deficient mice and ensuring the integrity of the intestinal mucosal barrier.

[0079] 4.1.2 Detection results of tight junction proteins in test animals in each group

[0080] Figure 2 The immunohistochemical staining diagram of colon tissues of each group of test animals is shown. Figure 3 The schematic diagram shows the expression of three tight junction proteins in the colon tissue of each group of test animals. Claudin-1, Occludin and ZO-1 are all tight junction proteins, which play an important role in maintaining the barrier function of the intestinal mucosa. Claudin-1 is involved in regulating cell permeability to prevent the invasion of pathogens and harmful substances. Occludin interacts with proteins such as ZO-1 to form stable tight junctions.

[0081] Figure 2 The brownish yellow area in the middle is the positive protein expression area. Figure 2 It can be seen that the three tight junction proteins are all positively expressed on the cell membrane of colon tissue epithelial cells. Specifically, for the three tight junction proteins Claudin-1, Occludin, and ZO-1, the blank group can see more brown-yellow positive staining areas, and the staining intensity is stronger; the model group has fewer brown-yellow positive staining areas and the staining is weaker; the combination group also has more brown-yellow positive staining areas, and the staining intensity is stronger.

[0082] The average optical density of three tight junction proteins was detected to quantify the expression of positive proteins. Figure 3 shown. Figure 3 In the table, ## indicates that there is a very significant difference between this group and the blank group (P < 0.01), ### indicates that there is a very significant difference between this group and the blank group (P < 0.001); * indicates that there is a significant difference between this group and the model group (P < 0.05), and ** indicates that there is a very significant difference between this group and the model group (P < 0.01). Figure 3It can be seen that, compared with the blank group, the positive expression level of Claudin-1 in the model group was extremely significantly decreased (P<0.001); compared with the model group, the positive expression level of Claudin-1 in the composition group was extremely significantly increased (P<0.01). Compared with the blank group, the positive expression level of Occludin in the model group was extremely significantly decreased (P<0.001); compared with the model group, the positive expression level of Occludin in the composition group was extremely significantly increased (P<0.01). Compared with the blank group, the positive expression level of ZO-1 in the model group was extremely significantly decreased (P<0.01); compared with the model group, the positive expression level of ZO-1 in the composition group was significantly increased (P<0.05).

[0083] In summary, the composition can significantly increase the expression of tight junction proteins, stabilize the intestinal barrier, reduce the occurrence of "intestinal leakage", and ultimately significantly improve the damage between cells in spleen-deficient mice, ensuring the normal function of the intestinal mucosal barrier.

[0084] 4.2 Appearance signs of each group of test animals

[0085] As mentioned above, "during the 3rd week, the appearance signs, behavior status, food intake, and body weight of each group of mice were recorded daily". According to the above scoring criteria, the hair conditions of mice in each group were scored, and after scoring, statistical analysis was performed using GraphPad Prism 8.0 software. The final data was expressed as mean ± standard deviation (one-way ANOVA was used for comparison among multiple groups); similarly, the behavior status, food intake, and body weight of each group of mice were also analyzed statistically using GraphPad Prism 8.0 software (one-way ANOVA was used for comparison among multiple groups). The results are as Figures 4 - 7 shown. Figures 4 - 7 follows. # indicates a significant difference between this group and the blank group (P<0.05), ## indicates an extremely significant difference between this group and the blank group (P<0.01); * indicates a significant difference between this group and the model group (P<0.05), ** indicates an extremely significant difference between this group and the model group (P<0.01).

[0086] Figure 4 Figure shows the statistical analysis schematic diagram of the hair condition scores of each group of test animals within the 3rd week. From Figure 4It can be seen that most of the test animals in the blank group had smooth, shiny, and non-yellowing hair. Compared with the blank group, some of the test animals in the model group had messy, dull, and slightly yellowing hair, some had dirty, dry, and yellowing hair, and individual test animals even had piloerection. For the hair condition score, there was a highly significant difference between the model group and the blank group (P<0.01). Compared with the model group, the test animals in the composition group also had messy, dull, and slightly yellowing hair at the end of the 1st week, but after the experiment ended, the aforementioned hair condition was significantly improved. Except for some individual test animals still having messy and yellowing hair, the hair condition of the remaining test animals was basically comparable to that of the blank group. For the hair condition score, there was a significant difference between the composition group and the model group (P<0.05). Thus, it can be known that the hair of spleen-deficiency mice was messy, dull, yellowing, dirty, and dry. After intervention with the composition for 2 weeks, it was able to significantly reverse the "messy, dull, yellowing, dirty, and dry hair" condition of spleen-deficiency mice, making the hair of the mice basically return to smooth, shiny, and non-yellowing.

