Application of flaccid knotweed herb alcohol extract in preparation of medicine for treating enteritis

Abstract

The invention discloses application of a flaccid knotweed herb alcohol extract in preparation of a medicine for treating enteritis, and relates to the field of traditional Chinese medicine, and a preparation method of the flaccid knotweed herb alcohol extract comprises the following steps: adding powdery flaccid knotweed herb into ethyl alcohol for soaking, heating and refluxing, centrifugally collecting supernate, concentrating the supernate, removing ethyl alcohol through rotary evaporation, and freeze-drying to obtain the flaccid knotweed herb alcohol extract. The invention shows that the red-knees herb alcohol extract is a potential natural enteritis treatment medicine, regulates and controls an integration mechanism of oxidative stress, inflammation and ferroptosis through a KEAP1-Nrf2 axis, and provides a theoretical basis for developing a novel multi-target therapy.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine, and in particular to application of an alcohol extract of Polygonum hydropiper in preparing a medicine for treating enteritis. Background Art

[0002] Enteritis is gastroenteritis, enteritis and colitis caused by bacteria, viruses, fungi and parasites. Clinical manifestations include nausea, vomiting, abdominal pain, diarrhea, loose watery stools or mucus, pus and blood in the stools. Some patients may have fever and a feeling of tenesmus, so it is also called infectious diarrhea. Enteritis is divided into acute and chronic types according to the length of the disease.

[0003] Polygonum hydropiper L. is the dried whole herb of Polygonum hydropiper L., a plant of the Polygonaceae family. It is also known as willow grass, Polygonum hydropiper, bat grass, etc. It is pungent and warm in nature. It has the effects of dispelling wind and dampness, dispersing blood stasis and relieving pain, detoxifying and reducing swelling, killing insects and relieving itching. Polygonum hydropiper L. is widely distributed in my country and has rich plant resources. Its main chemical components include flavonoids, volatile oils, tannins, triterpenes, glycosides, etc. At present, Polygonum hydropiper L. extracts are mainly used in botanical pesticides. In the application of botanical pesticides, Polygonum hydropiper L. has antifeedant activity against aphids, armyworms, diamondback moths, cabbage worms, rice planthoppers and other pests. At present, there are no reports on its application in the preparation of enteritis drugs. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide an application of an alcohol extract of Polygonum hydropiper in preparing a drug for treating enteritis.

[0005] The technical solution of the present invention is as follows:

[0006] Application of an alcohol extract of Polygonum hydropiper in preparing a drug for treating enteritis, wherein the preparation method of the alcohol extract of Polygonum hydropiper is as follows:

[0007] The powdered Polygonum hydropiper is soaked in ethanol, heated to reflux, and the supernatant is collected by centrifugation. The supernatant is concentrated, and the ethanol is removed by rotary evaporation, and freeze-dried to obtain the Polygonum hydropiper ethanol extract.

[0008] Furthermore, the weight ratio of the powder material to the volume of ethanol is 1:6-8. The heating reflux is performed for 4-6 hours, and the number of reflux extractions is 2-4 times. The centrifugal speed is 5000-7000 rpm.

[0009] Furthermore, the drug has the alcohol extract of Polygonum hydropiper as a pharmaceutically active ingredient and contains a pharmaceutically acceptable carrier, wherein the alcohol extract of Polygonum hydropiper accounts for 0.01-99.99% by weight in the preparation, and the rest is a pharmaceutically acceptable carrier;

[0010] Furthermore, the ethanol extract of Polygonum hydropiper can inhibit the pro-inflammatory factors TNF-α and IL-6 and activate the Nrf2 / ARE pathway, enhance GSH synthesis, and scavenge reactive oxygen species.

[0011] Furthermore, the Polygonum hydropiper ethanol extract can upregulate tight junction proteins Zo-1, Occludin and mucin Muc2.

[0012] Furthermore, the ethanol extract of Polygonum hydropiper can reduce Fe 2+ level, regulate the COX2 / GPX4 balance, and block the ferroptosis process.

[0013] The beneficial effects of the present invention are as follows: the alcohol extract of Polygonum hydropiper can effectively improve DSS-induced intestinal barrier damage and colitis in mice, reduce DSS-induced colonic inflammatory response and oxidative stress in mice, and improve DSS-induced colonic ferroptosis in mice, indicating that the alcohol extract of Polygonum hydropiper can improve inflammatory bowel disease to a certain extent. Therefore, the present invention provides a new application of the alcohol extract of Polygonum hydropiper in medicines for treating inflammatory bowel disease, laying a foundation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the total ion current diagram of Polygonum hydropiper, where A is the positive ion mode and B is the negative ion mode.

