Application of polyglutamic acid in preparation of product for treating slow transit constipation

Abstract

The invention provides application of polyglutamic acid in preparation of a product for treating slow transit constipation, and belongs to the technical field of biological medicines. The polyglutamic acid can treat the slow transit constipation by adjusting aquaporin, promoting intestinal peristalsis, repairing intestinal tissue damage, repairing intestinal barrier function, inhibiting expression of intestinal proinflammatory factors and adjusting intestinal flora, which is of great significance to prevention and treatment of the slow transit constipation.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of polyglutamic acid in preparing a product for treating slow-transmission constipation. Background Art

[0002] Constipation is a common digestive disease in clinical practice. With the changes in people's lifestyle and dietary structure, the incidence of constipation has increased year by year. According to the causes of constipation, constipation can be divided into two types: organic constipation and functional constipation. Among them, slow transit constipation (STC) is a common type of functional constipation, which is mainly manifested by symptoms such as weakened colon transmission capacity, prolonged retention of intestinal contents, dry and hard stools, low defecation frequency, and labored defecation. Constipation is caused by many different reasons, such as insufficient intake of low-fiber foods, low water intake, abuse or unreasonable use of laxatives, etc. The pathogenesis of STC is complex. The more recognized pathogenic mechanisms include the nervous system theory, smooth muscle theory, gastrointestinal peptide hormone theory, interstitial cell theory, and psychological factor theory. Long-term constipation not only affects the quality of life of patients, but also affects the mental state of patients, thereby inducing neurasthenia, insomnia, and cardiovascular and cerebrovascular diseases. At present, STC patients mainly defecate by taking laxatives or enema for a long time. There are many types of laxatives, such as bulk laxatives, osmotic laxatives, stimulant laxatives, lubricant laxatives, and epsom salt drugs. Polyethylene glycol 4000 is an osmotic laxative commonly used in clinical practice, which can significantly improve the frequency of bowel movements and stool characteristics of patients with chronic constipation. Although the above laxatives can effectively relieve constipation, long-term use of such drugs can make patients dependent, and the side effects of the drugs are large, which can easily lead to repeated recurrence of constipation. Therefore, it is very important to improve the treatment level of STC and develop safer, more convenient and effective STC treatment drugs.

[0003] Poly-γ-glutamic acid (γ-PGA) is a glutamic acid polymer formed by the dehydration condensation of the α-amino group of D-glutamic acid and L-glutamic acid and the γ-carboxyl group of the adjacent glutamic acid. It is an anionic polymer, usually composed of about 500~5000 glutamic acid monomers, and the molecular weight is generally between 10~1000kDa. Some bacteria, archaea, and eukaryotes can produce γ-PGA, among which Bacillus bacteria are the main microorganisms producing γ-PGA. As a natural polyamino acid in nature, γ-PGA has a structure similar to natural polypeptides. Because of its good water solubility, biodegradability, immunogenicity, and non-toxicity to humans and the natural environment, it has been widely used in biomedicine, food, cosmetics, and agriculture in recent years.

[0004] Aquaporins (AQPs) are a class of cell membrane channel proteins that selectively transport water molecules. Studies have found that the expression of AQPs in the colon tissue of patients with constipation increases significantly, causing excessive absorption of water and reduced intestinal fluid secretion, which in turn causes dry and hard stools that are difficult to excrete. In addition, intestinal flora can exchange substances and information with the host to maintain the stability of the intestinal environment. Studies have found that the occurrence of constipation is closely related to intestinal microbial disorders, a decrease in beneficial bacteria, and an increase in harmful bacteria. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of polyglutamic acid in the preparation of a product for treating slow-transmission constipation.

[0006] The technical solution of the present invention is as follows: Application of polyglutamic acid in the preparation of products for treating slow-transmission constipation.

[0007] Preferably, polyglutamic acid treats slow-transit constipation by regulating aquaporins.

[0008] Further preferably, the mechanism of regulating aquaporins is: inhibiting the expression of AQP3 and AQP4 in the intestine.

[0009] Preferably, polyglutamic acid treats slow-transmission constipation by promoting intestinal motility, repairing intestinal tissue damage, and repairing the intestinal barrier.

[0010] Preferably, polyglutamic acid treats slow-transit constipation by inhibiting the expression of intestinal pro-inflammatory factors.

