Platycodon stem cellulose nanocrystal, preparation method thereof and application thereof in relieving intestinal injury

Abstract

The application discloses a luffa stem cellulose nanocrystal, a preparation method thereof and application thereof in relieving intestinal injury, and belongs to the technical field of plant active ingredients, and particularly relates to a preparation method of the luffa stem cellulose nanocrystal, which comprises the following steps: crushing and screening luffa stems, mixing and treating luffa stem powder with a NaOH solution, and taking a precipitate; bleaching the precipitate by using a NaClO2 solution, and then re-bleaching the precipitate by using a NaOH solution to obtain a pre-product; washing the pre-product until neutral to obtain luffa stem cellulose; dispersing the luffa stem cellulose in a H2SO4 solution, acidizing, adding deionized water to terminate the reaction, and obtaining a reaction product; centrifuging and repeatedly washing the reaction product, and dialyzing for 5 days until pH is stable, so that the luffa stem cellulose nanocrystal is obtained. The luffa stem cellulose nanocrystal (PGCNC) prepared by the application can regulate intestinal flora and metabolism, improve lipid metabolism disorder of a host, and promote colon injury repair by relying on a butyric acid-IL-10-MUFA related path, and has potential application value in regulating intestinal health.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient technology, and particularly relates to a cellulose nanocrystal from Platycodon grandiflorus stem, its preparation method, and its application in alleviating intestinal damage. Background Technology

[0002] Dietary fiber is an essential dietary component for maintaining gut health and a key nutritional basis for regulating colonic homeostasis. Numerous studies have shown that adequate dietary fiber intake helps improve gut microbiota structure, promotes short-chain fatty acid (SCFA) production, maintains intestinal barrier integrity, and reduces local inflammatory responses. In studies related to colonic injury or inflammatory bowel disease, appropriate dietary fiber intervention can alleviate the phenotype of colonic tissue damage. Its mechanism mainly involves multiple aspects, including the restoration of microbiota homeostasis, enhancement of the intestinal epithelial barrier, and improvement of the inflammatory microenvironment. From a mechanistic perspective, dietary fiber, after entering the colon, can be fermented and utilized by gut microbiota to produce SCFAs such as butyric acid, propionic acid, and acetic acid. Butyric acid is not only an important energy source for colonic epithelial cells but also promotes TJ expression, maintains mucus layer stability, and participates in local immune regulation, thus facilitating the repair of damaged colon. Meanwhile, different types of dietary fiber exhibit significant differences in physicochemical properties, fermentability, and their ability to regulate the microbiota; therefore, their effects on improving colonic injury are not entirely consistent. Overall, the protective effect of dietary fiber against colonic injury has been supported by numerous studies and is believed to exert its protective effect through the regulation of the gut microbiota.

[0003] While dietary fiber plays a crucial role in alleviating colonic damage, traditional dietary fiber interventions still have certain limitations. First, dietary fibers from different sources and with different structures vary significantly in terms of solubility, viscosity, water-holding capacity, fermentability, and surface physicochemical properties. These structural and property differences directly affect their transport, fermentation, and utilization processes in the gastrointestinal tract, thus influencing the final intervention effect. Second, the regulatory effect of dietary fiber on the gut microbiota exhibits individual variability. Differences in the original gut microbiota composition, dietary structure, and metabolic state of different hosts may lead to variations in the fermentation efficiency and metabolite profile of dietary fiber, resulting in instability in its effect on improving colonic damage.

[0004] Furthermore, traditional dietary fiber has limited ability to retain in the colon, contact the mucosal surface, and interact with the interfacial environment. Its effects largely rely on passive fermentation after ingestion, making it difficult to sustainably and stably regulate the colonic microenvironment. For pathological conditions like colonic injury, which are accompanied by intestinal mucosal barrier disruption, microbial imbalance, and metabolic disorders, simply relying on traditional dietary fiber supplementation may not fully meet the needs for efficient, stable, and sustained intervention. Therefore, based on research into traditional dietary fiber, developing novel dietary fibers that are safe in origin, structurally sound, stable in function, and possess good potential for local intestinal action has become an important direction for product innovation related to improving colonic injury. Among these, nanofibers, with their large specific surface area, strong interfacial interaction capabilities, and good potential for intestinal microenvironment interaction, offer new research ideas for colonic injury intervention. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a Platycodon grandiflorus stem cellulose nanocrystal, its preparation method, and its application in alleviating intestinal damage. The Platycodon grandiflorus stem cellulose nanocrystal (PGCNC) prepared by this invention can promote colonic damage repair by regulating intestinal flora and its metabolism, improving host lipid metabolism disorders, and relying on butyrate-IL-10-MUFA-related pathways, thus having potential application value in regulating intestinal health.

[0006] To achieve the above objectives, the present invention provides a method for preparing cellulose nanocrystals from Platycodon grandiflorus stems, comprising the following steps: 1) The bellflower stem is crushed and sieved to obtain bellflower stem powder. The bellflower stem powder is mixed with NaOH solution and the precipitate is collected. 2) The precipitate obtained in step 1) is bleached with NaClO2 solution and then rebleached with NaOH solution to obtain the preproduct; 3) The preproduct obtained in step 2) was washed until neutral and freeze-dried to obtain Platycodon grandiflorus stem cellulose; 4) Disperse the cellulose from the bellflower stems obtained in step 3) in H2SO4 solution, acid hydrolyze it, and add deionized water to terminate the reaction to obtain the reactants; 5) Centrifuge and wash repeatedly the reactants obtained in step 4) to obtain a suspension of Platycodon grandiflorum stem cellulose nanocrystals. Dialyze the suspension of Platycodon grandiflorum stem cellulose nanocrystals for 5 days until the pH is stable. The retentate is Platycodon grandiflorum stem cellulose nanocrystals.

[0007] Preferably, the particle size of the Platycodon grandiflorus stem powder in step 1) is 30-50 mesh; the mixing ratio of the Platycodon grandiflorus stem powder and NaOH solution in step 1) is 1g:15-25mL, and the concentration of the NaOH solution is 5%-15% (w / v); the temperature for mixing the Platycodon grandiflorus stem powder and NaOH solution in step 1) is 80-90℃, and the time is 20-40min.

[0008] Preferably, the concentration of the NaClO2 solution in step 2) is 2%~4% (w / v); the pH of the precipitate bleached with NaClO2 solution in step 2) is 3~4, the bleaching temperature is 75~85℃, the number of bleaching cycles is 3~5, and the bleaching time for each cycle is 50~70min.

[0009] Preferably, the concentration of the NaOH solution in step 2) is 0.4%~0.6% (w / v); the re-bleaching temperature of the NaOH solution in step 2) is 80~90℃, and the time is 80~100min.

[0010] Preferably, the washing in step 3) uses deionized water, and the freeze-drying in step 3) is carried out at a temperature of -60~-40℃ for 24~48h.

[0011] Preferably, in step 4), the ratio of Platycodon grandiflorus stem cellulose to H2SO4 solution is 1g:15~25mL, and the concentration of H2SO4 solution is 62%~66% (w / v); the acid hydrolysis temperature in step 4) is 45~55℃, and the acid hydrolysis time is 40~50min; the deionized water in step 4) is deionized water pre-cooled at 4℃.

[0012] Preferably, the centrifugation temperature in step 5) is 4°C, the rotation speed is 10000~14000 r / min, and the time is 8~12 min; the dialysis in step 5) uses a dialysis bag with a molecular weight cutoff of 8000~14000 Da.

[0013] The present invention also provides the cellulose nanocrystals of Platycodon grandiflorus stem prepared by the aforementioned preparation method.

[0014] The present invention also provides the application of the aforementioned Platycodon grandiflorus stem cellulose nanocrystals in the preparation of products that alleviate intestinal damage.

