Application of golden-edge phellinus igniarius powder preparation in preparation of medicine for treating ulcerative colitis

Abstract

The invention relates to the technical field of traditional Chinese medicine. The invention discloses application of a golden-edge phellinus igniarius powder preparation in preparation of a medicine for treating ulcerative colitis. The golden-edge phellinus igniarius powder preparation is prepared from the following raw material medicines in parts by weight: 20-40 parts of phellinus igniarius, 0.6-1.2 parts of sanguisorba officinalis, 0.6-1.2 parts of coptis chinensis, 20-40 parts of angelica sinensis, 10-20 parts of scutellaria baicalensis and 10-20 parts of honey-fried licorice root. The preparation method of the medicine comprises the following steps: mashing and sieving the raw material medicines to prepare powder. The golden-edge phellinus igniarius powder preparation can up-regulate ZO-1, Occludin and goblet cells and repair intestinal barriers; glutamic acid can be used for up-regulating GPX4, SLC7A11 and FTH1, and inhibiting intestinal epithelial ferroptosis. Meanwhile, cGAS / STING protein is down-regulated, proinflammatory factors such as TNF-alpha, IL-1beta and IL-6 are reduced, and intestinal inflammation is inhibited; the traditional Chinese medicine composition can reduce the content of MDA and Fe < 2 + > in colon, improve GSH, relieve oxidative stress, break vicious circle and efficiently treat ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to the application of a Jinbian Sanghuang powder preparation in the treatment of ulcerative colitis. Background Technology

[0002] Ulcerative colitis is a chronic, nonspecific inflammatory bowel disease with an incompletely understood etiology. It primarily affects the colorectal mucosa and submucosa, with typical clinical manifestations including recurrent diarrhea, bloody and purulent stools, and abdominal pain. In severe cases, it can be accompanied by complications such as intestinal perforation and cancer, significantly impacting patients' quality of life. In recent years, influenced by lifestyle changes and environmental factors, the global incidence of this disease has been rising continuously, becoming a common and intractable digestive system disease, with an increasingly urgent need for clinical treatment. Its pathogenesis is complex, closely related to multiple factors such as impaired intestinal barrier function, disordered immune inflammatory response, excessive oxidative stress, and abnormal death of intestinal epithelial cells. Disruption of the intestinal barrier integrity is a key initiating factor, leading to dysbiosis, antigen invasion, activation of inflammatory pathways, and the formation of a vicious cycle of barrier destruction, inflammation activation, oxidative stress, and cell damage.

[0003] Current clinical treatment focuses on controlling inflammation, alleviating symptoms, and preventing recurrence. Commonly used drugs include aminosalicylic acids, glucocorticoids, immunosuppressants, and biologics, but these have significant limitations. Aminosalicylic acids have limited efficacy in moderate to severe patients and are prone to drug resistance; glucocorticoids have significant side effects; and immunosuppressants and biologics are expensive, pose potential infection risks, and exhibit large individual variability in efficacy. Existing drugs mostly focus on inhibiting single inflammatory pathways, making it difficult to comprehensively cover the complex pathophysiological processes of the disease, resulting in low clinical cure rates and persistently high recurrence rates. Traditional Chinese medicine compound formulas, with their unique advantages of multiple components, multiple targets, and holistic regulation, show promising application prospects in the treatment of ulcerative colitis. Single herbs such as Sanghuang, Danggui, and Huangqin have been proven to have regulatory effects on related pathological processes, but there is still no systematic research on combining them into compound formulas for multi-target synergistic treatment targeting intestinal barrier repair, inflammation suppression, oxidative stress relief, and ferroptosis regulation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention discloses the application of a Jinbian Sanghuang powder preparation for the treatment of ulcerative colitis, which can target multiple pathological aspects of ulcerative colitis and provide an effective treatment plan for the disease.

[0005] This invention discloses the application of a Jinbian Sanghuang powder preparation in the treatment of ulcerative colitis, comprising: The Jinbian Sanghuang Powder preparation is made from the following raw materials in parts by weight: Phellinus linteus 20-40 parts, Sanguisorba officinalis 0.6-1.2 parts, Coptis chinensis 0.6-1.2 parts, Angelica sinensis 20-40 parts, Scutellaria baicalensis 10-20 parts, Glycyrrhiza uralensis (processed) 10-20 parts; The preparation method of the drug includes: crushing and sieving the above-mentioned raw materials to make a powder.

[0006] Furthermore, the Jinbian Sanghuang powder preparation is used in the form of its water extract, alcohol extract, polysaccharide-rich fraction, or total flavonoid-rich fraction.

[0007] Further applications include upregulating the expression of intestinal epithelial tight junction proteins ZO-1 and / or Occludin to repair intestinal barrier function.

[0008] Further uses include downregulating the expression of cGAS and / or STING proteins, and / or reducing the levels of one or more of tumor necrosis factor-α, interleukin-1β, and interleukin-6 to suppress intestinal inflammatory responses.

[0009] Further applications include upregulating the expression of glutathione peroxidase 4, solute carrier family 7 member 11, and / or ferritin heavy chain 1. It inhibits ferroptosis in intestinal epithelial cells.

[0010] Further applications include reducing malondialdehyde and / or ferrous ions in colon tissue, and / or increasing reduced glutathione levels to mitigate oxidative stress damage.

[0011] Further uses include reducing disease activity index scores and / or colon histopathology scores.

[0012] Furthermore, its uses include increasing the number of goblet cells in the colon.

[0013] Furthermore, its uses include lowering the spleen index.

[0014] The beneficial effects of this invention are: Jinbian Sanghuang Powder can effectively repair the intestinal barrier function and protect intestinal epithelial cells. It can upregulate the expression of intestinal epithelial tight junction proteins ZO-1 and Occludin, enhancing intestinal barrier integrity, reducing intestinal permeability, and preventing the invasion of harmful substances. This Jinbian Sanghuang Powder compound can upregulate the expression of glutathione peroxidase 4, solute carrier family 7 member 11, and ferritin heavy chain 1, inhibiting ferroptosis in intestinal epithelial cells and reducing cell damage. It can also increase the number of colonic goblet cells, promote mucus secretion, further strengthen the protective function of the intestinal barrier, and build a robust defense system for the intestinal mucosa.

[0015] This compound of Sanghuang powder with golden edges exhibits remarkable effects in inhibiting intestinal inflammatory responses and alleviating oxidative stress damage. It can downregulate the expression of cGAS and STING proteins, block the activation of related inflammatory pathways, and simultaneously reduce the levels of pro-inflammatory factors such as tumor necrosis factor-α, interleukin-1β, and interleukin-6 in serum, thus reducing intestinal inflammatory infiltration. In regulating oxidative stress, it can reduce the content of malondialdehyde and ferrous ions in colonic tissue, decrease the accumulation of oxidative products, and simultaneously increase the content of reduced glutathione, enhancing the body's antioxidant capacity, effectively alleviating oxidative damage to the intestinal mucosa, and breaking the vicious cycle of inflammation and oxidative stress. Attached Figure Description

[0016] Figure 1 The image shows the protein expression bands of cGAS, STING, ZO-1, Occludin, GPX4, SLC7A11, and FTH1 in Caco-2 cells.

[0017] Figure 2 The expression of cGAS, STING, ZO-1, and Occludin proteins in Caco-2 cells ( (±S, n=3) Figure.

[0018] Figure 3 The expression of GPX4, SLC7A11, and FTH1 proteins in Caco-2 cells ( (±S, n=3) Figure.

[0019] Figure 4 Figures showing histopathological changes in mouse colon tissue (100x, 200μm; 400x, 50μm).

[0020] Figure 5 The average number of goblet cells per crypt in mouse colon tissue ( (±s, n=3) Figure.

[0021] Figure 6 The levels of LPS, TNF-α, IL-1β, and IL-6 in mouse serum ( (±s, n=6) Figure.

[0022] Figure 7 Fe in mouse colon tissue 2+ MDA and GSH content ( (±s, n=3) Figure.

[0023] Figure 8 This is a gross morphological observation of the spleen tissue of a mouse.

[0024] Figure 9 Changes in the spleen index of mice ( (±s, n=6) Figure.

[0025] Figure 10 Changes in body weight of mice in each group ( (±s, n=6) Figure.

[0026] Figure 11 Figure showing the expression level of ZO-1 in mouse colon tissue (200x: 100μm; 400x: 50μm).

[0027] Figure 12 Figure showing the expression level of occludin in mouse colon tissue (200x: 100μm; 400x: 50μm).

[0028] Figure 13 The expression levels of ZO-1 and occludin in mouse colon tissue ( (±s, n=8) Figure.

[0029] Figure 14 Image showing histopathological changes in mouse colon tissue (40x: 500μm; 200x: 100μm).

[0030] Figure 15 Semi-quantitative HE scoring of mouse colon tissue ( (±s, n=8) Figure.

[0031] Figure 16 MPO activity in mouse colon tissue ( (±s, n=8) Figure.

[0032] Figure 17 This is a diagram of a mouse colon.

[0033] Figure 18 The colon length and intestinal weight index of mice ( (±s, n=8) Figure.

[0034] Figure 19 Mouse weight status ( (±s, n=8) Figure.

[0035] Figure 20 DAI score of mice ( (±s, n=8) Figure. Detailed Implementation

[0036] To enable those skilled in the art to better understand the present invention, the technical solutions in the specific embodiments of the present invention will be clearly and completely described below.

[0037] This invention discloses the application of a Jinbian Sanghuang powder preparation in the treatment of ulcerative colitis, comprising: The Jinbian Sanghuang Powder preparation is made from the following raw materials in parts by weight: Phellinus linteus 20-40 parts, Sanguisorba officinalis 0.6-1.2 parts, Coptis chinensis 0.6-1.2 parts, Angelica sinensis 20-40 parts, Scutellaria baicalensis 10-20 parts, Glycyrrhiza uralensis (processed) 10-20 parts; The source of the *Sanghuang* fungus in this invention is artificially cultivated *Sanghuangporus vaninii* QJF-8, belonging to the genus *Sanghuang* of the family Phlomycetes. The strain of *Sanghuangporus vaninii* is QJF-8, and its preservation number is CGMCCNo.40120.

[0038] The preparation method of the drug includes: crushing and sieving the above-mentioned raw materials to make a powder. After the raw materials are proportioned by weight, they are mixed using an equal-incremental method: first, pulverize Sanguisorba officinalis and Coptis chinensis and sieve them through an 80-mesh sieve, then add an equal weight of roasted licorice powder and mix evenly. Then, add the pulverized powders of Scutellaria baicalensis, Angelica sinensis, and Phellinus linteus successively, mixing thoroughly for 5-10 minutes after each addition to ensure uniform distribution of the powders and avoid agglomeration of small components. This formulation has been verified by orthogonal experiments. Phellinus linteus and Angelica sinensis are the principal herbs, and a dosage of 20-40 parts can fully exert their core anti-inflammatory and repairing effects; Sanguisorba officinalis and Coptis chinensis are the adjuvant herbs, and a dosage of 0.6-1.2 parts can synergistically enhance the heat-clearing and hemostatic effects of the principal herbs while avoiding gastrointestinal irritation caused by excessive dosage.

[0039] First, pulverize Sanguisorba officinalis and Coptis chinensis and pass them through an 80-mesh sieve. Mix them evenly to obtain mixed powder A. Take an equal weight of roasted licorice powder and mix it thoroughly with mixed powder A for 5 minutes to obtain mixed powder B. Then, take an equal weight of Scutellaria baicalensis powder and add it to mixed powder B. Mix for 8 minutes to obtain mixed powder C. Continue to take an equal weight of Angelica sinensis powder and mix for 10 minutes to obtain mixed powder D. Finally, add the remaining Phellinus linteus powder and mix for 10 minutes to ensure uniformity. The contents of berberine and Sanguisorba officinalis saponins in three randomly selected batches of powders were determined by HPLC. RSD ≤ 1.

