Use of a Lianmei granule in the preparation of a medicament for preventing or treating nephropathy

Abstract

The application discloses application of Lianmei granules in preparation of medicines for preventing or treating nephropathy, and belongs to the technical field of medicines. The application proposes the application of the Lianmei granules in preparation of medicines for improving nephropathy, and specifically, the Lianmei granules can be used for preparing medicines for improving at least one of glomerular hypertrophy, tubular epithelial cell vacuolar degeneration, tubular lumen stenosis, focal tubular atrophy, local glomerular basement membrane thickening and foot process fusion, reducing deposition of renal interstitial collagen fibers and / or improving intestinal microenvironment and reinforcing intestinal mucosal barrier. The application provides a new medicine approach for treating and preventing nephropathy, and has high popularization and application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of Lianmei granules in the preparation of drugs for the prevention or treatment of kidney disease. Background Technology

[0002] Nephropathy refers to various diseases of the kidneys. The clinical manifestations of chronic kidney disease (CKD) vary at different stages. Before stage 3 CKD, patients may have no symptoms or only mild discomfort such as fatigue, lower back pain, and increased nocturia; a few patients may experience loss of appetite, metabolic acidosis, and mild anemia. After stage 3 CKD, the above symptoms become more pronounced, and further aggravated after entering the renal failure stage. Sometimes, acute heart failure, severe hyperkalemia, gastrointestinal bleeding, central nervous system disorders, and even life-threatening conditions may occur.

[0003] Lianmei granules are a traditional Chinese medicine made from dried plum, coptis root, rehmannia root, ophiopogon root or lotus seed heart, donkey-hide gelatin, astragalus root, yam, scrophularia root, and atractylodes rhizome. Currently, this medicine is used clinically for the prevention or treatment of diabetes; its application in the preparation of medicines for the prevention or treatment of kidney disease has not been observed.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an application of Lianmei granules in the preparation of drugs for the prevention or treatment of kidney disease. This application provides a new drug approach for the treatment and prevention of kidney disease and has high promotion and application value.

[0006] This invention can be implemented as follows:

[0007] This invention provides the application of Lianmei granules in the preparation of drugs for the prevention or treatment of kidney disease.

[0008] In an optional embodiment, the above-mentioned drug is a drug used to improve glomerular hypertrophy.

[0009] In an optional embodiment, the above-mentioned drug is a drug used to improve vacuolar degeneration of renal tubular epithelial cells.

[0010] In an optional embodiment, the above-mentioned drug is a drug used to improve renal tubular lumen stenosis.

[0011] In an optional implementation, the above-mentioned drug is a drug used to improve focal renal tubular atrophy.

[0012] In an optional embodiment, the above-mentioned drug is a drug used to improve local glomerular basement membrane thickening.

[0013] In an optional embodiment, the above-mentioned drug is a drug used to improve foot process fusion.

[0014] In an optional embodiment, the above-mentioned drug is a drug for reducing the deposition of collagen fibers in the renal interstitium.

[0015] In an optional embodiment, the above-mentioned drug is a drug used to improve the intestinal microenvironment.

[0016] In an optional embodiment, the drug is a drug for promoting the proliferation of at least one of Prevotella spp., Parabacterium spp., Bacteroides oxytocinoides, and Akkermansia myxophilus in the intestine, and / or, the drug is a drug for reducing the content of at least one of Helicobacter pylori and Helicobacter pylori in the intestine.

[0017] In an optional embodiment, the above-mentioned drug is a drug used to strengthen the intestinal mucosal barrier.

[0018] In optional embodiments, the drug is a drug for upregulating at least one of TNFα, IL-6 and IL-1β levels; and / or, the drug is a drug for downregulating LPS levels; and / or, the drug is a drug for enhancing the expression of at least one of ZO-1, OCLN and CLDN1 molecules.

[0019] The beneficial effects of this invention include:

[0020] This invention proposes for the first time the application of Lianmei granules in the preparation of drugs for improving kidney disease. Specifically, it can be used, for example, to prepare drugs that improve at least one of the following: glomerular hypertrophy, vacuolar degeneration of renal tubular epithelial cells, luminal stenosis, focal tubular atrophy, localized glomerular basement membrane thickening, and foot process fusion; reduce the deposition of renal interstitial collagen fibers; and / or improve the intestinal microenvironment and strengthen the intestinal mucosal barrier. This novel application of Lianmei granules provides a new pharmacological approach for the treatment and prevention of kidney disease and has high potential for promotion and application. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a microscopic examination result of the kidney after HE staining in Example 1;

[0023] Figure 2 This is a microscopic image of the kidney after Masson staining in Example 1;

[0024] Figure 3This is a statistical chart of the results of Masson staining microscopic examination of the kidneys in Example 1;

[0025] Figure 4 This is an electron micrograph of the kidney from Example 1;

[0026] Figure 5 The figure shows the effect of Lianmei granules on the lipopolysaccharide level in mice in Example 3;

[0027] Figure 6 The figure shows the effect of Lianmei granules on TNFα, IL-6 and IL-1β in mouse serum in Example 3.

[0028] Figure 7 This is a graph showing the expression of Zona occludens in the colon tissue in Example 3;

[0029] Figure 8 This is a graph showing the expression of Occludin in the colon tissue in Example 3;

[0030] Figure 9 This is a graph showing the expression of Claudin (CLDN1) in the colon tissue in Example 3. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0032] The following is a detailed description of the application of the Lianmei granules provided by this invention in the preparation of drugs for the prevention or treatment of kidney disease.

[0033] This invention proposes the application of Lianmei granules in the preparation of drugs for the prevention or treatment of kidney disease.

