Granules capable of clearing heat, tonifying kidney and eliminating diseases and application of granules in treatment of diabetic nephropathy

Abstract

The invention discloses granules for clearing heat, tonifying kidney and eliminating diseases and application of the granules in treatment of diabetic nephropathy, and belongs to the field of biological medicine. The invention provides granules for clearing heat, tonifying kidney and eliminating diseases. The granules are prepared from the following raw materials: 30 g of raw radix astragali, 10 g of radix pseudostellariae, 10 g of radix angelicae sinensis, 15 g of radix rehmanniae, 15 g of semen cuscutae, 10 g of fructus forsythiae, 15 g of fructus arctii, 10 g of radix scutellariae, 3 g of rhizoma coptidis, 10 g of turtle shells, 3 g of leeches and 10 g of seaweed. Experiments prove that the granules for clearing heat, tonifying kidney and eliminating diseases can play a role in treating diabetic nephropathy by means of improving renal function, relieving renal injury, improving renal fibrosis, relieving kidney cell apoptosis, improving the form and function of kidney mitochondria, resisting oxidative stress, inhibiting iron overload, relieving ferroptosis degree and the like. In conclusion, the heat-clearing, kidney-tonifying and mass-eliminating granules provided by the invention can remarkably relieve diabetic nephropathy, and a new thought is provided for clinical treatment of diabetic nephropathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a heat-clearing, kidney-tonifying and symptom-eliminating granule and an application thereof in treating diabetic nephropathy. Background Art

[0002] Diabetic kidney disease (DKD) refers to a type of chronic kidney disease (CKD) caused by diabetes. Its pathogenesis has been hypothesized to include various mechanisms, such as oxidative stress, inflammatory response, and hemodynamic changes, but these mechanisms remain unclear. The main clinical manifestations include persistent increased albuminuria excretion and / or a progressive decrease in estimated glomerular filtration rate (eGFR).

[0003] Currently, the incidence of kidney diseases caused by diabetes, hypertension, and other factors is rapidly increasing. Diabetic nephropathy is associated with complications of diabetes mellitus: spleen deficiency, edema, kidney atrophy, obstruction of the spleen, and drowning. The term "spleen deficiency" was first recorded in the Yellow Emperor's Classic of Internal Medicine. "Suwen: Discussion on Strange Diseases, Chapter 47" states: "There is a disease in which a person experiences a sweet taste in the mouth. What is the name? How is it caused? Qi Bo said: This is the overflow of the five elements, and it is called spleen deficiency." Spleen heat, the pathogenesis of spleen deficiency, can lead to diabetes mellitus over time. "Edema" is mentioned in many medical works throughout history. The "Shuiqi Bing" section of the "Golden Chamber Essentials" states that people with edema have puffy eyelids resembling bags under their eyes, swollen facial features, and a weak pulse, indicating diabetes mellitus. The "Shengji Zonglu" states: "With prolonged diabetes mellitus, kidney qi is damaged... resulting in edema." These texts clarify the pathogenesis of edema and the clinical manifestations of edema associated with diabetes mellitus. "Shenxiao" (kidney consumption) is considered the lower form of the three types of consumption. It is more severe than the upper and middle forms and more consistent with diabetic keratin syndrome (DKD). The "Yi Fang Lei Ju - Xiao Ke" (Medical Prescriptions Collection) discusses kidney consumption as a condition characterized by persistent thirst. This condition, in which pathogenic qi damages the kidneys in the lower jiao (burner), causing the kidneys to lose their ability to retain nutrients and fluids, leading to the downward flow of food and drink, resulting in spermatorrhea, residual urine, and thin vaginal discharge. Long-lasting diabetes can develop into the more serious complications of "guan ge" (a form of constipation) and "ni du" (a form of urination). The term "guan ge" was first recorded in the "Treatise on Febrile Diseases," with "guan" referring to urinary obstruction and "ge" to vomiting. The "Zheng Zhi Hui Bu" (Compendium of Symptoms and Treatments) further elaborates on the concept, stating that both guan and ge can lead to a dissociation of yin and yang, resulting in a critically ill condition. This is because turbid pathogenic qi obstructs the triple jiao (burner), stagnates qi flow, and disrupts ascending and descending functions. Impaired qi transformation in the kidneys and bladder results in urinary obstruction, while upward reversal of stomach qi causes vomiting. The term "drowning poison" was first seen in He Lianchen's "Revised Guang Wenre Lun". The book said that "drowning poison" is often manifested as headache, dizziness, blurred vision, nausea and vomiting, hearing loss, itchy skin, dark face, and even thick black tongue coating. The clinical manifestations of the terminal stage of DKD are similar to it.

[0004] In order to further delay the progression of chronic kidney disease and understand the pathogenesis, treatment methods and prognosis of DKD in Western medicine, it is necessary to develop a Chinese medicine compound composition with the characteristics of traditional Chinese medicine on the basis of existing technology, which can effectively prevent and treat diabetic nephropathy. Summary of the Invention

[0005] The purpose of the present invention is to provide a heat-clearing, kidney-tonifying and symptom-eliminating granule and its application in the treatment of diabetic nephropathy, so as to solve the problems existing in the above-mentioned prior art. The heat-clearing, kidney-tonifying and symptom-eliminating granule provided by the present invention can significantly alleviate diabetic nephropathy, providing a new idea for the clinical treatment of diabetic nephropathy.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a heat-clearing, kidney-tonifying and symptom-eliminating granule, which comprises the following raw materials: 30g of raw astragalus, 10g of pseudoginseng, 10g of angelica, 15g of raw rehmannia, 15g of dodder seed, 10g of forsythia, 15g of burdock fruit, 10g of scutellaria, 3g of coptis root, 10g of turtle shell, 3g of leech and 10g of seaweed.