[0011] More preferably, the pro-inflammatory factors are Caspase-1, IL-1β, IL-6, and TLR2.

[0012] Preferably, polyglutamic acid treats slow-transit constipation by regulating intestinal flora.

[0013] Further preferably, the regulating the intestinal flora is to increase the abundance of beneficial bacteria in the intestinal flora and reduce the abundance of harmful bacteria in the intestinal flora.

[0014] Further preferably, the beneficial bacteria are Firmicutes , Lachnospiraceae , Rikenellaceae , Clostridia_UCG-014_unclassified , Oscillospiraceae , Clostridiales_unclassified , Ruminococcaceae One or more of .

[0015] Further preferably, the harmful bacteria are Bacteroidota , Muribaculaceae One or more of .

[0016] Beneficial effects: The present invention provides the use of polyglutamic acid in the preparation of a product for treating slow-transmission constipation, which is of great significance for the prevention and treatment of slow-transmission constipation. The present invention has found that γ-PGA can effectively promote the fecal output characteristics of STC mice, increase fecal water content and enhance intestinal peristalsis; can effectively improve the intestinal tissue damage of STC mice; can downregulate the colon in STC mice. AQP3 , AQP4 , ENaC-β、SERT The expression level of STC mice can maintain the moisture in the intestine and promote intestinal peristalsis; it can upregulate the intestinal barrier-related genes of STC mice Claudin-1 , Occludin , ZO-1 The expression level of STC mice was reduced, restoring the intestinal barrier function of mice; it was able to downregulate pro-inflammatory factors in the colon of STC mice Caspase-1, IL-1β, IL-6, TLR2 The expression level of AQP3 and AQP4 can be maintained to maintain intestinal homeostasis; it can alleviate the abnormal expression of AQP3 and AQP4 in the colon of STC mice, thereby inhibiting the excessive absorption of water by the colon, and relieve constipation symptoms by regulating the secretion of intestinal fluid and increasing the water content of feces; it can improve the Alpha diversity and Beta diversity of the intestinal flora of STC mice; it shows good effects in restoring the intestinal flora structure of STC mice, increasing the relative abundance of beneficial bacteria, and reducing the relative abundance of harmful bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 3 is the improvement effect of γ-PGA on the defecation characteristics of STC mice; Figure A is the number of defecation times of each group of mice within 8 hours from the first gavage during the experiment, Figure B is the fecal water content of each group of mice on the 18th day of the experiment, Figure C is the time of the first black stool of each group of mice on the 19th day of the experiment, Figure D is the distance that activated carbon pushes the small intestine, and Figure E is the intestinal transit rate of each group of mice.

[0018] Figure 2 This is the improvement effect of γ-PGA on intestinal tissue damage in STC mice.

[0019] Figure 3 The effect of γ-PGA on the expression of related genes in the colon tissue of STC mice; A to D are water, Na + Absorption and intestinal motility-related genes AQP3 , AQP4 , ENaC-β , SERT Expression of genes related to intestinal barrier function. Figures E to G are Claudin-1 , Occludin , ZO-1 Expression of pro-inflammatory cytokines in colon Caspase-1、IL-1β , IL-6, TLR2 expression of.

[0020] Figure 4 Figure 3 shows the effect of γ-PGA on the expression of AQP3 and AQP4 in the colon tissue of STC mice; Figure A shows the Western-Blot results of AQP3 and AQP4, and Figure B shows the Western-Blot quantitative analysis results of AQP3 and AQP4.

[0021] Figure 5 The intermolecular docking results of γ-PGA and AQPs; Figure A is a schematic diagram of the 2D structure of γ-PGA, Figure B is a schematic diagram of the 3D structure of γ-PGA, Figure C is a schematic diagram of the AQP3-γ-PGA complex structure (left) and binding site (right), and Figure D is a schematic diagram of the AQP4-γ-PGA complex structure (left) and binding site (right).

[0022] Figure 6 The effect of γ-PGA on the Alpha diversity of intestinal flora in STC mice.

[0023] Figure 7 This is the effect of γ-PGA on the Beta diversity of intestinal flora in STC mice.

[0024] Figure 8 Figure 2 shows the distribution of intestinal flora at the phylum level in healthy mice.

[0025] Fig. 9 Firmicutes / Bacteroidetes in the intestinal flora of mice in each group ( Firmicutes / Bacteroidota , F / B) value.