[0040] As one implementation method, the Jinbian Sanghuang powder preparation is used in the form of its water extract, alcohol extract, polysaccharide-rich fraction, or total flavonoid-rich fraction.

[0041] For the preparation of the water extract, the raw material of Jinbian Sanghuang San compound was taken, and 12 times the amount of purified water was added. The mixture was refluxed twice, 1.5 hours each time. The extracts were combined and concentrated under reduced pressure to a relative density of 1.10-1.15 (60℃). The water extract powder was obtained by spray drying. The alcohol extract was extracted using 75% ethanol as solvent, following the same process described above. The polysaccharide content of the polysaccharide-rich fraction was ≥50% (calculated as glucose), and the total flavonoid content of the total flavonoid-rich fraction was ≥30% (calculated as rutin), both determined by UV-Vis spectrophotometry. The baicalin content of the water extract and alcohol extract was ≥1.2%, and the berberine content was ≥0.3%, determined by high performance liquid chromatography (HPLC).

[0042] This invention discovers the synergistic multi-target effect of the Jinbian Sanghuang Powder compound, composed of Sanghuang, Diyu, Huanglian, Danggui, Huangqin, and Gancao in a specific ratio, in the treatment of ulcerative colitis. Traditionally, this Jinbian Sanghuang Powder compound is used in the form of a powder for decoction. However, to meet the needs of modern drug research and development and production, and to improve the quality controllability, enrichment of active ingredients, and convenience of administration of the preparation, the raw materials of this specific Jinbian Sanghuang Powder compound can be prepared into a more refined form through modern extraction and separation technology.

[0043] The water extract / alcohol extract refers to the extract or powder obtained by using water or ethanol of appropriate concentration as solvents, with a specific ratio of the Jinbian Sanghuang powder compound medicinal materials of this invention, through conventional extraction methods such as decoction, reflux, and percolation, collecting the extract, concentrating and drying it. This process aims to enrich the comprehensive group of water-soluble or alcohol-soluble active ingredients in the compound and remove most of the inactive substances such as medicinal fiber.

[0044] The polysaccharide-enriched fractions / total flavonoid-enriched fractions refer to the selective enrichment of a group of chemical components with definite biological activity by further refining techniques such as macroporous adsorption resins, membrane separation, precipitation, and chromatographic separation based on the above-mentioned total extract. Polysaccharide-enriched fractions refer to preparations rich in polysaccharide components derived from multiple medicinal herbs in the compound, such as Sanghuang and Danggui; total flavonoid-enriched fractions refer to preparations rich in flavonoid components derived from multiple medicinal herbs in the compound, such as Huangqin and Sanghuang. These fractions represent an effective group of fractions between traditional crude extracts and single compounds, retaining the synergistic effect of multiple components in traditional Chinese medicine, while having a clearer material basis. The polysaccharide enrichment fraction was prepared using D101 macroporous adsorption resin at a loading concentration of 1 g / mL (crude drug concentration), an adsorption time of 2 h, followed by elution with 50% ethanol after impurity removal with distilled water, concentration, and drying, with a polysaccharide content ≥50% (calculated as glucose). The total flavonoid enrichment fraction was prepared using AB-8 macroporous adsorption resin at a loading concentration of 1 g / mL, an adsorption time of 1.5 h, followed by elution with 70% ethanol after impurity removal with 30% ethanol, concentration, and drying, with a total flavonoid content ≥30% (calculated as rutin), and deproteinized using the Sevag method.

[0045] It is important to note that whether it is the water extract, alcohol extract, or the polysaccharide or total flavonoid fraction obtained through further purification, the source of the substances must be the complete compound with the specific ratio defined in this invention. The preparation premise is to maintain the integrity of the substances in the original compound's principal, assistant, adjuvant, and guide relationships. The pharmacodynamic data provided by this invention, such as repair of the intestinal barrier, inhibition of inflammatory pathways, and anti-ferroptosis, confirm the overall therapeutic efficacy of this specific compound. Preparing it into the various purified forms described above is a conventional and foreseeable technical extension undertaken by those skilled in the art to optimize the formulation after confirming the overall pharmacodynamics. These purified forms aim to deliver the active substances in the compound more efficiently, and their expected therapeutic effects derive from and are attributed to the specific compound combination itself.

[0046] As one implementation method, its use includes upregulating the expression of intestinal epithelial tight junction proteins ZO-1 and / or Occludin to repair intestinal barrier function. This refers to the core mechanism by which Jinbian Sanghuang San preparations regulate molecular signaling pathways, promoting the synthesis and localization of key junction proteins in intestinal epithelial cells, thereby restoring the integrity of the intestinal mucosal physical barrier. In the pathological state of ulcerative colitis, the tight junction structure of the intestinal epithelium is damaged, and the expression of its main functional proteins is often significantly downregulated, leading to abnormally increased intestinal permeability. This allows harmful substances such as bacteria and endotoxins to easily cross the intestinal mucosa, thereby continuously activating the immune inflammatory response and forming a vicious cycle. Therefore, actively upregulating the expression of these key junction proteins is an important strategy for repairing the intestinal barrier and fundamentally blocking disease progression.

[0047] The experimental data of this invention fully demonstrate the specific role of Jinbian Sanghuang Powder in this process: In a lipopolysaccharide (LPS)-induced Caco-2 intestinal epithelial cell injury model, LPS stimulation significantly reduced the protein expression levels of ZO-1 and Occludin. However, after intervention with serum containing Sanghuang powder, the expression of both proteins showed a dose-dependent significant upregulation, even recovering to or exceeding normal levels. This result directly demonstrates at the cellular and molecular level that the Jinbian Sanghuang powder compound of this invention has the ability to directly enhance the synthesis of tight junction proteins in intestinal epithelial cells. In a mouse UC model induced by dextran sulfate sodium (DSS), immunohistochemical staining showed that the expression levels of ZO-1 and Occludin proteins in the colonic tissue of the model group mice were significantly lower than those in the normal group. After treatment with Jinbian Sanghuang powder, the expression levels of these two proteins in the colonic mucosa were increased. This indicates that even in the complex pathological environment of living animals, the Jinbian Sanghuang powder compound of this invention can effectively promote the expression and localization of key proteins of the intestinal barrier, achieving structural repair.

[0048] Upregulating ZO-1 and Occludin expression is one of the definite targets of Jinbian Sanghuang Powder in repairing the intestinal barrier and reducing intestinal permeability in ulcerative colitis. This mechanism, together with the anti-inflammatory and antioxidant effects of Jinbian Sanghuang Powder, constitutes the scientific basis for its multi-target and multi-level treatment of ulcerative colitis.

[0049] As one implementation method, the use includes downregulating the expression of cGAS and / or STING proteins, and / or reducing the levels of one or more of tumor necrosis factor-α, interleukin-1β, and interleukin-6 to inhibit intestinal inflammatory responses. This refers to the core anti-inflammatory mechanism of Jinbian Sanghuang San preparation, which inhibits abnormal immune activation in ulcerative colitis (UC) at multiple levels by intervening in innate immune signaling and reducing the release of key pro-inflammatory mediators.

[0050] In the pathogenesis of ulcerative colitis (UC), damage-associated molecular patterns (DAMPs) released by damaged intestinal epithelial cells can activate the intracellular cGAS-STING signaling pathway, thereby driving the production of pro-inflammatory factors such as type I interferon and amplifying the inflammatory cascade. Simultaneously, activated immune cells secrete large amounts of core pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, directly leading to intestinal mucosal inflammatory infiltration, tissue destruction, and clinical symptoms. Therefore, simultaneously targeting both the upstream cGAS-STING signaling axis and downstream effector cytokines can more comprehensively and synergistically suppress the intestinal inflammatory network.

[0051] The experimental data of this invention confirm the anti-inflammatory effect of the Jinbian Sanghuang Powder compound from the following two aspects: In a lipopolysaccharide (LPS)-stimulated Caco-2 cell model, LPS significantly upregulated the protein expression of cGAS and STING, activating related inflammatory pathways. Serum intervention containing Sanghuang powder dose-dependently downregulated the expression of these two key signaling proteins. This indicates that the Jinbian Sanghuang powder compound of this invention can intervene in the overactivation of innate immunity at its signaling source.

[0052] Inhibition of downstream pro-inflammatory mediators: In a DSS-induced mouse UC model, serum levels of endotoxin (LPS) and pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly elevated in the model group. After treatment with Jinbian Sanghuang Powder, the levels of these key inflammatory mediators showed a significant dose-dependent decrease. This result confirms at the whole-animal level that the Jinbian Sanghuang Powder compound of this invention can effectively reduce systemic inflammatory burden.

[0053] It should be noted that the decrease in serum LPS levels also indirectly confirms the repair of intestinal barrier function and reduces the translocation of intestinal endotoxins.

[0054] In summary, Jinbian Sanghuang Powder regulates innate immune signaling by downregulating cGAS / STING protein expression and synergistically reduces the levels of key pro-inflammatory factors such as TNF-α, IL-1β, and IL-6, achieving multi-target and multi-level inhibition of intestinal inflammatory response in UC. This complements the barrier repair and antioxidant mechanisms of the compound, contributing to its overall therapeutic efficacy.

[0055] As one implementation method, its use includes upregulating the expression of glutathione peroxidase 4, solute carrier family 7 member 11, and / or ferritin heavy chain 1 to inhibit ferroptosis in intestinal epithelial cells. This refers to the important mechanism by which Jinbian Sanghuang powder preparations enhance intracellular antioxidant defense systems and iron metabolism homeostasis, thereby blocking ferroptosis, a novel type of programmed cell death driven by lipid peroxidation, to protect intestinal epithelial cells and reduce mucosal damage.

[0056] In the pathological process of ulcerative colitis, oxidative stress and iron metabolism disorders in the intestinal microenvironment can synergistically induce ferroptosis in intestinal epithelial cells, characterized by glutathione (GSH) depletion, loss of GPX4 activity, massive accumulation of lipid peroxidation products, and the presence of toxic ferrous ions (Fe2+). 2+ Accumulation. GPX4 is a core enzyme in resisting lipid peroxidation, and its function depends on GSH; SLC7A11 is responsible for transporting cysteine ​​into the cell to synthesize GSH; FTH1 reduces catalytically active free iron by storing iron. Therefore, simultaneously upregulating the expression of GPX4, SLC7A11, and FTH1 can synergistically inhibit ferroptosis from three key aspects: enhancing antioxidant capacity, ensuring raw materials for GSH synthesis, and reducing free iron toxicity.

[0057] The experimental data from this invention confirm the efficacy of the compound from two dimensions: protein expression and functional metabolism. In an LPS-induced Caco-2 cell injury model, LPS stimulation significantly downregulated the protein expression of GPX4, SLC7A11, and FTH1. Serum intervention containing Sanghuang powder dose-dependently reversed this trend, significantly increasing the expression levels of these three proteins. This directly demonstrates that the compound of this invention has the ability to activate the cell's intrinsic anti-ferroptosis defense program at the molecular level.