[0015] Preferably, it regulates gut health by modulating gut microbiota and its metabolism, improving host lipid metabolism disorders, and promoting colonic injury repair via butyrate-IL-10-MUFA-related pathways.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides a method for preparing Platycodon grandiflorus stem cellulose nanocrystals and their application in alleviating intestinal injury. It was found that Platycodon grandiflorus stem cellulose nanocrystals (PGCNCs) can alleviate colonic injury through multiple pathways, including repairing colonic tissue structure, increasing goblet cell count, upregulating TJ expression, and inhibiting inflammatory responses. Furthermore, PGCNCs exhibit structural stability, good safety, and can remain in the colon for a relatively long time, demonstrating good potential for colonic injury intervention. This invention shows that PGCNCs can synergistically improve intestinal barrier damage, inflammatory responses, and metabolic disorders by remodeling the gut microbiota structure, enhancing the microbiota's carbohydrate metabolism function, promoting butyrate production, and further mediating host colonic MUFA metabolic remodeling through IL-10. These results suggest that the "gut microbiota-butyrate-IL-10-MUFA" pathway may be an important pathway for PGCNCs to alleviate colonic injury and provide new theoretical basis for the use of plant-derived nanofibers in colonic injury intervention. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The effects of CNCs from different sources on general physiological indicators of mice with colon injury are shown in Figure 1. In Figure 2, A represents body weight and B represents DAI score. Figure 2 The effects of CNCs from different sources on fecal bleeding and colon length in mice with colon injury are shown in Figure A, where A represents fecal bleeding and B represents colon length. Figure 3 The damage to colon tissue is shown in Figure A, which represents morphological observation (100×, 200×) with scale bars of 200μm and 100μm, and Figure B represents the damage score. Figure 4 To show the integrity of the mucus layer in colonic tissue, where A represents morphological observation (100×, 200×), with scale bars at 200μm and 100μm, and B represents the number of goblet cells in each crypt; Figure 5 The effects of CNC from different sources on the mRNA expression levels of different inflammatory factors were investigated. Among them, A represents Tnf-α, B represents Il-10, C represents Il-6, and D represents Il-1β. Figure 6 Characterization of PGCNC, where A is macroscopic appearance, B is FTIR curve, C is TEM image, scale bar is 500 nm, D is Zeta potential, E is biocompatibility, scale bar is 100 μm; Figure 7 HE staining images of heart, liver, kidney, lung, spleen, and colon of mice after gavage administration of high doses of PGCNC dispersion (100×, 200×), scale bars at 100μm (200×) and 200μm (100×); Figure 8 For mouse blood biochemical analysis, A is ALT, B is AST, C is BUN, D is CREA, and E is ALP; Figure 9 In vivo imaging results of mice after oral administration of PGCNC; Figure 10 This is a map showing the gastrointestinal tract distribution of mice after oral administration of PGCNC. Figure 11 Distribution of brain, heart, liver, kidney, lung, and spleen in mice after oral administration of PGCNC; Figure 12 The effect of PGCNC on the expression of ZO-1, Claudin-1 and Occludin is shown in the figure. A is the immunofluorescence staining image (100×, scale bar is 200μm), B is the proportion of ZO-1 positive area, C is the proportion of Claudin-1 positive area, and D is the proportion of Occludin positive area. Figure 13 To verify the effect of antibiotics on intestinal flora depletion, where A represents before antibiotic gavage and B represents after antibiotic gavage. Figure 14 The effect of FMT on general physiological indicators of mice with colon injury, where A is body weight and B is DAI score; Figure 15 The effect of FMT on fecal bleeding and colon length in mice with colon injury is shown in Figure A, where A represents fecal bleeding and B represents colon length. Figure 16 The image shows the extent of damage to the colonic tissue. A represents morphological observation (100×, 200×), with scale bars of 100μm (200×) and 200μm (100×). B represents the damage score. Figure 17 To show the differences in species distribution, A is a bar chart of the horizontal distribution of phyla in each group, and B is a bar chart of the horizontal distribution of genera in each group. Figure 18 For Alpha diversity analysis, A represents Shannon diversity at the genus level and B represents the Chao1 index at the genus level. Figure 19 Beta diversity analysis was performed for each group of samples, where A is the genus-level PCoA and B is the genus-level NMDS. Figure 20 For LEfSe analysis, where A is the evolutionary clade diagram and B is the LDA score; Figure 21 For metastats difference analysis, where A is PGCNC vs DSS, B is Control vs DSS, and C is PGCNC vs Control; Figure 22 For KEGG functional genome composition analysis, A represents pathway annotation, B represents level 1, C represents level 2, and D represents level 3. Figure 23 This is a functional gene composition analysis of CAZy, where A represents Level 1, B represents Level 2, and C represents ec. Figure 24 The diagram shows the co-occurrence of microbial networks in each group, where A represents the Control group, B represents the DSS group, and C represents the PGCNC group. Figure 25 The abundance of four key species is represented by A. Xylanibacter rodentium B is Enterocloster clostridioformis C is Petralouisia muris D is Roseburia zhanii . Detailed Implementation

[0019] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. The specification and embodiments of the present invention are merely exemplary.

[0020] The ingredients used in this invention—Platycodon grandiflorus stem, ginseng stem, buckwheat bran, almond coating, apple and pear peel, and pine nut coating—were all commercially available in Yanji City. DSS (36000~40000Da) was purchased from Dalian Meilun Biotechnology Co., Ltd.; AB-PAS staining kit and HE staining kit were purchased from Beijing Solarbio Science & Technology Co., Ltd.; specific amplification primers were purchased from Jilin Kumei Biotechnology Co., Ltd.; SuperReal fluorescence quantitative premixed reagent (enhanced version) was purchased from Beijing Tiangen Biotech Co., Ltd.; FastKing one-step genomic cDNA first-strand synthesis premixed reagent was purchased from Beijing Tiangen Biotech Co., Ltd.; ECOPLATE48 qPCR reaction plate was purchased from Bibby Scientific, UK; Brain and Heart Infusion Broth (BHI) was purchased from MedChemExpress, USA; Fluorescence In Situ Hybridization Staining Kit and EUB338 were purchased from Suzhou Jima Gene Co., Ltd.; ZO-1, Occludin, Claudin-1, and MUC2 primary antibodies were purchased from Cell Signaling Technology, USA; and immunofluorescence staining kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0021] Example 1 1) The Platycodon root is crushed and sieved to obtain Platycodon root powder with a particle size of 40 mesh. The Platycodon root powder is mixed with 10% (w / v) NaOH solution at a ratio of 1g:20mL. The mixing temperature is 85℃ and the time is 30min. The supernatant is discarded and the precipitate is collected.

[0022] 2) The precipitate was bleached with a 3% (w / v) NaClO2 solution at a pH of 3-4 and a temperature of 80℃ for 4 bleaching cycles, each lasting 60 min. Then, it was rebleached with a 0.5% (w / v) NaOH solution at a temperature of 85℃ for 90 min to obtain the preproduct.

[0023] 3) The preproduct was washed with deionized water until neutral, and then freeze-dried at a temperature of -60 to -40°C for 24 to 48 hours to obtain Platycodon grandiflorus stem cellulose.

[0024] 4) Disperse the cellulose from the platycodon root stem in a 64% (w / v) H2SO4 solution at a ratio of 1 g: 20 mL. Perform acid hydrolysis at 50°C for 45 min. Terminate the reaction by adding 10 times the volume of pre-cooled deionized water at 4°C to obtain the reactants.

[0025] 5) The reactants were centrifuged at 4°C and 12000 r / min for 10 min to obtain a suspension of Platycodon grandiflorum stem cellulose nanocrystals. The suspension was dialyzed for 5 days using a dialysis bag with a molecular weight cutoff of 8000~14000 Da until the pH stabilized. The precipitate was Platycodon grandiflorum stem cellulose nanocrystals (PGCNC). The particle size of the prepared PGCNC was 183±5.13 nm.

[0026] Example 2 1) The Platycodon root is crushed and sieved to obtain Platycodon root powder with a particle size of 30 mesh. The Platycodon root powder is mixed with 5% (w / v) NaOH solution at a ratio of 1g:15mL. The mixing temperature is 80℃ and the time is 20min. The supernatant is discarded and the precipitate is collected.

[0027] 2) The precipitate was bleached with a 2% (w / v) NaClO2 solution at a pH of 3-4 and a temperature of 75℃ for 3 times, each time for 50 min. Then it was bleached again with a 0.4% (w / v) NaOH solution at a temperature of 80℃ for 80 min to obtain the preproduct.

[0028] 3) The preproduct was washed with deionized water until neutral, and then freeze-dried at a temperature of -60 to -40°C for 24 to 48 hours to obtain Platycodon grandiflorus stem cellulose.

[0029] 4) Disperse the cellulose from the platycodon root stem in a 62% (w / v) H2SO4 solution at a ratio of 1 g: 15 mL. Perform acid hydrolysis at 45°C for 40 min. Terminate the reaction by adding 10 times the volume of pre-cooled deionized water at 4°C to obtain the reactants.

[0030] 5) Centrifuge the reactants at 4°C and 10,000 r / min for 8 min to obtain a suspension of Platycodon grandiflorum stem cellulose nanocrystals. Dialyze the suspension of Platycodon grandiflorum stem cellulose nanocrystals using a dialysis bag with a molecular weight cutoff of 8,000~14,000 Da for 5 days until the pH stabilizes. The precipitate is Platycodon grandiflorum stem cellulose nanocrystals.

[0031] Example 3 1) The Platycodon root is crushed and sieved to obtain Platycodon root powder with a particle size of 50 mesh. The Platycodon root powder is mixed with 15% (w / v) NaOH solution at a ratio of 1g:25mL. The mixing temperature is 90℃ and the time is 40min. The supernatant is discarded and the precipitate is collected.

[0032] 2) The precipitate was bleached with a 4% (w / v) NaClO2 solution at a pH of 3-4 and a temperature of 85℃ for 5 bleaching cycles, each lasting 70 min. Then, it was rebleached with a 0.6% (w / v) NaOH solution at a temperature of 90℃ for 100 min to obtain the preproduct.

[0033] 3) The preproduct was washed with deionized water until neutral, and then freeze-dried at a temperature of -60 to -40°C for 24 to 48 hours to obtain Platycodon grandiflorus stem cellulose.