[0026] Fig.10 This is the distribution of intestinal flora in healthy mice at the family level.

[0027] Fig.11 Heat map analysis of intestinal flora of healthy mice at the genus level.

[0028] Fig.12 is the relative abundance of intestinal flora of each group of mice at the family level; Figures A to G are the relative abundance of intestinal flora of each group of mice at the family level. Muribaculaceae , Lachnospiraceae , Rikenellaceae , Clostridia_UCG- 014_unclassified , Clostridiales_unclassified , Oscillospiraceae , Ruminococcaceae relative abundance. DETAILED DESCRIPTION

[0029] The following is explained in conjunction with specific embodiments: Source of experimental materials: BALB / c male mice: purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with production license number SCXK(Beijing)2021-0006.

[0030] Polyglutamic acid (10 kDd) was purchased from Nanjing Xuankai Biotechnology Co., Ltd.

[0031] Loperamide hydrochloride: purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0032] Polyethylene glycol 4000: purchased from Bofu-Ipsen Pharmaceutical Co., Ltd.

[0033] RNA extraction kit, PrimeScript™ RT reagent Kit with gDNA Eraser kit, SYBR® Premix Ex Taq™ II: all purchased from Bio-Rad Biotechnology Co., Ltd.

[0034] BCA protein assay kit: purchased from Beijing Solebow Biotechnology Co., Ltd.

[0035] AQP3 primary antibody: purchased from Abimat Pharmaceutical Technology Co., Ltd.

[0036] AQP4 primary antibody: purchased from Wuhan Fine Biotechnology Co., Ltd.

[0037] Example 1: Effect of γ-PGA on the improvement of defecation characteristics and intestinal tissue damage in STC mice 1. Effect of γ-PGA on the improvement of defecation characteristics of STC mice Loperamide hydrochloride can inhibit intestinal fluid secretion and promote the absorption of water and other electrolytes in the intestinal cavity, thereby reducing the water content of feces and increasing the difficulty of defecation. In this study, loperamide hydrochloride was used to construct the STC mouse model.

[0038] Thirty-six 6-8-week-old BALB / c male mice (20±2g) were housed in IVC cages, with room temperature at 23±2℃, relative humidity at 45%-55%, 12h light-dark switching, and free access to food and water. After one week of adaptive culture, six mice were randomly selected and gavaged with 0.25mL of normal saline as the normal control group (CK). The remaining 30 mice were randomly divided into 5 groups, namely, constipation model group (LOP), polyethylene glycol 4000 group (PEG), low-dose polyglutamic acid group (γ-PGAL), medium-dose polyglutamic acid group (γ-PGAM), and high-dose polyglutamic acid group (γ-PGAH); the mice in the 5 groups were gavaged with 0.25 mL of 0.08 g / mL loperamide hydrochloride suspension once a day for 12 days; on the 12th day, all mice fasted for 16 h, and then gavaged with 0.25 mL of activated carbon solution, and the time when the mice first had black stools was recorded. When the time of the first black stool in the 5 groups of mice was significantly higher than that in the CK group, it indicated that the STC mouse model was successfully constructed.

[0039] On days 13 to 19, all mice were treated as follows: mice in CK group were gavaged with 0.25 mL of normal saline every day; mice in LOP group were gavaged with loperamide hydrochloride (10 mg / kg / d); mice in PEG group were first gavaged with loperamide hydrochloride (10 mg / kg / d), and then gavaged with polyethylene glycol 4000 (3 g / kg / d) 1 hour later; mice in γ-PGAL group, γ-PGAM group, and γ-PGAH group were first gavaged with loperamide hydrochloride (10 mg / kg / d), and then gavaged with γ-PGA (2.5 g / kg / d, 5 g / kg / d, and 10 g / kg / d), respectively, 1 hour later.

[0040] During the experiment, the number of defecations of mice within 8 hours from the first gavage was recorded daily. On the 18th day of the experiment, fresh feces of mice were collected in sterile polyethylene tubes, weighed and thoroughly dried, and the feces water content was calculated. Feces water content (%) = (feces wet weight - feces dry weight) / feces wet weight × 100%. On the 19th day of the experiment, all mice fasted for 16 hours and then gavaged with 0.25 mL of activated carbon solution. Three mice were observed in each group and the time of the first black stool was recorded; the intestinal transit rate of the remaining three mice was measured. Intestinal transit rate (%) = (the distance of the small intestine pushed by activated carbon / the total length of the small intestine) × 100%.

