A method for promoting intestinal flora against cell fibrosis based on polysaccharides and an experimental method thereof

Abstract

The application discloses a method for improving the anti-cellular fibrosis of intestinal flora based on polysaccharides, and the steps of the method include: preparing Yunnan rhizoma polygonati mutant polysaccharide PKVP-1; and placing the Yunnan rhizoma polygonati mutant polysaccharide PKVP-1 in an intestinal environment to improve the anti-cellular fibrosis of intestinal flora. The steps of an experimental method for improving the anti-cellular fibrosis of intestinal flora based on polysaccharides include: selecting experimental animals and performing random grouping, and then performing drug administration; collecting the intestinal contents and kidney tissues of the experimental animals; taking the kidney tissues of the experimental animals to prepare paraffin sections for trichrome staining, and evaluating the inflammation degree of the kidney tissues according to the inflammatory cell infiltration; performing sequencing; detecting metabolites and obtaining a conclusion according to metabolite analysis. The method for improving the anti-cellular fibrosis of intestinal flora based on polysaccharides provided by the application improves the anti-cellular fibrosis ability of intestinal flora by placing the mutant polysaccharide in the intestinal environment of the experimental animals.

Description

Technical Field

[0001] This invention relates to the field of microbial control technology, and in particular to a method and experimental approach for enhancing the intestinal flora's resistance to cell fibrosis based on polysaccharides. Background Technology

[0002] Currently, anti-fibrosis strategies based on gut-kidney axis regulation mainly include the following categories: 1. Probiotic supplementation: Oral administration of specific probiotics to restore gut microbiota balance. However, probiotic colonization efficiency is low, and the microbiota easily returns to an dysbiotic state after discontinuation. Furthermore, its effect on reversing fibrosis in patients with severe dysbiosis is limited, often only delaying rather than significantly reversing the fibrosis process. 2. Prebiotics: Selectively promote the growth of beneficial bacteria. They can increase SCFAs production and lower intestinal pH, inhibiting pathogenic bacteria. However, their direct improvement effect on renal fibrosis is weak, and individual responses to prebiotics vary greatly, making the effect unpredictable. 3. Synbiotics: Slightly more effective than single-drug formulations, but still have colonization and stability issues, and long-term use may cause discomfort such as bloating and diarrhea.

[0003] 4. Fecal microbiota transplantation (FMT): It can rapidly remodel the microbial community structure. However, FMT faces obstacles such as difficulties in donor selection, infection risks, ethical issues, and unknown long-term safety, making it difficult to promote as a routine anti-fibrotic method.

[0004] Although the above-mentioned strategies for regulating gut microbiota have shown some anti-fibrotic potential in experiments, the following key technical limitations still exist: The results are not ideal: existing methods have limited effectiveness in improving renal fibrosis, with most only showing a slight decrease in biochemical indicators.

[0005] The mechanism is unclear, making it difficult to guide optimization: Most existing technologies are limited to changes in the number of bacteria, that is, they only detect the relative abundance of a few common bacteria, and lack systematic correlation analysis of the overall structural diversity of the bacterial community, metabolite profiles, and key signaling pathways of the gut-kidney axis.

[0006] Lack of effective enhancement strategies: Due to the lack of clarity regarding the above mechanisms, there are currently no clear technical solutions to guide how to further enhance the anti-fibrotic function of the gut microbiota.

[0007] Polygonatum is a traditional Chinese medicinal and edible plant. Its active ingredient, Polygonatum polysaccharide, possesses various pharmacological activities, including antioxidant, immunomodulatory, and hypoglycemic effects. Recent studies have shown that the polysaccharide exhibits multiple biological activities, such as immunomodulation, antioxidant activity, and regulation of gut microbiota.

[0008] Therefore, there is an urgent need for a method for polysaccharides to resist intestinal cell fibrosis and a verification experimental method. Summary of the Invention

[0009] In view of the current shortcomings, the present invention provides a method and experimental method for enhancing the intestinal flora's resistance to cell fibrosis based on polysaccharides, and verifies the effect of Polygonatum polysaccharides in restoring the inhibitory effect of renal fibrosis on growth and organs, protecting organs, and restoring the diversity of intestinal flora.

[0010] To achieve the above objectives, the embodiments of the present invention adopt the following technical solutions: A method for enhancing the intestinal flora's resistance to cell fibrosis based on polysaccharides, the method comprising the steps of: preparing a polysaccharide PKVP-1 from a variant of Polygonatum yunnanense; and placing the polysaccharide PKVP-1 from the variant of Polygonatum yunnanense in the intestinal environment to enhance the intestinal flora's resistance to cell fibrosis.

[0011] According to one aspect of the present invention, the identification certificate number of the Polygonatum yunnanense variant polysaccharide PKVP-1 is 2024002.