[0015] Figure 2 Figure 1 shows the improvement of intestinal barrier damage and colitis induced by DSS in mice by LAL. A: Food intake; B: Body weight change (%); C: Disease activity index; D: Rectal bleeding score; E: Stool consistency score; F: Colon morphology and length; G: HE staining of colon tissue; H: Relative mRNA levels of Zo-1, Occludin, Claudin-1 and Muc2 in colon tissue were determined by qPCR; Data are expressed as mean ± SD. Compared with the DSS model group, * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001.

[0016] Figure 3 Figure 1 shows how LAL alleviates DSS-induced colonic inflammation and oxidative stress in mice. A: mRNA levels and secretion of TNF-α, IL-1β, and IL-6 in mouse colon tissues were measured by qPCR; B: mRNA levels and secretion of TNF-α, IL-1β, and IL-6 in mouse colon tissues were measured by ELISA; C: CAT enzyme activity; D: SOD enzyme activity; E: glutathione level (GSH); F: malondialdehyde (MDA); G: 4-HNE and lipid peroxidation (LPO, H) in mouse colon tissues; data are expressed as mean ± standard deviation. Compared with the DSS model group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0017] Figure 4 Figure 1 shows the effect of LAL on improving DSS-induced colon ferroptosis in mice. A: GO enrichment; B: KEGG pathway; C: trace elements (Cu 2+ , Fe 2+ , Ca 2+ Mg 2+ 、Zn 2+ , Mn 2+ 、Se 2+ ) detection; D: Western blot analysis of COX2, ACSL4, FTH1, GPX4 and GAPDH in mouse colon tissues, and quantitative analysis of protein levels by densitometry using ImageJ software. Data are expressed as mean ± standard deviation, compared with the DSS model group, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

[0018] Figure 5 Figure 1 shows the effect of LAL on erastin-induced ferroptosis in Caco-2 cells. A: Western blot analysis of COX2, ACSL4, FTH1, GPX4, and GAPDH in Caco-2 cells; B: MDA levels in Caco-2 cells; C: 4-HNE levels in Caco-2 cells; D: GSH levels in Caco-2 cells; E: Fe 2+ The data were expressed as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 compared with the Erastin group.

[0019] Figure 6 Activate Nrf2 / ARE signaling pathway for LAL to increase GSH synthesis and metabolism Figure 1 A: Heat map of genes related to lipid metabolism, iron ion transport, and oxidative stress in the transcriptome; B: GSEA analysis; C: Relative mRNA levels of genes related to glutathione metabolism (Gclc, Gclm, Gsta1, Gsta3, Gpx4, Hmox1, and Slc7a11) were determined in mouse colon tissue by qPCR.

[0020] Figure 7 Activate Nrf2 / ARE signaling pathway for LAL to increase GSH synthesis and metabolism Figure 2, where D: Western blot of KEAP1, p-Nrf2, Nrf2, and GAPDH in mouse colon tissue, E: Western blot of KEAP1, p-Nrf2, Nrf2, and GAPDH in Caco-2 cells, quantitative analysis of protein levels using ImageJ software; F: Luciferase reporter gene assay in Caco-2 cells. G: Relative mRNA levels of genes related to glutathione metabolism determined by qPCR in Caco-2 cells. Data are expressed as mean ± SD. Compared with the DSS model group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0021] Figure 8 The effect of LAL in alleviating colitis disappeared in Nrf2 knockout mice. A: Animal experiment design diagram; B: Body weight change (%); C: Food intake; D: Disease activity index; E: Rectal bleeding score; F: Fecal consistency score; G: Colon tissue HE staining; H: Colon length; I: Colon tissue MDA; J: 4-HNEL level; K: GSH level, L: Fe2+ level; M is Western blot analysis of COX2, ACSL4, FTH1, GPX4 and GAPDH in mouse colon tissue; Data are expressed as mean ± standard deviation. Compared with DSS Nrf2+ / - Compared with the above, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0022] Fig. 9 UPLC-LTQ-orbitrap-MS / MS was used to analyze the chemical composition of LAL in positive and negative ion modes.

[0023] Fig.10 LAL binds to KEAP1 to antagonize the Keap1-Nrf2 complex. Among them, B: Molecular docking of coumarin, epicatechin, quercetin and catechin with Keap1. C: The effect of LAL on the thermal stability of KEAP1 in Caco-2 cells was analyzed by CETSA-coupled western blot assay. D: The effect of LAL on the susceptibility of Keap1 proteolysis was analyzed by DARTS assay. E: Co-immunoprecipitation. Data are expressed as mean ± SD. Compared with the Ctrl group, * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001.