[0041] The results are as follows Figure 1As shown (data are expressed as mean±SD, n=3 or 6, #P<0.05, ##P<0.01 compared with the CK group, *P<0.05, **P<0.01 compared with the LOP group), where A is the number of defecation times of mice in each group within 8 hours from the first oral gavage during the experiment, B is the fecal water content of mice in each group on the 18th day of the experiment, C is the time of the first black stool of mice in each group on the 19th day of the experiment, D is the distance pushed by activated carbon in the small intestine, and E is the intestinal transit rate of mice in each group.

[0042] Depend on Figure 1 As shown in Figure A, starting from the 13th day of the experiment, compared with the LOP group, the PEG group, γ-PGAL group, γ-PGAM group, and γ-PGAH group all significantly increased the number of defecations in constipated mice, among which γ-PGAM and γ-PGAH gavage could effectively alleviate the symptom of low defecation frequency in constipated mice, and the overall effect was PEG>γ-PGAH>γ-PGAM>γ-PGAL. Figure 1 According to B, compared with the CK group, the fecal water content of the LOP group mice decreased significantly; compared with the LOP group, the fecal water content of the PEG group increased significantly by 25.42%, and the fecal water content of the γ-PGAL group, γ-PGAM group, and γ-PGAH group increased by 16.16%, 27.08%, and 34.23%, respectively. Among them, the fecal water content of the γ-PGAH group was 1.35 times that of the PEG group. The overall effect was γ-PGAH>PEG>γ-PGAM>γ-PGAL. In addition, Figure 1 As shown in Figures C~E, compared with the CK group, the time to the first black stool in the LOP group was significantly prolonged and the intestinal transit rate was significantly reduced; compared with the LOP group, the PEG group, γ-PGAL group, γ-PGAM group, and γ-PGAH group all significantly shortened the time to the first black stool in constipated mice and increased the intestinal transit rate.

[0043] The above results indicate that γ-PGA can effectively promote the fecal output characteristics of STC mice, increase the fecal water content and enhance their intestinal motility.

[0044] 2. The improvement effect of γ-PGA on intestinal tissue damage in STC mice After the above experiments, the colon tissues of mice in each group were collected, fixed with 4% paraformaldehyde, embedded in paraffin, cut into 5-mm serial sections, stained with hematoxylin-eosin (HE) according to standard procedures, and histopathologically observed under an optical microscope.

[0045] The staining results are as follows Figure 2 As shown. Figure 2It can be seen that the intestinal tissue structure of the CK group was normal, the mucosal villi were long, neat and closely arranged, and the epithelial cells did not undergo obvious degeneration and shedding; compared with the CK group, the intestinal tissue structure of the LOP group was abnormal, the muscle layer thickness was significantly reduced, the epithelial cells were eroded and shedding (as shown by the yellow arrow), and inflammatory cell infiltration was visible in the tissue (as shown by the red arrow); compared with the LOP group, the intestinal tissue structure of the PEG group, γ-PGAL group, γ-PGAM group, and γ-PGAH group was basically normal, and the integrity of the colon wall and villi was restored.

[0046] The above results indicate that γ-PGA can effectively improve intestinal tissue damage in STC mice.

[0047] Example 2: Effect of γ-PGA on the expression of related genes in colon tissue of STC mice Three mice were randomly selected from the CK group, LOP group, PEG group, γ-PGAL group, γ-PGAM group, and γ-PGAH group in Example 1, and the expression of related genes in the colon tissue of each group of mice at the mRNA level was detected by RT-qPCR. The specific operation steps are as follows: Total RNA from mouse colon tissue was extracted using an RNA extraction kit, and cDNA was synthesized using a PrimeScript™ RT reagentKit with gDNA Eraser kit. β-actin was used as an internal reference, and SYBR® Premix Ex Taq™ II was used for real-time fluorescence quantitative PCR detection. AQP3 , AQP4 , ENaC-β , SERT , Claudin-1 , Occludin , ZO-1 , Caspase-1、IL-1β , IL-6 , TLR2 Relative expression of genes at the mRNA level.