[0034] 4) Disperse the cellulose from the platycodon root stem in a 66% (w / v) H2SO4 solution. The ratio of platycodon root stem cellulose to the 66% (w / v) H2SO4 solution is 1 g: 25 mL. Acid hydrolysis is performed at 55°C for 50 min. The reaction is terminated by adding 10 times the volume of pre-cooled deionized water at 4°C to obtain the reactants.

[0035] 5) Centrifuge the reactants at 4°C and 14000 r / min for 12 min to obtain a suspension of Platycodon grandiflorum stem cellulose nanocrystals. Dialyze the suspension of Platycodon grandiflorum stem cellulose nanocrystals using a dialysis bag with a molecular weight cutoff of 8000~14000 Da for 5 days until the pH stabilizes. The precipitate is Platycodon grandiflorum stem cellulose nanocrystals.

[0036] Comparative Example 1 The difference from Example 1 is that the bellflower stem was replaced with buckwheat bran to prepare buckwheat cellulose nanocrystals (FTCNC) with a particle size of 207.21±7.44nm.

[0037] Comparative Example 2 The difference from Example 1 is that the platycodon root stem was replaced with ginseng root stem to prepare ginseng root stem cellulose nanocrystals (AGCNC) with a particle size of 291±9.13 nm.

[0038] Comparative Example 3 The difference from Example 1 is that the platycodon root stem was replaced with almond coating to prepare almond coating cellulose nanocrystals (ACCNC) with a particle size of 472±30.16nm.

[0039] Comparative Example 4 The difference from Example 1 is that the bellflower stem was replaced with apple pear peel to prepare apple pear peel cellulose nanocrystals (PPCNC) with a particle size of 518±50.53 nm.

[0040] Comparative Example 5 The difference from Example 1 is that the platycodon root stem was replaced with pine kernel coating to prepare pine kernel coated cellulose nanocrystals (PNCNC) with a particle size of 605.68±172.25nm.

[0041] Example 4 Screening of the intervention effects of cellulose nanocrystals (CNCs) from different sources on colonic injury in mice: I. Experimental Methods: 1. Animal experiment design: Eight-week-old SPF-grade female Balb / c mice (experimental license number: NO.SCXK(Liaoning)2025-0001) were routinely housed in an IVC system. The ambient temperature was maintained at 21–25℃, relative humidity at (50±10)%, and a 12-hour light-dark cycle. Mice had free access to food and water during the experiment. All animal experiments were approved by the Ethics Committee of Yanbian University. After one week of acclimatization, mice were randomly divided into eight groups of 12 mice each: a control group, a DSS (Digital Supersonic Strain) model group, a PGCNC (Polydioxanone Conjugate) group, an AGCNC (Augmented Genetic Controlled Strain) group, an FTCNC (Fractional Controlled Strain) group, an ACCNC (Acute Capillary Controlled Strain) group, a PPCNC (Polydioxanone Conjugate) group, and a PNCNC (Polydioxanone Conjugate) group. During the experiment (7 days), the Control and DSS groups were administered physiological saline by gavage, while the other six groups were administered 200 mg / kg of PGCNC, AGCNC, FTCNC, ACCNC, PPCNC, or PNCNC by gavage, respectively. Except for the Control group, all other groups received 3% DSS in the oral solution for 7 days to establish a colonic injury model.

[0042] 2. Analysis of general mouse phenotype and DAI score: During the experiment, the daily weight, mental state, fecal characteristics, and fecal blood loss of mice in each group were observed and recorded. The DAI score was calculated according to the scoring criteria in Table 1, and the calculation formula is as follows: DAI = (weight loss score + fecal characteristics score + fecal occult blood score) / 3.

[0043] Table 1 DAI Scoring Criteria

[0044] 3. Biological sample collection: After the experiment, mice were euthanized and quickly dissected. Colonic tissue was harvested for photography and colonic length measurement. Cecal contents and colonic tissue were collected, preserved in EP tubes, labeled, and temporarily stored in liquid nitrogen before being transferred to a -80°C freezer for later use. Colonic tissue used for hematoxylin-eosin (HE) staining was fixed in 4% paraformaldehyde fixative (all tissue was taken from the distal colon), while colonic tissue used for alricin blue-periodic acid-Schiff (AB-PAS), immunofluorescence (IF), and fluorescence in situ hybridization (FISH) staining was fixed in Carnoy's fixative (all tissue was taken from the proximal colon).

[0045] 4. Observation of colonic tissue morphology: Colonic tissue fixed in 4% paraformaldehyde fixative was rinsed with running water and then sequentially subjected to graded ethanol dehydration, xylene clearing, and paraffin embedding (soft paraffin for 1 hour, hard paraffin for 1.5 hours). After embedding, the tissue was sectioned to a thickness of 5 μm, stained with hematoxylin and eosin (HE), and the extent of colonic tissue damage was observed under a microscope. The degree of colonic damage was scored according to the scoring criteria shown in Table 2.

[0046] Table 2 Tissue Damage Scoring Table

[0047] 5. Assessment of colonic mucus layer integrity: Colonic tissue fixed in Carnoy's fixative was rinsed with running water and then dehydrated sequentially with a gradient of ethanol (70%, 80%, 90%, 100%). The tissue was then embedded in paraffin and sectioned. After dewaxing and hydration, the sections were stained using the AB-PAS method, which included periodic acid oxidation, Schiff's reagent staining, Alicin blue staining, and treatment with acidic differentiation solution. After staining, the sections were dehydrated, cleared, and mounted with neutral resin. The integrity of the colonic mucus layer was observed under an optical microscope.

[0048] 6. Measurement of mRNA expression levels of colonic inflammatory factors: Total RNA extraction from colon tissue: Colon tissue was placed in an EP tube, and an appropriate amount of lysis buffer was added. The tissue was then ground and allowed to stand at room temperature before centrifugation at 4°C. The supernatant was collected, and RNA dilution buffer was added and mixed. After centrifugation for 5 min, the supernatant was collected and 0.5 volumes of anhydrous ethanol were added. The mixture was transferred to a centrifuge column, centrifuged for 1 min, and the filtrate was discarded. RNA washing buffer was then added to the centrifuge column, and the filtrate was discarded after centrifugation. DNase I incubation buffer was added, and the mixture was allowed to stand at room temperature for 15 min. RNA washing buffer was added, and the filtrate was discarded after centrifugation. This process was repeated twice. The centrifuge column was then placed back onto the collection tube and centrifuged for 2 min. The centrifuge column was transferred to the elution tube, and nuclease-free water was added to the center of the column membrane. The mixture was allowed to stand at room temperature for 2 min, centrifuged for 1 min, and the collected RNA was stored at -70°C.

[0049] cDNA synthesis: Prepare the reverse transcription reaction solution as shown in Table 3. Mix the collected RNA with the reaction solution evenly and place it in a PCR instrument for reaction. Set the instrument reaction conditions as follows: 42℃ for 2 min and 95℃ for 1 min.

[0050] Table 3 Reverse transcription reaction procedure

[0051] Real-time quantitative PCR analysis: The PCR reaction system was prepared as shown in Table 4, and the entire process was performed at low temperature. The PCR reaction conditions were: pre-denaturation at 95℃ for 15 min, followed by 40 cycles (95℃ for 10 s, 60℃ for 30 s). The melting curve was set at 95℃, 60℃, and 95℃ for 15 s each. mRNA expression levels were calibrated using β-actin mRNA expression levels, using 2... -ΔΔCt Results of the method calculation. Primers are shown in Table 5.

[0052] Table 4 PCR reaction system

[0053] Table 5 Primer Sequences

[0054] II. Statistical Analysis: Experimental data were statistically analyzed using SPSS 25.0 software, including one-way ANOVA and Waller-Duncan multiple comparison analysis. P < 0.05 was considered statistically significant. GraphPad Prism 10 software was used for plotting.

[0055] III. Results and Analysis: 1. Effects of CNCs from different sources on the general phenotype and DAI score of mice with colon injury: Observations revealed that, starting from day 2 of modeling, mice in all groups that drank 3% DSS exhibited varying degrees of disheveled fur, decreased mental state, weight loss, and diarrhea, consistent with signs of colonic injury. Figure 1 Compared with the DSS group, all six different sources of CNC improved weight loss and alleviated diarrhea and bloody stool symptoms in mice. The degree of colonic injury in each group of mice was assessed based on the DAI score. Figure 1 In the study (B), it was found that the DAI scores of all six CNC groups were significantly lower than those of the DSS group (P < 0.0001), indicating that CNC intervention can alleviate weight loss, diarrhea, and bloody stools in mice caused by colon injury to some extent. These results suggest that although all six CNCs showed potential to alleviate colon injury, comprehensive analysis showed that PGCNC had a more significant advantage.

[0056] 2. Effects of CNCs from different sources on fecal bleeding and colon length in mice with colon injury: Compared with the control group, mice in the DSS group showed severe rectal bleeding and significant shortening of the colon. After CNC intervention, the rectal bleeding in the mice was significantly improved, and the colon length was also significantly restored. Figure 2 China A and Figure 2 (B) These results preliminarily indicate that CNC has a beneficial effect on colonic injury, with PGCNC showing the best improvement.