[0034] Lianmei granules are prepared from raw materials including dried plum, coptis, rehmannia root, ophiopogon root or lotus seed heart, donkey-hide gelatin, astragalus root, yam, scrophularia root, and atractylodes rhizome. For reference, by mass parts, the raw materials of Lianmei granules may include 8-16 parts dried plum, 3-10 parts coptis root, 6-15 parts rehmannia root, 6-16 parts ophiopogon root or lotus seed heart, 0.5-9 parts donkey-hide gelatin, 6-16 parts astragalus root, 6-18 parts yam, 5-13 parts scrophularia root, and 2-9 parts atractylodes rhizome.

[0035] The preparation method of the Lianmei granules can be referred to the relevant existing technology, and will not be elaborated here.

[0036] In some embodiments, the aforementioned drug can be a drug for improving glomerular hypertrophy, that is, Lianmei granules can be used to prepare a drug for improving glomerular hypertrophy.

[0037] In some embodiments, the aforementioned drug can be a drug for improving vacuolar degeneration of renal tubular epithelial cells, that is, Lianmei granules are used to prepare a drug for improving vacuolar degeneration of renal tubular epithelial cells.

[0038] In some embodiments, the above-mentioned drug can be a drug for improving renal tubular lumen stenosis, that is, Lianmei granules are used to prepare a drug for improving renal tubular lumen stenosis.

[0039] In some embodiments, the above-mentioned drug can be a drug for improving focal renal tubular atrophy, that is, Lianmei granules can be used to prepare a drug for improving focal renal tubular atrophy.

[0040] In some embodiments, the above-mentioned drug can be a drug for improving local glomerular basement membrane thickening, that is, Lianmei granules are used to prepare a drug for improving local glomerular basement membrane thickening.

[0041] In some embodiments, the aforementioned drug can be a drug for improving foot process fusion, that is, Lianmei granules can be used to prepare a drug for improving foot process fusion.

[0042] In some embodiments, the aforementioned drug can be a drug for reducing the deposition of collagen fibers in the renal interstitium, that is, Lianmei granules are used to prepare a drug for reducing the deposition of collagen fibers in the renal interstitium.

[0043] Furthermore, in other embodiments, the aforementioned drug may also be a drug for improving the intestinal microenvironment and / or strengthening the intestinal mucosal barrier. This drug achieves the effect of preventing or treating kidney disease by improving the intestinal microenvironment and / or strengthening the intestinal mucosal barrier.

[0044] In some embodiments, the drug may be a drug for promoting the proliferation of at least one of Prevotella spp., Parabacterium spp., Bacteroides oxytocinoides, and Akkermansia myxophilus in the intestine.

[0045] In some embodiments, the drug may be a drug for reducing the content of at least one of Helicobacter pylori and Helicobacter filamentosa in the intestine.

[0046] In some embodiments, the drug may be a drug for upregulating at least one of TNFα, IL-6 and IL-1β levels.

[0047] In some implementations, the drug may be a drug used to downregulate LPS levels.

[0048] In some embodiments, the drug may be a drug that enhances the expression of at least one molecule selected from ZO-1, OCLN and CLDN1.

[0049] Based on the above, the present invention provides for the first time the use of Lianmei Granules in preparing a medicament for improving nephropathy. Specifically, it can be used, for example, to prepare a medicament for improving at least one of glomerular hypertrophy, vacuolar degeneration of renal tubular epithelial cells, lumen stenosis, focal renal tubular atrophy, local glomerular basement membrane thickening, and foot process fusion, reducing the deposition of renal interstitial collagen fibers and / or improving the intestinal microenvironment, and strengthening the intestinal mucosal barrier. The proposal of the new use of Lianmei Granules provides a new drug approach for the treatment and prevention of nephropathy, and has high promotion and application value.

[0050] The features and properties of the present invention are further described in detail below with reference to examples.

[0051] The Lianmei Granules used in the following examples are all prepared by the following method: 90 g of Fructus Mume, 60 g of Coptidis Rhizoma, 90 g of Rehmanniae Radix, 90 g of Ophiopogonis Radix, 150 g of Astragali Radix, 150 g of Dioscoreae Rhizoma, 100 g of Scrophulariae Radix and 60 g of Atractylodis Rhizoma are mixed to obtain a traditional Chinese medicine mixture; then water 6 times the mass of the traditional Chinese medicine mixture is added for extraction twice, 1.5 hours for each extraction, the filtrates are combined, then 60 g of Colla Corii Asini is added, the mixture is concentrated into a thick paste, an appropriate amount of auxiliary materials are added, the mixture is mixed uniformly, and granules are prepared.

[0052] Example 1

[0053] ① Experimental animals: 50 db / db mice, SPF grade, 7 weeks old, male, 6 db / m mice, body weight 40 g to 60 g, purchased from Changzhou Cavens Laboratory Animal Co., Ltd., the production license number of experimental animals is SCXK (Su) 2021-0013, and the quality certificate number of experimental animals is No.202360754. During the 1-week adaptive feeding of 7-week-old male db / db mice, the animals had free access to food and water, and bedding was changed every other day. From the date of receipt, all mice were adaptively fed and observed once a day, including mental state, whether the fur is glossy, whether the stool is loose, whether the eyes are congested, whether there is abnormal secretion, whether there is abnormal secretion in the urinary tract, whether there is death and other conditions.

[0054] ② Feed and feeding environment: SPF grade sterilized mouse feed was purchased from Guangdong Medical Laboratory Animal Center. Purified water was provided by the purified water system of the laboratory animal room of Guangzhou University of Chinese Medicine Science and Technology Industrial Park Co., Ltd. The animals were raised in the barrier environment animal laboratory of Guangzhou University of Chinese Medicine Science and Technology Industrial Park. The temperature of the barrier animal room is 25°C, the humidity is 40-70%, and the room is in good ventilation and light conditions. During the experimental operation, all experimental materials or personnel entering and exiting the barrier animal room, including mouse bedding, cages and other items, all implement sterile disinfection.