[0087] Figure 5 Figure 4 shows the average daily food intake of the test animals in each group. From Figure 5 It can be seen that compared with the blank group, the average daily food intake of the test animals in the model group was extremely significantly reduced (P<0.01). Compared with the model group, the test animals in the composition group also had a reduced average daily food intake at the end of the 1st week, but after the experiment ended, the average daily food intake of the test animals in the composition group was extremely significantly increased (P<0.01). Thus, it can be known that the average daily food intake of spleen-deficiency mice was significantly reduced. After intervention with the composition for 2 weeks, it was able to significantly increase the average daily food intake of spleen-deficiency mice.

[0088] Figure 6 Figure 5 shows the changes in the body weight of the test animals in each group before and after the experiment. From Figure 6 It can be seen that the weight gain of the test animals in the model group was significantly lower than that in the blank group (P<0.05). The weight gain of the test animals in the composition group was significantly higher than that in the model group (P<0.05) during the experiment and was similar to that in the blank group. Thus, it can be known that the weight gain rate of spleen-deficiency mice was significantly slowed down. After intervention with the composition for 2 weeks, it was able to significantly increase the weight gain of spleen-deficiency mice, making it basically return to the level of the blank group.

[0089] Figure 7 Figure 6 shows the evaluation of the spontaneous activities of the test animals in each group, specifically the number of activities of the mice within 5 minutes. From Figure 7It can be seen that compared with the blank group, the number of activities of the test animals in the model group decreased extremely significantly within 5 minutes (P<0.01). Compared with the model group, the number of activities in the composition group increased significantly (P<0.05), basically returning to the level of the blank group. Thus, it can be known that the number of activities per unit time of spleen-deficient mice decreased significantly. After 2 weeks of intervention with the composition, the number of activities per unit time of spleen-deficient mice can be significantly increased, basically returning to the level of the blank group.

[0090] In addition, Figure 8 Photos showing the behavioral states of the test animals in each group were taken on the first day after the end of the experiment. Figure 8 It can be seen that the mice in the blank group were active normally; the mice in the model group were listless, lazy to move, and liked to huddle together, which corroborated that the mice in the model group had significant spleen-deficiency symptoms; after 2 weeks of intervention with the composition, the behaviors of "listlessness, laziness to move, and liking to huddle together" of the mice could be significantly improved.

Claims

1. Use of a composition in the preparation of a product for improving damaged intestinal mucosal barrier of spleen deficiency type, wherein the composition comprises the following raw materials: 33.7 wt% of pumpkin peel, 20.8 wt% of pumpkin pedicel, 12.9 wt% of pumpkin vein, 7.9 wt% of Poria cocos, 9.0 wt% of Tremella fuciformis, and 15.7 wt% of xylo-oligosaccharide.

2. The application according to claim 1, wherein: The improvement of the damaged intestinal mucosal barrier of spleen deficiency type is to improve the damaged intestinal mechanical barrier of spleen deficiency type.

3. The application according to claim 1, wherein: The improvement of the intestinal mucosal barrier of spleen deficiency type includes improving the apoptosis of intestinal epithelial cells in spleen-deficient mice.

4. The application according to claim 1, wherein: The improvement of the intestinal mucosal barrier of spleen deficiency type includes improving the intercellular tight junction structure in spleen-deficient mice.

5. The application according to claim 4, characterized in that: The improvement of the intestinal mucosal barrier of spleen deficiency type includes significantly increasing the expression levels of intestinal tight junction proteins Claudin-1, Occludin, and ZO-1.

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