[0008] Optionally, it is prepared by the following method:

[0009] Weigh all raw materials according to the formula, mix them, extract with water, filter and concentrate to obtain extract, dry and grind to obtain dry powder, add dextrin and granulate to obtain the extract.

[0010] Optionally, the extraction with water is performed by adding 5 times the weight of water and boiling for 2 hours, extracting twice, and combining the decoctions.

[0011] Optionally, the weight ratio of the dry powder to the dextrin is 1:4.

[0012] The present invention also provides the use of the heat-clearing, kidney-tonifying and symptom-reducing granules in preparing medicine for treating diabetic nephropathy.

[0013] The present invention also provides a medicine for treating diabetic nephropathy, comprising the heat-clearing, kidney-tonifying and symptom-eliminating granules.

[0014] Optionally, the Qingre Yishen Xiaozheng granules play a role in treating diabetic nephropathy by resisting oxidative stress.

[0015] Optionally, the Qingre Yishen Xiaozheng granules play a role in treating diabetic nephropathy by inhibiting iron overload and reducing the degree of ferroptosis.

[0016] The present invention discloses the following technical effects:

[0017] The present invention provides a heat-clearing, kidney-tonifying and symptom-eliminating granule, comprising the following raw materials: 30g of raw astragalus, 10g of pseudoginseng, 10g of angelica, 15g of raw rehmannia, 15g of dodder seed, 10g of forsythia, 15g of arctium, 10g of scutellaria, 3g of coptis root, 10g of turtle shell, 3g of leech and 10g of seaweed. Experiments have confirmed that the heat-clearing, kidney-tonifying and symptom-eliminating granule can play a role in treating diabetic nephropathy by improving renal function, alleviating renal damage, improving renal fibrosis, alleviating renal cell apoptosis, improving renal mitochondrial morphology and function, resisting oxidative stress, inhibiting iron overload, alleviating the degree of ferroptosis and inhibiting the HIF-1a / HO-1 signaling pathway. In summary, the heat-clearing, kidney-tonifying and symptom-eliminating granule provided by the present invention can significantly alleviate diabetic nephropathy and provide a new idea for the clinical treatment of diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. The drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Figure 2 shows the body weight and kidney weight to body weight ratio of rats; A: Body weight statistics from 0 to 12 weeks; B: Body weight at 12 weeks; C: Kidney weight to body weight ratio at 12 weeks;

[0020] Figure 2 Comparison of blood glucose levels in rats of each group; A: Blood glucose statistics from week 0 to week 12; B: Blood glucose at week 12;

[0021] Figure 3 Comparison of renal function indicators in rats of each group; A: Scr; B: BUN; C: Cys-C; D: 24h-UTP;

[0022] Figure 4 Comparison of renal injury indicators in rats of each group; A: KIM-1; B: NGAL;

[0023] Figure 5 Comparison of renal tissue pathological damage in rats of each group;

[0024] Figure 6 Comparison of FN immunohistochemical staining of renal tissues of rats in each group;

[0025] Figure 7 Comparison of TGF-β protein expression levels in renal tissue of rats in each group; A: WB band; B: quantitative statistical graph of WB band;

[0026] Figure 8 Comparison of immunohistochemical staining of Caspase 3 in renal tissues of rats in each group;

[0027] Figure 9 Comparison of Caspase3 expression levels in renal tissues of rats in each group; A: WB bands; B: Relative expression of Caspase3 in WB bands; C: PCR detection of Caspase3 expression levels;

[0028] Figure 10 Comparison of mitochondrial damage in renal tissue of rats in each group;

[0029] Figure 11 Comparison of oxidative stress indicators in renal tissue of rats in each group; A: GSH-Px; B: MDA; C: SOD;

[0030] Figure 12 Comparison of iron-related indexes in renal tissue of rats in each group; A: SI; B: SF; C: TF;

[0031] Figure 13 Comparison of GPX4 and ACSL4 expression levels in renal tissues of rats in each group; A: WB strips; B: relative expression of GPX4 in WB strips; C: relative expression of ACSL4 in WB strips; D: PCR detection of GPX4 expression level; E: PCR detection of ACSL4 expression level;

[0032] Figure 14 Comparison of FTH1 and NCOA4 expression levels in renal tissues of rats in each group; A: WB strips; B: Relative expression of FTH1 in WB strips; C: Relative expression of NCOA4 in WB strips; D: PCR detection of FTH1 expression level; E: PCR detection of NCOA4 expression level;

[0033] Figure 15 Comparison of HIF-1a and HO-1 expression levels in renal tissues of rats in each group; A: WB strips; B: relative expression of HIF-1a in WB strips; C: relative expression of HO-1 in WB strips; D: PCR detection of HIF-1a expression level; E: PCR detection of HO-1 expression level;

[0034] In the above figures, Control is the sham operation group, Model is the model group, QRYSXZF is the Qingre Yishen Xiaozheng prescription group, and Dapagliflozin is the dapagliflozin group. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1: Heat-clearing, kidney-tonifying and symptom-reducing granules and their preparation method

[0041] Qingre Yishen Xiaozheng Granules contain the following ingredients: 30g of raw astragalus, 10g of pseudoginseng, 10g of angelica, 15g of raw rehmannia, 15g of dodder seed, 10g of forsythia, 15g of burdock fruit, 10g of scutellaria, 3g of coptis root, 10g of turtle shell, 3g of leech, and 10g of seaweed. Raw astragalus, pseudoginseng, and angelica are monarch herbs, raw rehmannia and dodder seed are assistant herbs, forsythia, burdock fruit, scutellaria, and coptis root are adjuvant herbs, and turtle shell, leech, and seaweed are guiding herbs.