[0012] An experimental method for enhancing the anti-cellular fibrosis effect of gut microbiota based on polysaccharides, the steps of which include: Experimental animals were selected and randomly grouped for drug administration; Collect intestinal contents and kidney tissue from laboratory animals; Kidney tissue from experimental animals was prepared into paraffin sections and stained with trichrome staining. The degree of inflammation in the kidney tissue was assessed based on the infiltration of inflammatory cells. Perform sequencing; The metabolites are detected and conclusions are drawn based on the metabolite analysis.

[0013] According to one aspect of the present invention, the random grouping includes: a renal fibrosis model group, a normal control group, a low-dose PKVP-1 group, a medium-dose PKVP-1 group, a high-dose PKVP-1 group, and a fosinopril group.

[0014] According to one aspect of the present invention, the renal fibrosis model group, i.e., the RF group, uses adenine and adds physiological saline; the normal control group, i.e., the NC group, uses an equal volume of physiological saline.

[0015] According to one aspect of the present invention, the low-dose PKVP-1 group, namely the L-PKVP-1 group, uses adenine and adds 200 mg / kg PKVP-1; the medium-dose PKVP-1 group, namely the M-PKVP-1 group, uses adenine and adds 400 mg / kg PKVP-1; the high-dose PKVP-1 group, namely the H-PKVP-1 group, uses adenine and adds 800 mg / kg PKVP-1; and the fosinopril group, namely the FOS group, uses adenine and adds 10 mg / kg fosinopril.

[0016] According to one aspect of the invention, the administration is performed by gavage.

[0017] According to one aspect of the invention, the collection of intestinal contents and kidney tissue from experimental animals requires continuous gavage and fasting before the collection of contents and kidney tissue.

[0018] According to one aspect of the invention, the tricolor staining includes hematoxylin-eosin staining and Masson staining.

[0019] According to one aspect of the invention, the sequencing is performed by extracting genomic DNA from fecal samples of laboratory animals.

[0020] Advantages of this invention: Through the above technical solutions, (1) the polysaccharide PKVP-1 of the variant of Polygonatum yunnanense can reduce collagen deposition, restore the growth inhibition induced by renal fibrosis in rats, restore the inhibitory effect of renal fibrosis on organs, restore organ index, and play a significant protective role on organs, especially the kidneys; (2) the polysaccharide PKVP-1 of the variant of Polygonatum yunnanense can reduce the degree of inflammation in kidney tissue and protect the kidneys; (3) positively regulate the structural diversity of intestinal flora; (4) improve the intestinal metabolite spectrum, upregulate the content of beneficial metabolites such as lactucin, and reduce the content of harmful metabolites such as streptomycin 6-phosphate; (5) significantly inhibit the fibrotic phenotype of the intestine, reduce the protein expression of fibrosis-related genes TGF-β1 and α-SMA, and significantly promote the protein expression of E-calcified protein, directly inhibiting the renal tubular epithelial-mesenchymal transition at the cellular level. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying 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.