[0057] 3. The effects of CNC from different sources on colon tissue morphology: The degree of colonic injury in mice was assessed by combining HE staining observation with tissue damage scoring. For example... Figure 3 China A and Figure 3 As shown in Figure B, the colonic mucosa of mice in the Control group was intact, with regular epithelial cell arrangement and clear crypt structure. In contrast, the colonic mucosa of mice in the DSS group was severely damaged, with disordered epithelial cell arrangement, disappearance of crypts, and extensive inflammatory cell infiltration in the lamina propria. Compared with the DSS group, colonic tissue damage in all CNC intervention groups was alleviated to varying degrees. The PGCNC group showed the most significant improvement (P<0.0001), with its overall colonic mucosa morphology approaching that of the Control group, exhibiting regular crypt arrangement, a significantly increased number of goblet cells, reduced inflammatory cell infiltration in the lamina propria, and reduced mucosal epithelial cell shedding. This indicates that PGCNC has a good protective effect against DSS-induced colonic damage.

[0058] 5. The impact of CNC from different sources on the integrity of the colonic mucus layer: AB-PAS staining clearly reveals changes in colonic goblet cells and the mucus layer. Figure 4 China A and Figure 4As shown in Figure B, the colonic epithelial mucus layer of mice in the Control group was continuous and intact, with abundant goblet cells that stained distinctly blue-purple, indicating that both acidic and neutral mucus could be secreted normally in the colon. In contrast, the colonic epithelial mucus layer of the DSS group was significantly damaged or even nearly disappeared, and the number of goblet cells was significantly reduced, indicating that its mucus barrier function was severely impaired. Compared with the DSS group, the colonic epithelial mucus layer structure and goblet cell count of mice in the six CNC groups were restored to varying degrees, indicating that CNC has a certain alleviating effect on DSS-induced mucus barrier damage. Among them, the PGCNC group showed the best improvement effect, with its colonic epithelial mucus layer morphology approaching that of the Control group, indicating that PGCNC has the best protective potential for the mucus barrier.

[0059] 6. Effects of CNCs from different sources on the expression of inflammatory cytokine mRNA: like Figure 5 China A~ Figure 5 As shown in Figure D, compared with the Control group, the mRNA expression of Il-6, Il-1β, and Tnf-α in the colonic tissue of mice in the DSS group was significantly increased, while the mRNA expression of Il-10 was significantly decreased (P<0.0001), indicating that DSS-induced colonic injury led to disordered expression of local inflammatory factors in the colon. Compared with the DSS group, the mRNA expression of pro-inflammatory factors in all CNC intervention groups showed a decreasing trend, while the mRNA level of Il-10 significantly rebounded, with the PGCNC group showing the most significant improvement (P<0.0001). These results indicate that CNC can alleviate the DSS-induced local inflammatory response in the colon of mice to a certain extent, with PGCNC showing the most significant regulatory effect on the disordered expression of local inflammatory factors in the colon.

[0060] Six different types of colonic neuron (CNC) from various sources all alleviated DSS-induced colonic injury in mice to some extent, specifically by reducing weight loss, alleviating diarrhea and bloody stools, and improving colonic shortening. Furthermore, each CNC intervention repaired damaged colonic mucosal structure, enhanced mucus layer integrity, increased goblet cell number, and regulated inflammatory cytokine mRNA expression levels. Comprehensive comparison revealed that PGCNC showed the best intervention effect across all evaluation indicators. Therefore, PGCNC was ultimately selected and identified as the key target for subsequent research on its mechanism of action.

[0061] Example 5 Evaluation of the Application Basis of PGCNC and its Protective Effect on Colonic Barrier Function: The occurrence and development of colonic injury are usually accompanied by impaired intestinal barrier function. The intestinal barrier mainly includes the mechanical barrier maintained by TJs such as ZO-1, Occludin, and Claudin-1, the mucus barrier with MUC2 as its core, and the biological barrier formed by the participation of gut microbiota. These three are interconnected and work together to maintain local colonic homeostasis. The results of Example 4 show that PGCNC has a good intervention effect in alleviating colonic injury.

[0062] However, whether the protective effect of PGCNCs primarily derives from their direct action due to their nanoscale size and physicochemical properties, or whether it relies on gut microbiota mediation, remains to be clarified. Therefore, this study evaluated the physicochemical properties, in vivo safety, and distribution characteristics of PGCNCs after oral administration to determine whether they possess the basis for exerting effects locally in the gastrointestinal tract, particularly the colon. Subsequently, the effects of PGCNCs on colonic barrier function were analyzed by examining changes in TJs expression and MUC2 protein expression. Finally, the mediating role of gut microbiota in the process of PGCNCs alleviating colonic injury was confirmed, thereby further clarifying whether the colonic protective effect of PGCNCs mainly depends on their direct action or is achieved through gut microbiota mediation.

[0063] I. Experimental Methods: 1. PGCNC characterization: The PGCNC suspension was diluted approximately 1:1000 and dropped onto the surface of a carbon support membrane, then allowed to air dry overnight at room temperature. After the sample was fully dried, its microstructure was observed using TEM at 80 kV. A PGCNC suspension with a mass fraction of approximately 0.1% (w / v) was prepared using water as the dispersion medium. After thorough dispersion, its zeta potential was measured using a nanolaser particle size analyzer at room temperature. The functional group characteristics of PGCNC were determined using Fourier transform infrared spectroscopy. Before testing, the sample was prepared using the potassium bromide pellet method. The measurement conditions were: resolution 4 cm⁻¹, scanning range 4000–500 cm⁻¹, and a total of 32 scans. To evaluate the cell compatibility of PGCNC, the effect of PGCNC intervention on RAW264.7 cells was observed using the Calcein-AM / PI live / dead cell staining method. RAW264.7 cells were seeded into 6-well plates. After cell adhesion and reaching a suitable confluence, the culture medium was replaced with complete culture medium containing 0, 10, 100, and 1000 μg / mL PGCNC, respectively, and incubated at 37°C and 5% CO2 for 24 h. After treatment, the culture medium was discarded, and the cells were washed twice with PBS. Calcein-AM / PI staining working solution was prepared according to the kit instructions and added to the 6-well plates, and incubated at 37°C in the dark. After staining, the cells were washed with PBS, and images were observed and acquired using a fluorescence inverted microscope.

[0064] 2. Animal experiment design: In vivo safety evaluation of PGCNC: Referring to GB15193.22—2014 "National Food Safety Standard 28-day oral toxicity test", 8-week-old SPF-grade female Balb / c mice were randomly divided into two groups of 6 mice each after one week of acclimatization: a normal control group (Control group) and a high-dose PGCNC intervention group (PGCNC-H group). Mice in the PGCNC-H group were administered 1000 mg / kg PGCNC dispersion by gavage daily for 28 days, while mice in the Control group were administered the same volume of physiological saline by gavage. The mice's condition was recorded daily during the experiment.

[0065] In vivo tracing of PGCNC: PGCNC was dispersed in 100 mM phosphate buffer (pH 4.5), and NaIO4 was added to a final concentration of 10 mM. Oxidation was carried out at room temperature in the dark for 2 h to introduce aldehyde groups. Ethylene glycol was then added for quenching for 5 min, and the pH was adjusted to 6.0. Cy5-hydrazide was then added, and the reaction was carried out at room temperature in the dark for 2 h. After the reaction, residual dye was removed by centrifugation to obtain stable Cy5-PGCNC for subsequent in vivo distribution studies. The distribution of PGCNC in vivo was investigated. Six- to eight-week-old SPF-grade female BALB / c mice were selected and acclimatized for one week before in vivo imaging. The mice were fasted for 12 h before imaging, and abdominal hair was removed before the experiment. 0.2 mL of Cy5-PGCNC (14.45 mg / kg) was administered by gavage, and in vivo fluorescence images were collected at 1, 2, 4, 8, 12, 24, 48 and 72 h after gavage. Mice were anesthetized and placed in a small animal in vivo imaging system, and then the main tissues were dissected and separated for in vitro imaging analysis.