[0042] Astragalus root is sweet and warm in nature, entering the spleen and lung meridians. It has the effects of tonifying qi, strengthening the exterior, and promoting diuresis and reducing edema. Its qi-tonifying effect can elevate yang qi and consolidate the essence, effectively improving qi deficiency in DKD patients while also alleviating edema symptoms through its diuretic effect. Astragalus root and Pseudostellaria radix have a yin-based effect of tonifying middle qi, while astragalus root has a yang-based effect of consolidating defensive qi. Together, they work together to strengthen qi and strengthen the body's internal and external yin and yang. As a key blood-tonifying herb, angelica root also promotes blood circulation and unblocks the meridians, earning it the title of "blood-Qi herb." Based on the theory of "essence and blood share a common origin," kidney deficiency can lead to blood deficiency, resulting in symptoms such as fatigue, insomnia, and pale complexion. Angelica root can improve renal anemia in DKD patients. Blood stasis runs through the entire course of DKD. Angelica sinensis can promote blood circulation and remove blood stasis, promote new blood production, moisten the intestines and promote bowel movements, and expel dampness and turbidity; Rehmannia glutinosa and Cuscuta chinensis nourish the kidneys and yin, and when combined with Astragalus membranaceus, they can enhance the effects of invigorating qi and tonifying the kidneys and reducing proteinuria, and are especially suitable for the early and middle stages of DKD; seaweed, first recorded in the "Shennong Bencao Jing", is bitter, salty, and cold in nature, and enters the liver, stomach, and kidney meridians, with the effects of resolving phlegm, softening hard masses, dispersing nodules, and promoting diuresis; turtle shell has the effects of softening hard masses, dispersing nodules, nourishing yin and suppressing yang, and is suitable for symptoms of phlegm and blood stasis. It forms a pair of medicines with raw Astragalus membranaceus, clearing away heat, dispersing nodules, and eliminating symptoms while also nourishing kidney qi; leeches promote the recovery of renal blood flow by breaking up blood, removing blood stasis, and dissolving microthrombi, further delaying renal dysfunction; the pathogenesis of DKD is based on yin deficiency and symptomatic with dryness and heat, and is easily accompanied by dampness, which manifests as edema, proteinuria, and other symptoms. Scutellaria baicalensis and Coptis chinensis can improve the internal state of dampness and heat, clear the interior and relieve the exterior, and reduce the burden on the kidneys by clearing heat and dampness, detoxifying and promoting salivation; Forsythia suspensa and Arctium lappa, etc., as heat-clearing and detoxifying drugs with anti-inflammatory and detumescent effects, can enhance the ability to eliminate kidney meridian masses.

[0043] Preparation method: Weigh the raw material according to the above ratio and extract it with water twice, add 5 times the weight of water each time and boil for 2 hours, then combine the decoctions, filter, concentrate the filtrate to an extract with a relative density of 1.38, vacuum dry at 80℃, grind, and pass through a 65-mesh sieve to obtain dry powder, add 4 times the weight of dextrin, mix well, use 85% ethanol as a binder, granulate with a 16-mesh sieve, dry, and granulate, and package into 7-gram packages.

[0044] Example 2 Animal Experiment Verification of the Application of Qingre Yishen Xiaozheng Granules in Diabetic Nephropathy

[0045] 1. Experimental Animals

[0046] The experimental animals used in this example were SPF male Sprague-Dawley rats (6-8 weeks old, weighing 200 ± 20 g) provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The animals were housed in a barrier environment (24 ± 2°C, 60 ± 5% humidity, 12-h photoperiod) with free access to standard chow and sterilized water. The experiments were conducted in accordance with the Animal Care Guidelines of Beijing University of Chinese Medicine and were approved by the Ethics Committee (No. DZMYY24-19).

[0047] 2. Experimental drugs

[0048] Qingre Yishen Xiaozheng granules were prepared as in Example 1, and dapagliflozin tablets (10 mg / tablet, national medicine standard number H20170206) were purchased from AstraZeneca.