[0055] ③ Experimental reagents and their preparation.

[0056] Preparation of the test drug: Lianmei granules (provided by Guangzhou University of Chinese Medicine Science and Technology Industrial Park; each gram of powder is equivalent to 2.6053g of crude drug; the proposed clinical dose is 50.5g of crude drug per day; calculated based on an adult weight of 60kg, the proposed dose of Lianmei granules is 0.842g of crude drug per kg per day). -1 The equivalent dose for mice was 7.6 g crude drug per kg. -1 ·d -1 Before use, dissolve the granules in pure water to prepare low-dose, medium-dose, and high-dose solutions containing 0.2 g / mL, 0.4 g / mL, and 0.8 g / mL, respectively, which means each gram of mouse requires 0.01 mL of solution for gavage. The specific preparation method is as follows: Take 20 g of Lianmei granules, place them in a 150 mL volumetric flask, add pure water to make up to 100 mL, stir thoroughly with a glass rod, and sonicate in a CNC ultrasonic cleaner for 10 minutes to prepare a 0.2 g / mL Lianmei suspension; prepare Lianmei suspensions with concentrations of 0.4 g / mL and 0.8 g / mL in the same way.

[0057] Preparation of the positive control drug: Metformin hydrochloride tablets (trade name: Glucophage, Merck Pharmaceuticals (Jiangsu) Co., Ltd., batch number: ACH5727), specification 0.5g / tablet. The drug was crushed and dissolved in pure water to prepare a solution with a concentration of 30mg / mL, i.e., 0.01mL per gram of mouse administered by gavage. The specific preparation method is as follows: Take 2 metformin tablets, place them in a 50mL centrifuge tube, add pure water to a final volume of 33.3mL, thus preparing a 30mg / mL solution. Prepare and use immediately.

[0058] ④ Experimental methods.

[0059] A. Grouping of Experimental Animals: Fifty male db / db mice (SPF grade) were acclimatized for one week (8 weeks old). Fasting blood glucose and urinary protein were measured in the mice. The DKD model criteria were: elevated blood glucose that was stable for more than 8 weeks, and the following requirements were met: ① Fasting blood glucose greater than 11.1 mmol / L. ② Positive urinary protein. Thirty db / db mice meeting the requirements were selected and randomly divided into the following groups according to their fasting blood glucose values: normal group (Control group, db / m, n=6), model group (Model group, db / db, n=6), metformin tablet group (db / db, n=6, positive drug group), low-dose Lianmei granules group (LM low-dose group, db / db, n=6), medium-dose Lianmei granules group (LM medium-dose group, db / db, n=6), and high-dose Lianmei granules group (LM high-dose group, db / db, n=6).

[0060] B. This experiment included three dosage groups: low, medium, and high, equivalent to 0.5, 1, and 2 times the clinically intended dose, respectively. That is, the three doses of Lianmei granules were 1.9g crude drug·kg. -1 ·d -1 3.8g crude drug·kg -1 ·d -1 7.6g crude drug·kg -1 ·d -1 The maximum clinical dose of the positive control drug metformin is 2000 mg, and the equivalent dose in mice is 300 mg compound / kg. -1 ·d -1 As shown in Table 1.

[0061] Table 1 Dosage Design for the Pharmacodynamic Test of Lianmei Granules in Mice

[0062]

[0063] C. Administration method: Gavage administration, once a day, for 8 weeks.

[0064] D. Sample Collection Method: After collecting blood from mice, immediately remove both kidneys, wash with physiological saline, absorb surface blood with filter paper, remove the membrane, weigh the wet weight, and then separate them with a scalpel. The left kidney is fixed by immersion in 10% formaldehyde solution for observation of renal pathological changes. The right kidney is halved, and two pieces of kidney tissue are taken from the cortex of one half of the kidney. These are placed in autoclaved EP tubes and frozen at -80℃ for later use. Another small piece of kidney tissue is taken and immediately placed in 4℃ fixative within 1–3 minutes. Then, immediately in an ice box, the sample is cut into 1mm cross-sections. 2 It is a long strip no more than 3 mm in length, used for transmission electron microscopy observation.

[0065] E. Pathological morphological observation of mouse kidney tissue.

[0066] Preparation and staining of kidney tissue specimens: Kidney tissue was fixed in 10% neutral formalin. The pathological condition of kidney tissue in each group of mice was observed using both HE and Masson staining methods.

[0067] The specific operating steps are as follows:

[0068] a. Rinsing and dehydration: Rinse the fixed kidney tissue with pure water; after rinsing, dehydrate the kidney tissue in the following order: 70% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, anhydrous ethanol for 30 min to 1 hour, xylene I solution for 15 min, xylene II solution for 15 min, paraffin I at 56-58℃, paraffin II at 56-58℃, and paraffin III at 56-58℃.

[0069] b. Embedding: Adjust the temperature of the paraffin chamber in the embedding machine to 63℃. After the paraffin melts, adjust the temperature of the embedding chamber to 62℃ and place the tissue to be embedded into the chamber. Select an appropriate embedding mold according to the size of the tissue block. First, pour a small amount of paraffin into the embedding mold, then immediately clamp the tissue block into the embedding membrane. Gently press the tissue cut surface to the bottom of the embedding mold with forceps, and then place a cooling platform to fix the tissue to the bottom of the embedding mold. Place the dehydration box on the embedding mold and pour more paraffin. Cool again for at least 30 minutes, then separate the dehydration box from the embedding mold. The kidney tissue is now embedded in paraffin.