[0024] Fig.11Figure 2 shows the weakened ability of LAL to alleviate ferroptosis when KEAP1 is overexpressed in Caco-2 cells. A: Western blot analysis of COX2, ACSL4, FTH1, GPX4, and GAPDH in CACO-2 cells. B: MDA levels in Caco-2 cells; C: 4-HNE levels in Caco-2 cells; D: GSH levels in Caco-2 cells; E: Fe 2+ The data are expressed as mean ± SD. * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001 compared with the Erastin group. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below with reference to specific embodiments.

[0026] Example 1 Preparation of Polygonum multiflorum extract

[0027] Polygonum aviculare was purchased from Changda Chinese Medicine Pieces Co., Ltd. (Changda, China). Powdered Polygonum aviculare (100 g) was soaked in 95% ethanol (1:7, w / v) in a 1000 mL round-bottom flask overnight. The mixture was heated to reflux for 4-6 hours the next day, and then centrifuged at 6000 rpm to collect the supernatant. The supernatant was concentrated to 100 mL using a rotary evaporator to obtain an extract with a crude drug concentration of 1 μg / mL, which was stored at 4°C. This process was repeated 3 times, the combined ethanol extracts were filtered, the ethanol was removed by rotary evaporation, and the residue was freeze-dried to obtain the ethanol extract (LAL).

[0028] The ethanol extract (LAL) obtained in Example 1 was subjected to structural identification and performance testing, as follows:

[0029] (1) UPLC-LTQ-ORBITRAP-MS / MS identification

[0030] The mass of each secondary metabolite in LAL was measured using a UPLC-LTQ-Orbitrap-MS / MS system. The lyophilized powder (0.50 g) was dissolved in 80% methanol by volume in a 10 mL volumetric flask, diluted to scale, centrifuged, and the supernatant was filtered through a 0.22 μm membrane to obtain the test solution. The analysis was performed on a (2.1×100 mm, 1.8 μm) (Waters Corp. Milford, MA, United States) column using 0.1% formic acid (a) and acetonitrile (B) as mobile phases. The gradient elution was applied as follows: 0-5 min, 5-30% B; 5-18 min, 30-5% B; 18-30 min, 65-95% B; 30-32 min, 95% B. The column temperature was set to 40°C, the flow rate was 0.2 ml / min, and the injection volume was 3 μl. Electrospray ionization source (ESI), positive and negative ion modes, scanning range m / z100-1250; nebulizing gas (Ion source gas1) 55psi, drying gas (Ion source gas2) 55psi, curtain gas (Curtai on gas) 35psi, fragmentation voltage (DP) 60eV, collision energy (CE) 30eV; collision voltage difference (CES) 15eV; ion source temperature is 550℃; spray voltage 5500V / -5500V.

[0031] Base peak diagram of 80% (v / v) methanol extract of Polygonum hydropiper in positive and negative ion modes (see Figure 1 ). The mass spectrometry data were viewed using PeakView software, and combined with the comparative analysis of relevant literature and mass spectrometry library data, a total of 45 chemical components were identified from Polygonum hydropiper. The detailed information is shown in Table 1. The results showed that the components of Polygonum hydropiper were complex and varied. The identified components included 19 flavonoids and their glycosides, 6 organic acids, 3 amino acids, 3 phenols, 2 fatty acids, 2 nucleosides, 1 glycoside and 9 other components.

[0032] Table 1 Identification of chemical components of Polygonum hydropiper by UPLC-LTQ-Orbitrap-MS / MS

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] (2) Animal experiments

[0039] Male C57BL / 6J mice (2 months old, weighing 18-20 g, specific pathogen-free grade) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and housed under controlled conditions of temperature 22±2°C, relative humidity 50%±10%, and a 12-hour light / dark cycle. All mice had free access to food and water throughout the experiment. In order to evaluate the alleviating effect of LAL on colitis, the mice were randomly divided into five groups (n=8 in each group): normal control group (NCD), DSS-treated group (DSS), DSS+5-ASA (100 mg / kg), DSS+LAL (20 mg / kg), and DSS+LAL (60 mg / kg) groups. The NCD group received a standard diet and water as a negative control, and the remaining groups received a 2.5% DSS aqueous solution. The DSS+LAL group was gavaged with LAL every day, and the DSS+5-ASA group was gavaged with 5-ASA every day. All mice had free access to food and water throughout the experiment. To investigate the pharmacological effects of LAL mediated by the Nrf2 pathway, the mice were divided into three groups (6 mice in each group): wild-type DSS group, wild-type DSS+LAL group, and DSS+LAL group. Nrf2- / - The wild-type DSS group was given 2.5 wt% DSS aqueous solution and drinking water; the wild-type DSS+LAL group was given 2.5 wt% DSS aqueous solution and gavage with LAL (60 mg / kg); DSS+LAL Nrf2 - / - The mice were given 2.5 wt% DSS aqueous solution and LAL (60 mg / kg) by gavage. The daily food intake, body weight, disease activity index (DAI) and stool consistency of the mice were recorded. The experiment lasted for 8 days. The mice were euthanized under anesthesia and colon tissues were collected. Some colon tissues were fixed with 4 wt% paraformaldehyde, and the rest were stored at -80°C.