[0048] The results are as follows Figure 3 As shown (data are expressed as mean±SD, n=3, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 are compared with the CK group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 are compared with the LOP group), where A to D are water, Na + Absorption and intestinal motility-related genes AQP3 , AQP4 , ENaC-β , SERT Expression of genes related to intestinal barrier function. Figures E to G are Claudin-1 , Occludin , ZO-1 Expression of pro-inflammatory cytokines in colon Caspase-1、IL-1β , IL-6 , TLR2 expression of.

[0049] Depend on Figure 3 As shown in Figures A to D, compared with the CK group, the number of AQP3 , AQP4 (Transports water from the lumen to the blood vessels in the intestine), ENaC-β (Overexpression can lead to mucosal dehydration) 、SERT (overexpression will inhibit intestinal propulsion) was significantly upregulated; compared with the LOP group, the expression levels of γ-PGAL, γ-PGAM, and γ-PGAH in the colon of mice in the PEG group AQP3 , AQP4, ENaC-β, SERT The expression levels of Figure 3 As shown in Figures E-G, compared with the CK group, the number of Claudin-1 , Occludin , ZO-1 The expression level of γ-PGAL, γ-PGAM, and γ-PGAH in the colon of mice in the PEG group and the γ-PGAL group decreased compared with the LOP group. Claudin-1 , Occludin, ZO-1 The expression levels of Claudin-1 The expression levels of , ZO-1 were significantly upregulated, and the expression of intestinal barrier-related genes in mice was improved. Figure 3 The H~K diagram in Figure 2 shows that compared with the CK group, the pro-inflammatory factors in the colon of the LOP group mice Caspase-1, IL-1β, IL-6, TLR2 The expression level of γ-PGAL, γ-PGAM, and γ-PGAH in the colon of mice in the PEG group and the γ-PGAL group increased compared with the LOP group. Caspase-1, IL-1β, IL-6, TLR2 The expression levels of IL-1β, TLR2 The expression level of Caspase-1 The expression level was significantly downregulated.

[0050] The above results indicate that γ-PGA can downregulate the expression of AQP3 , AQP4 , ENaC-β、SERT The expression level of γ-PGA can maintain the moisture in the intestine and promote intestinal peristalsis; γ-PGA can upregulate the intestinal barrier-related genes in STC mice Claudin-1 , Occludin , ZO-1The expression level of STC mice was reduced, restoring the intestinal barrier function of mice; γ-PGA could downregulate the pro-inflammatory factors in the colon of STC mice Caspase-1, IL-1β, IL-6, TLR2 expression levels to maintain intestinal homeostasis.

[0051] Example 3: Effect of γ-PGA on the expression of aquaporins AQP3 and AQP4 in colon tissue of STC mice Aquaporins (AQPs) are cell membrane channel proteins that form "pores" to control the flow of water molecules into and out of cells and are known as the "water pumps" of cells. In the colon, AQP3 not only promotes the absorption of water by colon cells, but also enhances the transport of urea and glycerol by colon cells; AQP4 plays an important role in maintaining the integrity of the gastric mucosa, and is also involved in the colon's permeability and transport of water and the secretion of colonic mucus.

[0052] Three mice were randomly selected from the CK group, LOP group, PEG group, γ-PGAL group, γ-PGAM group, and γ-PGAH group in Example 1, and β-actin was used as a reference to detect the relative expression of AQP3 and AQP4 at the protein level in the colon tissue of each group of mice by Western-Blot analysis. The specific steps are as follows: 50 mg of mouse colon tissue was placed in a grinding tube, and PIPA lysis buffer and grinding beads were added for tissue grinding. The tissue was centrifuged at 4°C and 12000g for 10 min, and the supernatant, i.e., the total protein solution, was collected. The total protein was quantified using a BCA protein assay kit, and then separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 h, and then incubated with the primary antibody AQP3 (1:500) and AQP4 (1:500) at 4°C overnight, and the secondary antibody was incubated at room temperature for 1 h. The PVDF membrane was stained with an ECL chemiluminescent solution, and the protein bands were displayed using a gel imaging analysis system.

[0053] The results are as follows Figure 4 As shown (data are expressed as mean±SD, n=3, ##P<0.01 indicates comparison with the CK group, **P<0.01, ***P<0.001, ****P<0.0001 indicate comparison with the LOP group), where Figure A is the Western-Blot band results of AQP3 and AQP4, and Figure B is the Western-Blot quantitative analysis results of AQP3 and AQP4.