[0022] Figure 1 This is a schematic diagram of the structure of the polysaccharide PKVP-1 of Polygonatum yunnanense variant described in this invention; Figure 2 This is a graph showing the changes in the body weight of rats during the experiments of this invention; Figure 3 This is an organ index diagram of the rat spleen during the experimental process of this invention; Figure 4 This is an organ index diagram of the rat heart during the experiments of this invention; Figure 5 This is an organ index diagram of rat kidneys during the experiments of this invention; Figure 6 This is an organ index diagram of the rat liver during the experiments of this invention; Figure 7 This is an organ index diagram of the rat spleen during the experimental process of this invention; Figure 8 This is an organ index diagram of the rat lungs and thymus during the experiments of this invention; Figure 9 These are representative images of rat kidney tissue stained with hematoxylin and eosin (HE) during the experiments of this invention; Figure 10 These are representative images of rat kidney tissue stained with Masson staining during the experiments of this invention; Figure 11 This is a diagram showing the degree of renal interstitial fibrosis in rats during the experiments of this invention. Figure 12 This is a bar chart showing the fold change (FC) of metabolites between the normal control group and the renal fibrosis model group during the experimental process of this invention. Figure 13 This is a bar chart showing the fold change (FC) of metabolites between the low-dose PKVP-1 group and the renal fibrosis model group during the experiments of this invention. Figure 14 This is a bar chart showing the fold change (FC) of metabolites in the PKVP-1 medium-dose group compared to the renal fibrosis model group during the experiments of this invention. Figure 15 This is a bar chart showing the fold change (FC) of metabolites between the high-dose PKVP-1 group and the renal fibrosis model group during the experiments of this invention. Figure 16 This is a bar chart showing the fold change (FC) of metabolites between the fosinopril group and the renal fibrosis model group during the experiments of this invention. Figure 17 This is a vitality plot of the TGF-β1 model group during the experiments of this invention; Figure 18 This is a bioactivity diagram of the TGF-β1+PKVP-1 group during the experiments of this invention; Figure 19 This is a vitality graph of the HMBA group during the experiment of this invention; Figure 20 This is a bioactivity graph of the TGF-β1+HMBA group during the experiments of this invention; Figure 21 This is a cell morphology diagram of the TGF-β1+PKVP-1 group during the experiments of this invention; Figure 22 This is a cell morphology diagram of the TGF-β1+HMBA group during the experiments of this invention; Figure 23 These are the protein blot bands of the PKVP-1 dosage group during the experiments of this invention; Figure 24This is a protein quantification diagram of α-SMA in the PKVP-1 dosage group during the experiments of this invention; Figure 25 This is a protein quantification diagram of E-cadherin in the PKVP-1 dosage group during the experiment of this invention; Figure 26 This is a protein quantification diagram of TGF-β1 in the PKVP-1 dosage group during the experiments of this invention; Figure 27 The protein blot bands of the HMBA dose group during the experiment of this invention; Figure 28 This is a protein quantification diagram of α-SMA in the HMBA dosage group during the experiment of this invention; Figure 29 This is a protein quantification diagram of E-cadherin in the HMBA dosage group during the experiment of this invention; Figure 30 This is a protein quantification diagram of TGF-β1 in the HMBA dosage group during the experiment of this invention; Where ns indicates no significant difference; . Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] A method for enhancing the intestinal flora's resistance to cell fibrosis based on polysaccharides, the method comprising the steps of: preparing a polysaccharide PKVP-1 from a variant of Polygonatum yunnanense; and placing the polysaccharide PKVP-1 from the variant of Polygonatum yunnanense in the intestinal environment to enhance the intestinal flora's resistance to cell fibrosis.

[0025] In practical applications, the structure of the polysaccharide PKVP-1 from the Yunnan Polygonatum variety is as follows: Figure 1 As shown.

[0026] In practical applications, the polysaccharide PKVP-1 of Polygonatum yunnanense variant is administered via gavage, allowing it to enter the intestinal environment of experimental animals.

[0027] In practical applications, all experimental animals comply with the regulations of the Experimental Animal Ethics Committee of Hunan University of Traditional Chinese Medicine, approval number: HNUCM 21-2410-02.

[0028] An experimental method for enhancing the anti-cellular fibrosis effect of gut microbiota based on polysaccharides, the steps of which include: Experimental animals were selected and randomly grouped for drug administration; Collect intestinal contents and kidney tissue from laboratory animals; Kidney tissue from experimental animals was prepared into paraffin sections and stained with trichrome staining. The degree of inflammation in the kidney tissue was assessed based on the infiltration of inflammatory cells. Perform sequencing; The metabolites are detected and conclusions are drawn based on the metabolite analysis.

[0029] In practical applications, SPF-grade male SD rats with a body weight of 180-220g were selected from Hunan Slack Jingda Experimental Animal Co., Ltd.

[0030] In practical applications, rats are housed in a constant temperature and humidity environment with a 12-hour light-dark cycle, an ambient temperature of 22±2℃, a relative humidity of 60%-70%, and free access to food and water.

[0031] In practical applications, 36 SPF-grade male SD rats were selected and divided into 6 groups for experiments.

[0032] In practice, all drugs were administered by gavage, with adenine administered by gavage for 42 consecutive days.

[0033] In practical applications, the first 21 days after drug administration are the modeling stage, and the next 21 days are the drug intervention stage.

[0034] In practical applications, rats are fasted for 24 hours after the last administration.

[0035] In practical applications, after fasting for 24 hours, rats were anesthetized by intraperitoneal injection of sodium pentobarbital, and their intestinal contents and kidney tissue were collected.

[0036] In practical applications, rat kidney tissue is used to prepare paraffin sections.

[0037] In practical applications, the prepared paraffin sections are stained with hematoxylin-eosin (HE) and Masson trichrome staining, respectively.

[0038] In practical applications, the degree of renal fibrosis can be quantitatively evaluated by observing the extent of collagen fiber staining.

[0039] In practical applications, TransStart Fastpfu DNA polymerase is used for low-cycle PCR amplification with barcodes, and the amplification products are purified using AgencourtAMPure XP magnetic beads.

[0040] In practical applications, a library is constructed by connecting Y-type adapters, enriched by PCR, and then denatured with sodium hydroxide to obtain a single-stranded library.

[0041] In practical applications, bridge PCR is performed on the Illumina sequencing platform to generate DNA clusters, and paired-end sequencing is completed using synthetic sequencing technology.