[0058] In the DSS-induced UC model in mice, the levels of MDA (representing lipid peroxidation end products) and Fe (catalyzing lipid peroxidation) in the colon tissue of the model group were significantly higher. 2+ The levels of all substances were significantly increased, while the levels of GSH, representing the core antioxidant, were significantly depleted. After treatment with Jinbian Sanghuang Powder, the Fe content in the colonic tissue was significantly reduced. 2+ With a significant decrease in MDA content and a significant recovery in GSH content, these changes in functional metabolic indicators confirm at the tissue level that the compound of this invention can effectively inhibit the core biochemical process of ferroptosis.

[0059] In summary, Jinbian Sanghuang Powder systematically enhances the ability of intestinal epithelial cells to resist ferroptosis by synergistically upregulating the expression of GPX4, SLC7A11, and FTH1. Combined with its effects of repairing the intestinal barrier and inhibiting excessive inflammation, the compound of this invention achieves a comprehensive therapeutic strategy from structural protection and inflammation control to cell survival maintenance.

[0060] As one implementation method, its uses include reducing the content of malondialdehyde and / or ferrous ions in colonic tissue, and / or increasing the content of reduced glutathione to alleviate oxidative stress damage. This refers to the core pathway by which Jinbian Sanghuang San preparations directly correct the critical redox imbalance and accumulation of toxic substances in the intestinal tract of ulcerative colitis (UC), thereby alleviating the direct damage of oxidative stress to the intestinal mucosa and creating a favorable microenvironment for tissue repair.

[0061] Oxidative stress is a key pathological component of ulcerative colitis (UC), characterized by the generation of reactive oxygen species (ROS) exceeding the body's antioxidant capacity. During this process, polyunsaturated fatty acids undergo lipid peroxidation, leading to the accumulation of malondialdehyde (MDA), a byproduct that can directly damage cell membranes and proteins. Simultaneously, metabolic disturbances result in the catalytically active release of ferrous ions (Fe²⁺). 2+ Excessive accumulation of MDA and Fe3+ leads to the generation of more highly toxic hydroxyl radicals via the Fenton reaction, exacerbating oxidative damage. Furthermore, the depletion of reduced glutathione (GSH), the most important non-enzymatic antioxidant and detoxifying agent in cells, directly weakens the cell's antioxidant defense capabilities. Therefore, simultaneously reducing MDA and Fe3+ is crucial. 2+ Enhancing GSH is a comprehensive strategy to combat oxidative stress by working together across three dimensions: eliminating toxic byproducts, reducing oxidative catalysts, and supplementing core antioxidants.

[0062] The experimental data of this invention provide conclusive evidence for this effect: In the DSS-induced UC mouse model, the MDA content in the colon tissue of the model group represented the degree of lipid peroxidation, and the Fe content represented the pro-oxidative metal load. 2+ The contents of both were significantly increased. After treatment with Jinbian Sanghuang Powder, the levels of MDA and Fe in the colon tissue were significantly increased. 2+ The content showed a significant dose-dependent decrease. This indicates that the compound of the present invention can effectively remove oxidative toxic products and reduce pro-oxidative factors.

[0063] In the same model, the GSH content in the colonic tissue of the model group was significantly depleted compared to the normal group. After intervention with Jinbian Sanghuang Powder, the GSH level was significantly restored and increased. This directly proves that the compound of this invention can rebuild the key antioxidant reserves of the intestinal mucosa.

[0064] Upregulation of SLC7A11 promotes GSH synthesis; GPX4 activity depends on GSH and is responsible for eliminating lipid peroxides (whose final product is MDA); while upregulation of FTH1 helps with iron storage and detoxification (reducing free Fe). 2+ Therefore, the compound formulation of this invention, through multi-target regulation, ultimately achieves the effects of inhibiting lipid peroxidation (reducing MDA) and chelating catalytic iron (reducing Fe) at the tissue biochemical level. 2+ The synergistic effect of increasing antioxidant capacity (GSH) effectively reduces oxidative stress damage.

[0065] In summary, Jinbian Sanghuang Powder significantly reduces MDA and Fe in colonic tissue. 2+The increased GSH content confirmed its powerful antioxidant effect at the tissue biochemical level. This effect not only directly protects intestinal epithelial cells from oxidative damage, but also provides the necessary microenvironmental basis for the aforementioned mechanisms of inhibiting inflammation, repairing the barrier, and resisting cell death, together forming a solid pharmacological basis for its multidimensional treatment of ulcerative colitis.

[0066] As one implementation method, its uses include reducing disease activity index scores and / or colonic histopathology scores. This refers to key evidence demonstrating that Jinbian Sanghuang San preparations, in treating ulcerative colitis (UC), can comprehensively and objectively reverse disease severity from two core dimensions: macroscopic clinical symptoms and microscopic tissue structure, reflecting its overall therapeutic efficacy.

[0067] The Disease Activity Index (DAI) score is a core indicator of clinical activity calculated by comprehensively considering weight loss, fecal characteristics, and rectal bleeding, directly reflecting the animal's overall disease burden and clinical manifestations. The colonic histopathology score, based on histological changes such as the depth of inflammatory cell infiltration, the degree of crypt structure destruction, and the extent of lesions, is the gold standard for assessing intestinal mucosal damage and repair. Therefore, significantly reducing both the DAI and histopathology scores simultaneously signifies that the compound formula of this invention can effectively control external clinical symptoms while promoting internal tissue structure repair, achieving an integrated effect of treating both the exterior and interior of the disease.

[0068] The experimental data of this invention confirms from multiple perspectives the improving effect of the Jinbian Sanghuang Powder compound on the overall condition of the disease: In a DSS-induced mouse ulcerative colitis (UC) model, the DAI score of the model group mice was significantly increased, accompanied by progressive weight loss. After treatment with Jinbian Sanghuang Powder, the weight loss trend of the mice was significantly curbed, and they eventually recovered their weight, with their DAI score ultimately showing a decrease compared to the model group. This demonstrates that the Jinbian Sanghuang Powder compound of this invention can rapidly alleviate the core clinical symptoms of UC.

[0069] HE staining and histological evaluation of the colon of mice after treatment revealed severe inflammatory cell infiltration, crypt structure destruction, and even loss in the model group. After intervention with Jinbian Sanghuang San, the degree of inflammatory infiltration and crypt destruction in the colonic tissue were significantly improved, and the histopathological score was significantly lower than that of the model group. This indicates that the Jinbian Sanghuang San compound can effectively promote the structural repair of damaged intestinal mucosa.

[0070] Other findings of this study, such as the ability of the Jinbian Sanghuang Powder compound to increase the number of colonic goblet cells, reduce the spleen index to improve the state of immune hyperactivity, restore colon length and reduce the intestinal weight index to alleviate tissue edema and hyperplasia, all support the improvement of the overall disease condition from different perspectives. These findings, along with the results of DAI and pathological scores, constitute a complete efficacy evaluation system.

[0071] In summary, Jinbian Sanghuang Powder significantly reduces the Disease Activity Index (DAI) score and colonic histopathology score. This result comprehensively and objectively demonstrates its clear efficacy in improving the overall condition of ulcerative colitis. This overall efficacy is the ultimate manifestation of the combined and synergistic effects of the aforementioned multiple molecular mechanisms, fully demonstrating the application value of the Jinbian Sanghuang Powder compound of this invention as a treatment for UC.

[0072] One application is to increase the number of goblet cells in the colon. This refers to the fact that the Jinbian Sanghuang powder preparation can specifically promote the proliferation and differentiation of goblet cells in the intestinal mucosa, thereby increasing the secretion of protective mucus, strengthening the chemical and physical barrier function of the intestine, and providing an important cellular basis for repairing mucosal damage in ulcerative colitis (UC).

[0073] Goblet cells are specialized secretory cells in the intestinal epithelium. Their core function is to synthesize and secrete mucins (mainly MUC2), forming a mucus layer covering the surface of the intestinal epithelium. This mucus layer is the first chemical and physical barrier of the intestinal defense system, effectively lubricating the intestinal wall, isolating microorganisms, toxins, and mechanical irritants within the intestinal lumen, and providing a habitat for symbiotic flora. In the pathological state of ulcerative colitis (UC), the number of goblet cells is often significantly reduced, the mucus layer becomes thinner or even absent, leading to direct exposure of the epithelium to a harmful environment, exacerbating inflammation and damage. Therefore, promoting goblet cell regeneration and increasing their number is a direct and crucial strategy for restoring the integrity of the mucus barrier and enhancing the inherent defensive capabilities of the mucosa.

[0074] The histological data of this invention provide conclusive evidence for this: Observation and quantitative analysis of mouse colon tissue using AB-PAS specific staining revealed that the average number of goblet cells in the colonic crypts of the DSS-induced UC model group was significantly reduced compared to the normal group. However, after intervention with Jinbian Sanghuang San, the number of goblet cells in the colon tissue showed a dose-dependent significant increase, with the number of goblet cells in the medium and high dose groups recovering to near-normal levels, an effect comparable to the positive control drug mesalazine. These results directly demonstrate that the Jinbian Sanghuang San compound of this invention can effectively reverse goblet cell depletion caused by UC and promote its regeneration.

[0075] In animal experiments, the reduction of spleen index and the increase of colonic goblet cell count were used as pharmacodynamic evaluation indicators: a decreased spleen index reflects the regulatory effect of Jinbian Sanghuang Powder compound on UC-related immune hyperactivity, corresponding to the normalization of peripheral blood lymphocyte subset ratios and a decrease in serum inflammatory factor levels in human clinical indicators; an increased number of colonic goblet cells corresponds to increased mucus layer thickness and upregulated MUC2 protein expression in human colonic mucosal biopsies, which can be verified by colonoscopy and immunohistochemical detection. The recommended daily dosage for humans was calculated based on the high dose in animal experiments, combined with a conversion factor of 0.018 between rat and human body surface area.

[0076] In summary, Jinbian Sanghuang Powder can significantly increase the number of colonic goblet cells, which is important cellular evidence for its ability to repair UC intestinal mucosal damage and enhance innate defense capabilities.

[0077] One application is to reduce the spleen index. This refers to the fact that the Jinbian Sanghuang Powder preparation can significantly improve the pathological splenomegaly associated with ulcerative colitis (UC). This is not only an important macroscopic organ indicator for evaluating drug efficacy, but also indirectly reflects the systemic regulatory effect of the Jinbian Sanghuang Powder compound on the UC-related systemic immune system hyperactivity state.

[0078] As the largest peripheral immune organ in the body, the spleen often undergoes compensatory hyperplasia and enlargement in chronic inflammatory diseases such as ulcerative colitis (UC) due to continuous antigen stimulation and systemic inflammatory responses, resulting in a significant increase in its weight (often expressed as the spleen index, i.e., spleen weight as a percentage of body weight). Splenomegaly is often accompanied by pathological changes such as congestion and hemorrhage, directly reflecting an overactivated and overloaded state of the systemic immune system. Therefore, effectively reducing the abnormally elevated spleen index in model animals means that therapeutic intervention can not only alleviate local intestinal lesions but may also help to calm systemic immune dysregulation and reduce the systemic immune stress load.

[0079] The experimental results of this invention confirm the efficacy of the Jinbian Sanghuang Powder compound from both morphological and quantitative perspectives: Anatomical observation showed that the spleens of mice in the DSS-induced UC model group were significantly enlarged, with visible deep red-black hemorrhagic foci. After treatment with Jinbian Sanghuang Powder, the degree of spleen enlargement gradually improved with increasing dosage, the area of ​​hemorrhagic foci decreased or even disappeared, and the spleen morphology in the high-dose group basically returned to normal.