[0070] c. Sectioning, mounting, drying, and baking: Before sectioning, refrigerate the wax block in a refrigerator for 0.5-1 hour. Use an electric rotary microtome to section, adjusting the blade angle (4°-6°). Place the wax block on the holder and lock it in place. Press the wax block advance / retreat button with your left hand and rotate the handwheel with your right hand to repeatedly adjust the wax block to ensure that the embedded tissue surface is exposed before sectioning. Lay the sections flat and separate them with tweezers, smooth side down, into the water tank of a slide spreader. After the sections have fully spread in the water tank, observe for any damage or wrinkles. Maintain the water temperature in the slide spreader at approximately 43°C-48°C. Mount the retrieved tissue sections onto glass slides. Place the mounted slides on a slide dryer at 56°C for 1 hour. Transfer the sections to a slide rack and place them in a drying oven at approximately 60°C for 1-1.5 hours.

[0071] d. HE staining: Dewaxing with xylene I for 10-20 min; dewaxing with xylene II for 10-20 min; anhydrous ethanol I for 1-2 min; anhydrous ethanol II for 1-2 min; 95% ethanol I for 2-1 min; 95% ethanol II for 2-1 min; 90% ethanol for 2-1 min; 80% ethanol for 2-1 min; washing with tap water for 2-1 min; Harris hematoxylin staining for 5-12 min; washing with tap water for 2-1 min; differentiation with 1% hydrochloric acid ethanol for 2-5 s (until a pale purple-red color is visible to the naked eye); tap water... Blueing should be performed for at least 30 minutes (in urgent cases, 1% ammonia solution can be used for blueing in about 30 seconds); 1% eosin staining for 2-5 minutes; rinsing with tap water for 1-2 seconds; dehydration with 80% ethanol for 1-2 seconds; dehydration with 90% ethanol for 1-2 seconds; 95% ethanol I for 2-1 minutes; 95% ethanol II for 2-1 minutes; anhydrous ethanol I for 2-5 minutes; anhydrous ethanol II for 2-5 minutes; xylene carbolic acid for 1-3 minutes; TO clearing agent I for 5-15 minutes; TO clearing agent II for 5-15 minutes; and then sealing with neutral resin or Canada balsam.

[0072] e. Masson staining: Paraffin sections are routinely dewaxed and washed with purified water for 10 seconds. They are then stained with prepared Weigert iron-hematoxylin solution for 5-10 minutes, followed by Masson's blue solution for 5 minutes. The sections are first rinsed with purified water, then with distilled water. Finally, they are stained with Ponceau S and fuchsin solution for 8 minutes. A weak acid working solution is prepared at a ratio of distilled water to weak acid solution of 2:1. The sections are washed with the working solution for 1 minute. They are then washed with phosphomolybdic acid solution for 1-2 minutes. They are then washed with the previously prepared weak acid working solution for 1 minute. The sections are directly immersed in aniline blue staining solution for 1-2 minutes. They are then washed with the prepared weak acid working solution for 1 minute. Rapid dehydration is performed with 95% ethanol for 2-3 seconds, followed by dehydration three times with anhydrous ethanol for 5-10 seconds each time. Clearing is done three times with xylene for 1-2 minutes each time, followed by mounting with neutral resin.

[0073] Electron microscopy of the kidneys: Kidney tissue was fixed in 2.5% glutaraldehyde for at least 4 hours and stored on ice at 4°C. It was washed three times with PBS buffer for 10 minutes each time. Then, it was fixed with -1% osmium tetroxide for 1.5 hours and stored at 4°C. It was then washed three more times with PBS buffer for 10 minutes each time. The tissue was dehydrated twice with 50%, 70%, 80%, 90%, and 100% ethanol concentrations, for 12 minutes each time. It was then incubated in 100% acetone and embedding buffer (3:1 ratio) at room temperature for 0.5 hours, followed by incubation in 100% acetone and embedding buffer (1:1 ratio) at room temperature for 4 hours. Finally, it was incubated overnight at 4°C in pure embedding buffer. The next day, the samples were embedded in embedding plates and placed in a 37°C oven for 24 hours. The oven temperature was then increased to 60°C and incubated for 48 hours. The paraffin block was cut into ultrathin sections (100 nm thick) using an ultramicrotome, and stained with uranyl acetate and lead citrate for 20 min and 12 min, respectively. Finally, the differences were observed by imaging with a transmission electron microscope.

[0074] ⑤ Results.

[0075] HE staining and microscopic examination showed that the model group mice had atrophied renal tubules and narrowed lumens; the renal tissue pathology of the mice in the Lianmei granule group showed good improvement. The glomerular capsule structure was clearly visible, the renal tubular epithelium was intact, and no abnormalities were observed in the epithelial cells (e.g., ...). Figure 1 (As shown).

[0076] Masson staining revealed extensive collagen fiber deposition in the renal interstitium of mice in the model group, while the renal interstitium collagen fiber deposition in the Lianmei granule group showed a slightly greater improvement, with only a small amount of collagen fiber deposition in the renal tubular interstitium (e.g., Figure 2 and Figure 3 As shown, ΔΔΔΔ represents P < 0.0001 compared to the normal group; ** represents P < 0.01 compared to the model group.

[0077] Under electron microscopy, compared with the normal group, the model group mice showed localized thickening of the glomerular basement membrane and partial fusion of foot processes. The renal tissue of mice in the Lianmei granule administration group showed significant improvement (e.g., ...). Figure 4 (As shown).