[0042] In practical applications, after the raw sequencing data is filtered for quality, the UPARSE algorithm is used to cluster and obtain operational taxonomic units (OTUs), and species classification annotation is performed based on the 16S rRNA reference database.

[0043] In practical applications, microbial diversity analysis is performed on the QIIME2 platform.

[0044] In practical applications, genomic DNA is extracted from rat fecal samples by vortexing with pre-cooled methanol-acetonitrile-water solution.

[0045] In practical applications, the extracted genomic DNA needs to be treated with ultrasound, precipitated at low temperature, centrifuged to collect the supernatant, dried and concentrated with nitrogen, and then reconstituted with acetonitrile-water solution.

[0046] In practical applications, ultra-high performance liquid chromatography and high-resolution mass spectrometry are used for combined detection.

[0047] In practical applications, the Thermo Fisher Vanquish UPLC is used for ultra-high performance liquid chromatography.

[0048] In practical applications, the high-resolution mass spectrometer used is the Thermo Fisher Q Exactive HFX.

[0049] In practical applications, the chromatographic column is an HSS T3 column, and gradient elution is performed using an aqueous phase containing 0.1% formic acid and acetonitrile as the mobile phase.

[0050] In practical applications, mass spectrometry uses an electrospray ionization source and performs full scan-data-dependent secondary mass spectrometry (Full MS-ddMS²) acquisition in both positive and negative ion modes.

[0051] In practical applications, the raw data obtained from the processing is subjected to peak extraction, peak alignment, and normalization using Progenesis QI software.

[0052] In practical applications, HK-2 cells are used for toxicity and detoxification tests.

[0053] In practical applications, HK-2 cells are human renal tubular epithelial cells, cell number: AC112.

[0054] In practical applications, the polysaccharide and HMBA toxicity test group included a blank control group, a PKVP-1 concentration gradient group, and an HMBA concentration gradient group.

[0055] In practical applications, the polysaccharide and HMBA combined modeling agent toxicity test group was set up with a blank control group, a TGF-β1 model group, a 10 ng / mL TGF-β1 and different concentrations of PKVP-1 group, and a 10 ng / mL TGF-β1 and different concentrations of HMBA group.

[0056] In practical applications, HMBA, or N,N'-diacetyl-1,6-diaminohexane, is selected from Yuanye Biotechnology Co., Ltd., batch number: JY240173.

[0057] In practical applications, the CCK8 kit is used for cell counting and viability detection.

[0058] In practical applications, CCK8, or Cell Counting Kit 8, is selected from Abbkine, product number: ATYF16131.

[0059] In practical applications, microscopes are used to observe cell morphology.

[0060] In practical applications, the BCA method is used to determine protein concentration in Western blotting experiments.

[0061] In practical applications, TGF-β1, α-SMA, E-Cadherin and GAPDH were used for primary antibody incubation.

[0062] In practical applications, TGF-β1, or transforming growth factor-β1 antibody, is selected from Selleck, Inc., product number: F16240.

[0063] In practical applications, α-SMA, or α-smooth muscle actin antibody, is selected from Selleck, Inc., product number: F001701.

[0064] In practical applications, E-Cadherin, an epithelial cadherin antibody, was selected from Selleck, Inc., product number F351701.

[0065] In practical applications, GAPDH, or internal reference, is selected from Bioworld, product number: AB56131.

[0066] In practical applications, all experimental data are expressed as mean ± standard deviation (x ± s).

[0067] In practical applications, all data are obtained through analysis after three independent repeated experiments.

[0068] Example 1

[0069] Effects of PKVP-1 on body weight and organ index in rats with renal fibrosis Thirty-six rats were randomly divided into six groups of six each: renal fibrosis model group, normal control group, low-dose PKVP-1 group, medium-dose PKVP-1 group, high-dose PKVP-1 group, and fosinopril group.

[0070] The renal fibrosis model group (RF) was administered adenine by gavage with added saline; the normal control group (NC) was administered an equal volume of saline by gavage; the low-dose PKVP-1 group (L-PKVP-1) was administered adenine by gavage with added 200 mg / kg PKVP-1; the medium-dose PKVP-1 group (M-PKVP-1) was administered adenine by gavage with added 400 mg / kg PKVP-1; the high-dose PKVP-1 group (H-PKVP-1) was administered adenine by gavage with added 800 mg / kg PKVP-1; and fosinopril (FOS) was administered adenine by gavage with added 10 mg / kg fosinopril. All drugs were administered by gavage. All six groups of mice received adenine by gavage for 42 consecutive days, with the first 21 days constituting the modeling phase and the last 21 days constituting the drug intervention phase. Rats were fasted for 24 hours after the last administration. Rats were anesthetized via intraperitoneal injection of sodium pentobarbital. Intestinal contents and kidney tissue were collected for subsequent experiments. Body weight changes in the rats were recorded during the experiment, and organ indices were calculated. Body weight changes in the rats during the experiment are shown below. Figure 2 As shown in the figure, weight monitoring results indicate that PKVP-1 can effectively restore renal fibrosis-induced growth inhibition in rats. Organ indices of the spleen, heart, kidneys, liver, spleen, lungs, and thymus in rats are shown in the figure. Figure 3-8 As shown, this indicates that PKVP-1 exhibits a significant protective effect on the liver, a major metabolic organ in the body.