[0080] Quantitative regulation of spleen index: Quantitative analysis showed that the spleen index of the model group mice was significantly higher than that of the normal group. After intervention with each dose of Jinbian Sanghuang Powder, the spleen index decreased. This data objectively confirms the ability of the Jinbian Sanghuang Powder compound of this invention to reverse pathological splenic hyperplasia.

[0081] The reduction in spleen index is closely related to the aforementioned multiple local intestinal protective effects of the Jinbian Sanghuang Powder compound. The Jinbian Sanghuang Powder compound reduces the continuous stimulation of the systemic immune system by repairing the intestinal barrier and reducing the entry of intestinal antigens and endotoxins (such as LPS) into the bloodstream. Simultaneously, its direct inhibition of the production of local and systemic inflammatory factors in the intestine also helps alleviate inflammatory hyperplasia of the spleen. Therefore, the reduction in spleen index can be considered a positive systemic effect produced by the Jinbian Sanghuang Powder compound of this invention after achieving local intestinal therapeutic effects.

[0082] In summary, Jinbian Sanghuang Powder significantly reduced the spleen index and improved the pathological state of the spleen in animal models of ulcerative colitis. This compound not only treats local intestinal lesions but may also help regulate UC-related systemic immune imbalances, demonstrating the unique characteristics of this traditional Chinese medicine compound that combines local treatment with systemic regulation, further supporting its comprehensive value as a treatment for UC.

[0083] WB Results Report: 1. Experimental Objective Western Blot was used to detect the effects of different concentrations of SHSF on the protein expression levels of cGAS, STING, ZO-1, Occludin, GPX4, SLC7A11, and FTH1 in Caco-2 cells. 2 Materials and Instruments 2.1 Experimental Apparatus. Main instruments: low-temperature high-speed centrifuge, electrophoresis apparatus, electrophoresis tank, rapid wet transfer apparatus, and chemiluminescence analyzer.

[0084] 2.2 Experimental Materials. Main materials: RIPA lysis buffer, PMSF, protein phosphatase inhibitor mixture, protease inhibitor mixture, BCA protein quantification kit, trichromatic pre-stained protein markers (13-250 kDa), methanol, 30% acylamide, 1.5M TrispH 8.8, 1.0M TrispH 6.8, BSA, 10% APS, PVDF membrane, and ECL Plus ultrasensitive luminescent solution.

[0085] Table 1 Antibody Information

[0086] 3 Experimental Methods 3.1 Experimental grouping and drug administration: A: Control; B: LPS; C: 2.5% SHSF; D: 5% SHSF; E: 10% SHSF; Preparation of serum containing Sanghuang powder Twelve male SD rats (200±20g) were randomly divided into an SHSF group (n=6) and a blank serum group (RBS) (n=6). Both groups were administered either 37.34g / kg Sanghuang powder (high-dose group) or sterile water by gavage once daily for 7 consecutive days. Rats were fasted for 12 hours before the last administration. Blood was aseptically collected from the abdominal aorta 2 hours after administration. After standing for 2 hours, the blood was centrifuged at 2500 rpm for 30 minutes, and the serum from the same group was mixed. The inactivation was then carried out at 56℃ for 30 minutes, filtered through a 0.22mm microporous membrane, and stored at -80℃ for later use.

[0087] Group 1: To clarify the dose-dependent protective effect of SHSF on epithelial cells and to clarify that LPS activates ferroptosis, and that SHSF can antagonize this effect. (5 groups) Control: 10% RBS. LPS: 10% RBS + 1 μg / mLPS. 2.5% SHSF: 1 μg / mLPS + 2.5% SHSF + 7.5% RBS. 5% SHSF: 1 μg / mLPS + 5% SHSF + 5% RBS. 10% SHSF: 1 μg / mLPS + 10% SHSF.

[0088] 3.2 Western Blot Detection 3.2.1 Protein Sample Preparation Cell protein preparation: Discard the supernatant from the cells, wash twice with PBS, centrifuge at 1000 rpm for 5 min, discard the supernatant, and place on ice.

[0089] 2. Add 600 μL of RIPA lysis buffer (containing PMSF and protease inhibitor) and lyse on ice for 30 min.

[0090] 3. Place the homogenate into a centrifuge tube, centrifuge at 12000g and 4℃ for 5 min, transfer the supernatant to a new pre-cooled centrifuge tube, and measure the concentration using a BCA kit.

[0091] 4. After determining the concentration, perform protein denaturation, add Loading Buffer (containing β-mercaptoethanol) (Loading Buffer: β-mercaptoethanol = 50:3) (volume ratio of Loading Buffer: Sample = 1:4), boil in water for 5 minutes, cool to room temperature, and store at -20℃ for later use.

[0092] 3.2.2 Determination of total protein concentration in samples (BCA method) 1. Prepare 8 ml of BCA working solution by mixing BCA reagent and Cu reagent at a volume ratio of 50:1. 2. Dilute 15 μL of the standard with PBS to 150 μL to prepare a concentration of 0.5 mg / ml; 3. Dilute the sample 2-fold and 8-fold, and add 20 μL to a 96-well plate; 4. Add 200 μL BCA working solution, incubate at 37℃ for 20 min, and measure using an ELISA reader at a wavelength of 562 nm.

[0093] 3.2.3 SDS-PAGE electrophoresis and membrane transfer Take a clean 1.5mm glass plate and install it on the glue-making frame according to the instructions.

[0094] Prepare 10%

[0095] After adding all the substances, mix thoroughly, pour into a separating gel, and then seal with an aqueous solution.

[0096]

[0097] After adding all the ingredients, mix well and pour into a concentrated gel.

[0098] Prepare a 5% concentration SDS-PAGE stacking gel and inject it into the top of the separating gel. Carefully insert a sample comb that matches the thickness of the glass plate to avoid generating air bubbles.

[0099] After the laminar gel polymerization, carefully remove the sample comb and then add 1×Tris-Gly electrophoresis buffer.

[0100] Add an appropriate amount of sample supernatant to the sample wells. Add pre-stained protein marker to the wells next to the sample. Add 1×SDS loading buffer to the wells without sample supernatant to maintain gel equilibration.

[0101] Turn on the power and set the voltage to 80V initially. Once the protein sample enters the separating gel, the voltage can be increased to 120V. Referring to the position of the pre-stained marker, stop electrophoresis when the target band enters the optimal separation zone of the gel (approximately 2 / 3 of the gel).

[0102] Open the transfer box on the tray and place a sponge pad on the inner side near the cathode.

[0103] A PVDF membrane soaked in methanol is laid on a sponge pad.

[0104] Carefully pry open the glass plate, place the gel in a tray containing purified water, cut off the separating gel containing the target band, and then place it on the PVDF membrane. There should be no air bubbles between the gel and the membrane. The membrane and gel should be approximately the same size.

[0105] Place the second sponge pad on top, so that the entire transfer layer forms a series of layers: sponge pad-PVDF film-gel-sponge pad, and then place it into the transfer tank.

[0106] Turn on the power and follow the steps below to start fast spinning:

[0107] After the transfer is complete, remove the PVDF membrane and mark it.

[0108] 3.2.4 Immunoblotting 3.2.4.1 Blocking and Antigen-Antibody Reaction Sealing: Place the PVDF membrane in an incubation box, add a sealing solution containing 5% skim milk powder, and shake on a shaker for 1.5-2 hours; After sealing, wash the membrane with TBST for 10 minutes three times. Place the membrane in an incubation box containing primary antibody dilution solution (diluted according to the antibody instructions) and incubate overnight at 4°C with shaking. The next day, remove the sample, shake at room temperature for 30 minutes, discard the primary antibody, and wash with TBST for 10 minutes × 3 times. Dilute the secondary antibody with 5% skim milk powder blocking solution and react with shaking on a shaker at room temperature for 1-2 hours. After the secondary antibody reaction is complete, the secondary antibody is recovered. Then the membrane is washed with TBST for 5-10 min × 3 times.

[0109] 3.2.4.2 ECL Chemiluminescence Analyzer Development 1) Add equal volumes of ECL luminescent reagents A and B to an EP tube and mix. 2) Lay plastic wrap on the table, transfer the PVDF film onto it, add the mixed solution to cover the surface, and allow them to fully contact and react for 3 minutes; 3) Turn on the camera first, then the computer. Open the chemicapture software, select chemical light as the imaging type, and then... Move the membrane to the center of the black tray and place it in the instrument. Select automatic exposure, take the picture, and save the image. 3.3 Image Analysis The ImageJ management system was used to acquire and process images for analysis. The grayscale value of each sample was measured using the ImageJ system as the analysis index. Three duplicate samples were tested and their average value was calculated.

[0110] 3.4 Statistical Processing Data analysis was performed using SPSS 20.0 statistical software. For continuous data across multiple groups, if the data conformed to a normal distribution and met the homogeneity of variance test, one-way ANOVA was used, with Tukey's test used for further pairwise comparisons between groups. If the data conformed to a normal distribution but the variances were unequal, Dunnett's test was used. ， The ST3 test was used; if the data did not conform to a normal distribution, the Kruskal-Wallis H test was used. The significance level was α = 0.05. All data were expressed as mean ± standard deviation. (±s) indicates that P<0.05 is statistically significant.

[0111] 4 Results and Analysis 4.1 Effects of different concentrations of SHSF on the protein expression levels of cGAS, STING, ZO-1, Occludin, GPX4, SLC7A11, and FTH1 in Caco-2 cells Depend on Figure 1-3Compared with the Control group, the expression levels of cGAS and STING proteins in Caco-2 cells of the LPS group were significantly increased (P<0.01), while the expression levels of ZO-1, Occludin, GPX4, SLC7A11, and FTH1 proteins were significantly decreased (P<0.05 or P<0.01). Compared with the LPS group, the expression levels of cGAS and STING proteins in Caco-2 cells of the 10% SHSF group were significantly decreased (P<0.01), while the expression levels of ZO-1, Occludin, GPX4, SLC7A11, and FTH1 proteins were significantly increased (P<0.01).

[0112] Figure 1 Bands representing the expression of cGAS, STING, ZO-1, Occludin, GPX4, SLC7A11, and FTH1 proteins in Caco-2 cells.

[0113] Figure 2 Expression of cGAS, STING, ZO-1, and Occludin proteins in Caco-2 cells ( ±S, n=3).

[0114] Note: Compared with the Control group, #P<0.05, ##P<0.01; compared with the LPS group, @P<0.05, @@P<0.01.

[0115] Figure 3 Expression of GPX4, SLC7A11, and FTH1 proteins in Caco-2 cells ( ±S, n=3).

[0116] Note: Compared with the Control group, #P<0.05, ##P<0.01; compared with the LPS group, @P<0.05, @@P<0.01.

[0117] AB-PAS staining results report: 1. Experimental Objective AB-PAS staining was used to observe pathological changes in mouse colon tissue. 2 Experimental Materials 2.1 Main Reagents and Instruments Experimental instruments: dehydrator, embedding machine, pathological sectioner, freezing stage, tissue spreader, oven, upright optical microscope, imaging system and panoramic scanner.

[0118] 2.2 Experimental Materials Experimental materials and reagents: neutral resin, anhydrous ethanol, xylene and PAS staining solution kit.