[0078] Based on the above, it can be explained that Lianmei granules can improve glomerular hypertrophy, vacuolar degeneration of renal tubular epithelial cells, luminal stenosis, focal tubular atrophy, as well as local glomerular basement membrane thickening and foot process fusion in db / db mice, while reducing the deposition of renal interstitial collagen fibers.

[0079] Example 2

[0080] The experimental animals, drugs, grouping, and administration were all carried out in accordance with Example 2.

[0081] Eight weeks after drug administration, feces were collected. Wearing disposable gloves, the mice were held by their tails with the right hand and placed on their cages to stabilize them. Gently pressing the lower abdomen stimulated defecation. Feces (at least three pellets) were collected and placed in sterile cryovials, which were then capped and sealed. The alpha diversity index was calculated using Simpson's Index of Diversity. The beta diversity index was calculated using Qiime (V1.9.1); PCoA plotting was performed using the WGCNA, stats, and ggplot2 functions in R software; and AMOVA was performed using the amova function in Mothur software. Species differences between groups were analyzed using R software and LEfSe (v1.1.2).

[0082] result:

[0083] ① Alpha diversity analysis

[0084] Alpha diversity was assessed using the Chao 1 index, ACE index, Shannon index, Simpson index, Observed species index, and PD_whole_tree index to evaluate the species richness and diversity changes of the gut microbiota in Yangnei. Alpha diversity was used to analyze within-community microbial community diversity. Single-sample diversity analysis (Alpha diversity) can reflect the richness and diversity of the microbial community within a sample, including using species accumulation box plots, species diversity curves, and a series of statistical indices to assess the differences in species richness and diversity of the microbial community among samples.

[0085] The results of the alpha diversity study showed that there were certain differences in the composition and structure of the gut microbiota among the groups, suggesting that Lianmei granules can alter the structure of the gut microbiota in the model animals.

[0086] ② Beta diversity analysis

[0087] PCoA analysis was performed based on weighted and unweighted unifrac distances, and the principal coordinate combination with the highest contribution rate was selected for plotting. The closer the sample distances, the more similar the species composition structure. Therefore, samples with high community structural similarity tend to cluster together, while samples with large community differences will be far apart.

[0088] The results showed that there were significant differences in community structure between the Model group and the Control group, and between the LM group and the Model group (P < 0.05); indicating that Lianmei granules have a significant effect on the composition of intestinal flora in model animals.

[0089] ③ Species composition analysis

[0090] Based on the species annotation results, the top 10 species with the highest abundance at each taxonomic level (phylum, class, family, order, genus, species) for each sample or group are selected, and a cumulative bar chart of relative abundance of species is generated to visually view the species with higher relative abundance and their proportion at different taxonomic levels for each sample.

[0091] At the phylum level, the gut microbiota of all three groups were mainly composed of Bacteroidetes (p_Bacteroidota), Firmicutes (p_Firmicutes), and Verrucomicrobiota (p_Verrucomicrobiota). In the normal group, the relative abundances of these three phyla were 58.38%, 36.32%, and 1.04%, respectively; in the Model group, the relative abundances were 68.56%, 20.21%, and 8.60%, respectively; and in the LM group, the relative abundances were 62.16%, 16.68%, and 18.82%, respectively.

[0092] At the class level, the gut microbiota of all three groups were mainly composed of Bacteroidetes, Clostridia, and Verrucomicrobiae. In the normal group, the relative abundances of these three classes were 58.38%, 28.61%, and 1.04%, respectively; in the Model group, the relative abundances were 68.56%, 11.85%, and 8.60%; and in the LM group, the relative abundances were 62.17%, 10.31%, and 18.82%.

[0093] At the family level, the gut microbiota of all three groups was mainly composed of Bacteroidetes (f. Muribaculaceae), Prevotellaceae, and Lachnospiraceae. In the normal group, the relative abundances of these three families were 43.15%, 9.26%, and 21.25%, respectively; in the Model group, the relative abundances were 43.34%, 18.40%, and 5.81%, respectively; and in the LM group, the relative abundances were 34.49%, 17.09%, and 6.62%, respectively.

[0094] At the order level, the order with the largest proportion of gut microbiota in the three groups were Bacteroidetes, Lachnospirales, Verrucomicrobiales, and Lactobacillales. In the Control group, the relative abundances of these four orders were 58.37%, 21.25%, 1.04%, and 6.52%, respectively; in the Model group, they were 68.51%, 5.81%, 8.60%, and 8.00%; and in the LM group, they were 62.01%, 6.26%, 18.82%, and 6.18%.

[0095] At the genus level, the genera with the largest proportions of gut microbiota in the three groups were *Akkermansia* (g.), *Ligilactobacillus* (g.), *Alloprevotella* (g.), *Bacteroides* (g.), and *Parabacteroides* (g.). In the control group, the relative abundances of these five phyla were 1.04%, 4.67%, 0.90%, 1.06%, and 1.14%, respectively; in the model group, they were 8.60%, 5.30%, 1.90%, 1.81%, and 0.54%; and in the LM group, they were 18.82%, 4.39%, 7.91%, 5.57%, and 2.27%, respectively.

[0096] At the species level, the gut microbiota of all three groups were mainly composed of *Akkermansia muciniphila*, *Lactobacillus murinus*, and *Bacteroides acidifacicense*. In the Control group, the relative abundances of these three species were 1.04%, 4.66%, and 0.50%, respectively; in the Model group, they were 8.59%, 5.29%, and 1.35%; and in the LM group, they were 18.82%, 4.38%, and 2.48%.

[0097] The analysis at different levels of enterobiota classification shows that Lianmei granules can significantly influence the structure of enterobiota. In particular, the levels of *Akkermansia muciniphila* and *Bacteroides acidifacicum*, as beneficial intestinal bacteria, are significantly increased under the influence of Lianmei granules.