[0066] Study on the role of gut microbiota in regulating colonic injury by PGCNC: 300 mg of fresh fecal particles from PGCNC mice were collected in a sterile laminar flow hood and rapidly transferred to a sterile centrifuge tube containing 2 mL of pre-cooled PBS. After vortexing for 10 seconds to mix thoroughly, the tube was centrifuged at 1000 rpm for 8 minutes, and the supernatant was collected as the fecal microbiota transplantation suspension. The entire process was performed under low temperature conditions to maintain bacterial activity as much as possible. Eight-week-old SPF-grade female Balb / c mice were pretreated with a broad-spectrum antibiotic mixture after one week of acclimatization. Ampicillin (1 g / L), neomycin (1 g / L), metronidazole (1 g / L), and vancomycin (0.5 g / L) were dissolved in sterile drinking water, thoroughly mixed, and prepared as an antibiotic cocktail solution. The solution was prepared immediately and stored away from light. Mice in each group were administered 100 μL / day by gavage for 9 consecutive days. After antibiotic treatment, fresh feces were collected under aseptic conditions and mixed thoroughly with sterile PBS buffer at a ratio of 1:10 (w / v). 100 μL of the fecal suspension was spread onto Brain Heart Infusion (BHI) agar plates and incubated at 37°C for 48 h. Colony growth was observed to verify the effect of intestinal flora depletion. After confirming the successful construction of pseudo-germ-free mice, mice were given free access to sterile water for 24 h to remove residual antibiotics and reduce their impact on subsequent experiments. Pseudo-germ-free mice were randomly divided into three groups of six mice each: the DSS model group (ABX-DSS), the PGCNC group (ABX-PGCNC), and the fecal microbiota transplantation (FMT) group. The ABX-DSS group received saline by gavage; the ABX-PGCNC group received PGCNC (200 mg / kg) by gavage daily; and the FMT group received 200 μL of PGCNC donor bacterial suspension by gavage daily. During the experiment, the mice's condition was recorded daily. Starting from day 16, mice in each group were allowed free access to 3% DSS solution for 7 days to induce colonic injury (day 23).

[0067] 3. Analysis of general mouse phenotype and DAI score: The detection of indicators such as mouse weight and DAI score was carried out according to the method described in Example 4.

[0068] 4. Biological sample collection: In vivo safety evaluation of PGCNC: After the experiment, whole blood was collected from mice in EDTA 2K anticoagulant tubes and non-anticoagulant blood collection tubes. EDTA 2K anticoagulant whole blood was used for routine blood tests, while non-anticoagulant whole blood was centrifuged to separate serum for blood biochemical index detection. The mice were then euthanized, and the heart, liver, spleen, lung, kidney, and colon tissues were quickly dissected and fixed in 4% paraformaldehyde solution.

[0069] Study on the role of gut microbiota-mediated PGCNC in regulating colonic injury: biological sample collection was conducted using the method described in Example 4.

[0070] 5. Serum marker testing: Whole blood samples from mice were collected and subjected to routine blood tests using an automated blood analyzer. Separately collected mouse serum was used for blood biochemistry tests. Blood biochemistry indicators included liver function indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP); and kidney function indicators: blood urea nitrogen (BUN) and creatinine (CREA). These blood biochemistry indicators were measured using appropriate kits, following the instructions provided.

[0071] 6. Histological observation: The methods for observing the histological morphology of mouse heart, liver, spleen, lung, kidney and colon tissues are the same as in Example 4.

[0072] 7. IF staining: Paraffin sections were baked in a 60°C oven for 1 hour, followed by dewaxing, hydration, and washing with PBS. They were then placed in EDTA antigen retrieval solution (pH 9.0) and incubated on high for 9 minutes, then on low for 7 minutes, followed by medium for 7 minutes for antigen retrieval. Afterward, BSA was added and incubated at room temperature for 30 minutes for blocking. After removing the blocking solution, ZO-1 antibody was added and incubated overnight at 4°C, followed by HRP-labeled secondary antibody and incubation at room temperature for 50 minutes. Finally, TSA Tyramide-488 was added and incubated at room temperature in the dark for 10 minutes. After washing to remove unbound antibodies, the sections were incubated again with Claudin-1 primary antibody, HRP-labeled secondary antibody, and TSA Tyramide-647 for the same duration. After eluting the antibodies again, the sections were incubated sequentially with Occludin primary antibody, HRP-labeled secondary antibody, and TSA Tyramide-555. After eluting the antibodies, autofluorescence quenching was performed, and finally, DAPI dye was added to the sections for counterstaining at room temperature for 10 minutes. The slides were washed three times in PBS buffer for 5 minutes each time to remove excess liquid, and then mounted with anti-fluorescence quenching mounting medium. The immunofluorescence stained slides were observed and photographed using a fluorescence microscope.

[0073] II. Statistical Analysis: The experimental data were statistically analyzed using SPSS 25.0 software. One-way ANOVA and Waller-Duncan multiple comparison analyses were performed. P < 0.05 was considered statistically significant. GraphPad Prism 10 software was used for graphing.

[0074] III. Results and Analysis: 1. Characterization of PGCNC: The characterization results of PGCNC show that PGCNC possesses the typical physicochemical properties and biocompatibility of cellulose nanocrystals. Figure 6 China A~ Figure 6(Middle E). Macroscopic observation shows that PGCNCs are uniformly dispersed and have a stable appearance. FTIR spectroscopy revealed that PGCNCs are at 3414 cm⁻¹. -1 The presence of an OH stretching vibration peak at 2900 cm⁻¹ indicates that its surface contains abundant hydroxyl groups; -1 The peak at 1448 cm⁻¹ corresponds to the C–H stretching vibration, indicating that it retains the cellulose unit structure. -1 and 1123cm -1 The absorption peaks at 603 cm⁻¹ correspond to the –CH₂ bending vibration and the C–O–C / C–O stretching vibration, respectively, indicating that it possesses a typical cellulose skeletal structure. -1 The nearby peaks may be related to the vibration of the sugar ring skeleton. Overall, this indicates that the nanocrystals retain the characteristic functional groups of cellulose. PGCNCs exhibit the main functional group characteristics of CNCs, and Zeta potential measurements show that their surface is negatively charged and they possess good stability. TEM imaging shows that the PGCNCs have a length of 183±5.13 nm and exhibit a rod-like crystal structure. In vitro biocompatibility results showed that no large-scale cell death was observed under PGCNC intervention conditions ranging from 0 to 1000 μg / mL, further confirming that PGCNCs have no significant cytotoxicity. Overall, the prepared PGCNCs possess a stable structure, good dispersibility, and excellent biocompatibility, providing a foundation for subsequent in vivo experiments.

[0075] 2. Security evaluation of PGCNC: Effects on the morphology of major organs and tissues: To evaluate the in vivo safety of PGCNCs, this study, referring to the relevant requirements of GB15193.22—2014 "National Food Safety Standard 28-day Oral Toxicity Test", conducted a preliminary evaluation of the potential toxicity of PGCNCs from three levels: histopathology, hematology, and serum biochemistry. HE staining results showed ( Figure 7 In both the Control and PGCNC-H groups, the major organs (heart, liver, spleen, lungs, kidneys, and colon) of mice showed normal overall morphology with no abnormalities, and there were no significant differences between the two groups. Further observation revealed that after high-dose PGCNC intervention, the heart muscle fibers of mice were neatly arranged and clearly structured; the liver lobule structure was intact, and the hepatocyte morphology was normal; the white and red pulp of the spleen were clearly structured and normally demarcated; the alveoli were intact with clear septa; the glomeruli and renal tubules were intact and regularly arranged; and the colonic mucosa was intact with neatly arranged crypts and no damage was observed. These results indicate that high-dose PGCNC intervention did not cause significant histological damage to the major organs of mice and has good biocompatibility.

[0076] Effects on complete blood count: Table 6 shows the results of complete blood count in mice. There were no significant differences in any of the complete blood count indicators between the PGCNC-H group and the Control group (P>0.05), indicating that high-dose PGCNC intervention did not have a significant adverse effect on the hematological indicators of mice.

[0077] Table 6. Complete Blood Count of Mice

[0078] Note: (M±SD, n=6), compared with the Control group, ns indicates no statistically significant difference.

[0079] Among the parameters, WBC, Gran#, Lymph#, Mon#, and their percentages remained generally stable, indicating that high-dose PGCNC did not cause significant inflammation or abnormal immune responses. RBC, HGB, HCT, MCV, MCH, MCHC, RDW-CV, and RDW-SD showed no abnormalities, indicating that PGCNC had minimal impact on erythrocytes. PLT, MPV, PDW, PCT, P-LCC, and P-LCR were all within the normal range, indicating that PGCNC did not adversely affect platelet-related parameters. Overall, high-dose PGCNC had minimal impact on mouse hematological parameters and demonstrated good biocompatibility.

[0080] Effects on blood biochemistry: To further evaluate the effects of high-dose PGCNC on liver and kidney function in mice, relevant serum biochemical indicators were measured. Results are as follows: Figure 8 China A~ Figure 8 As shown in Figure E, compared with the Control group, the serum levels of ALT, AST, BUN, CREA, and ALP in the PGCNC-H group mice showed no significant changes, with each indicator's variation being less than 8% and remaining within the normal physiological range. These results indicate that high-dose PGCNC did not cause significant adverse effects on liver and kidney function in mice, demonstrating its good in vivo safety. Combined with histological and blood routine results, PGCNC did not exhibit significant systemic toxicity and possesses excellent in vivo biocompatibility.