[0049] 3. Modeling and grouping

[0050] 3.1 Establishing the Animal Model

[0051] To establish a DKD rat model with unilateral nephrectomy combined with STZ injection, 60 healthy male SD rats (weighing 200-250g) were first selected and weighed and numbered after one week of adaptive feeding. Based on body weight and blood glucose levels, 10 rats were randomly selected as the sham-operated group, and the remaining 40 rats were included in the unilateral nephrectomy model group. After random blood glucose testing and confirmation of negative urine protein in all rats, unilateral nephrectomy surgery was performed: all rats were fasted for 12 hours before surgery. During the surgery, the rats were first anesthetized intraperitoneally with 1% sodium pentobarbital (40-50mg / kg, 1mL). After they completely lost the ability to move independently, they were fixed on the operating table and placed in the lateral decubitus position. The abdominal surgical area was disinfected with iodine cotton balls. Subsequently, a sterile scalpel was used to make a dorsal longitudinal incision approximately 1.5cm long, 1cm from the midline of the rat's back and 1-2cm below the left rib cage. The skin and subcutaneous tissue were cut open to expose the abdominal wall muscles. The muscle fibers were carefully separated using ophthalmic scissors, and the abdominal cavity was entered to expose the kidneys. The left kidney was located and the renal pedicle was gently clamped with hemostats to minimize bleeding. The renal pedicle was ligated with 5-0 sutures, and the vessels and ureter were cut along the base of the pedicle. The left kidney was then completely removed. After the surgical wound was carefully inspected to confirm the absence of significant bleeding, the abdominal wall muscles were sutured with absorbable sutures. Finally, the skin incision was sutured intradermally to ensure a tight closure. An appropriate amount of cephalosporin powder was sprinkled over the wound to reduce the risk of postoperative infection. Two weeks after surgery, rats in the DKD group received an intraperitoneal injection of 50 mg / kg of STZ, while the sham-operated group received the same dose of citrate buffer. 72 hours after STZ injection, the rats' blood glucose levels were measured. If non-fasting blood glucose reached or exceeded 16.7 mmol / L for three consecutive times, the model was considered successfully established. Patients whose blood glucose levels did not reach the target were retested one week later. If their blood glucose levels remained below 16.7 mmol / L, an additional intraperitoneal injection of STZ (55 mg / kg) was administered.

[0052] 3.2 Grouping

[0053] After the rat model was successfully established, the experimental animals were randomly divided into groups based on their blood glucose levels. After excluding two rats that failed to establish the model and eight rats that died, the remaining rats with successful modeling were divided equally into a model group, a Qingre Yishen Xiaozheng Recipe group, and a dapagliflozin group. A sham operation group was also set up, with 10 rats in each group. All rats were subjected to gavage intervention starting from the day after the successful modeling, and the duration was 12 weeks. Among them, the dosage of the Qingre Yishen Xiaozheng Recipe group and the dapagliflozin group was based on the "Pharmacological Experimental Methods". The specific dosage regimen was: Qingre Yishen Xiaozheng Granules daily dose of 11.34g / kg, dapagliflozin daily dose of 0.9mg / kg.

[0054] ① Sham operation group: 3 mL of normal saline was administered orally once a day;

[0055] ② Model group: 3 mL of normal saline was administered orally once a day;

[0056] ③ Qingre Yishen Xiaozheng prescription group: model + Qingre Yishen Xiaozheng prescription, Qingre Yishen Xiaozheng granules were given orally once a day;

[0057] ④Dapagliflozin group: model + dapagliflozin, dapagliflozin dissolved in normal saline was administered orally once a day.

[0058] 4. Specimen collection

[0059] 4.1 Serum / Plasma Samples

[0060] At the end of the experiment, the rats were anesthetized and blood was collected from the abdominal aorta. The whole blood samples were centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected to obtain serum samples. The serum samples were aliquoted and stored at -80°C until use.

[0061] 4.2 Urine samples

[0062] Before and after administration and every four weeks after successful model establishment, 24-hour urine was collected from rats. The urine volume was measured and the urine was centrifuged at 3000 rpm for 15 minutes. The supernatant was collected to obtain urine samples. The urine samples were aliquoted and stored at -80°C until use.

[0063] 4.3 Kidney tissue samples

[0064] At the end of the experiment, blood was drawn from the abdominal aorta, and renal tissue was removed and weighed. The renal capsule was removed, and the renal cortex was longitudinally sectioned into four sections, each of which was treated differently. One section was fixed with 4% paraformaldehyde and embedded in paraffin and OCT; a smaller section was placed directly in a 2.5% glutaraldehyde tube for subsequent electron microscopy; the remaining two sections were directly frozen in liquid nitrogen for subsequent molecular biology analysis, such as PCR and western blotting.

[0065] 5. Index detection

[0066] 5.1 Hematuria testing

[0067] Twenty-four-hour urine protein (24h-UTP), blood urea nitrogen (BUN), serum creatinine (Scr), cystatin C (Cys-C), transferrin (TF), and serum iron (SI) were measured by the Department of Laboratory Medicine of Dongzhimen Hospital, Beijing University of Chinese Medicine. Serum ferritin (SF) was measured according to the kit instructions (Ferritin (FE) ELISA kit, purchased from Shanghai Jianglai Biotechnology Co., Ltd.).

[0068] 5.2 Renal tissue biochemical analysis

[0069] The levels of glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) in renal tissue were detected according to the instructions of the kits ((GSH) ELISA kit, (MDA) ELISA kit, (SOD) ELISA kit, (Kim-1) ELISA kit, and (NGAL) ELISA kit, all purchased from Shanghai Jianglai Biotechnology Co., Ltd.).

[0070] 5.3 Pathological examination

[0071] 5.3.1 Masson trichrome staining

[0072] After dewaxing and hydrating the sections, stain with coal at 60°C for 1 hour and rinse with running water for 10 minutes. Stain with lapis lazuli blue for 2-3 minutes, hematoxylin for 3 minutes, and hydrochloric acid-ethanol for differentiation until the tissue appears red. Stain with Ponceau red for 12 minutes and treat with phosphomolybdic acid for 15-20 minutes until the collagen fades. Stain with aniline blue for 5 minutes and wash with a weak acid solution for 2 minutes. Dehydrate using increasing concentrations of ethanol (70% to 100%) for 1 minute each. Finally, clear the sections with xylene and mount with neutral gum.