[0098] LEfSe analysis (LDA value > 4) revealed 14 species that significantly contributed to the differences between the Model and LM groups. Among them, 3 species were significantly elevated in the LM group, such as *Lactobacillus johnsonnii*, *Flavobacteriales*, and *Flavobacteriaceae*. Eleven species were significantly enriched in the Control group, including *Enterorhabdus* and *Parvibacter*.

[0099] Example 3

[0100] The experimental animals, drugs, grouping, administration and sample collection were all carried out in accordance with Example 2.

[0101] Experimental methods:

[0102] (1) Detection method of mouse lipopolysaccharide (LPS) in serum

[0103] Before starting the experiment, remove the kit from the refrigerator and allow it to cool to room temperature.

[0104] Preparation of standards: Take out the lyophilized standard tube and add 1 mL of sample diluent. Use a pipette to gently pipette repeatedly until the lyophilized powder is completely dissolved, and prepare a stock solution with a concentration of 10 pg / mL. Then, dilute the stock solution in half with the sample diluent in a 1:1 ratio to prepare a standard concentration gradient of 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.312 ng / mL, 0.156 ng / mL, and 0 ng / mL.

[0105] Preparation of antigen working solution: Take out the concentrated antibody, centrifuge at 1000 rpm for 1 min, and then dilute the concentrated antibody with antibody dilution buffer to a working concentration of 1×. Prepare and use immediately, and store away from light.

[0106] Preparation of washing solution: Take 10 mL of 20× washing solution, add distilled water to 200 mL, and dilute to prepare 1× working solution. Equilibrate to room temperature and protect from light for later use.

[0107] Sampling and testing

[0108] Sample loading: Add 50 μL of serum sample and the known concentration of the standard to be tested to each well, and add 50 μL of universal diluent to the blank well as a control. Then add 50 μL of antigen working solution to each well, cover with sealing film, and incubate in a 37°C incubator for 1 h.

[0109] Washing: After incubation, vigorously shake off the supernatant and repeat the antibody washing steps, washing the plate 5 times in total;

[0110] Add substrate: Add 90 μL of reaction substrate to each well, seal the plate tightly with the film, and place it in a 37°C incubator in the dark for 15 min;

[0111] Termination of reaction: After adding 50 μL of stop solution directly to each well, the OD value of each reaction well was measured at a wavelength of 450 nm, and the trend of index change was analyzed by plotting.

[0112] (2) Methods for detecting the level of inflammation in mice in serum

[0113] Whole blood was collected from the fundus venous plexus of mice, centrifuged at 3000g for 10 minutes, and the supernatant was separated. Serum samples from 6 mice in each group were randomly selected, and the levels of TNF-α, IL-1β and IL-6 in the serum were detected by ELISA.

[0114] The specific operating steps are as follows:

[0115] Taking the detection of TNF-α levels in serum as an example, before the assay, the test kit is removed from the 4°C refrigerator and left at room temperature for about 1 hour until it reaches equilibration with room temperature before the experiment.

[0116] The specific testing procedures are as follows:

[0117] Reagent preparation before experiment

[0118] Preparation of standards: Take out the lyophilized standard tube and centrifuge briefly to concentrate the powder at the bottom; add 1 mL of sample diluent, and gently pipette repeatedly until the lyophilized powder is completely dissolved to prepare a stock solution with a concentration of 1000 pg / mL. Dilute the stock solution in half sequentially with sample diluent at a 1:1 ratio to prepare a standard concentration gradient of 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.62 pg / mL, and 0 pg / mL.

[0119] Antibody working solution preparation: Take out the concentrated antibody, centrifuge at 1000 rpm for 1 min, and then dilute the concentrated antibody to 1× working concentration with antibody diluent. Prepare and use immediately, and store away from light.

[0120] Preparation of enzyme conjugate working solution: Prepare 100× concentrated enzyme conjugate, centrifuge at 1000 rpm for 1 min, then dilute with universal diluent to the working concentration. Prepare and use immediately, store at room temperature away from light.

[0121] Preparation of washing solution: Take 10 mL of 20× washing solution, add distilled water to 200 mL, and dilute to prepare 1× working solution. Equilibrate to room temperature and protect from light for later use.

[0122] Sampling and testing

[0123] Sample loading: Add 100 μL of serum sample and the known concentration of the standard to be tested to each well, and add 100 μL of universal diluent as a control to the blank well. Cover with sealing film and incubate in a 37°C incubator for 1 h.

[0124] Antibody incubation: Remove the ELISA plate, discard the supernatant, add 100 μL of biotinylated antibody working solution to each well, seal with a sealing film, and incubate at 37°C for 1 hour.

[0125] Antibody washing: Take out the microplate, add 300 μL of 1× washing buffer to each well, let stand for 1 min, shake off the washing buffer forcefully, and then turn the plate face down and tap it on absorbent paper until no droplets are visible in each well. Repeat this washing operation 3 times.

[0126] Enzyme conjugate reaction: Add 100 μL of enzyme conjugate working solution to each well of the microplate after patting off the washing solution, cover with sealing film, and incubate at 37°C for 30 min.

[0127] Washing: After incubation, vigorously shake off the supernatant and repeat the antibody washing steps, washing the plate 5 times in total;

[0128] Add substrate: Add 90 μL of reaction substrate to each well, seal the plate tightly with the film, and place it in a 37°C incubator in the dark for 15 min;

[0129] Termination of reaction: After adding 50 μL of stop solution directly to each well, the OD value of each reaction well was measured at a wavelength of 450 nm, and the trend of index change was analyzed by plotting.