[0054] Depend on Figure 4It can be seen that compared with those in CK group, the expression levels of AQP3 and AQP4 in the colon of mice in LOP group were significantly increased; compared with those in LOP group, the expression levels of AQP3 and AQP4 in the colon of mice in PEG group, γ-PGAL group, γ-PGAM group and γ-PGAH group decreased to varying degrees, among which the expression level of AQP3 in γ-PGAH group was significantly downregulated, and the expression levels of AQP4 in γ-PGAL group, γ-PGAM group and γ-PGAH group were significantly downregulated.

[0055] The above results indicate that γ-PGA can alleviate the abnormal expression of AQP3 and AQP4 in the colon of STC mice, thereby inhibiting the excessive absorption of water by the colon and alleviating constipation symptoms by regulating the secretion of intestinal fluid and increasing the water content of feces.

[0056] Example 4: Interaction between γ-PGA and AQPs The γ-PGA structure diagram was produced using ChemDraw22.0 software. The 3D structures of AQP3 and AQP4 proteins were obtained through the PDB database (http: / / www.rcsb.org). Molecular docking analysis was performed using AutoDock Vina 1.5.6 software. The docking analysis results were visualized using PyMOL 2.4 software.

[0057] The results of molecular docking analysis are shown in Table 1 below: Table 1. Molecular docking analysis results

[0058] As shown in Table 1, the binding energies of γ-PGA with AQP3 and AQP4 are -6.4 kcal / mol and -6.8 kcal / mol, respectively, which indicates that γ-PGA has good binding activity with both AQP3 and AQP4.

[0059] The visual display results are as follows Figure 5 As shown, Figure A is a schematic diagram of the 2D structure of γ-PGA, Figure B is a schematic diagram of the 3D structure of γ-PGA, Figure C is a schematic diagram of the AQP3-γ-PGA complex structure (left) and binding site (right), and Figure D is a schematic diagram of the AQP4-γ-PGA complex structure (left) and binding site (right).

[0060] Depend on Figure 5As shown in Figure C, the active pocket of AQP3 is in the form of a channel, and γ-PGA can be embedded in the hydrophobic cavity of AQP3. The hydrophobic force plays an important role in stabilizing the structure of the complex. In addition, γ-PGA is surrounded by the amino acid residues of AQP3 and forms hydrogen bonds with the surrounding amino acid residues ARG-218, GLY-145, ALA-148, GLY-207, SER-152, and TYR-150. These hydrogen bonds strengthen the non-covalent force between the two, making the combination more stable. Figure 5 As shown in Figure D, γ-PGA binds to the active pocket of AQP4, similar to AQP3, and forms hydrogen bonds with the surrounding amino acid residues.

[0061] The above results show that γ-PGA has good binding activity with AQP3 and AQP4.

[0062] Example 5: Effect of γ-PGA on the intestinal flora of STC mice 1. Effect of γ-PGA on Alpha and Beta Diversity of Intestinal Microbiota in STC Mice Alpha diversity can be used to evaluate the species richness and uniformity of the intestinal flora. The Shannon index was used to evaluate the Alpha diversity of the intestinal flora of mice in the CK group, LOP group, PEG group, γ-PGAL group, γ-PGAM group, and γ-PGAH group in Example 1. The results are shown in Table 1. Figure 6 shown.

[0063] Depend on Figure 6 It was found that compared with the CK group, the Shannon index of the mice in the LOP group was reduced, which indicated that the Alpha diversity of the intestinal flora of the STC mice was reduced. The reason for this phenomenon may be that the inflammation caused by STC destroyed the intestinal barrier, weakened the inhibition of the growth of pathogenic bacteria, and reduced the number of beneficial intestinal bacteria; at the same time, STC caused the intestinal peristalsis rate to slow down, resulting in the continuous fermentation of metabolites, and produced substances that were not conducive to maintaining the homeostasis of intestinal flora. Compared with the LOP group, the Shannon index of the PEG group and the γ-PGAL group was significantly increased, and the Shannon index of the γ-PGAH group was reduced, but there was no statistical difference (P>0.05).