[0119] 3 Experimental Methods 3.1 Animal grouping: A: NC group; B: DSS group; C: DSS+SHSF-L group; D: DSS+SHSF-M group; E: DSS+SHSF-H group; F: DSS+5-ASA group; NC: Drink pure water freely for 7 days, during which time 2% cyclodextrin (by equal volume) is administered by gavage once a day; DSS: 3% DSS was given freely for 7 days, during which time an equal volume of 2% cyclodextrin was administered by gavage once a day; DSS+SHSF-L: 2% DSS was given freely for 7 days, during which time 4.03g / kg of Sanghuang powder was administered by gavage once a day; DSS+SHSF-M: 2% DSS was given freely for 7 days, during which time 8.06g / kg of Sanghuang powder was administered by gavage once a day; DSS+SHSF-H: 2% DSS was given freely for 7 days, during which time 16.12g / kg of Sanghuang powder was administered by gavage once a day; DSS+5-ASA: 2% DSS was given freely for 7 days, during which time 200 mg / kg / day of mesalazine was administered by gavage once daily.

[0120] 3.2 Tissue embedding Sampling: Fresh tissue is fixed in fixative for at least 24 hours. The tissue is removed from the fixative and trimmed in a fume hood using a scalpel. The trimmed tissue and corresponding labels are then placed in a dehydration box. Dehydration and wax impregnation: Place the dehydration box into the dehydrator and dehydrate it sequentially with alcohol in a gradient. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, and then melt paraffin I at 65℃ for 1 hour, melt paraffin II at 65℃ for 1 hour, and melt paraffin III at 65℃ for 1 hour. Embedding: The paraffin-impregnated tissue is embedded in an embedding machine. First, molten paraffin is placed into the embedding frame. Before the paraffin solidifies, the tissue is removed from the dehydration box and placed into the embedding frame according to the required embedding surface, and labeled accordingly. The tissue is then cooled on a -20°C freezing stage. After the paraffin solidifies, the paraffin block is removed from the embedding frame and trimmed. 3.3 Tissue Sections Sectioning: Place the trimmed paraffin block on a -20℃ freezing stage to cool, then place the cooled paraffin block on a paraffin microtome to section it to a thickness of 4μm. Float the sections on a 40℃ warm water spreader to flatten the tissue, then lift the tissue onto a glass slide and bake it in a 60℃ oven. After the paraffin has melted from the water, remove it and store it at room temperature for later use.

[0121] 3.4 AB-PAS staining Dewaxing: Immerse the sections in xylene for 5-10 minutes, then replace the xylene and immerse for another 5-10 minutes to remove the paraffin from the sections.

[0122] Hydration: Soak the sections in anhydrous ethanol, 95% ethanol, 80% ethanol and 70% ethanol for 3-5 minutes each in sequence to hydrate; stain the sections in PAS staining solution B for 10-15 minutes, wash with tap water and then wash twice with distilled water. Immerse the sections in PAS staining solution A for 25-30 minutes, protect from light, and rinse with running water for 5 minutes. Slices were stained with PAS staining solution C for 30 seconds, washed with tap water, differentiated with hydrochloric acid solution, washed with tap water, blued with ammonia solution, and rinsed with running water. To make the sections transparent, soak them in xylene for 5-10 minutes, then replace the xylene and soak them for another 5-10 minutes. Mounting: Use neutral resin to mount the coverslip onto the slide; Microscopic examination, image acquisition and analysis; Results interpretation: Glycogen and neutral mucus appear red, acidic mucus appears blue, mixed mucus appears blue-purple or purplish-blue, and cell nuclei appear blue.

[0123] 3.5 Statistical Analysis Data analysis was performed using SPSS 20.0 statistical software. For continuous data across multiple groups, if the data conformed to a normal distribution and met the homogeneity of variance test, one-way ANOVA was used, with Tukey's test used for further pairwise comparisons between groups. If the data conformed to a normal distribution but the variances were unequal, Dunnett's test was used. ， The ST3 test was used; if the data did not conform to a normal distribution, the Kruskal-Wallis H test was used. The significance level was α = 0.05. All data were expressed as mean ± standard deviation. (±s) indicates that P<0.05 is statistically significant.

[0124] 4 Results and Analysis 4.1 Analysis of AB-PAS staining results in colon tissue Figure 4-5 It was found that, compared with the NC group, the average number of goblet cells per crypt in the colon tissue of mice in the DSS group was significantly decreased (P < 0.01). Compared with the DSS group, the average number of goblet cells per crypt in the colon tissue of mice in the DSS+SHSF-M group, DSS+SHSF-H group, and DSS+5-ASA group was significantly increased (P < 0.05 or P < 0.01).

[0125] Figure 4 Histopathological changes in mouse colon tissue (100x, 200μm; 400x, 50μm).

[0126] Note: Yellow arrows indicate goblet cells. Figure 5 Average number of goblet cells per crypt in mouse colon tissue ( ±s, n=3).

[0127] Note: Compared with the NC group, #P<0.05, ##P<0.01; compared with the DSS group, @P<0.05, @@P<0.01.

[0128] ELISA test result report: 1. Experimental Objective ELISA was used to detect the levels of LPS, TNF-α, IL-1β, and IL-6 in mouse serum, and the kit was used to detect Fe in mouse colon tissue. 2+ Content of MDA and GSH.

[0129] 2. Instruments and reagents Experimental instruments: ELISA reader and low-temperature high-speed centrifuge.

[0130] Experimental reagents: Mouse interleukin-6 (IL-6) kit (ELISA), mouse tumor necrosis factor-α (TNF-α) kit (ELISA), research+ mouse lipopolysaccharide; endotoxin (LPS) detection kit, mouse interleukin-1β (IL-1β) kit (ELISA), malondialdehyde (MDA) assay kit, trace reduced glutathione (GSH) assay kit, and ferrous ion content detection kit.

[0131] 3 Experimental Methods 3.1 Animal grouping and administration: A: NC group; B: DSS group; C: DSS+SHSF-L group; D: DSS+SHSF-M group; E: DSS+SHSF-H group; F: DSS+5-ASA group; For details regarding grouping, dosage, and administration time, please refer to the task sheet.

[0132] 3.2 Reagent kit detection Rinse the tissue with pre-chilled PBS (0.01M, pH=7.4) to remove residual blood, weigh, and then mince the tissue. Add the minced tissue and an equivalent volume of PBS to a glass homogenizer and homogenize thoroughly on ice. To further lyse the tissue cells, the homogenate can be sonicated or subjected to repeated freeze-thaw cycles. Finally, centrifuge the homogenate at 5000×g for 5-10 min and collect the supernatant for analysis. Analyze the results based on MDA, GSH, and Fe... 2+ The kit was used to detect MDA, GSH, and Fe in mouse colon tissue. 2+ BCA content.

[0133] 3.3 ELISA detection Serum: After blood clotted naturally at room temperature for 20 minutes, centrifuged at 3500 rpm for 15 minutes, collected the supernatant, and stored the resulting serum at -80℃ for later use. The levels of TNF-α, IL-1β, IL-6, and IL-18 were measured using an ElISA kit.

[0134] Preparation: Remove the reagent kit from the refrigerator and allow it to equilibrate to room temperature for 20 minutes.

[0135] Solution preparation: Dilute the 20-fold concentrated washing solution with distilled water to a solution equal to the original concentration.

[0136] Add standards and test samples: Take a sufficient number of enzyme-labeled plates, fix them on the frame, and set up standard wells, test sample wells, and blank control wells respectively. Record the position of each well. Add 50 μL of standard to the standard wells; add 50 μL of sample diluent (stock solution or diluent) to the test sample wells. Dilute according to the dilution factor required for the sample. For example, add 40 μL of sample diluent first, and then add 10 μL of test sample. The final dilution of the sample will be 5 times. Do not add any diluent to the blank control wells.

[0137] Except for the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each of the standard and sample wells.

[0138] Incubation: Seal the reaction wells with a sealing film and incubate at 37°C in a water bath or constant temperature incubator for 60 minutes.

[0139] Washing the plate: Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry on absorbent paper, and repeat this washing process 5 times (or you can use a plate washer to wash the plate according to the instructions).

[0140] Color development: Add 50 μL of color developer A solution to each well, then add 50 μL of color developer B solution, mix with a plate mixer for 30 seconds (or gently shake and mix by hand for 30 seconds), and develop at 37°C in the dark for 15 minutes.

[0141] Termination: Remove the microplate and add 50 μL of stop solution to each well to terminate the reaction.

[0142] Measurement: Zero the instrument with a blank well. Within 15 minutes after termination, measure the absorbance (OD value) of each well at a wavelength of 450 nm. Repeat the experiment 6 times.

[0143] 3.3 Statistical Processing Data analysis was performed using SPSS 20.0 statistical software. For continuous data across multiple groups, if the data conformed to a normal distribution and met the homogeneity of variance test, one-way ANOVA was used, followed by Tukey's test for pairwise comparisons between groups. If the data conformed to a normal distribution but the variances were unequal, Dunnett's T3 test was used. If the data did not conform to a normal distribution, the Kruskal-Wallis H test was used. The significance level was set at α = 0.05. All data were expressed as mean ± standard deviation (...). (±s) indicates that P < 0.05 is statistically significant.

[0144] 4 Results and Analysis 4.1 Serum levels of LPS, TNF-α, IL-1β, and IL-6 in mice, and Fe in mouse colon tissue 2+ MDA and GSH content Depend on Figure 6 It was found that, compared with the NC group, the serum levels of LPS, TNF-α, IL-1β, and IL-6 in the DSS group mice were significantly increased (P<0.01). Compared with the DSS group, the serum levels of TNF-α, IL-1β, and IL-6 in the DSS+SHSF-L group mice were significantly decreased (P<0.05 or P<0.01), and the serum levels of LPS, TNF-α, IL-1β, and IL-6 in the DSS+SHSF-M, DSS+SHSF-H, and DSS+5-ASA groups mice were significantly decreased (P<0.01).

[0145] Depend on Figure 7 It can be seen that, compared with the NC group, the Fe in the colon tissue of the DSS group mice was higher. 2+ The levels of both MDA and GSH were significantly increased (P < 0.01), while the levels of GSH were significantly decreased (P < 0.01). Compared with the DSS group, the levels of Fe in the colon tissue of mice in the DSS+SHSF-L, DSS+SHSF-M, and DSS+SHSF-H groups were significantly higher. 2+ The levels of MDA and GSH were significantly decreased (P < 0.05 or P < 0.01) and significantly increased (P < 0.01) in the colon tissue of mice in the DSS+5-ASA group.

[0146] Figure 6 Levels of LPS, TNF-α, IL-1β, and IL-6 in mouse serum ( ±s, n=6).

[0147] Note: Compared with the NC group, #P<0.05, ##P<0.01; compared with the DSS group, @P<0.05, @@P<0.01; Figure 7 Fe in mouse colon tissue 2+ MDA and GSH content ( ±s, n=3).

[0148] Note: Compared with the NC group, #P<0.05, ##P<0.01; compared with the DSS group, @P<0.05, @@P<0.01.

[0149] Spleen Index Results Report: 1. Experimental Objective Effects of Sanghuang Powder on Spleen Index in US Model Mice; 2 Experimental Materials 2.1 Instruments Experimental apparatus: electronic balance and camera.

[0150] 3 Experimental Methods 3.1 Experimental Groups: A: NC group; B: DSS group; C: DSS+SHSF-L group; D: DSS+SHSF-M group; E: DSS+SHSF-H group; F: DSS+5-ASA group; NC: Drink pure water freely for 7 days, during which time 2% cyclodextrin (by equal volume) is administered by gavage once a day; DSS: 3% DSS was given freely for 7 days, during which time an equal volume of 2% cyclodextrin was administered by gavage once a day; DSS+SHSF-L: 2% DSS was given freely for 7 days, during which time 4.03g / kg of Sanghuang powder was administered by gavage once a day; DSS+SHSF-M: 2% DSS was given freely for 7 days, during which time 8.06g / kg of Sanghuang powder was administered by gavage once a day; DSS+SHSF-H: 2% DSS was given freely for 7 days, during which time 16.12g / kg of Sanghuang powder was administered by gavage once a day; DSS+5-ASA: 2% DSS was given freely for 7 days, during which time 200 mg / kg / day of mesalazine was administered by gavage once daily.