[0130] (3) Immunohistochemical experimental methods for colon tissue

[0131] The immunohistochemical (IHC) experimental method is as follows:

[0132] Paraffin-embedded sections: After fixing the large intestine with 4% paraformaldehyde for 1 week, it was embedded in paraffin, sliced ​​into 10μm thickness by a microtome, spread out and fixed with an adhesive glass slide, and placed in a 55℃ oven for min to promote adhesion of the sections to the glass slide.

[0133] Dewaxing and rehydration of sections: Paraffin sections were placed in three separate xylene dewaxing solutions for 10 minutes each, then in three separate anhydrous ethanol solutions for 5 minutes each, and finally in distilled water.

[0134] Antigen retrieval: Place the rehydrated slides in an autoclave, add antigen retrieval solution until the liquid level covers the tissue section, heat until boiling for 5 minutes, then allow to cool naturally to room temperature. Place the slides in PBS buffer and wash three times with a shaker for 5 minutes each time.

[0135] Blocking endogenous peroxidase: Using an immunohistochemical pen, draw a continuous closed circle along the tissue edge, and add 3% H2O2 solution to the tissue. Incubate at room temperature in the dark for 25 minutes, and then repeat the washing method in step 3) 3 times.

[0136] Serum blocking: After absorbing excess water from the histology zone with absorbent paper, add 3% BSA solution to the histology zone until it covers the tissue section, and block at room temperature for 30 minutes.

[0137] Primary antibody incubation: Gently shake off the blocking solution, add about 60 μL of antibody diluted 1:200 to each tissue circle, place the slide in a humidified chamber, and incubate overnight at 4°C.

[0138] Secondary antibody incubation: Aspirate the primary antibody incubation solution, wash the slide 3 times according to the washing method in step 3), blot off excess water with absorbent paper, add 100 μL of HRP-labeled secondary antibody corresponding to the species of the primary antibody, and incubate at room temperature in the dark for 50 min.

[0139] DAB staining: Discard the secondary antibody incubation solution, wash the slide 3 times according to the washing method in step 3), and after slightly drying the slide, add 50 μL of freshly prepared DAB staining solution. Observe the staining changes under a microscope. When the staining turns brownish-yellow, immediately rinse the slide with distilled water to stop the staining process.

[0140] Counterstaining cell nuclei: After cleaning the slides, hematoxylin was added to the tissue area and stained for about 3 minutes. The slides were then rinsed with distilled water, placed in hydrochloric acid alcohol differentiation solution for about 10 seconds, and then stained with hematoxylin blue solution. The slides were then rinsed with running water.

[0141] Dehydration and mounting: Place the slides in 75% alcohol, 85% alcohol, 100% alcohol, 100% alcohol solution, n-butanol, and xylene in sequence. Let each solution stand for 5 minutes. Remove the slides, let them dry slightly, add 1 drop of neutral resin, cover the slide with a coverslip, remove air bubbles, and let it stand at room temperature until the resin solidifies.

[0142] Section scanning: The entire slide was scanned, and the positive staining area of ​​each section was analyzed using Caseview 2.4 software.

[0143] result:

[0144] ① Effect of Lianmei Granules on Lipopolysaccharide (LPS) Levels in Mice.

[0145] The results are as follows Figure 5 As shown in the figure, compared with the normal group, △ represents P < 0.05, and △△ represents P < 0.01; compared with the model group, * represents P < 0.05, and ** represents P < 0.01. The results showed that compared with the normal group, the LPS level of mice in the model group was significantly different (P < 0.05). Compared with the model group, the high-dose Lianmei granules group and the metformin tablet group significantly reduced the serum LPS level of db / db mice (P < 0.05 or P < 0.01).

[0146] ② Effects of Lianmei Granules on serum TNFα, IL-6 and IL-1β in db / db mice.

[0147] The results are as follows Figure 6As shown in the figure, compared with the normal group, △ represents P < 0.05 and △△ represents P < 0.01; compared with the drug-treated group, * represents P < 0.05 and ** represents P < 0.01. The results showed that compared with the normal group mice, the serum TNFα level of the model group mice was significantly higher than that of the normal group, with a statistically significant difference (P < 0.05). Compared with the model group, serum TNFα levels in the medium- and high-dose groups of Lianmei granules were significantly decreased (P < 0.01). Compared with the normal group of mice, serum IL-1β levels in the model group were significantly higher than those in the normal group (P < 0.01). Compared with the model group, serum IL-1β levels in the low- and medium-dose groups of Lianmei granules were significantly decreased (P < 0.05). Compared with the normal group of mice, serum IL-6 levels in the model group were higher than those in the normal group, but not statistically significant (P > 0.05). Compared with the model group, serum IL-6 levels in the low-, medium-, and high-dose groups of Lianmei granules showed a decreasing trend, but not statistically significant (P > 0.05). Compared with the metformin tablet group, the low-, medium-, and high-dose groups of Lianmei granules were superior to the metformin tablet group in downregulating TNFα, IL-6, and IL-1β levels. Therefore, Lianmei granules can downregulate serum TNFα, IL-6, and IL-1β levels.

[0148] ③ Expression of Zona occludens (ZO-1) in colon tissue.

[0149] Tight junction proteins are mainly expressed on the cell membrane and cytoplasm, and positive staining shows brownish-yellow granules on the cell membrane. ZO-1 is located on the lower side of the cell membrane and, through interaction with other proteins, constitutes the intercellular junction and sealing structure, which can affect the function of the intestinal mucosal barrier.