[0040] (3) Cell culture and treatment

[0041] Caco-2 cells were purchased from ATCC (Manassas, VA, USA) and maintained in DMEM (Gibco BRL, Rockville, MD, United States) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C, 95% humidity and 5% carbon dioxide. The FBS used was purchased from Gibco (MD, USA).

[0042] In order to study the effect of LAL on Caco-2 cells, different LAL concentrations and treatment times were selected for cell activity experiments. The cells were incubated with 10 μL CCK-8 (Beyotime, Shanghai, China) for 1 hour. The absorbance was measured at 450 nm using a microplate reader (SynergyHT, Bio-Tek, USA). In order to study the inhibitory effect of LAL on cell ferroptosis, erastin (10 μM) and LAL (2 mg / kg) were co-incubated in cells for 24 hours. The corresponding index was then determined, and each determination was performed at least three times.

[0043] (4) Histopathological evaluation

[0044] The samples were embedded in paraffin and sectioned (3 μm thick). The sections were stained with hematoxylin and eosin according to standard procedures. Histological scores were performed according to the degree of colon damage, ranging from 0 to 4. The scoring criteria are described in Table 2.

[0045] Table 2 Disease Activity Index

[0046]

[0047] (5) Transcriptomic analysis

[0048] RNA-seq was performed by Lianchuan Biotechnology (Hangzhou, China). To prepare cDNA libraries for RNA-seq, total RNA was isolated from colon tissues of WT, DSS control, and DSS plus LAL group mice using an RNA extraction kit. Agilent Technologies 2100 Bioanalyzer (Agilent Technologies) played a major role in assessing RNA integrity. The Illumina HiSeq 2500 platform was used to perform RNA-seq, and the Qubit 2.0 Fluorometer (Invitrogen) was used to visualize RNA-seq data. Fold changes > 2 and P < 0.05 indicated differentially expressed genes (DE G).

[0049] (6) Trace element detection

[0050] Colon tissue was dried at 80°C and ground into powder. The tissue was then digested using a microwave digester. 200 mg of liver powder and 4 mL of concentrated nitric acid were added to a digestion tube, and the liver powder was microwave digested at 150°C for 40 min. The digestion solution was diluted to 10 mL with water. The final element content was analyzed using a Multiwave GO microwave digestion system (HV750, Germany).

[0051] (7) Biochemical testing

[0052] IL-1β, IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioYangtian Biotechnology Co., Ltd. (Shanghai, China). Superoxide dismutase (SOD) assay kit, catalase assay kit, lipid peroxidation (LPO) assay kit, 4-hydroxynonene (4-HNE) assay kit, malondialdehyde (MDA) assay kit, and reduced glutathione (GSH) assay kit were purchased from Nanjing Jiancheng Bioengineering Research Institute, respectively. 2+ The assay kit was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0053] (8) Quantitative RT-PCR

[0054] Total RNA was extracted from colon tissue or cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The RNA was used to synthesize cDNA using a reverse transcription kit (Takara Biomedical Technology Co. LTD) according to the manufacturer's protocol. SYBR Green PCR Master Mix (Applied Biosystems, Foster City, California, USA) facilitates real-time PCR. Specific primer sequences are detailed in Table 2. PCR was performed using a LightCycler 480 qRT-PCR system (Roche, Mannheim, Germany) with the following procedure: initial denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 10 seconds, 40 cycles, and at 60°C for 1 minute. Gene expression levels were assessed using the 2-ΔΔCt method.

[0055] (9) Western blotting

[0056] Western blotting was performed according to a previously developed protocol. Primary antibodies included: anti-KEAP1 (ab227828), anti-ACSL4 (ab155282), anti-Nrf2 (ab62352), anti-phospho-Nrf2 (ab76026), and anti-GPX4 (ab125066), purchased from Abcam (Cambridge, UK); anti-COX2 (#73315), anti-FTH1 (#4393), and anti-GAPDH (#5174) were obtained from Cell Signaling Technology (Beverly, MA, USA). Band intensities were quantified using ImageJ software.