[0071] Example 2

[0072] Effects of PKVP-1 on the degree of renal tissue inflammation in rats with renal fibrosis Kidney tissues from six groups of rats that underwent 42 days of drug administration were prepared into paraffin sections and stained with hematoxylin-eosin (HE) and Masson's trichrome staining, respectively. The degree of kidney inflammation was assessed based on the extent of inflammatory cell infiltration; the degree of kidney fibrosis was quantitatively evaluated by observing the extent of collagen fiber staining. Representative images of hematoxylin-eosin (HE) staining from the six groups of rats are shown below. Figure 9As shown in the image. HE staining results showed that, compared with the normal control group, the kidney tissue of rats in the renal fibrosis model group exhibited significant pathological damage, manifested as incomplete glomerular structure, atrophy, sclerosis, a significant reduction in the number of glomeruli, inflammatory cell infiltration and vacuolar degeneration in the renal tubules, coexistence of tubular dilation and atrophy, brownish crystal deposition in the renal tubules and interstitium, extensive interstitial fibrosis, Bowman's capsule adhesion, basement membrane thickening, mesangial cell proliferation, ultimately leading to renal fibrosis. Compared with the model group, the pathological damage to the kidneys of rats in all four drug treatment groups was improved to varying degrees. Representative images of Masson staining of kidney tissue from the six groups of rats are shown in the image. Figure 10 As shown in the figure. Masson staining is a classic method for evaluating the degree of renal fibrosis, and it can simultaneously reflect the level of glomerular sclerosis and renal interstitial fibrosis. The results showed that in the normal control group, the cell nuclei of the rat renal tissue were blue-purple, the muscle fibers were red, and only a small amount of collagen fibers in the mesangial area and basement membrane area were blue, with no obvious collagen fiber aggregation; while the model group rats had extremely significant pathological fibrosis in their renal tissue, with a large amount of collagen fiber aggregation in the renal interstitium, and the Masson staining score was significantly increased; compared with the model group, the pathological degree of renal interstitial fibrosis in the four drug treatment groups was significantly reduced, and the area of ​​fibrotic region was significantly reduced. Both HE staining and Masson staining results showed that the improvement effect of the medium-dose PKVP-1 group was better than that of the low-dose group, while the intervention effect of the high-dose group was weaker than that of the low- and medium-dose groups. The degree of renal interstitial fibrosis in the six groups of rats is shown in the figure. Figure 11 As shown. Figure 3-8 Organ indices of the spleen, heart, kidneys, liver, spleen, lungs, and thymus in rats and Figure 11 The images of the degree of renal interstitial fibrosis in the six groups of rats showed that the renal fibrosis modeling process could inhibit the development of most organs in rats. PKVP-1 not only has a therapeutic effect on renal fibrosis, but also shows a significant protective effect on the liver, the body's main metabolic organ.

[0073] Example 3

[0074] Effects of PKVP-1 on the composition of gut microbiota in rats with renal fibrosis Genomic DNA was extracted from rat fecal samples from each group: Samples were removed from the refrigerator, and 0.2g of sample was quickly added to centrifuge tubes containing extraction lysis buffer and ground at a frequency of 60Hz. For pretreated samples, nucleic acids were extracted using the MagBeads FastDNA Kit for Soil (116564384) (MP Biomedicals, CA, USA). The extracted DNA was subjected to 0.8% agarose gel electrophoresis to determine molecular size, assess DNA purity and integrity, and quantify the DNA using Nanodrop. Low-cycle PCR amplification with barcodes was performed using TransStart Fastpfu DNA polymerase. The PCR amplification system included: 0.25 μL ABclonal DNA polymerase; 5 μL reaction buffer; 5 μL 5* high GC content enhancer; 2 μL dNTPs (10 mM); 2 μL template DNA; 1 μL forward primer (10 uM); 1 μL reverse primer (10 uM); and 8.75 μL water. The PCR reaction program was as follows: pre-denaturation at 98℃ for 5 minutes, followed by amplification cycling; 26 cycles of denaturation at 98℃ for 30 seconds, annealing at 52℃ for 30 seconds, and extension at 72℃ for 45 seconds; a final extension at 72℃ for 5 minutes; and storage at 12℃. The amplified products were detected by 2% agarose gel electrophoresis and purified using AgencourtAMPure XP magnetic beads.