[0150] like Figure 7 As shown, compared with the normal group, the number of ZO-1 positive staining granules in the large intestine tissue of mice in the model group was significantly reduced, and the villi were arranged in a disordered and sparse manner. Compared with the model group, the number of ZO-1 positive staining granules in the large intestine tissue of mice in the low-dose Lianmei group, high-dose Lianmei group and metformin group was increased, with the increase being more significant in the low-dose Lianmei group and metformin group.

[0151] ④ Expression of Occludin (OCLN) in colon tissue.

[0152] Claudin-1 and Occludin together form the tightly linked main body of the large intestine tissue, playing an important role in regulating cell permeability.

[0153] like Figure 8As shown, compared with the normal group, the number of occludin-positive staining granules in the large intestine tissue of mice in the model group was reduced, and the villi were sparsely arranged. Compared with the model group, the number of occludin-positive staining granules in the large intestine tissue of mice in the medium- and high-dose Lianmei groups and the metformin group was increased, with the increase being more obvious in the medium-dose Lianmei group and the metformin group.

[0154] ⑤ Expression of Claudin (CLDN1) in colon tissue.

[0155] Claudin-1 is mainly located in the cytoplasm and appears as brown granules. Its main functions include participating in the regulation of extracellular permeability and polarity, cell differentiation, proliferation, migration and apoptosis, or participating in physiological functions such as signal transduction. Mice lacking Claudin-1 protein expression will die due to severe barrier function impairment leading to epidermal dehydration.

[0156] like Figure 9 It can be seen that, compared with the normal group, the Claudin-1 brown granules in the large intestine tissue of the model group mice were significantly reduced, and the villi were sparsely arranged, with most of them appearing free. Compared with the model group, the Claudin-1 positive staining granules in the large intestine tissue of mice in the low-dose Lianmei group, high-dose Lianmei group, and metformin group were all increased to varying degrees, with the low-dose Lianmei group and metformin group showing more significant increases.

[0157] Therefore, Lianmei granules can upregulate the serum levels of TNFα, IL-6, and IL-1β in db / db mice, downregulate the serum level of LPS, and enhance the expression of tight junction proteins such as ZO-1, OCLN, and CLDN1, thereby strengthening the intestinal mucosal barrier.

[0158] Continuing from the above, Lianmei granules can improve renal pathological damage in db / db mice. For example, it can increase the number of beneficial bacteria such as Bacteroides and Acetobacter xanthipes in the intestine, reduce harmful bacteria such as Helicobacter pylori and Helicobacter pylori, improve the intestinal barrier integrity and chronic low-toxicity inflammation of the internal environment in db / db mice, and protect renal function. In addition, Lianmei granules can also upregulate the levels of inflammatory substances such as TNFα, IL-6 and IL-1β in the serum of db / db mice, downregulate the level of LPS, and enhance the expression of molecules such as tight junction proteins ZO-1, OCLN and CLDN1 to strengthen the intestinal mucosal barrier.

[0159] In summary, this invention proposes for the first time the application of Lianmei granules in the preparation of drugs for improving kidney disease. Specifically, it can be used, for example, to prepare drugs that improve at least one of the following: glomerular hypertrophy, vacuolar degeneration of renal tubular epithelial cells, luminal stenosis, focal tubular atrophy, localized glomerular basement membrane thickening, and foot process fusion; reduce the deposition of renal interstitial collagen fibers; and / or improve the intestinal microenvironment and strengthen the intestinal mucosal barrier. This novel application of Lianmei granules provides a new pharmaceutical approach for the treatment and prevention of kidney disease and has high potential for promotion and application.

[0160] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of Lianmei granules in the preparation of drugs for the prevention or treatment of kidney disease; The Lianmei granules are prepared by the following method: 90g of dried plum, 60g of Coptis chinensis, 90g of Rehmannia glutinosa, 90g of Ophiopogon japonicus, 150g of Astragalus membranaceus, 150g of Dioscorea opposita, 100g of Scrophularia ningpoensis and 60g of Atractylodes lancea are mixed to obtain a traditional Chinese medicine mixture; then, water is added at 6 times the mass of the traditional Chinese medicine mixture and extracted twice, each time for 1.5 hours, the filtrates are combined, and then 60g of donkey-hide gelatin is added, concentrated into a thick paste, and appropriate excipients are added, mixed well, and made into granules.

2. The application according to claim 1, characterized in that, The drug is used to improve glomerular hypertrophy.

3. The application according to claim 1, characterized in that, The drug is used to improve vacuolar degeneration of renal tubular epithelial cells.

4. The application according to claim 1, characterized in that, The drug is used to improve narrowing of the renal tubule lumen.

5. The application according to claim 1, characterized in that, The drug is used to improve focal renal tubular atrophy.

6. The application according to claim 1, characterized in that, The drug is used to improve local thickening of the glomerular basement membrane.

7. The application according to claim 1, characterized in that, The drug is used to improve foot process fusion.

8. The application according to claim 1, characterized in that, The drug is used to reduce the deposition of collagen fibers in the renal interstitium.

9. The application according to claim 1, characterized in that, The drug is used to improve the intestinal microenvironment.

10. The application according to claim 9, characterized in that, The drug is a drug for promoting the proliferation of at least one of Prevotella spp., Parabacterium spp., Bacteroides oxytocinae, and Akkermansia phili in the intestine, and / or, the drug is a drug for reducing the content of at least one of Helicobacter pylori and Helicobacter pylori in the intestine.

11. The application according to claim 1, characterized in that, The drug is used to strengthen the intestinal mucosal barrier.

12. The application according to claim 11, characterized in that, The drug is a drug for upregulating at least one of TNFα, IL-6 and IL-1β levels; and / or, the drug is a drug for downregulating LPS levels; and / or, the drug is a drug for enhancing the expression of at least one of ZO-1, OCLN and CLDN1 molecules.

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