[0057] (10) Luciferase activity assay

[0058] Caco-2 cells were cultured at 3 × 10 4The density of cells / well is in 24-well plates. Each well is transfected with 90ng of firefly luciferase reporter gene and 10ng of kidney fluorine luciferase reporter gene. Renill a luciferase reporter gene is used as a load control. After transfection, 12 hours, cells are processed for 2h, 6h and 12h in the absence of LAL (2mg / kg), and then the luciferase activity of the samples is measured using a dual luciferase assay system (Orion L, USA) according to the manufacturer's instructions.

[0059] (11) siRNA interference and overexpression

[0060] Nrf2 was knocked down in Caco-2 cells using the recessive RNAi kit (SR321100A-AUUGAUGUUUCUGA UCUAUCACUTT, China). Cells were seeded into six-well plates, cultured to approximately 80% and transfected with Lipofectamine 3000 (Invitrogen, Camarillo, CA, USA) according to the manufacturer's instructions. After 48 hours of incubation, Nrf2 protein levels were assessed by western blotting. Similarly, the KEAP1 expression plasmid was purchased from Addgene (pcDNA3-HA2-KEAP1) and transfected into Caco-2 cells using Megatran 1.0 transfection reagent (OriGene, China). After 36 hours of incubation, KEAP1 protein expression levels were assessed by western blotting.

[0061] (12) Cellular thermal shift assay (CETSA)

[0062] Cellular proteins were extracted in PBS (containing 1wt% protease inhibitors). After centrifugation at 12000rpm for 10 minutes, the supernatant was collected. Lysates were set up with DMSO control and LAL (2mg / kg) treated groups and incubated at 37°C for 30 minutes. Each lysate was heated at 45°C, 48°C, 51°C, 54°C, 57°C and 60°C for 3 minutes. The heated lysate was then centrifuged at 12000rpm for 10 minutes, and the supernatant was collected again. The supernatant was boiled and analyzed by SDS-PAGE and then by Western blotting.

[0063] (13) Drug Affinity Response Target Stability (DARTS) Assay

[0064] The cells were lysed using 600 μL M-PER (Cat#78503, Thermo Fisher Scientific) and 1% protease inhibitors, and the supernatant was collected after centrifugation. The protein content was mixed in 10× reaction buffer. The solution was divided equally into two parts and incubated with LAL (2 mg / kg) or PBS for 1 hour at 25°C. Subsequently, the samples were treated with pronase (CAT#10165921001; Roche), diluted with 1× buffer and placed at 25°C for 30 minutes. The samples were analyzed by Western blotting.

[0065] (14) Molecular docking

[0066] To investigate the interaction between LAL and 16 compounds in the active site of KEAP1, molecular docking simulations were performed using the Autodock online system (http: / / autodock.scripps.edu / ). The chemical structures of the compounds were obtained from Pubchem compounds (https: / / Pubchem.NCBI.nlm.nih.Gov / ). The published crystal structure of KEAP1 (PDB: IU6D) was retrieved from the RCSB protein database, and Pymol 2.3.0 software was used to remove protein crystal water, original ligands, etc. Hydrogenation, charge assignment and names of atoms were determined. Molecular docking was performed by Autodock software, and molecular docking data were visualized using Pymol2.6.0 software.

[0067] (15) Co-immunoprecipitation (CO-IP)

[0068] Cell lysates from Caco-2 cells were extracted using RIPA lysis buffer. Cell lysates were then incubated with Nrf2 or KEAP1 antibodies for 2 h at room temperature, followed by binding of antigen-antibody complexes to protein A / G magnetic beads (ThermoScientific, Rockford, USA) for 1 h. Subsequently, immune complexes were detected using KEAP1 or Nrf2 antibodies.

[0069] (16) Statistical analysis

[0070] The Shapiro-Wilk test was used to assess data distribution using SPSS (Version 21, IBM Corporation, Armonk, NY). One-way analysis of variance (ANOVA) with Tukey's post hoc test or unpaired t-test was performed. A significance level of P < 0.05 was considered statistically significant.

[0071] 2. Experimental results

[0072] (1) LAL improves DSS-induced intestinal barrier damage and colitis in mice

[0073] A total of 40 male mice were divided into 4 groups, namely, normal control group (NCD), DSS treatment group (DSS), low-dose LAL group (DSS+LAL, 20 mg / kg), high-dose LAL group (DSS+LAL, 60 mg / kg) and positive control group (DSS+5-ASA).