[0075] A library was constructed by ligating Y-connectors; self-ligated fragments of the adapters were removed using magnetic beads; after enriching the library template by PCR amplification, single-stranded libraries were obtained by denaturation with sodium hydroxide; DNA clusters were generated by bridging PCR on an Illumina sequencing platform, and paired-end sequencing was performed using synthetic sequencing technology. One end of the DNA fragment was complementary to the primer bases and immobilized on the chip; the other end was randomly complementary to the primer and immobilized to form a bridge; PCR amplification was performed to generate DNA clusters; the DNA amplicon was linearized into single strands; a modified DNA polymerase and four fluorescently labeled dNTPs were added, synthesizing only one base per cycle; the surface of the reaction plate was scanned with a laser to read the types of nucleotides polymerized in the first round of reaction for each template sequence; the fluorescent group and terminator were chemically cleaved to restore the 3' end adhesion, and the second nucleotide was polymerized; the fluorescence signal results collected in each round were counted to obtain the sequence of the template DNA fragment. Gut microbiota sequencing results showed that 5,971,958 raw sequences were obtained after CASAVA base identification, and 5,755,514 valid sequences were obtained after quality control and filtering. Representative sequences obtained from the assembly were length-statistically analyzed, and high-quality sequences were selected for subsequent analysis. Based on 97% sequence similarity, high-quality sequences were clustered into operational taxonomic units (OTUs), and amplicon variants (ASVs) were obtained by clustering based on DNA sequence differences, resulting in the identification of 2,356 characteristic sequences (OTUs / ASVs).

[0076] The common and unique distribution of characteristic sequences in fecal samples from six groups of rats was analyzed to determine the composition of common and unique intestinal flora in each group. The species richness and evenness of each group were analyzed. The composition of the top 20 species with the highest relative abundance of intestinal flora in each group of rats was analyzed sequentially according to phylum, class, order, family, genus, and species. This analysis revealed changes in the intestinal flora composition of the six groups of rats treated with different drugs, thus determining the effect of PKVP-1 on the intestinal flora composition of rats with renal fibrosis.

[0077] Example 4

[0078] Effects of PKVP-1 on intestinal flora metabolites in rats with renal fibrosis Based on the identification and differential analysis of metabolites, a total of 4192 metabolites were identified in both positive and negative ion modes, of which 3717 were retained after quality control filtration. All metabolites were classified into 95 chemical categories based on their correlations and chemical classification information. Differential compounds between different groups were screened and similarity enrichment was performed. The fold change (FC) histogram of differential metabolites obtained from different groups after fold analysis is shown below. Figure 12-16As shown in the bar chart of differential metabolite fold change (FC), compared with the normal control group, the model group showed significant upregulation of harmful metabolites such as anthracoline G and 2-hydroxy-N-methylpseudocarnitine, as well as significant upregulation of beneficial metabolites such as dihydroxycholic acid-chenodeoxychoyl-N-acetyl-aspartic-valine and lactucin. Compared with the model group, the low-dose PKVP-1 group significantly upregulated beneficial metabolites such as leucyl-asparagine and 3-hydroxyphenyl sulfate, and significantly downregulated harmful metabolites such as streptomycin 6-phosphate and Gnf-PI-4549. The medium-dose PKVP-1 group showed a more significant regulatory effect on differential metabolites, while the high-dose PKVP-1 group and the fosinopril group showed reduced regulatory precision and limited effect. Low and medium doses of PKVP-1 can effectively reverse the above-mentioned metabolic abnormalities, significantly upregulate beneficial metabolites such as leucyl-asparagine (an amino acid derivative) and 3-hydroxyphenyl sulfate (a short-chain fatty acid precursor), and downregulate toxic metabolites such as streptomycin 6-phosphate, with the medium dose showing the most significant regulatory effect.

[0079] Example 5

[0080] Effects of PKVP-1 and its metabolite HMBA on HK-2 cells HMBA was randomly selected from differentially expressed gut microbiota metabolites that decreased in the renal fibrosis model group but increased in the drug-treated group for cellular experiments. The cells were divided into two groups: a polysaccharide and HMBA toxicity testing group and a polysaccharide and HMBA combined modeling agent toxicity testing group.

[0081] The polysaccharide and HMBA toxicity assay included a blank control group, PKVP-1 concentration gradient groups, and HMBA concentration gradient groups. The blank control group was cultured in complete culture medium. The PKVP-1 concentration gradient groups were cultured in complete culture medium at concentrations of 25, 50, 75, 100, 150, 200, 300, and 400 mg / mL. The HMBA concentration gradient groups were cultured in complete culture medium at concentrations of 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, and 10 mmol / L.