[0081] 3. In vivo distribution characteristics of PGCNC: The distribution of PGCNCs in mice was analyzed using a small animal in vivo imaging system. Figure 9The results showed that after oral administration of Cy5-PGCNC, significant fluorescent signals were observed in the gastrointestinal tract of mice at 1 and 2 hours. The fluorescence signal then gradually diffused distally and weakened over time, disappearing almost completely by 48 hours. This indicates that PGCNC is mainly distributed in the gastrointestinal tract after oral administration and remains there for a certain period. This may be because after entering the digestive tract orally, PGCNC initially accumulates in the gastrointestinal tract and is gradually transported downstream with peristalsis before being excreted. The above results indicate that after oral administration, PGCNC is mainly distributed in the gastrointestinal tract and can remain there for a certain period. This phenomenon may be related to the accumulation of PGCNC in the gastrointestinal contents after oral administration and its gradual transport with intestinal peristalsis. The local retention characteristics of PGCNC in the gastrointestinal tract facilitate its full contact with the intestinal flora and intestinal mucosal barrier, providing an important basis for its subsequent regulation of the intestinal microenvironment and improvement of colonic injury.

[0082] To further clarify the distribution characteristics of PGCNCs in mice, the gastrointestinal tract, heart, liver, spleen, lungs, and kidneys were dissected and imaged in vitro. Figure 10 and Figure 11 The results showed that PGCNCs began to be transported in the intestines 1 hour after oral administration; by 4 hours, obvious fluorescence signals were visible in the colonic region, and a small amount of residual fluorescence was still detected until 48 hours, indicating that PGCNCs had a relatively long residence time in the colon. In addition, some fluorescence signals were observed in the liver, suggesting that some PGCNCs may have entered the systemic circulation. Overall, PGCNCs were mainly distributed in the gastrointestinal tract and exhibited a relatively long residence time in the colon, while a small amount entered the circulatory system. This distribution characteristic may be related to the large specific surface area of ​​nanofibers and their interaction with the intestinal mucus layer, thus enhancing their local retention in the gastrointestinal tract. Previous studies have reported that nanofibers have the characteristic of enhancing intestinal retention, and ginseng-derived nanofibers have also shown similar results. Combining the above distribution characteristics, it can be inferred that PGCNCs, as nanoscale dietary fibers, may interact with the gut microbiota in the colon and further participate in the regulation of gut microbiota and metabolism.

[0083] 4. Effects of PGCNC on the expression of TJs proteins ZO-1, Occludin, and Claudin-1: Normal expression of TJs can effectively prevent the invasion of harmful substances in the intestine and maintain intestinal barrier function. Immunofluorescence staining results showed ( Figure 12 China A~ Figure 12In the DSS group (Control group), the colonic epithelial structure of mice was intact, with strong positive expression of ZO-1, Claudin-1, and Occludin proteins, mainly located at the edge of epithelial cells, presenting a continuous and clear honeycomb structure. In contrast, the expression levels of these proteins in the colonic epithelium of mice in the DSS group were significantly reduced (P<0.0001). Morphological observation also revealed obvious breaks, diffusion, and discontinuities in protein distribution, indicating severe damage to the intestinal barrier structure during intestinal injury. After PGCNC intervention, the expression levels of ZO-1, Claudin-1, and Occludin proteins in the colonic tissue of mice significantly rebounded, and the protein distribution regained continuity and clear boundaries (P<0.05). This indicates that PGCNC can effectively upregulate the expression of TJs and improve their distribution, thereby enhancing the integrity of the intestinal barrier.

[0084] 5. Study on the role of gut microbiota-mediated PGCNC in regulating colonic injury: Construction of pseudo-germ-free mice: By comparing the results before and after gavage administration of broad-spectrum antibiotic cocktails ( Figure 13 (A) and (B) Figure 13 The colony growth on the BHI plates (B) confirmed that after 9 days of antibiotic treatment, the total intestinal bacteria in the mice were basically depleted, and pseudo-germ-free mice required for subsequent experiments were successfully established.

[0085] Effects on general phenotype and DAI score in mice: From Figure 14 It can be seen that the ABX-DSS group mice exhibited a typical phenotype of acute colonic injury, mainly manifested as continuous weight loss and a sustained increase in DAI scores due to worsening diarrhea and bloody stools. In contrast, the FMT group showed a significant intervention effect. After PGCNC donor microbiota transplantation, the trend of weight loss in mice was alleviated (P<0.0001), the DAI score was significantly reduced (P<0.0001), and the overall physiological state was significantly improved. It is worth noting that compared with the FMT group, the ABX-PGCNC group did not show a significant advantage in improving colonic injury, indicating that under conditions of depleted gut microbiota, PGCNC is unlikely to directly exert a protective effect. Therefore, it is speculated that the effect of PGCNC in alleviating colonic injury may depend on gut microbiota mediation, exerting its effect by restoring or regulating gut microbial function.

[0086] Effects on rectal bleeding and colon length: such as Figure 15 China A and Figure 15As shown in Figure B, the colon length in the FMT group was significantly increased compared to the ABX-DSS and ABX-PGCNC groups (P<0.0001), and diarrhea and rectal bleeding were also alleviated, indicating that FMT intervention can alleviate DSS-induced colon shortening and damage to some extent. Recovery of colon length usually reflects a reduction in intestinal inflammation and tissue damage; therefore, this result suggests that gut microbiota reconstruction helps improve the colonic damage caused by DSS. In contrast, the colon length in the ABX-PGCNC group did not show significant recovery, and diarrhea and rectal bleeding remained severe, indicating that the alleviating effect of PGCNC on colonic damage was significantly affected after the gut microbiota was interfered with by antibiotics. This result further suggests that the colon-protective effect of PGCNC may depend on the participation of the gut microbiota, which plays a crucial role in regulating the intestinal microenvironment, alleviating inflammatory responses, and promoting colonic damage repair through PGCNC.

[0087] Effects on colon tissue morphology: such as Figure 16 China A and Figure 16 As shown in Figure B, the colonic mucosa structure of the FMT group was intact, with regular arrangement of epithelial cells and clear crypt structures. In contrast, the colonic mucosa structure of mice in the ABX-DSS and ABX-PGCNC groups was severely damaged, with missing crypt structures, disordered intestinal lumen structure, and obvious inflammatory cell infiltration in the lamina propria, indicating that the repair effect of PGCNC on damaged colon depends on gut microbiota mediation.

[0088] In summary, PGCNCs are structurally stable and well-dispersed, exhibiting typical CNC structural characteristics, and showed no significant cytotoxicity. PGCNCs did not adversely affect the structure of major organs, hematological parameters, or liver and kidney function in mice, demonstrating excellent overall in vivo biocompatibility. After oral administration, PGCNCs are mainly distributed in the gastrointestinal tract and remain in the colon for a relatively long time, with a small amount entering the circulatory system. PGCNCs can improve colonic injury in mice at multiple levels: they can improve the mRNA expression of mucus barrier-related factors; enhance MUC2 protein expression, maintaining normal spatial isolation between intestinal bacteria and epithelium; and regulate the expression and distribution of TJs such as ZO-1, Occludin, and Claudin-1, thereby enhancing intestinal barrier integrity. Under conditions of intestinal flora depletion, PGCNCs are unable to continue to exert their protective effect, and their colonic protective effect is closely related to intestinal flora mediation.

[0089] Example 6 The Regulatory Role of PGCNC in the Gut Microbiota of Mice with Colon Injury: The gut microbiota plays a crucial role in maintaining intestinal homeostasis and barrier function. Reshaping gut microbiota homeostasis helps repair intestinal barrier function, reduce inflammatory responses, and thus effectively alleviate colon injury. PGCNC has excellent repair effects on colon injury in mice, and its protective effect depends on the mediation of the gut microbiota. However, the regulatory mechanisms of PGCNC on the structure and function of the gut microbiota, as well as the key bacterial species playing a core role, remain unclear. Therefore, using mice with DSS-induced colon injury as the research subject, this study analyzed the effects of PGCNC intervention on the composition, diversity, and community differences of the gut microbiota. Through microbiota function and correlation analysis, key bacterial species that may be involved in the repair of colon injury were screened, elucidating the regulatory role of PGCNC on the gut microbiota from a microecological perspective, and laying a theoretical foundation for subsequent mechanistic research.

[0090] I. Experimental Methods: 1. Library Construction and Sequencing: Total genomic DNA from collected cecal contents was extracted using a rapid DNA spin extraction kit. One μg of genomic DNA from the sample was randomly fragmented into approximately 350 bp fragments using a Covaris ultrasonic disruptor. Library construction was then performed, including end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification. After construction, the integrity and insert size of the library fragments were first checked using AATI to ensure they met the expected requirements. Subsequently, the effective concentration of the library was quantified using Q-PCR (effective concentration > 3 nM) to ensure library quality. Once the libraries were deemed acceptable, different libraries were mixed according to the effective concentration and target data volume requirements, and then sequenced using PE150 sequencing.

[0091] 2. Sequencing information analysis: Sequencing result preprocessing: FastP was used to preprocess the raw data obtained from the NovaSeq sequencing platform to obtain valid data for subsequent analysis. The specific processing steps are as follows: Reads containing adapter sequences were removed; reads containing more than 50% low-quality (Q≤5) bases were removed; and reads containing more than 10% N bases were removed.