[0074] Results Figure 2 Compared with the normal control group, the daily food intake, body weight change rate, and colon length of the DSS model group mice were significantly decreased, and the disease activity index (DAI), rectal bleeding score, and stool consistency score were significantly increased, indicating that the DSS mouse model was successfully established. Compared with the DSS model group mice, the low- and high-dose LAL administration groups could significantly increase (p<0.01) the daily food intake, body weight change rate, and colon length of the mice, and significantly reduce the disease activity index (DAI), rectal bleeding score, and stool consistency score (p<0.05). The intervention effect of the LAL treatment group was positively correlated with the concentration.

[0075] HE staining results showed that the colon structure and tissue of mice in the normal control group were intact; after DSS modeling, the colon crypts of mice disappeared, a large number of inflammatory cells infiltrated, and the colon tissue was severely damaged; compared with the DSS model group, the colon villus morphology and crypt damage in the LAL group were significantly restored, the inflammatory cell infiltration was reduced, and the colon tissue morphology was improved.

[0076] After LAL administration intervention, the relative mRNA expression levels of Zo-1, Occludin, Claudin-1 and Muc2 in the DSS model group mice were significantly increased (p<0.05, p<0.01 or p<0.001), among which Claudin-1 expression was significantly increased only in the high-dose LAL group (p<0.05), indicating that higher doses of LAL can better play a therapeutic role by upregulating tight junction proteins to repair the intestinal barrier.

[0077] (2) LAL alleviates DSS-induced colonic inflammation and oxidative stress in mice

[0078] Methods Referring to animal experiments, the mRNA levels of TNF-α, IL-1β, and IL-6 in mouse colon tissue were measured by qPCR and ELISA. The severity of colon inflammation in DSS mice was directly correlated with the expression of inflammatory factors. Figure 3As shown in A and B, the concentrations and mRNA expressions of inflammatory factors IL-1β, IL-6, and TNF-α in the colon tissue of DSS-induced mice were significantly higher than those in the normal group (p<0.05, p<0.01, or p<0.001). In contrast, LAL intervention can effectively reduce the concentrations and mRNA expressions of IL-1β, IL-6, and TNF-α (p<0.05, p<0.01, or p<0.001), and the intervention efficiency is positively correlated with the concentration.

[0079] To investigate the effect of LAL on the antioxidant enzyme activity and peroxide content in the colon of DSS mice. Figure 3 As shown in Figure CE, compared with the normal control group, the CAT enzyme activity, SOD enzyme activity, and GSH level in the colon of mice in the DSS model group were significantly reduced; compared with the DSS model group, the low- and high-dose LAL groups could significantly increase the CAT enzyme activity, SOD enzyme activity, and GSH level (p<0.05, p<0.01, or p<0.001), and significantly reduce the MDA content (p<0.01 or p<0.001); Figure 3 As shown in Figures FH, compared with the normal control group, the production of MDA, 4-HNE and LPO in the DSS model group was significantly increased; compared with the DSS model group, the content of MDA, 4-HNE and LPO was significantly decreased (p<0.01 or p<0.001), indicating that LAL has a significant effect on alleviating oxidative stress and reducing lipid peroxidation.

[0080] (3) LAL can improve DSS-induced colon ferroptosis in mice

[0081] Comparing the differences in the genomes of mice in the DSS group and the DSS+LAL group, GO enrichment showed that the differentially expressed genes were mainly enriched in biological processes such as iron ion sequestration, blood clot formation, and lipid transport, and existed in cell components such as blood microparticles, extracellular space, extracellular region, and platelet α granules. The molecular functions mainly involved metal ion binding, antioxidant activity, hemoglobin binding, etc. KEGG analysis mainly included apoptosis, diabetic complications AGE-RAGE signaling pathway, JAK-STAT signaling pathway, Wnt signaling pathway, tryptophan metabolism, etc.

[0082] The results of colon tissue trace element detection showed that the low-dose LAL group only had a negative effect on Fe 2+ The level of Cu was significantly affected (p<0.001). The high-dose LAL group could significantly reduce (p<0.05 or p<0.001) 2+ , Fe 2+ , Ca 2+ Levels, LAL intervention had an effect on Mg 2+ 、Zn 2+ , Mn 2+ 、Se 2+There was no significant effect. Combined with the results of GO and KEGG analysis, it was inferred that LAL may improve colitis by regulating the ferroptosis process.