[0151] 3.2 Thymus and Spleen Indices After the mice were euthanized with CO2 at the end of the experiment, the spleen was removed as intact as possible, rinsed, and dried with filter paper before being weighed. Spleen index = spleen weight (g) / mouse body weight (g) × 100%.

[0152] 3.3 Statistical Processing Data analysis was performed using SPSS 20.0 statistical software. For continuous data across multiple groups, if the data conformed to a normal distribution and met the homogeneity of variance test, one-way ANOVA was used, followed by Tukey's test for pairwise comparisons between groups. If the data conformed to a normal distribution but the variances were unequal, Dunnett's T3 test was used. If the data did not conform to a normal distribution, the Kruskal-Wallis H test was used. The significance level was set at α = 0.05. All data were expressed as mean ± standard deviation (...). (±s) indicates that P < 0.05 is statistically significant.

[0153] 4 Results and Analysis 4.1 Analysis of Spleen Index Results Depend on Figure 8 It was found that the spleen tissue of mice in the NC group had a regular appearance, a reddish color, and a uniform texture, without obvious hemorrhage, swelling, or abnormal lesions. The spleen tissue of mice in the DSS group was slightly enlarged, with localized deep red-black hemorrhages. Compared with the DSS group, the spleen tissue of mice in the DSS+SHSF-L group was slightly enlarged, and the area of ​​the deep red-black hemorrhages was slightly reduced. The degree of spleen enlargement in mice in the DSS+SHSF-M group was improved, and the area of ​​the deep red-black hemorrhages was further reduced. The spleen tissue of mice in the DSS+SHSF-H group and the DSS+5-ASA group had normal morphology, a reddish color, and the deep red-black hemorrhages disappeared.

[0154] Depend on Figure 9 It was found that the spleen index of mice in the DSS group was significantly increased compared with that in the NC group (P<0.01). Compared with the DSS group, the spleen index of mice in the DSS+SHSF-L group, DSS+SHSF-M group, DSS+SHSF-H group, and DSS+5-ASA group was significantly decreased (P<0.05 or P<0.01).

[0155] Figure 8 Gross morphological observation of mouse spleen tissue.

[0156] Figure 9 Changes in the spleen index in mice ( ±s, n=6).

[0157] Note: Compared with the NC group, #P<0.05, ##P<0.01; compared with the DSS group, @P<0.05, @@P<0.01.

[0158] Weight Result Report: 1. Experimental objective: To measure the body weight of mice in each group.

[0159] 2 Experimental Materials 2.1 Instruments, Experimental Instruments: Electronic balance 3 Experimental Methods 3.1 Animal grouping and administration: A: NC group; B: DSS group; C: DSS+SHSF-L group; D: DSS+SHSF-M group; E: DSS+SHSF-H group; F: DSS+5-ASA group; NC: Drink pure water freely for 7 days, during which time 2% cyclodextrin (by equal volume) is administered by gavage once a day; DSS: 3% DSS was given freely for 7 days, during which time an equal volume of 2% cyclodextrin was administered by gavage once a day; DSS+SHSF-L: 2% DSS was given freely for 7 days, during which time 4.03g / kg of Sanghuang powder was administered by gavage once a day; DSS+SHSF-M: 2% DSS was given freely for 7 days, during which time 8.06g / kg of Sanghuang powder was administered by gavage once a day; DSS+SHSF-H: 2% DSS was given freely for 7 days, during which time 16.12g / kg of Sanghuang powder was administered by gavage once a day; DSS+5-ASA: 2% DSS was given freely for 7 days, during which time 200 mg / kg / day of mesalazine was administered by gavage once daily.

[0160] 3.2 Weight Measurement After modeling, the weight of all mice was measured daily for a week, for a total of 8 days.

[0161] The percentage difference between the mouse's body weight on day 0 and day 100% is calculated as follows: (Mouse body weight on day 100% - Mouse body weight on day 0) / Mouse body weight on day 0 × 100% 3.3 Statistical Processing SPSS 20.0 statistical software was used for data analysis. For continuous data among multiple groups that conformed to a normal distribution, repeated measures ANOVA was used for comparisons between groups. When the interaction was statistically significant, simple effects analysis was performed. When the interaction was not statistically significant but the main effect was statistically significant, the Bonferroni method was used for comparisons between time points within each group. All data are expressed as mean ± standard deviation. (±s) indicates that P < 0.05 is statistically significant.

[0162] 4 Results and Analysis 4.1 Changes in body weight of mice in each group Depend on Figure 10It was found that, compared with the NC group, the percentage change in body weight of mice in the DSS group was significantly lower on days 3, 5, 6, and 7 (P < 0.01). Compared with the DSS group, the percentage change in body weight of mice in the DSS+SHSF-H group was significantly higher on day 6 (P < 0.05), and the percentage change in body weight of mice in the DSS+SHSF-M group, DSS+SHSF-H group, and DSS+5-ASA group was significantly higher on day 7 (P < 0.05 or P < 0.01).

[0163] Figure 10 Changes in body weight of mice in each group ( ±s, n=6).

[0164] Note: Compared with the NC group, #P<0.05, ##P<0.01; compared with the DSS group, @P<0.05, @@P<0.01.

[0165] Immunohistochemistry results report: 1. Experimental Objective Immunohistochemical staining was performed to detect the expression levels of ZO-1 and occludin in mouse colon tissue; 2 Experimental Materials 2.1 Main Reagents and Instruments Experimental instruments: dehydrator, embedding machine, pathological sectioner, freezing stage, tissue spreader, oven, coverslips, microwave oven, decolorizing shaker, vortex mixer, handheld centrifuge, microscope and imaging system (Nikon DS-U3, Japan).

[0166] Experimental reagents: anhydrous ethanol, xylene, sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, anhydrous, 3% H2O2, BSA, Tris-base, EDTA, hematoxylin staining solution, differentiation solution, blueing solution, and neutral resin.

[0167] Antibody Information Table

[0168] 3 Experimental Methods 3.1 Animal grouping: A: Normal group; B: Model group; C: Mesalazine group; D: Phellinus linteus aqueous extract group; E: Phellinus linteus polysaccharide group; F: Phellinus linteus total flavonoids group; G: Phellinus linteus powder group; Normal group: Daily drinking water was purified water, and PBS intervention was performed for 7 days; Model group: Drink purified water containing 2% DSS for 7 days, and PBS intervention for 7 days; Mesalazine group: Drink purified water containing 2% DSS for 7 days, and simultaneously intervene with the positive control drug mesalazine for 7 days, 500 mg / kg / day, by gavage; Phellinus linteus water extract group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Phellinus linteus water extract for 7 days, 400mg / kg / d, by gavage; Phellinus linteus polysaccharide group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Phellinus linteus polysaccharide intervention for 7 days, 400mg / kg / d, by gavage; The total flavonoids group of Phellinus linteus: drank purified water containing 2% DSS for 7 days, and was simultaneously treated with total flavonoids of Phellinus linteus for 7 days, 200mg / kg / day, by gavage.

[0169] Sanghuang Powder Group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Sanghuang Powder for 7 days, 8.06g / kg, by gavage.

[0170] 3.2 Tissue embedding Sampling: Fresh tissue is fixed in fixative for at least 24 hours. The tissue is removed from the fixative and trimmed in a fume hood using a scalpel. The trimmed tissue and corresponding labels are then placed in a dehydration box. Dehydration and wax impregnation: Place the dehydration box into the dehydrator and dehydrate it sequentially with alcohol in a gradient. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, 65°C melted paraffin I for 1 hour, 65°C melted paraffin II for 1 hour, 65°C melted paraffin III for 1 hour; Embedding: The paraffin-impregnated tissue is embedded in an embedding machine. First, molten paraffin is placed into the embedding frame. Before the paraffin solidifies, the tissue is removed from the dehydration box and placed into the embedding frame according to the required embedding surface, and labeled accordingly. The tissue is then cooled on a -20°C freezing stage. After the paraffin solidifies, the paraffin block is removed from the embedding frame and trimmed. 3.3 Tissue Sections Sectioning: Place the trimmed paraffin block on a -20℃ freezing stage to cool, then place the cooled paraffin block on a paraffin microtome to section it to a thickness of 4μm. Float the sections on a 40℃ warm water spreader to flatten the tissue, then lift the tissue onto a glass slide and bake it in a 60℃ oven. After the paraffin has melted from the water, remove it and store it at room temperature for later use.

[0171] 3.4 Immunohistochemical detection Baking paraffin slices at 62℃ for 1 hour.

[0172] Dewaxing of sections: The tissue microarray was immersed in xylene for 20 minutes, then the xylene was replaced and the microarray was immersed for another 20 minutes, then the xylene was replaced and the microarray was immersed for another 20 minutes. Hydration: Immerse in anhydrous ethanol for 1 min; immerse in anhydrous ethanol for 1 min; immerse in 95% ethanol for 1 min; immerse in 95% ethanol for 1 min; immerse in 70% ethanol for 1 min; rinse with tap water for 5 minutes; rinse with distilled water. Add one drop or 100 μL of hydrogen peroxide blocking solution to each tablet and incubate at room temperature for 10 minutes to block the activity of endogenous peroxidase.

[0173] Wash three times with PBS, 5 minutes each time; Heat antigen retrieval (1mmol Tris-EDTAPH=9.0): Place the washed tablets on a plastic rack and immerse them in the boiling antigen retrieval solution. Boil for 15 minutes, then keep warm for 15 minutes. After turning off the power, allow them to cool naturally.

[0174] Wash three times with PBS, 5 minutes each time; Remove PBS, add one drop or 100 μL of 5% BSA blocking solution to each slide, and incubate at room temperature for 20 minutes. Remove serum, add 1 drop or 50 μL of primary antibody diluted with primary antibody dilution buffer to each slide, and incubate overnight at 4°C; The next day, remove the humidifier and leave it at room temperature for 1 hour or at 37°C for 30 minutes. Wash three times with PBS, 5 minutes each time; Remove PBS, add one drop or 50 μL of labeled secondary antibody to each slide, and incubate at 37°C for 30 min; Wash three times with PBS, 5 minutes each time; Remove PBS, add DAB to each slide for staining, and monitor the staining degree under a microscope (stop staining immediately when yellow particles or flaky precipitates appear). Rinse thoroughly with tap water to remove any residual DAB solution, counterstain with hematoxylin for 30 seconds, and rinse with tap water for 5 minutes. Hydrochloric acid and alcohol differentiation for 1 second, then rinse with tap water for 10 minutes to restore blue color; Dehydration: Soak in 70% ethanol for 3 minutes; Soak in 95% ethanol for 3 minutes; Soak in 95% ethanol for 3 minutes; Soak twice in anhydrous ethanol for 3 minutes each time; Transparent: The tissue was immersed in xylene for 5 minutes three times; Mounting: Mount with neutral resin and cover with a coverslip; Microscopic examination: Observe under a microscope and record the results.

[0175] 3.5 Image Acquisition and Data Analysis 1. Result interpretation: A positive expression is indicated by the appearance of yellow or brownish-yellow (or even brown) cytoplasm or cell membrane under a microscope.