[0092] Metagenome assembly: The cleandata was assembled and analyzed using MEGAHIT software. Then, the assembled scaffolds were broken at the connections containing N to obtain scaffolds without N.

[0093] Gene prediction and abundance analysis: MetaGeneMark was used to predict the ORF of scaftigs (≥500bp) for each sample, and information shorter than 100nt in the prediction results was filtered out, all using default parameters. The ORF prediction results were deredundantd using CD-HIT software to obtain a non-redundant initial gene catalog, and the nucleic acid sequences encoded by these non-redundant genes were referred to as genes. Subsequently, Bowtie2 was used to align the clean data of each sample to the initial gene catalog, and the number of aligned reads for each gene in each sample was calculated. Genes with ≤2 reads in each sample were filtered out, resulting in the final non-redundant gene set used for subsequent analysis. Finally, based on the number of aligned reads and gene length, the abundance information of each gene in different samples was calculated.

[0094] Species annotation: Unigenes were compared with the Micro_NR database using DIAMOND software. Micro_NR consists of bacterial, fungal, archaea, and viral sequences extracted from the NCBI NR database.

[0095] Common Functional Database Annotation: Using DIAMOND software, unigenes were aligned with functional databases with parameters set to blastp, -e1e-5. Functional databases included the KEGG and CAZy databases. For each sequence alignment, the highest-scoring alignment was selected for subsequent analysis. Based on the alignment results, the relative abundance of different functional levels was calculated. The relative abundance of each functional level was equal to the sum of the relative abundances of genes annotated at that functional level. Based on the functional annotation results and gene abundance tables, a gene count table for each sample at each taxonomic level was obtained (the number of genes for a specific function in a sample is equal to the number of genes with a non-zero abundance among those annotated for that function). Furthermore, based on the abundance tables at the functional taxonomic levels, the number of annotated genes was statistically analyzed, relative abundance overviews were displayed, and abundance clustering heatmaps were generated.

[0096] 3. Co-occurrence Network Construction and Key Species Identification: To explore the synergistic or antagonistic relationships among key species in the microbial community, a co-occurrence network was constructed and key species were identified. First, based on the abundance table obtained from metagenomic sequencing, low abundance (relative abundance <0.01%) and low frequency (occurrence frequency <20%) features were removed, and a standardized gene abundance table was used for network analysis. The SparCC algorithm was used to calculate the correlation coefficients between species, and significant co-occurrence relationships (|r|>0.6, P<0.05) were selected for constructing the co-occurrence network. Network construction and visualization were performed using Cytoscape (v3.9.1), where nodes represent species and edges represent significant correlations between them. Topological parameter analysis was performed on the co-occurrence network, including indicators such as degree, betweenness centrality, and compactness. In summary, key nodes with high degree values ​​and high centrality were selected as key species.

[0097] 4. Statistical Analysis: Experimental data were statistically analyzed using SPSS 25.0 software. One-way ANOVA and Waller-Duncan multiple comparison analyses were performed. P < 0.05 was considered statistically significant. GraphPad Prism 10 software was used for graphing. Bioinformatics analysis was performed using QIIME.

[0098] II. Results and Analysis: 1. Metagenomic data quality control, assembly, and analysis: Gene set basic information statistics: After quality control of the sequencing data, the quality control statistics and assembly results of each group of sequencing data are detailed in Table 7. The results show that a total of 281.37G of clean reads were obtained in this experiment, of which more than 98% were effective reads, indicating a high overall effectiveness rate and good sequencing data quality. Clean_Q20 and Clean_Q30 were both at high levels, indicating high accuracy in sequencing base recognition; at the same time, the GC content (Clean_GC) of each sample was between 45.96% and 49.87%, with a relatively stable distribution and no obvious abnormalities. After removing the host sequence, each sample still retained a certain amount of non-host data, which can meet the needs of subsequent species annotation and functional analysis. Overall, the sequencing data in this study is of reliable quality and can be used for subsequent metagenomic analysis.

[0099] Table 7. Quality control and assembly results of sample sequencing data

[0100] 2. Characteristics of the gut microbiota and analysis of differential flora: Species composition and abundance analysis: A total of 5 kingdoms, 166 phyla, 161 classes, 325 orders, 705 families, 2567 genera, and 12308 species were identified. The focus was on exploring the differences in gut microbiota composition among the samples at both the phylum and genus levels. Figure 17 China A~ Figure 17 In the study, it was found that at the phylum classification level, after PGCNC intervention, the candidate phylum of saccharified bacteria (…) Candidatus Saccharibacteria ), Actinobacteria ( Actinomycetota ), Thermo-desulfurobacteria ( Thermodesulfobacteriota ), deferrobacteria ( Deferribacterota Campylobacteria ( Campylobacterota ), Verrucous microbes ( Verrucomicrobiota ), Pseudomonas ( Pseudomonadota Bacteroidetes ( Bacteroidota ) and Bacillus phylum ( Bacillota All of them recovered to the Control group level. At the genus level, after PGCNC intervention, Aristoptera spp. ( Alistipes ), Duncanella ( Duncaniella ), Procardia ( Phocaeicola ), Parabacteroides ( Parabacteroides ), acetic acid-producing bacteria ( Acetatifactor Escherichia coli ( ) Escherichia ), Helicobacter spp. Helicobacter Akkermania ( ) Akkermansia ), genus *Vibrio* ( Oscillibacter ) and Bacteroides ( Bacteroides All of them recovered to the Control group level. Among them, the genus *Oscillatoria* ( Oscillibacter ) and Bacteroides ( Bacteroides These bacteria were the dominant genus in all three groups. Most of these bacteria can break down complex carbohydrates in the colon, stimulating butyrate production. These results indicate that PGCNC can not only correct abnormal gut microbiota composition caused by colonic injury, but also promote the remodeling of the dominant microbiota structure towards a healthy state, with most of the dominant bacteria having the ability to promote SCFAs production.

[0101] Alpha diversity analysis: Alpha diversity reflects the richness and evenness of the gut microbiota, including Shannon diversity and the Chao1 index. Figure 18 As can be seen, compared with the Control group and the PGCNC group, the alpha index of the DSS group was lower, indicating that colonic injury led to the dysregulation of the gut microbiota ecosystem. Meanwhile, both the Control group and the PGCNC group showed higher values ​​for the aforementioned alpha diversity index. This result indicates that PGCNC intervention inhibited the gut microbiota dysregulation caused by DSS and maintained a high level of microbial diversity.

[0102] Beta diversity analysis: Unlike alpha diversity analysis, beta diversity measures structural differences by calculating the similarity between samples, reflecting changes in species composition among different samples. Through principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS), it was found that at the genus level, such as... Figure 19 China A and Figure 19 As shown in Figure B, the Control group and the DSS group were clearly separated along the first principal component (PC1), and PGCNC intervention caused a significant migration of samples towards the Control group. PCoA based on Bray-Curtis distance showed a clear clustering pattern among the three groups, with the microbial structure of the DSS group deviating from that of the Control group, indicating a disordered gut microbiota structure. However, PGCNC intervention caused the microbial community to move away from the DSS group and towards the Control group, indicating a restoration of the gut microbiota structure. NMDS further confirmed this structural change, with the DSS group forming an independent set far from the Control group. Notably, the PGCNC group was clearly distinguishable from the DSS group and showed a trend of regression towards the Control group. In summary, PGCNC intervention remodeled the overall gut microbiota structure towards a healthy state.

[0103] LEfSe differential analysis: LEfSe analysis is used to screen for differentially expressed bacterial communities between different groups. The LDA value is a key indicator in LEfSe analysis; a higher LDA value indicates a stronger ability to distinguish between different groups. Figure 20 China A and Figure 20 B shows the phylogenetic clade diagram of LEfSe analysis. The PGCNC group is mainly enriched in Bacteroidetes ( Bacteroidales ), Mouse-bacteriaceae ( Muribaculaceae Akkermaniaceae ( Akkermansiaceae ), Akkermansia These microbiota, including those related to complex carbohydrate utilization, mucus barrier maintenance, and SCFAs production, indicate that PGCNC can effectively remodel gut microbiota dysbiosis caused by colonic injury. In contrast, the DSS group mainly enriched... Bacteroides , Parabacteroides and Escherichia coli ( Escherichiacoli The presence of bacteria such as [list of bacteria] indicates a significant imbalance in the gut microbiota after DSS induction, accompanied by an increase in inflammation-related bacteria. In summary, PGCNC can regulate DSS-induced gut microbiota imbalance, promote the enrichment of core functional bacteria, and inhibit the proliferation of inflammation-related bacteria.