[0073] 5.3.2 HE staining

[0074] After the sections were baked at 60°C for 1 hour, they were placed in xylene I, II, and III for 10 minutes each for dewaxing. Subsequently, they were hydrated with ethanol solutions of decreasing concentration (100%, 95%, 90%, 80%, and 70%) for 3 minutes each level, and finally rinsed with distilled water for 5 minutes. After hematoxylin staining for 10 minutes, the sections were differentiated with 1% hydrochloric acid ethanol for 15 seconds and rinsed with running water for 10 minutes to complete the blueing. Eosin staining was performed for 15 minutes and rinsed with running water for 1 minute. The subsequent dehydration, transparency, and sealing steps were the same as Masson trichrome staining.

[0075] 5.3.3 PAS staining

[0076] After dewaxing and hydrating tissue sections, oxidize with 1% periodic acid for 8–10 minutes and rinse with running water for 3 minutes. Stain with Schiff's reagent in the dark for 1 hour and rinse with running water for 10 minutes. Nuclear staining procedures are the same as for HE staining. Subsequent dehydration, clearing, and mounting procedures follow standard procedures.

[0077] 5.3.4 IHC staining

[0078] After deparaffinization and hydration, sections were antigen-retrieved in citrate buffer (pH 6.0) at 95°C for 20 minutes. Endogenous peroxidases were blocked by incubation with 3% hydrogen peroxide for 20 minutes at room temperature. Primary antibodies were incubated overnight at 4°C and then rewarmed at room temperature for 1 hour the following day. Sections were incubated in reaction enhancement solution at 37°C for 20 minutes, followed by incubation with secondary antibodies at 37°C for 20 minutes. DAB color development was followed by hematoxylin counterstaining, followed by routine dehydration, clearing, and mounting.

[0079] 5.3.5 Transmission Electron Microscopy Sample Preparation

[0080] Fresh kidney tissue was cut into 1 mm 3 Small sections were fixed with 2.5% glutaraldehyde overnight at 4°C. After rinsing with 0.1M phosphate buffer, the sections were fixed with 1% osmium sulfate for 2 hours at 4°C. Dehydration was performed using graded ethanol, followed by transition with propylene oxide, and embedding in epoxy resin. 70 nm sections were cut and double-stained with UA and Pb for 10 minutes each. Transmission electron microscopy was performed and photographed.

[0081] 5.4 RT-qPCR

[0082] 5.4.1 Primer Design

[0083] The primer sequences are shown in Table 1.

[0084] Table 1 Primer sequences

[0085]

[0086]

[0087] 5.4.2 Extraction of total RNA from samples

[0088] Quickly freeze a 30-50 mg sample of kidney tissue in liquid nitrogen and grind to a powder. Transfer the ground tissue to an RNase-free centrifuge tube and add an appropriate amount of TRIzol reagent to thoroughly lyse and mix. Add chloroform in a 5:1 ratio and shake vigorously for 15 seconds. Then let it stand at room temperature for 2 minutes and centrifuge at 12,000 rpm at 4°C for 10 minutes. The solution will then separate into three layers, with RNA primarily present in the colorless upper aqueous phase. Pipette the upper aqueous phase into a new RNase-free centrifuge tube, add an equal volume of isopropanol, mix, and let it stand at room temperature for 10 minutes to precipitate the RNA. Centrifuge again at 12,000 rpm at 4°C for 10 minutes, discard the supernatant, and retain the RNA pellet. Wash with 75% ethanol, gently shake, and centrifuge at 7,500 rpm at 4°C for 5 minutes. Discard the supernatant and repeat the wash. Incubate at 20-30°C for 15 minutes. Dissolve the pellet in RNase-free water and determine RNA concentration and purity using a spectrophotometer.

[0089] 5.4.3 RNA electrophoresis

[0090] 5 μL of RNA was electrophoresed on 1% agarose gel to detect the integrity of the RNA.

[0091] 5.4.4 Reverse transcription

[0092] Dissolve the indicated components in the kit and keep on ice. Add the components according to Table 2 to the reaction tube to a total volume of 30 μL and mix thoroughly. Incubate the reaction at 42°C for 50 minutes, then transfer to 85°C and incubate for another 5 minutes. After the reaction is complete, briefly centrifuge the reaction tube to ensure that any liquid adhering to the tube walls has settled to the bottom. The resulting cDNA product should be stored at -20°C until ready for use.

[0093] Table 2 Reverse transcription system

[0094]

[0095]

[0096] 5.4.5 Real-Time PCR

[0097] The mRNA / lncRNA qPCR kit (catalog number: GPQ1808, purchased from: GenePool) was used for the analysis. The specific operation was carried out according to the instructions. The reaction program was set to 95℃ pre-denaturation for 30 seconds, followed by 45 cycles of amplification (95℃ for 5 seconds, 60℃ for 30 seconds), and the melting curve was collected in the range of 60-95℃. The components of the reaction system and their dosage are detailed in Table 3; in the primer optimization stage, the sample cDNA was mixed in equal amounts and diluted 5-fold. 2μL of the dilution was used as a template and amplified using target gene-specific primers (Table 4) and the melting curve was analyzed. Primer pairs with amplification efficiency (90% to 110%) and a single-peak melting curve were screened. When testing the samples, the cDNA was diluted 10 times and 2μL was taken as a template. The target gene and internal reference gene primers (Table 5) were used for amplification, and the melting curve was analyzed to ensure specificity. The internal reference gene was used as a normalization reference. -△△CT The expression levels of target genes were analyzed by relative quantitative analysis.