[0082] The toxicity testing of the polysaccharide and HMBA combined modeling agent included a blank control group, a TGF-β1 model group, TGF-β1 + different concentrations of PKVP-1 groups, and TGF-β1 + different concentrations of HMBA groups. The blank control group was cultured in complete culture medium. The TGF-β1 model group was cultured with 10 ng / mL TGF-β1 in complete culture medium. The TGF-β1 + different concentrations of PKVP-1 groups were cultured with 10 ng / mL TGF-β1 and PKVP-1 at concentrations of 25, 50, 75, 100, 150, 200, 300, and 400 mg / mL in complete culture medium. The TGF-β1 + different concentrations of HMBA groups were cultured with 10 ng / mL TGF-β1 and HMBA at concentrations of 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, and 10 mmol / L in complete culture medium.

[0083] Cell suspensions from each group (100 μL / well) were seeded into 96-well plates and pre-cultured in a humidified incubator at 37°C and 5% CO2. After adding CCK-8 reagent, the cells were cultured for another 48 hours. After 48 hours, cell morphology was observed and recorded under an inverted microscope. Western blotting experiments were performed, with the cells divided into: a blank control group, a TGF-β1 model group, a TGF-β1+PKVP-1 group, and a TGF-β1+HMBA group (10 ng / mL TGF-β1 + 0.5, 1.0, and 2.0 mmol / L HMBA). The TGF-β1 model group received 10 ng / mL TGF-β1. The TGF-β1+PKVP-1 group was further divided into low-dose, medium-dose, and high-dose TGF-β1+PKVP-1 groups. The TGF-β1+PKVP-1 low-dose group was supplemented with 10 ng / mL TGF-β1 and 50 mg / mL PKVP-1; the TGF-β1+PKVP-1 medium-dose group was supplemented with 10 ng / mL TGF-β1 and 100 mg / mL PKVP-1; and the TGF-β1+PKVP-1 high-dose group was supplemented with 10 ng / mL TGF-β1 and 200 mg / mL PKVP-1. The TGF-β1+HMBA group was further divided into low-dose, medium-dose, and high-dose groups. The TGF-β1+HMBA low-dose group was supplemented with 10 ng / mL TGF-β1 and 0.5 mmol / L HMBA; the TGF-β1+HMBA medium-dose group was supplemented with 10 ng / mL TGF-β1 and 1.0 mmol / L HMBA; and the TGF-β1+HMBA high-dose group was supplemented with 10 ng / mL TGF-β1 and 2.0 mmol / L HMBA. CCK8 results showed that HK-2 cell viability was not significantly abnormal after treatment with PKVP-1 and HMBA alone or in combination with TGF-β1 modeling agent. Viability graphs are shown below. Figure 17-20 As shown. Cell morphology was observed; the cell morphology diagram of the TGF-β1+PKVP-1 group is shown below. Figure 21 As shown, the cell morphology diagram of the TGF-β1+HMBA group is as follows. Figure 22 As shown in the figure. Cell morphology observation results showed that TGF-β1 could induce HK-2 cells to transform into a spindle-shaped fibrotic phenotype, while PKVP-1 and HMBA treatment could significantly improve this morphological change.

[0084] After cell drug treatment, total cell protein was extracted for Western blotting experiments. The extraction steps included: washing adherent cells after discarding the culture medium; preparing fresh lysis buffer using RIPA strong lysis buffer containing protease inhibitors; lysing on ice with gentle shaking; scraping cell debris and centrifuging at 12000g for 15 min at 4°C; and using the supernatant for BCA quantification to determine protein concentration. BCA quantification was performed using the Sever BCA kit. Standards were diluted, working solutions were prepared, and samples were added and incubated at 37°C in the dark for 30 min. OD values ​​were measured at 562 nm using a microplate reader. After SDS-PAGE gel electrophoresis, transfer, and blocking, primary antibodies against TGF-β1, α-SMA, E-Cadherin, and GAPDH were added and incubated. After washing, the corresponding secondary antibodies were added and incubated. ECL chemiluminescence imaging was used, and the gray values ​​of the protein bands were analyzed using ImageJ software. The protein blot bands of the PKVP-1 dosage group are shown below. Figure 23 As shown in the figure, the protein quantification diagrams of α-SMA, E-cadherin, and TGF-β1 in the PKVP-1 dosage group are as follows. Figure 24-26 As shown. The protein blot bands of the HMBA dose group are as follows. Figure 27 As shown in the figure. The protein quantification graphs of α-SMA, E-cadherin, and TGF-β1 in the HMBA dose group are as follows. Figures 28-30 As shown in the results of Western blot experiments, both PKVP-1 and HMBA can inhibit the protein expression of TGF-β1 and α-SMA in a dose-dependent manner, while significantly promoting the protein expression of E-calcified protein. This indicates that PKVP-1 can not only regulate the gut microbiota and metabolome in vivo, but also directly inhibit the renal tubular epithelial-mesenchymal transition at the cellular level. Its metabolite HMBA may be a key molecule mediating the gut-kidney axis interaction.