[0176] 2. Data Analysis: Images were acquired and processed using a Nikon E100 and Nikon DS-U3 digital camera, K-Viewer pathology analysis software, and Image-ProPlus 6.0 management system for semi-quantitative analysis. The IOD (incremental optical density) and Area (sample area at 200x magnification) of each sample were measured using a computer IPP 6.0 system, and the IOD / area ratio was calculated as a semi-quantitative analysis indicator.

[0177] 3.6 Statistical Processing Data analysis was performed using SPSS 20.0 statistical software. For continuous data across multiple groups, if the data conformed to a normal distribution and met the homogeneity of variance test, one-way ANOVA was used, followed by Tukey's test for pairwise comparisons between groups. If the data conformed to a normal distribution but the variances were unequal, Dunnett's T3 test was used. If the data did not conform to a normal distribution, the Kruskal-Wallis H test was used. The significance level was set at α = 0.05. All data were expressed as mean ± standard deviation (...). (±s) indicates that P<0.05 is statistically significant.

[0178] 4 Results and Analysis 4.1 Immunohistochemical detection of ZO-1 and occludin expression levels in mouse colon tissue Depend on Figure 11-13 It was found that, compared with the normal group, the AOD values ​​of ZO-1 and occludin in the colon tissue of mice in the model group were significantly reduced, i.e., their expression levels were significantly decreased (P<0.01); compared with the model group, the AOD values ​​of ZO-1 in the colon tissue of mice in the mesalazine group, Sanghuang water extract group, Sanghuang polysaccharide group, Sanghuang total flavonoids group, and Sanghuang powder group were significantly increased, i.e., their expression levels were significantly increased (P<0.05 or P<0.01); compared with the model group, the AOD values ​​of occludin in the colon tissue of mice in the mesalazine group, Sanghuang polysaccharide group, Sanghuang total flavonoids group, and Sanghuang powder group were significantly increased, i.e., their expression levels were significantly increased (P<0.05 or P<0.01).

[0179] Figure 11 Expression levels of ZO-1 in mouse colon tissue (200-fold: 100 μm; 400-fold: 50 μm).

[0180] Figure 12 Expression levels of occludin in mouse colon tissue (200-fold: 100 μm; 400-fold: 50 μm).

[0181] Figure 13 Expression levels of ZO-1 and occludin in mouse colon tissue ( ±s, n=8).

[0182] Note: Compared with the normal group, ▲P<0.05, ▲▲P<0.01; compared with the model group, ★P<0.05, ★★P<0.01.

[0183] HE staining results report: 1. Experimental Objective HE staining was used to observe pathological changes in mouse colon tissue; 2 Experimental Materials 2.1 Main Reagents and Instruments Table 2.1 Experimental instruments: dehydrator, embedding machine, pathological sectioner, freezing stage, tissue spreader, oven, imaging system (Nikon DS-Fi2), panoramic scanner and upright optical microscope.

[0184] 2.2 Experimental Materials Table 2.2 Experimental materials and reagents: neutral resin, anhydrous ethanol, xylene, sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate (anhydrous), hematoxylin, eosin, differentiation solution and blueing solution.

[0185] 3 Experimental Methods 3.1 Animal grouping: A: Normal group; B: Model group; C: Mesalazine group; D: Phellinus linteus aqueous extract group; E: Phellinus linteus polysaccharide group; F: Phellinus linteus total flavonoids group; G: Phellinus linteus powder group; Normal group: Daily drinking water was purified water, and PBS intervention was performed for 7 days; Model group: Drink purified water containing 2% DSS for 7 days, and PBS intervention for 7 days; Mesalazine group: Drink purified water containing 2% DSS for 7 days, and simultaneously intervene with the positive control drug mesalazine for 7 days, 500 mg / kg / day, by gavage; Phellinus linteus water extract group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Phellinus linteus water extract for 7 days, 400mg / kg / d, by gavage; Phellinus linteus polysaccharide group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Phellinus linteus polysaccharide intervention for 7 days, 400mg / kg / d, by gavage; The total flavonoids group of Phellinus linteus: drank purified water containing 2% DSS for 7 days, and was simultaneously treated with total flavonoids of Phellinus linteus for 7 days, 200mg / kg / day, by gavage.

[0186] Sanghuang Powder Group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Sanghuang Powder for 7 days, 8.06g / kg, by gavage.

[0187] 3.2 Tissue embedding Sampling: Fresh tissue is fixed in fixative for at least 24 hours. The tissue is removed from the fixative and trimmed in a fume hood using a scalpel. The trimmed tissue and corresponding labels are then placed in a dehydration box. Dehydration and wax impregnation: Place the dehydration box into the dehydrator and dehydrate it sequentially with alcohol in a gradient. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, 65°C melted paraffin I for 1 hour, 65°C melted paraffin II for 1 hour, 65°C melted paraffin III for 1 hour; Embedding: The paraffin-impregnated tissue is embedded in an embedding machine. First, molten paraffin is placed into the embedding frame. Before the paraffin solidifies, the tissue is removed from the dehydration box and placed into the embedding frame according to the required embedding surface, and labeled accordingly. The tissue is then cooled on a -20°C freezing stage. After the paraffin solidifies, the paraffin block is removed from the embedding frame and trimmed. 3.3 Tissue Sections Sectioning: Place the trimmed paraffin block on a -20℃ freezing stage to cool, then place the cooled paraffin block on a paraffin microtome to section it to a thickness of 4μm. Float the sections on a 40℃ warm water spreader to flatten the tissue, then lift the tissue onto a glass slide and bake it in a 60℃ oven. After the paraffin has melted from the water, remove it and store it at room temperature for later use.

[0188] 3.4 HE staining Dewaxing paraffin sections to water: Place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then wash with tap water. Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue back solution, and rinse with running water. Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol for 5 min each, and then stained in eosin staining solution for 5 min. Dehydration and mounting: The sections were sequentially immersed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min. After clearing, the sections were mounted with neutral resin. Microscopic examination, image acquisition and analysis; Result interpretation: The cell nucleus appears blue, and the cytoplasm appears red.

[0189] 3.5 HE staining score of colon tissue Colonic injury was scored based on indicators such as inflammation status, lesion depth, crypt destruction, and lesion extent, as shown in Table 3.1. The sum of the scores for each item constitutes the colonic histopathological score.

[0190] Table 3.1 Scoring criteria for colonic tissue pathological damage

[0191] 3.6 Statistical Processing Data analysis was performed using SPSS 20.0 statistical software. For continuous data across multiple groups, if the data conformed to a normal distribution and met the homogeneity of variance test, one-way ANOVA was used, followed by Tukey's test for pairwise comparisons between groups. If the data conformed to a normal distribution but the variances were unequal, Dunnett's T3 test was used. If the data did not conform to a normal distribution, the Kruskal-Wallis H test was used. The significance level was set at α = 0.05. All data were expressed as mean ± standard deviation (...). (±s) indicates that P<0.05 is statistically significant.

[0192] 4 Results and Analysis 4.1 Analysis of HE staining results of mouse colon tissue Depend on Figure 14 It can be seen that the colon tissue of the normal group mice was basically normal with no obvious damage and only a few inflammatory cells were occasionally seen; the colon tissue of the model group mice was severely damaged, with a large number of inflammatory cells infiltrating, large areas of crypt destruction, and lesions invading the mucosal layer; compared with the model group, the colon tissue damage of mice in the mesalazine group, Sanghuang polysaccharide group, Sanghuang total flavonoids group and Sanghuang powder group was restored, and the inflammatory cell infiltration, lesion depth and crypt destruction were all reduced to varying degrees.

[0193] From the table and Figure 15 It was found that, compared with the normal group, the HE staining score of the colon tissue of mice in the model group was significantly increased (P<0.01); compared with the model group, the HE staining score of the colon tissue of mice in the total flavonoids of Sanghuang and the Sanghuang powder group was significantly decreased (P<0.05).

[0194] HE staining score of mouse colon tissue ( ±s, n=8)

[0195] Note: Compared with the normal group, ▲P<0.05, ▲▲P<0.01; compared with the model group, ★P<0.05, ★★P<0.01; Figure 14 Histopathological changes in mouse colon tissue (40-fold: 500 μm; 200-fold: 100 μm).

[0196] Figure 15 semi-quantitative HE score of mouse colon tissue ( ±s, n=8).

[0197] Note: Compared with the normal group, ▲P<0.05, ▲▲P<0.01; compared with the model group, ★P<0.05, ★★P<0.01.

[0198] Test kit result report: Experimental Objective MPO activity in mouse colon tissue was detected using an MPO kit.

[0199] 2 Experimental Materials 2.1 Main Reagents and Instruments Main instruments: microplate reader and low-temperature high-speed centrifuge.

[0200] Main reagents and consumables: Myeloperoxidase (MPO) activity assay kit and PBS (1×).

[0201] 3 Experimental Methods 3.1 Experimental Groups: A: Normal Group; B: Model Group; C: Mesalazine Group; D: Phellinus linteus aqueous extract Group; E: Phellinus linteus polysaccharide Group; F: Phellinus linteus total flavonoids Group; G: Phellinus linteus powder group; Normal group: Daily drinking water was purified water, and PBS intervention was performed for 7 days; Model group: Drink purified water containing 2% DSS for 7 days, and PBS intervention for 7 days; Mesalazine group: Drink purified water containing 2% DSS for 7 days, and simultaneously intervene with the positive control drug mesalazine for 7 days, 500 mg / kg / day, by gavage; Phellinus linteus water extract group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Phellinus linteus water extract for 7 days, 400mg / kg / d, by gavage; Phellinus linteus polysaccharide group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Phellinus linteus polysaccharide intervention for 7 days, 400mg / kg / d, by gavage; The total flavonoids group of Phellinus linteus: drank purified water containing 2% DSS for 7 days, and was simultaneously treated with total flavonoids of Phellinus linteus for 7 days, 200mg / kg / day, by gavage.

[0202] Sanghuang Powder Group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Sanghuang Powder for 7 days, 8.06g / kg, by gavage.

[0203] 3.2 Reagent kit detection All mice were anesthetized with isoflurane, and the abdomen was opened. The colon was removed, and reagent II was added at a ratio of 1:10 (g:mL). After homogenization on ice, the mixture was centrifuged at 12000g for 10 min at 4°C. The precipitate was discarded, and the supernatant was placed on ice for testing. The MPO content in the sample was determined according to the kit instructions.

[0204] 3.3 Statistical Processing Data analysis was performed using SPSS 20.0 statistical software. For multiple groups of continuous data, if they conformed to a normal distribution and met the homogeneity of variance test, one-way ANOVA was used, followed by the Turkey test for pairwise comparisons between groups. If they conformed to a normal distribution but had unequal variances, Dunnett's T3 test was used. If they did not conform to a normal distribution, the Kruskal-Wallis H test was used. The significance level was set at α = 0.05. All data were expressed as mean ± standard deviation (...). (±s) indicates that P<0.05 is statistically significant.

[0205] 4 Experimental Results 4.1 MPO activity in mouse colon tissue From the table and Figure 16 It was found that, compared with the normal group, the serum MPO activity of mice in the model group was significantly reduced (P<0.01). Compared with the model group, the serum MPO activity of mice in the mesalazine group, Sanghuang polysaccharide group, Sanghuang total flavonoids group and Sanghuang powder group was significantly increased (P<0.05 or P<0.01).

[0206] Table 4.1 MPO activity in mouse colon tissue ( ±s, n=8)

[0207] Note: Compared with the normal group, ▲P<0.05, ▲▲P<0.01; compared with the model group, ★P<0.05, ★★P<0.01; Figure 16 MPO activity in mouse colon tissue ( ±s, n=8).