[0064] Beta diversity can be used to compare the similarity of different samples in terms of species diversity. The Beta diversity of the intestinal flora of mice in the CK group, LOP group, PEG group, γ-PGAL group, γ-PGAM group, and γ-PGAH group in Example 1 was analyzed by non-metric multidimensional scaling (NMDS) based on the Bray-Curtis distance. The results are shown in Figure 2. Figure 7 As shown, each point in the figure represents a sample, points of the same color come from the same group, and the distance between two points reflects the similarity of the samples.

[0065] Depend on Figure 7 It can be seen that the CK group is far away from the LOP group, indicating that there is a significant difference in the intestinal flora structure between the LOP group and the healthy mice. The distribution points of the PEG group, γ-PGAL group and γ-PGAM group tend to approach the CK group, and are significantly different from the flora of the LOP group, indicating that the intestinal flora is close to the healthy group and improves the disorder of the intestinal flora of STC mice; among them, the γ-PGAL group has a better improvement effect, with high similarity between group samples, small differences in species composition, and stable intestinal flora structure; although the γ-PGAH group did not approach the CK group, the intervention of γ-PGA can make the intestinal flora of the γ-PGAH group mice different from that of the LOP group.

[0066] The above results indicate that a certain dose of γ-PGA can improve the Alpha diversity and Beta diversity of the intestinal flora of STC mice.

[0067] 2. Effect of γ-PGA on the intestinal bacterial structure of STC mice By means of statistical analysis, the dominant species and their relative abundances at the phylum, family, and genus levels in the intestinal flora of mice in the CK group, LOP group, PEG group, γ-PGAL group, γ-PGAM group, and γ-PGAH group in Example 1 were analyzed to explore the effect of γ-PGA on the intestinal flora structure of STC mice.

[0068] The results are as follows Figures 8 to 12 As shown, Figure 8 The distribution of intestinal flora at the phylum level in mice in the CK group. Fig. 9 Firmicutes / Bacteroidetes in the intestinal flora of mice in each group ( Firmicutes / Bacteroidota , F / B) value, Fig.10 The distribution of intestinal flora of mice in the CK group at the family level. Fig.11 This is the heat map analysis of the intestinal flora of mice in the CK group at the genus level. Fig.12 is the relative abundance of different intestinal flora in each group of mice at the family level.

[0069] Depend on Figure 8 It was found that a total of 18 bacterial phyla were detected at the phylum level of the intestinal flora of healthy mice, and the phylum with the highest relative abundance was Bacteroidetes ( Bacteroidota )、Firmicutes( Firmicutes ), Actinobacteria ( Actinobacteriota ), Proteobacteria ( Patescibacteria ),in Bacteroidota and Firmicutes The relative abundance of is the highest, and the sum of the two accounts for about 95%. Fig. 9It can be seen that there are differences in the composition of dominant bacterial phyla among the mice groups. Specifically, in the CK group, Firmicutes and Bacteroidota The proportion of each group was about 50%; compared with the CK group, the F / B value of the LOP group was significantly reduced, Bacteroidota The relative abundance of the dominant phylum increased by 29.37%. Firmicutes The relative abundance of the phylum Bacteria decreased by 26.48%; compared with the LOP group, the F / B values ​​of the PEG group, γ-PGAL group, and γ-PGAM group increased. Bacteroidota The relative abundance of the fungi decreased by 6.24%, 15.51%, and 9.54%, respectively. Firmicutes The relative abundance of bacteria increased by 5.73%, 13.99% and 8.71% respectively. The above results show that a certain dose of γ-PGA can improve Firmicutes The relative abundance of bacterial phyla decreased Bacteroidota The relative abundance of Bacteria was increased, thereby improving the intestinal flora structure of STC mice.

[0070] Depend on Figure 10 It can be found that the top ten bacterial families with the highest relative abundance in the intestinal flora of healthy mice are Salviaceae of the Bacteroidetes phylum ( Muribaculaceae )、Bacteroidetes( Bacteroidaceae )、Prevotellaceae( Prevotellaceae )、RIKEN Bacteria Department( Rikenellaceae ) and the Lachnospiraceae family of the Firmicutes ( Lachnospiraceae )、Lactobacillusaceae( Lactobacillaceae ) 、Clostridia( Clostridia_UCG-014_ unclassifie d) Oscillatory Spiraceae ( Oscillospiraceae ), Clostridiales ( Clostridiales_unclassified )、Ruminococcaceae( Ruminococcaceae ).