[0098] Table 3 PCR reaction system (20 μL)

[0099]

[0100] Table 4 Primer screening standard curve Real Time PCR design

[0101]

[0102] Table 5 Sample Real Time PCR detection design

[0103]

[0104] 5.5WB Analysis

[0105] 5.5.1 Protein extraction

[0106] Freshly harvested kidney samples were quickly washed in pre-chilled PBS buffer and excess water was removed using sterile filter paper. An appropriate amount of RIPA lysis buffer containing protease inhibitors was added to the tissue and homogenized thoroughly on ice until the tissue was completely disrupted. The mixture was transferred to a centrifuge tube and centrifuged at 12,000 rpm for 15 minutes at 4°C to remove cell debris and insoluble impurities. After centrifugation, the supernatant was aspirated to obtain the protein sample.

[0107] 5.5.2 BCA protein quantification

[0108] Prepare the standard and working solution according to the BCA kit instructions, and dilute the standard protein into different concentrations (0, 0.125, 0.25, 0.5, 1, 2 mg / mL) in a gradient. Take an appropriate amount of the protein sample to be tested and dilute it 10 times with lysis buffer. Add 20 μL of the standard and the sample to be tested to a 96-well plate, and set up duplicate wells for each sample to reduce errors. Then add 200 μL of the pre-prepared BCA working solution to each well, mix gently, and incubate at 37°C for 30 minutes. Use a microplate reader to measure the absorbance of each well at a wavelength of 562 nm. Draw a standard curve and calculate the protein concentration of the sample to be tested by a linear regression equation. According to the experimental requirements, use 5× Loading Buffer volume and RIPA lysis buffer to adjust the sample to a uniform concentration. After denaturation in a metal bath above 95°C for 10 minutes, aliquot and store at -80°C for later use.

[0109] 5.5.3WB Experiment

[0110] Prepare an SDS-PAGE resolving gel of appropriate concentration based on the molecular weight of the target protein. Using a 1 mL pipette, draw up 5 mL of gel and slowly pour it along the glass plate. Once the gel surface has risen to the desired height, add water to seal it. Once a refraction line appears between the water and the gel, let it stand for 3 minutes to allow the gel to fully solidify. Pour off the top layer of water and blot dry with absorbent paper. Next, prepare a stacking gel, fill it to the remaining space, and insert a comb, keeping the comb level. After the stacking gel solidifies, rinse it with water and place it in the electrophoresis tank. Gently remove the comb. Before loading the sample, boil the sample for 5 minutes to denature the protein. After adding enough electrophoresis buffer, load 5 μg of protein per well, avoiding bubbles and cross-contamination. Run the electrophoresis at 60-100 V for approximately 2 hours, until the bromophenol blue runs out. Prepare four sheets of filter paper and one NC membrane or PVDF membrane for transfer. The membrane should be activated by soaking in transfer buffer or methanol. With the black side of the clip facing down, place the sponge pad, filter paper, gel, membrane, filter paper, and sponge pad in that order. Remove any bubbles, close the clip, and place in a transfer tank, black side facing black and red side facing red. Transfer the membrane at 200 mA for 2 hours, keeping it on ice. After transfer, block with 5% BSA or skim milk powder for 1 hour. Dilute the primary antibody proportionally and incubate at 4°C overnight. The next day, remove the primary antibody and wash the membrane three times with TBST (10 minutes each). Prepare a secondary antibody and dilute it with 5% skim milk powder. After incubation for 1 hour, wash the membrane again with TBST (10 minutes each). Detect by chemiluminescence.

[0111] 6. Statistical methods

[0112] GraphPad Prism 9.0.0 software was used for statistical analysis and graph drawing, and Image J software was used for WB experiments.

[0113] The results were quantitatively analyzed by grayscale value. Experimental data are expressed as mean ± standard error. For data that conformed to a normal distribution, one-way analysis of variance was used for comparisons between groups; for data that did not conform to a normal distribution, nonparametric tests were used. The significance level was set at α = 0.05, and differences were considered statistically significant when the P value was less than 0.05.

[0114] 7. Experimental results

[0115] 7.1 General Status Comparison

[0116] Observations of the general condition of the rats in each group during the experiment revealed that the sham-operated group exhibited good physiological status, with motor ability, reaction sensitivity, and hair color within normal ranges. The model group exhibited a distinct pathological state: lethargy, decreased activity, sluggish reaction time, emaciation, and dull hair, accompanied by significant polydipsia, polyphagia, and polyuria. After drug intervention, the mental status of the rats in the Qingre Yishen Xiaozheng Recipe and dapagliflozin groups improved, their activity increased, and their hair color turned light yellow. Their food intake, water intake, and urine output remained similar to those of the model group.

[0117] 7.2 Comparison of body weight and kidney weight to body weight ratio

[0118] As shown in Table 6 and Figure 1 As shown in Figure A, the weight of rats in each group at week 0 was approximately 250 g. The weight of rats in the sham-operated group continued to increase throughout the experiment, from week 0 (252.0 ± 4.26 g) to week 12 (542.5 ± 28.99 g). The weight of rats in the model group decreased slightly, and the weight of rats in the Qingre Yishen Xiaozheng Recipe group and the dapagliflozin group did not increase significantly. After 12 weeks of treatment, the weight of rats in the other three groups decreased significantly compared with the sham-operated group, with the model group being the most significant ( Figure 1 The kidney weight of rats in each group was measured and the kidney weight to body weight ratio was calculated. It was found that the kidney weight to body weight of rats in each drug-treated group was significantly higher than that in the sham-operated group ( Figure 1 Middle C).

[0119] Table 6 Body weight and kidney weight to body weight ratio of the four groups of rats before and after intervention ( n=6)

[0120]

[0121] Note: Compared with the sham-operated rats, *** P<0.001.