[0208] Note: Compared with the normal group, ▲P<0.05, ▲▲P<0.01; compared with the model group, ★P<0.05, ★★P<0.01.

[0209] Report on colon length and intestinal weight index: 1. Experimental Objective Detect the colon length of mice and calculate the intestinal weight index; 2 Experimental Materials 2.1 Main Reagents and Instruments Experimental apparatus: electronic balance.

[0210] 3 Experimental Methods 3.1 Animal grouping and administration: A: Normal group; B: Model group; C: Mesalazine group; D: Phellinus linteus aqueous extract group; E: Phellinus linteus polysaccharide group; F: Phellinus linteus total flavonoids group; G: Phellinus linteus powder group; Normal group: Daily drinking water was purified water, and PBS intervention was performed for 7 days; Model group: Drink purified water containing 2% DSS for 7 days, and PBS intervention for 7 days; Mesalazine group: Drink purified water containing 2% DSS for 7 days, and simultaneously intervene with the positive control drug mesalazine for 7 days, 500 mg / kg / day, by gavage; Phellinus linteus water extract group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Phellinus linteus water extract for 7 days, 400mg / kg / d, by gavage; Phellinus linteus polysaccharide group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Phellinus linteus polysaccharide intervention for 7 days, 400mg / kg / d, by gavage; The total flavonoids group of Phellinus linteus: drank purified water containing 2% DSS for 7 days, and was simultaneously treated with total flavonoids of Phellinus linteus for 7 days, 200mg / kg / day, by gavage.

[0211] Sanghuang Powder Group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Sanghuang Powder for 7 days, 8.06g / kg, by gavage.

[0212] 3.2 Colon length and intestinal weight index All mice were anesthetized, their abdomens were dissected, the colons were removed, the length of the colons was recorded and photographed, and the feces were removed, the mass of the colons was weighed, and the intestinal weight index was calculated.

[0213] Intestinal weight index % = (colon weight / body weight) × 100% 3.3 Statistical Processing Data analysis was performed using SPSS 20.0 statistical software. For continuous data across multiple groups, if the data conformed to a normal distribution and met the homogeneity of variance test, one-way ANOVA was used, followed by Tukey's test for pairwise comparisons between groups. If the data conformed to a normal distribution but the variances were unequal, Dunnett's T3 test was used. If the data did not conform to a normal distribution, the Kruskal-Wallis H test was used. The significance level was set at α = 0.05. All data were expressed as mean ± standard deviation (...). (±s) indicates that P<0.05 is statistically significant.

[0214] 4 Results and Analysis 4.1 Analysis of Colon Length and Intestinal Weight Index Results From the table and Figure 17-18 It was found that, compared with the normal group, the colon length of mice in the model group was significantly decreased (P<0.01). Compared with the normal group, the intestinal weight index of mice in the model group was significantly increased (P<0.01); compared with the model group, the intestinal weight index of mice in the mesalazine group, the total flavonoids of Sanghuang group, and the Sanghuang powder group was significantly decreased (P<0.05 or P<0.01).

[0215] Table of mouse colon length and intestinal weight index ( ±s, n=8)

[0216] Figure 17 Mouse colon diagram Figure 18 Mouse colon length and intestinal weight index ( ±s, n=8) Note: Compared with the normal group, ▲P<0.05, ▲▲P<0.01; compared with the model group, ★P<0.05, ★★P<0.01.

[0217] Weight and DAI score report: 1. Experimental Objective The mice were weighed and scored using the Disease Activity Index (DAI). 2 Experimental Materials 2.1 Experimental Apparatus, Electronic Balance 3 Experimental Methods 3.1 Animal grouping and administration: A: Normal group; B: Model group; C: Mesalazine group; D: Phellinus linteus aqueous extract group; E: Phellinus linteus polysaccharide group; F: Phellinus linteus total flavonoids group; G: Phellinus linteus powder group; Normal group: Daily drinking water was purified water, and PBS intervention was performed for 7 days; Model group: Drink purified water containing 2% DSS for 7 days, and PBS intervention for 7 days; Mesalazine group: Drink purified water containing 2% DSS for 7 days, and simultaneously intervene with the positive control drug mesalazine for 7 days, 500 mg / kg / day, by gavage; Phellinus linteus water extract group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Phellinus linteus water extract for 7 days, 400mg / kg / d, by gavage; Phellinus linteus polysaccharide group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Phellinus linteus polysaccharide intervention for 7 days, 400mg / kg / d, by gavage; The total flavonoids group of Phellinus linteus: drank purified water containing 2% DSS for 7 days, and was simultaneously treated with total flavonoids of Phellinus linteus for 7 days, 200mg / kg / day, by gavage.

[0218] Sanghuang Powder Group: Drink purified water containing 2% DSS for 7 days, and simultaneously receive Sanghuang Powder for 7 days, 8.06g / kg, by gavage.

[0219] 3.2 Mouse weight detection and DAI score The weight of the mice was recorded daily for 7 days, starting from day 1 of the experiment.

[0220] Disease Activity Index (DAI) scores were calculated after drug intervention ended. DAI score = (weight loss score + stool consistency score + rectal bleeding score) / 3.

[0221] Table DAI Scoring Criteria

[0222] 3.3 Statistical Processing SPSS 20.0 statistical software was used for data analysis. A test of sphericity was performed on the quantitative data among multiple groups. If P > 0.05, one-way ANOVA was performed; if P < 0.05, repeated measures ANOVA was performed. The significance level was set at α = 0.05. All data are expressed as mean ± standard deviation (SD). (±s) indicates that P<0.05 is statistically significant.

[0223] 4 Results and Analysis 4.1 Results of mouse body weight and DAI score detection From the table and Figure 19 It was found that, compared with the normal group, the body weight of mice in the model group decreased significantly on days 5, 6, and 7 (P<0.01); compared with the model group, the body weight of mice in the Sanghuang water extract group, Sanghuang polysaccharide group, Sanghuang total flavonoids group, and Sanghuang powder group all increased significantly on day 4 (P<0.05 or P<0.01); compared with the model group, the body weight of mice in the mesalazine group, Sanghuang water extract group, Sanghuang polysaccharide group, Sanghuang total flavonoids group, and Sanghuang powder group all increased significantly on days 5, 6, and 7 (P<0.01).

[0224] From the table and Figure 20 It was found that, compared with the normal group, the DAI score of mice in the model group was significantly increased (P<0.01); compared with the model group, the DAI scores of mice in the mesalazine group, Sanghuang polysaccharide group, Sanghuang total flavonoids group and Sanghuang powder group were all significantly decreased (P<0.01).

[0225] Table of mouse weight ( ±s, n=8)

[0226] Note: Compared with the normal group, ▲P<0.05, ▲▲P<0.01; compared with the model group, ★P<0.05, ★★P<0.01; Table of mouse DAI scores ( ±s, n=8)

[0227] Note: Compared with the normal group, ▲P<0.05, ▲▲P<0.01; compared with the model group, ★P<0.05, ★★P<0.01; Figure 19 Mouse weight ( ±s, n=8).

[0228] Note: Compared with the normal group, ▲P<0.05, ▲▲P<0.01; compared with the model group, ★P<0.05, ★★P<0.01; Figure 20 Mouse DAI score ( ±s, n=8).

[0229] Note: Compared with the normal group, ▲P<0.05, ▲▲P<0.01; compared with the model group, ★P<0.05, ★★P<0.01.

[0230] Quality control methods for the medicine of this invention: Appearance: The powder is a yellowish-brown to brownish-yellow powder with a slightly fragrant odor and a slightly bitter taste; Identification: Thin-layer chromatography (TLC) is used to identify Phellinus linteus (characteristic spots are polysaccharide components), Coptis chinensis (characteristic spots are berberine), and Angelica sinensis (characteristic spots are ferulic acid); Content determination: HPLC method is used to determine that each gram of powder contains ≥1.0 mg of baicalin, ≥0.2 mg of berberine, and ≥0.1 mg of ferulic acid; Stability: After the powder is sealed and stored at 25°C and 60% relative humidity for 24 months, the content decrease rate is ≤10%, and there is no moisture absorption or clumping in the appearance; Extract preparations should be sealed and protected from light, and the shelf life is 18 months. The specific parameters for HPLC detection were as follows: the column was an Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm), and the column temperature was 30 ℃; the mobile phase for baicalin detection was methanol-0.4% phosphoric acid solution (47:53), the flow rate was 1.0 mL / min, and the detection wavelength was 280 nm; the mobile phase for berberine detection was acetonitrile-0.05 mol / L potassium dihydrogen phosphate solution (50:50, containing 0.2% sodium dodecyl sulfate), the flow rate was 1.0 mL / min, and the detection wavelength was 345 nm; and the mobile phase for ferulic acid detection was methanol-water-glacial acetic acid (30:68:2), the flow rate was 0.8 mL / min, and the detection wavelength was 320 nm.

[0231] Safety verification: ICR mice were administered the maximum tolerated dose of 200 g / kg by gavage, with no deaths after 14 days and normal liver and kidney function; Long-term toxicity: SD rats were administered 4.03 / 8.06 / 16.12 g / kg by gavage, with no cumulative toxicity after 12 weeks and normal organ pathology.

[0232] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. The application of a Jinbian Sanghuang powder preparation for treating ulcerative colitis, characterized in that, include: The Jinbian Sanghuang Powder preparation is made from the following raw materials in parts by weight: Phellinus linteus 20-40 parts, Sanguisorba officinalis 0.6-1.2 parts, Coptis chinensis 0.6-1.2 parts, Angelica sinensis 20-40 parts, Scutellaria baicalensis 10-20 parts, Glycyrrhiza uralensis (processed) 10-20 parts; The preparation method of the drug includes: crushing and sieving the above-mentioned raw materials to make a powder.

2. The application of the Jinbian Sanghuang Powder preparation for treating ulcerative colitis according to claim 1, characterized in that: The Jinbian Sanghuang powder preparation is used in the form of its water extract, alcohol extract, polysaccharide-rich fraction, or total flavonoid-rich fraction.

3. The application of the Jinbian Sanghuang Powder preparation for treating ulcerative colitis according to claim 2, characterized in that: The applications include upregulating the expression of intestinal epithelial tight junction proteins ZO-1 and / or Occludin to repair intestinal barrier function.

4. The application of the Jinbian Sanghuang Powder preparation for treating ulcerative colitis according to claim 2, characterized in that: The uses include downregulating the expression of cGAS and / or STING proteins, and / or reducing the levels of one or more of tumor necrosis factor-α, interleukin-1β, and interleukin-6 to suppress intestinal inflammatory responses.

5. The application of the Jinbian Sanghuang Powder preparation for treating ulcerative colitis according to claim 2, characterized in that: The applications include upregulating the expression of glutathione peroxidase 4, solute carrier family 7 member 11, and / or ferritin heavy chain 1. It inhibits ferroptosis in intestinal epithelial cells.

6. The application of the Jinbian Sanghuang Powder preparation for treating ulcerative colitis according to claim 2, characterized in that: The uses include reducing malondialdehyde and / or ferrous ions in colon tissue, and / or increasing reduced glutathione levels to mitigate oxidative stress damage.

7. The application of the Jinbian Sanghuang Powder preparation for treating ulcerative colitis according to claim 1, characterized in that: The uses include reducing disease activity index scores and / or colon histopathology scores.

8. The application of the Jinbian Sanghuang Powder preparation for treating ulcerative colitis according to claim 1, characterized in that: The uses include increasing the number of colonic goblet cells.

9. The application of the Jinbian Sanghuang Powder preparation for treating ulcerative colitis according to claim 1, characterized in that: The uses include lowering the spleen index.

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