[0071] Depend on Figure 11 Heatmap and Figure 12 The one-way ANOVA showed that compared with the CK group, the LOP group Muribaculaceae The relative abundance of beneficial bacteria increased significantly by 39.32% (##p<0.01). Lachnospiraceae , Rikenellaceae , Clostridia_UCG-014_unclassified , Oscillospiraceae , Clostridiales_unclassified and Ruminococcaceae Compared with the LOP group, different doses of γ-PGA treatment reduced the relative abundance of Muribaculaceae In addition, the γ-PGAL group significantly increased the relative abundance of Lachnospiraceae , Rikenellaceae and Clostridia_UCG-014_unclassifiedThe relative abundance of Oscillospiraceae , Clostridiales_unclassified and Ruminococcaceae The above results show that γ-PGA has a good effect on restoring the intestinal flora structure of STC mice and increasing the relative abundance of beneficial bacteria. Among them, the effects of γ-PGAL group and γ-PGAM group on improving intestinal flora are better than those of γ-PGAH group. After high-dose γ-PGA treatment, the intestinal flora structure of STC mice was not significantly improved, and the relative abundance of beneficial bacteria was increased. Bacteroidota The relative abundance of Bacteria may be due to the fact that excessively high γ-PGA concentration increases intestinal permeability, which in turn leads to intestinal infection by pathogens and an increase in harmful bacteria, which is not conducive to the recovery of the intestinal flora structure.

[0072] In summary, γ-PGA can effectively promote the fecal output characteristics of STC mice, increase fecal water content and enhance intestinal peristalsis; it can effectively improve the intestinal tissue damage of STC mice; it can downregulate the expression of γ-PGA in the colon of STC mice. AQP3 , AQP4 , ENaC-β, SERT The expression level of STC mice can maintain the moisture in the intestine and promote intestinal peristalsis; it can upregulate the intestinal barrier-related genes of STC mice Claudin-1 , Occludin , ZO-1 The expression level of STC mice was reduced, restoring the intestinal barrier function of mice; it was able to downregulate pro-inflammatory factors in the colon of STC mice Caspase-1, IL-1β, IL-6, TLR2 The expression level of AQP3 and AQP4 can be maintained to maintain intestinal homeostasis; it can alleviate the abnormal expression of AQP3 and AQP4 in the colon of STC mice, thereby inhibiting the excessive absorption of water by the colon, and relieve constipation symptoms by regulating the secretion of intestinal fluid and increasing the water content of feces; it can improve the Alpha diversity and Beta diversity of the intestinal flora of STC mice; it shows good effects in restoring the intestinal flora structure of STC mice, increasing the relative abundance of beneficial bacteria, and reducing the relative abundance of harmful bacteria.

Claims

1. Application of polyglutamic acid in the preparation of products for treating slow-transmission constipation.

2. The use according to claim 1, characterized in that Polyglutamic acid treats slow-transit constipation by regulating aquaporins.

3. The use according to claim 2, characterized in that The mechanism of regulating aquaporins is: inhibiting the expression of AQP3 and AQP4 in the intestine.

4. The use according to claim 1, characterized in that Polyglutamic acid treats slow-transmission constipation by promoting intestinal motility, repairing intestinal tissue damage, and repairing the intestinal barrier.

5. The use according to claim 1, characterized in that Polyglutamic acid treats slow-transit constipation by inhibiting the expression of intestinal proinflammatory factors.

6. The use according to claim 5, characterized in that The pro-inflammatory factors are Caspase-1, IL-1β, IL-6, and TLR2.

7. The use according to claim 1, characterized in that Polyglutamic acid treats slow-transit constipation by regulating intestinal flora.

8. The use according to claim 7, characterized in that The intestinal flora is regulated to increase the abundance of beneficial bacteria in the intestinal flora and reduce the abundance of harmful bacteria in the intestinal flora.

9. The use according to claim 8, characterized in that The beneficial bacteria are Firmicutes , Lachnospiraceae , Rikenellaceae , Clostridia_UCG-014_unclassified , Oscillospiraceae , Clostridiales_unclassified , Ruminococcaceae One or more of .

10. The use according to claim 8, characterized in that The harmful bacteria are Bacteroidota , Muribaculaceae One or more of .

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