[0083] To verify this conclusion, the method was referenced to animal experiments, and Western blotting was used to explore the regulation of LAL on ferroptosis in the colon tissue of enteritis mice. RNA-Seq technology was used to analyze the differences in the genomes of mice in the DSS group and the DSS+LAL (60 mg / kg) group; the results showed that Figure 4 LAL could significantly downregulate (p<0.05, p<0.01 or p<0.001) the expression of COX2, ACSL4, FTH1 and GPX4 proteins. The results verified that LAL could improve DSS-induced colon ferroptosis in mice.

[0084] (4) LAL intervenes in erastin-induced ferroptosis in Caco-2 cells

[0085] The test method refers to cell culture and treatment, using erastin to induce ferroptosis in Caco-2 cells, and the results are shown in Figure 5 ,Depend on Figure 5 AC showed that LAL could significantly reduce (p<0.01 or p<0.001) the content of MDA and 4-HNE in cells and significantly up-regulate the level of GSH, indicating that the oxidative stress and damage of Caco-2 cells were improved. 2+ The level was significantly reduced (p<0.001), indicating that the cellular iron metabolism was restored to balance and the cell function was restored to a certain extent.

[0086] Erastin was used to establish a Caco-2 cell ferroptosis model and LAL was used for intervention treatment. Figure 5 As shown in E, compared with the Ctrl group, the expression of COX2 and ACSL4 proteins in Caco-2 cells in the erastin group was significantly increased (p<0.001), and the expression of FTH1 and GPX4 proteins was significantly decreased (p<0.001). After LAL intervention, compared with the erastin group, the expression of COX2 and ACSL4 proteins was significantly decreased (p<0.001), and the expression of FTH1 and GPX4 proteins was significantly increased (p<0.001). The expression levels of ferroptosis-related proteins were close to those of the Ctrl group, indicating that LAL can prevent erastin-induced ferroptosis in Caco-2 cells.

[0087] (5) LAL activates the Nrf2 / ARE signaling pathway to increase GSH synthesis and metabolism

[0088] Refer to animal experiments, the results are shown in Figure 6 Transcriptome and GSEA analysis showed that LAL was significantly enriched in the glutathione metabolic pathway ( Figure 6 qPCR and Western blotting confirmed that LAL upregulated the expression of Nrf2 and its downstream genes (Gclc, Gpx4, Slc7a11) and promoted Nrf2 phosphorylation ( Figure 7 Luciferase reporter gene experiments further showed that LAL enhanced the transcriptional activity of ARE elements ( Figure 7 F). This indicates that the Nrf2 / ARE pathway is the core mechanism of LAL antioxidant activity, but its activation mode (direct binding to KEAP1 or indirect regulation) needs further verification.

[0089] (6) The effect of LAL in alleviating colitis is lost in Nrf2 knockout mice

[0090] The experimental mice were divided into the following groups: wild-type DSS group (DSS Nrf2+ / - ), wild-type DSS+LAL group (DSS+LAL Nrf2+ / - ) and DSS+LAL Nrf2- / - The results are shown in Figure 8 In Nrf2 knockout mice, the improvement effect of LAL on colitis symptoms (food intake, DAI, colon length) disappeared ( Figure 8 AG), and ferroptosis markers (COX2, ACSL4) and oxidative damage indicators (MDA, 4-HNE, etc.) did not change significantly ( Figure 8 Nrf2 is an essential molecule for LAL to exert its therapeutic effect, suggesting that it targets the Nrf2 pathway specifically, but whether other synergistic pathways are involved needs to be explored.

[0091] (7) LAL binds to KEAP1 and antagonizes the Keap1-Nrf2 complex

[0092] The chemical composition of LAL was analyzed by UPLC-LTQ-orbitrap-MS / MS in positive and negative ion modes. Fig. 9 To investigate the interaction between LAL and 16 compounds in the active site of KEAP1, molecular docking simulations were performed using the Autodock online system; cell-based thermal shift analysis (CETSA); drug affinity response target stability (DARTS) assay; and co-immunoprecipitation (CO-IP) assays were performed, i.e., Caco-2 cells were treated with or without LAL (1.0 or 2.0 mg / mL), and the lysates were immunoprecipitated with the indicated antibodies, followed by western blotting. Results are shown in Fig.10 Molecular docking showed that the flavonoid components in LAL (quercetin, catechins, etc.) bind to the active site of KEAP1 ( Fig.10 CETSA and DARTS experiments confirmed that LAL enhanced the thermal stability of KE AP1 and reduced its proteolytic sensitivity ( Fig.10 CO-IP experiments further showed that LAL disrupted the binding of KEAP1 to Nrf2 ( Fig.10 Middle E). The results showed that LAL directly binds to KEAP1, releases Nrf2 and promotes its nuclear translocation to activate downstream genes, which is the core of its molecular mechanism.