[0122] 7.3 Qingre Yishen Xiaozheng Recipe Reduces Blood Glucose Levels in DKD Rats

[0123] As shown in Table 7, Figure 2 As shown in middle A, 12 weeks after the start of oral gavage, the blood glucose of rats in the dapagliflozin group decreased significantly, the blood glucose of rats in the Qingre Yishen Xiaozheng prescription group decreased to a lesser extent, and there was no significant change in the blood glucose of rats in the model group. Figure 2 As shown in Figure B, after 12 weeks of treatment, the blood glucose level in the sham group was approximately 6.62 mmol / L, while that in the model group was approximately 28.77 mmol / L, showing no significant change compared to week 0. The blood glucose level in the dapagliflozin group was approximately 16.15 mmol / L, a significant decrease compared to the model group (P < 0.001).

[0124] Table 7 Comparison of blood glucose levels in rats in each group ( n=6)

[0125]

[0126] Note: Compared with the sham-operated rats, *** P<0.001; compared with the model group rats, ### P<0.001.

[0127] 7.4 Qingre Yishen Xiaozheng Recipe Improves Renal Function and Alleviates Renal Damage in DKD Rats

[0128] The renal function indexes of rats in each group after 12 weeks of drug intervention (Table 8, Figure 3 ) found that there was no significant difference in the Scr concentration among the four groups of rats (P>0.05); the BUN and 24h-UTP levels in the model group were significantly higher than those in the sham operation group, and the differences were statistically significant (P<0.01); the BUN and Cys-C levels of the rats in the two drug treatment groups were significantly lower than those in the model group, and the differences were statistically significant (P<0.01); the total 24h-UTP level showed a downward trend.

[0129] Table 8 Comparison of renal function indicators in rats of each group ( n=6)

[0130]

[0131] Note: Compared with the sham-operated rats, ** P<0.01, *** P<0.001; compared with the model group rats, ## P<0.01, ### P<0.001.

[0132] By comparing the expression levels of KIM-1 and NGAL in the kidney tissues of rats in each group, as shown in Table 9, Figure 4As shown, KIM-1 and NGAL levels were low in the sham group, indicating no significant renal damage. However, KIM-1 and NGAL levels in the model group increased significantly, reaching approximately 5.62 ng / mL and 741.1 pg / mL, respectively, with statistically significant differences compared to the sham group, suggesting significant renal damage in the DKD model. KIM-1 (1.69±0.32 ng / mL) and NGAL (387.0±30.06 pg / mL) levels in the Qingre Yishen Xiaozheng Formula group were significantly lower than those in the model group (p<0.001), indicating that the formula significantly alleviated renal damage. While KIM-1 and NGAL levels in the dapagliflozin group were also significantly lower than those in the model group (p<0.01), the effect was slightly weaker than that in the Qingre Yishen Xiaozheng Formula group. This demonstrates that the Qingre Yishen Xiaozheng Formula exhibits a significant renal protective effect in the treatment of DKD.

[0133] Table 9 Comparison of renal injury indicators in rats of each group ( n=6)

[0134]

[0135] Note: Compared with the sham-operated rats, *** P<0.001; compared with the model group rats, ## P<0.01, ### P<0.001.

[0136] 7.5 Qingre Yishen Xiaozheng Recipe alleviates kidney pathological damage and improves renal fibrosis in DKD rats

[0137] The results of HE staining, Masson staining and PAS staining are as follows Figure 5 In the sham-operated group, HE staining revealed intact renal tubular morphology and regular arrangement of epithelial cells. Masson staining revealed no significant collagen fiber accumulation in the renal interstitium and tubular basement membrane, indicating normal tissue structure. PAS staining further confirmed that the tubular basement membrane was uniform, without thickening or abnormal glycogen accumulation. The sham-operated group showed no obvious pathological changes in renal tissue, indicating normal tubular structure.

[0138] In the model group, HE staining revealed significant swelling and degeneration of renal tubular epithelial cells, with some cells undergoing necrosis and shedding. The tubular lumen was significantly dilated, accompanied by inflammatory cell infiltration. Masson staining revealed a significant increase in collagen fibers in the renal interstitial and peritubular regions, indicating a significant increase in tissue fibrosis. PAS staining revealed significant thickening of the renal tubular basement membrane and increased glycogen deposition. Renal tissue in the model group exhibited significant pathological damage, including tubular epithelial cell damage, interstitial fibrosis, and basement membrane thickening.

[0139] Qingre Yishen Xiaozheng prescription group: HE staining results showed that the swelling and degeneration of renal tubular epithelial cells were significantly reduced compared with the model group, the tubular expansion phenomenon was not obvious, and the inflammatory cell infiltration was reduced; Masson staining observed that the collagen fiber deposition in the renal interstitium and around the renal tubules was significantly reduced, and the degree of fibrosis was significantly improved compared with the model group; PAS staining further showed that the thickening of the renal tubular basement membrane was reduced and the amount of glycogen deposition was reduced.

[0140] In the dapagliflozin group, HE staining showed that the swelling and degeneration of renal tubular epithelial cells were alleviated compared with the model group, but tubular expansion and inflammatory cell infiltration were still observed; Masson staining results showed that the accumulation of collagen fibers in the renal interstitium and around the renal tubules was reduced; PAS staining showed that the thickening of the renal tubular basement membrane was reduced.