[0085] Advantages of this invention: Through the above technical solutions, (1) the polysaccharide PKVP-1 of the variant of Polygonatum yunnanense can reduce collagen deposition, restore the growth inhibition induced by renal fibrosis in rats, restore the inhibitory effect of renal fibrosis on organs, restore organ index, and play a significant protective role on organs, especially the kidneys; (2) the polysaccharide PKVP-1 of the variant of Polygonatum yunnanense can reduce the degree of inflammation in kidney tissue and protect the kidneys; (3) positively regulate the structural diversity of intestinal flora; (4) improve the intestinal metabolite spectrum, upregulate the content of beneficial metabolites such as lactucin, and reduce the content of harmful metabolites such as streptomycin 6-phosphate; (5) significantly inhibit the fibrotic phenotype of the intestine, reduce the protein expression of fibrosis-related genes TGF-β1 and α-SMA, and significantly promote the protein expression of E-calcified protein, directly inhibiting the renal tubular epithelial-mesenchymal transition at the cellular level.

[0086] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for enhancing the gut microbiota's resistance to cellular fibrosis based on polysaccharides, characterized in that, The method for enhancing the intestinal flora's resistance to cell fibrosis based on polysaccharides includes the following steps: preparing a polysaccharide PKVP-1 from a variant of Polygonatum yunnanense; and placing the polysaccharide PKVP-1 from the variant of Polygonatum yunnanense in the intestinal environment to enhance the intestinal flora's resistance to cell fibrosis.

2. The method for enhancing intestinal flora's resistance to cellular fibrosis based on polysaccharides according to claim 1, characterized in that, The identification certificate number for the polysaccharide PKVP-1 of the *Polygonatum yunnanense* variant is 2024002.

3. An experimental method for enhancing the gut microbiota's resistance to cellular fibrosis based on polysaccharides, characterized in that, The experiment based on polysaccharides to enhance the gut microbiota's resistance to cell fibrosis includes the following steps: Experimental animals were selected and randomly grouped for drug administration; Collect intestinal contents and kidney tissue from laboratory animals; Kidney tissue from experimental animals was prepared into paraffin sections and stained with trichrome staining. The degree of inflammation in the kidney tissue was assessed based on the infiltration of inflammatory cells. Perform sequencing; The metabolites are detected and conclusions are drawn based on the metabolite analysis.

4. The experimental method for enhancing intestinal flora's resistance to cell fibrosis based on polysaccharides according to claim 3, characterized in that, The randomized groups included: renal fibrosis model group, normal control group, low-dose PKVP-1 group, medium-dose PKVP-1 group, high-dose PKVP-1 group, and fosinopril group.

5. The experimental method for enhancing intestinal flora's resistance to cell fibrosis based on polysaccharides according to claim 4, characterized in that, The renal fibrosis model group, i.e., the RF group, used adenine with added physiological saline; the normal control group, i.e., the NC group, used an equal volume of physiological saline.

6. The experimental method for enhancing intestinal flora's resistance to cell fibrosis based on polysaccharides according to claim 4, characterized in that, The low-dose PKVP-1 group, namely the L-PKVP-1 group, uses adenine with 200 mg / kg PKVP-1 added; the medium-dose PKVP-1 group, namely the M-PKVP-1 group, uses adenine with 400 mg / kg PKVP-1 added; the high-dose PKVP-1 group, namely the H-PKVP-1 group, uses adenine with 800 mg / kg PKVP-1 added; and the fosinopril group, namely the FOS group, uses adenine with 10 mg / kg fosinopril added.

7. The experimental method for enhancing intestinal flora's resistance to cell fibrosis based on polysaccharides according to claim 3, characterized in that, The medication was administered via gavage.

8. The experimental method for enhancing intestinal flora's resistance to cell fibrosis based on polysaccharides according to claim 3, characterized in that, The collection of intestinal contents and kidney tissue from experimental animals requires continuous gavage, and the animals must be fasted before the collection of contents and kidney tissue.

9. The experimental method for enhancing intestinal flora's resistance to cell fibrosis based on polysaccharides according to claim 3, characterized in that, The trichrome staining includes hematoxylin-eosin staining and Masson staining.

10. The experimental method for enhancing the intestinal flora's resistance to cell fibrosis based on polysaccharides according to claim 3, characterized in that, The sequencing was performed by extracting genomic DNA from fecal samples of experimental animals.

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