[0093] (8) KEAP1 overexpression weakens the anti-ferroptosis effect of LAL

[0094] Plasmids were transfected to construct CACO-2 cells overexpressing Keap1, and erastin (10 μM) and LAL (2 mg / kg) were co-incubated in the cells for 24 h, and then the corresponding index was determined. Fig.11 In Caco-2 cells overexpressing KEAP1, the improvement effect of LAL on ferroptosis markers (COX2, ACSL4, etc.) and oxidative damage indicators (MDA, 4-HNE, etc.) was significantly inhibited ( Fig.11 These results suggest that KEAP1 is a key target of LAL, and its overexpression blocks LAL's activation of Nrf2, further validating the central role of the KEAP1-Nrf2 axis.

[0095] In summary, the present invention systematically reveals the molecular mechanism of LAL extract in relieving enteritis through multiple targets and multiple pathways:

[0096] (1) Intestinal barrier repair: LAL upregulates tight junction proteins (Zo-1, Occludin) and mucin (Muc2), restoring the intestinal mechanical barrier function.

[0097] (2) Anti-inflammatory and antioxidant: LAL inhibits pro-inflammatory factors (TNF-α, IL-6) and activates the Nrf2 / ARE pathway, enhances GSH synthesis, and scavenges reactive oxygen species (ROS).

[0098] (3) Inhibition of ferroptosis: LAL blocks the ferroptosis process by reducing Fe2+ levels and regulating the COX2 / GPX4 balance.

[0099] (4) The core role of the KEAP1-axis: The flavonoid components in LAL directly bind to KEAP1, releasing and activating its downstream antioxidant genes, which is the cornerstone of its molecular mechanism.

[0100] This invention is the first to associate the anti-enteritis effect of LAL with ferroptosis inhibition and the KEAP1-pathway, revealing the pleiotropic mechanism of natural products. In addition, LAL is a potential natural enteritis treatment drug that regulates the integration mechanism of oxidative stress, inflammation and ferroptosis through the KEAP1-axis, providing a theoretical basis for the development of new multi-target therapies.

[0101] The above-mentioned embodiments merely express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the patent scope of the present invention.

Claims

1. The use of the alcohol extract of Polygonum hydropiper in preparing a drug for treating enteritis, characterized in that: The preparation method of the Polygonum hydropiper ethanol extract is as follows: The powdered Polygonum hydropiper is soaked in ethanol, heated to reflux, and the supernatant is collected by centrifugation. The supernatant is concentrated, and the ethanol is removed by rotary evaporation, and freeze-dried to obtain the Polygonum hydropiper ethanol extract.

2. The use of the ethanol extract of Polygonum hydropiper according to claim 1 in preparing a drug for treating enteritis, characterized in that: The addition ratio of the mass of the powder material to the volume of ethanol is 1:6-8.

3. The use of the ethanol extract of Polygonum hydropiper according to claim 1 in preparing a drug for treating enteritis, characterized in that: The heating reflux is performed for 4-6 hours, and the number of reflux extractions is 2-4 times.

4. The use of the ethanol extract of Polygonum hydropiper according to claim 1 in preparing a drug for treating enteritis, characterized in that: The centrifugal speed is 5000-7000 rpm.

5. The use of the ethanol extract of Polygonum hydropiper according to claim 1 in preparing a drug for treating enteritis, characterized in that: The medicine uses the alcohol extract of Polygonum hydropiper as the active ingredient and contains a pharmaceutically acceptable carrier, wherein the alcohol extract of Polygonum hydropiper accounts for 0.01-99.99% by weight in the preparation, and the rest is the pharmaceutically acceptable carrier.

6. The use of the ethanol extract of Polygonum hydropiper according to claim 1 in preparing a drug for treating enteritis, characterized in that: The alcohol extract of Polygonum hydropiper can inhibit pro-inflammatory factors TNF-α and IL-6, activate Nrf2 / ARE pathway, enhance GSH synthesis, and remove reactive oxygen species.

7. The use of the ethanol extract of Polygonum hydropiper according to claim 1 in preparing a drug for treating enteritis, characterized in that: The ethanol extract of Polygonum hydropiper can upregulate tight junction proteins Zo-1, Occludin and mucin Muc2.

8. The use of the ethanol extract of Polygonum hydropiper according to claim 1 in preparing a drug for treating enteritis, characterized in that: The Polygonum hydropiper ethanol extract can reduce Fe 2+ level, regulate the COX2 / GPX4 balance, and block the ferroptosis process.

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