[0104] MetagenomeSeq differential analysis: From Figure 21 China A~ Figure 21As shown in Figure C, colonic injury affected the gut microbiota structure in mice, altering the clustering pattern and leading to the enrichment of various inflammation-related genera. Significant differences in gut microbiota characteristics were observed between the PGCNC and DSS groups. Compared to the DSS group, the PGCNC group showed increased relative abundance of some bacteria associated with complex carbohydrate utilization and anaerobic fermentation, including *Isprevotella* spp. Alloprevotella ), genus *Plasmodium* Paludibacter ) and Ricnerella spp. ( Rikenella ), while Shigella spp. in the DSS group ( Shigella Salmonella ( Salmonella ), Fusobacterium genus ( Fusobacterium Clostridium ( Clostridium The abnormal enrichment of inflammation-related bacteria, such as those found in the control group, indicates that PGCNC can, to some extent, reverse the gut microbiota dysbiosis caused by colonic injury and inhibit the enrichment of harmful bacteria. On the other hand, the differentially expressed microbiota between the PGCNC group and the control group were mostly low-abundance or atypical core gut microbiota, suggesting that the overall gut microbiota state after PGCNC intervention was closer to a healthy state. In conclusion, MetagenomeSeq analysis further supports the claim that PGCNC can improve DSS-induced microecological imbalance by reshaping the gut microbiota structure.

[0105] 3. Analysis of functional genes in gut microbiota: To further explore the key metabolic pathways involved by differentially metabolites after PGCNC intervention, this study performed KEGG pathway enrichment analysis on the differentially metabolites. The results showed that the gut microbiota of mice in each group exhibited a relatively consistent functional composition pattern, reflecting the relative stability of the gut microbiota ecosystem at the functional level. Among them, the functional category with the highest relative abundance was carbohydrate metabolism, followed by amino acid metabolism and membrane transport. Figure 22 (A). The high abundance of carbohydrate metabolism pathways indicates that the gut microbiota possesses strong polysaccharide degradation potential, suggesting that PGCNCs can be degraded and utilized in the gut to exert probiotic effects. Furthermore, multi-level analysis of the KEGG database (level 1, level 2, etc.) revealed... Figure 22 B~ Figure 22PGCNC intervention significantly regulated the metabolic function of the gut microbiota and effectively restored the dysregulation of multiple physiological processes caused by colonic injury. PGCNC may alleviate colonic injury mainly by regulating DNA repair, cell cycle regulation, lipid metabolism, and intracellular signaling pathways. These results indicate that PGCNC improves intestinal barrier function and alleviates DSS-induced intestinal injury and related inflammatory damage through multidimensional mechanisms, demonstrating potential protective effects.

[0106] CAZy functional annotation can be used to assess the potential changes in the gut microbiota's ability to degrade, utilize, and transform dietary fiber and polysaccharides after PGCNC intervention. Figure 23 China A~ Figure 23 The results showed that colonic injury led to changes in the function of carbohydrate-active enzymes in the mouse gut microbiota. The relative abundance of the glycoside hydrolase (GH) family decreased in the DSS group, while the relative abundance of glycosyltransferases (GT), carbohydrate binding modules (CBM), carbohydrate esterases (CE), polysaccharide lyases (PL), and accessory enzymes (AA) families did not change significantly. Further classification analysis showed that the abundance of glycosyltransferases such as GT2 and GT4 recovered to the control group level after PGCNC intervention. Figure 23 The results showed that multiple glycosyltransferases exhibited a certain recovery trend after PGCNC intervention, indicating that PGCNC intervention can partially restore the carbohydrate metabolism function of gut microbiota.

[0107] 4. Construction of microbial co-occurrence network and analysis of key species: To assess the ecological interaction patterns within each group of gut microbiota, this experiment constructed a microbial co-occurrence network (MIC). Figure 24 The results showed that the Control group exhibited the most complex network topology. Figure 24 Group A, with its high number of nodes and edge density, and predominantly positively correlated connections, indicates a close mutualistic symbiotic relationship within the healthy microbial community, maintaining a healthy ecological homeostasis. In contrast, the network structure of the DSS group was significantly disrupted. Figure 24 In the middle B group, the reduced network density, decreased mean, and sparsification of connections demonstrated that colonic injury led to a more vulnerable microbiota interaction network. However, the PGCNC intervention group effectively inhibited this worsening trend. Figure 24 In the control group (C), network complexity significantly increased, the number of connections between key species increased, and the overall topology gradually converged towards that of the control group. This indicates that PGCNC not only modulates the abundance of specific bacterial genera, but more importantly, it reconstructs a stable gut microbiota network by reshaping interactions between microbiota, thereby enhancing the resistance and resilience of the microbiota to inflammatory environments.

[0108] To identify the core driving factors maintaining the stability of the gut microbiota network, we conducted in-depth analysis of the collinear graphs of various microbial networks using node degree centrality and Zi-Pi topological properties, and found... Xylanibacter rodentium , Enterocloster clostridioformis , Petralouisia muris , Roseburia zhanii It may be a core species that plays a key role in the process of PGCNC reshaping the gut microbiota. Figure 25 China A~ Figure 25 As can be seen from the data in the middle D, compared with the DSS group, PGCNC intervention promoted Xylanibacter rodentium , Enterocloster clostridioformis , Petralouisia muris , Roseburia zhanii Once the relative abundance of core functional bacterial genera returned to Control levels, they re-emerged as network hubs. This indicates that PGCNC not only modulates the relative abundance of specific bacterial communities, but more importantly, enhances the ability of the micro-ecosystem to resist inflammatory disturbances by rebuilding a robust interaction network centered on key functional bacteria.

[0109] The results showed that PGCNC could improve the intestinal microecological imbalance caused by colon injury in mice and reshape the intestinal flora: mainly by restoring flora diversity and community structure, and promoting Bacteroidota, Bacillota Core phylum and Oscillibacter , Bacteroides The core bacterial species returned to healthy levels. Simultaneously, PGCNC enhanced the carbohydrate metabolism and glycosyltransferase-related functions of the gut microbiota, and reconstructed the synergistic microecological network centered on key functional bacteria. In summary, PGCNC effectively alleviates colonic injury in mice through multiple mechanisms, including regulating microbiota structure, restoring microbiota function, and reshaping the microecological network.

[0110] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing cellulose nanocrystals from Platycodon grandiflorus stems, characterized in that, Includes the following steps: 1) The bellflower stem is crushed and sieved to obtain bellflower stem powder. The bellflower stem powder is mixed with NaOH solution and the precipitate is collected. 2) The precipitate obtained in step 1) is bleached with NaClO2 solution and then rebleached with NaOH solution to obtain the preproduct; 3) The preproduct obtained in step 2) was washed until neutral and freeze-dried to obtain Platycodon grandiflorus stem cellulose; 4) Disperse the cellulose from the bellflower stems obtained in step 3) in H2SO4 solution, acid hydrolyze it, and add deionized water to terminate the reaction to obtain the reactants; 5) Centrifuge and wash repeatedly the reactants obtained in step 4) to obtain a suspension of Platycodon grandiflorum stem cellulose nanocrystals. Dialyze the suspension of Platycodon grandiflorum stem cellulose nanocrystals for 5 days until the pH is stable. The retentate is Platycodon grandiflorum stem cellulose nanocrystals.

2. The preparation method according to claim 1, characterized in that, The particle size of the Platycodon grandiflorus stem powder in step 1) is 30-50 mesh; the mixing ratio of the Platycodon grandiflorus stem powder and NaOH solution in step 1) is 1g:15-25mL, and the concentration of NaOH solution is 5%-15% (w / v); the temperature for mixing the Platycodon grandiflorus stem powder and NaOH solution in step 1) is 80-90℃, and the time is 20-40min.

3. The preparation method according to claim 1, characterized in that, The concentration of the NaClO2 solution mentioned in step 2) is 2%~4% (w / v); the pH of the precipitate bleached with NaClO2 solution in step 2) is 3~4, the bleaching temperature is 75~85℃, the number of bleaching times is 3~5, and the bleaching time for each time is 50~70min.

4. The preparation method according to claim 1, characterized in that, The concentration of the NaOH solution in step 2) is 0.4%~0.6% (w / v); the re-bleaching temperature of the NaOH solution in step 2) is 80~90℃, and the time is 80~100min.

5. The preparation method according to claim 1, characterized in that, The washing process in step 3) uses deionized water, and the freeze-drying process in step 3) is carried out at a temperature of -60 to -40°C for 24 to 48 hours.

6. The preparation method according to claim 1, characterized in that, In step 4), the ratio of Platycodon grandiflorus stem cellulose to H2SO4 solution is 1g: 15~25mL, and the concentration of H2SO4 solution is 62%~66% (w / v); in step 4), the acid hydrolysis temperature is 45~55℃, and the acid hydrolysis time is 40~50min; in step 4), the deionized water is deionized water pre-cooled at 4℃.

7. The preparation method according to claim 1, characterized in that, The centrifugation temperature in step 5) is 4℃, the rotation speed is 10000~14000r / min, and the time is 8~12min; the dialysis in step 5) uses a dialysis bag with a molecular weight cutoff of 8000~14000Da.

8. Platycodon root cellulose nanocrystals prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the Platycodon grandiflorus stem cellulose nanocrystals as described in claim 8 in the preparation of products that alleviate intestinal damage.

10. The application according to claim 9, characterized in that, It regulates gut health by modulating gut microbiota and its metabolism, improving host lipid metabolism disorders, and promoting colonic injury repair through butyrate-IL-10-MUFA-related pathways.

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