[0141] Immunohistochemical staining results Figure 6 As shown in the results, the expression of FN protein in the kidney tissue of rats in the sham operation group was low, mainly confined to the glomerular basement membrane and the peritubular area, with a weak expression intensity; the expression level of FN protein in the kidney tissue of rats in the model group was significantly increased, widely distributed in the glomeruli, renal tubules and interstitial areas, and the expression intensity was significantly enhanced; the expression level of FN protein in the kidney tissue of rats in the Qingre Yishen Xiaozheng Recipe group and the dapagliflozin group was significantly decreased compared with the model group, mainly distributed in the glomeruli and peritubular areas, and the expression intensity was significantly weakened. WB experiment showed (see Figure 7 ), the expression of TGF-β in the kidneys of rats in the Qingre Yishen Xiaozheng prescription group was slightly lower than that in the model group, but there was no significant difference in the expression of TGF-β in the kidneys of rats in each group.

[0142] 7.6 Qingre Yishen Xiaozheng Recipe Alleviates Renal Cell Apoptosis in DKD Rats

[0143] Compared with the sham operation group, the expression level of Caspase-3 in the kidneys of rats in the model group increased. After intervention with the Qingre Yishen Xiaozheng recipe, the protein expression of Caspase-3 in the kidney tissues of rats was significantly reduced compared with the model group. Its distribution was mainly concentrated in the renal tubular epithelial cells and glomerular areas, and the expression intensity was significantly weakened ( Figure 8 ).like Figure 9 Western blot and PCR results showed that the expression levels of caspase-3 mRNA and protein in the kidney tissues of rats in the sham-operated group were low. The expression levels of caspase-3 mRNA and protein in the kidney tissues of rats in the model group were significantly higher than those in the sham-operated group (P < 0.01). Western blot analysis showed that the expression levels of caspase-3 protein in the Qingre Yishen Xiaozheng Recipe and dapagliflozin groups were significantly lower than those in the model group (P < 0.01). The expression level of caspase-3 mRNA in the Qingre Yishen Xiaozheng Recipe group was also significantly lower than that in the model group (P < 0.05). However, there was no statistically significant difference in the expression of caspase-3 mRNA in the kidneys of rats in the dapagliflozin group compared with the model group.

[0144] 7.7 Qingre Yishen Xiaozheng Recipe Improves Mitochondrial Morphology and Function in the Kidneys of DKD Rats

[0145] Under transmission electron microscopy, Figure 10 The results showed that the mitochondria in the kidney cells of rats in the sham operation group showed complete morphological characteristics, with clear inner membrane structure and orderly arrangement of cristae. No obvious swelling, vacuolation or cristae rupture was observed, indicating that their mitochondrial function was normal and the cell metabolism was in good condition. In contrast, the mitochondrial damage in the kidney cells of rats in the model group was more significant, mainly manifested by enlarged mitochondrial volume, rupture or disappearance of cristae structure, internal vacuolation, and even rupture of some mitochondrial membranes. In the Qingre Yishen Xiaozheng Recipe group and the dapagliflozin group, the degree of mitochondrial damage in the kidney cells of rats was significantly alleviated compared with the model group, with significantly reduced mitochondrial swelling and cristae rupture, and the overall structure tended to be complete, indicating that both interventions can effectively improve mitochondrial morphology and function.

[0146] 7.8 Qingre Yishen Xiaozheng Recipe Improves Oxidative Stress in DKD Rats

[0147] The oxidative stress products of rats in each group were detected (Table 10, Figure 11 ). Compared with the sham-operated group, the SOD and GSH-Px levels in the renal tissues of rats in the model group were significantly decreased, while the MDA level showed an upward trend. Compared with the model group, the GSH-Px and SOD levels in the renal tissues of rats in the Qingre Yishen Xiaozheng Recipe group were increased (P < 0.001); MDA levels were decreased (P < 0.001). Compared with the model group, the GSH-Px level in the renal tissues of rats in the dapagliflozin group was increased; the SOD level was increased; and the MDA level was decreased (P < 0.05).

[0148] Table 10 Comparison of oxidative stress indicators in rats of each group ( n=6)

[0149]

[0150] Note: Compared with the sham-operated rats, *** P<0.001; compared with the model group rats, # P<0.05, ### P<0.001.

[0151] 7.9 Qingre Yishen Xiaozheng Recipe to Inhibit Iron Overload

[0152] By comparing the iron-related indicators of rats in each group after the intervention (Table 11, Figure 12Serum iron (SI) and ferritin (SF) levels in the model group were elevated compared to normal, indicating significant iron overload. However, transferrin (TF) levels in the model group showed a significant decrease compared to the sham-operated group (P < 0.001). SI levels were significantly decreased in the Qingre Yishen Xiaozheng formula group compared to the model group. SF levels were significantly decreased in the Qingre Yishen Xiaozheng formula group (21.60 ± 2.08 pg / mL) and the dapagliflozin group (32.58 ± 2.31 pg / mL) compared to the model group (P < 0.01). TF levels in both groups were approximately 0.45 g / L, which was increased compared to the model group (P < 0.05).

[0153] Table 11 Comparison of iron-related indicators in rats in each group ( n=6)

[0154]

[0155] Note: Compared with the sham-operated rats, *** P<0.001; compared with the model group rats, # P<0.05, ## P<0.01, ### P<0.001.

[0156] 7.10 Qingre Yishen Xiaozheng Recipe Reduces Ferroptosis in DKD Rats

[0157] WB experiments showed (see Figure 13 Compared with the sham operation group, the expression of GPX4 in the kidneys of rats in the model group was decreased, while the expression of ACSL4 was significantly increased (P < 0.001). The expression of GPX4 in the kidneys of rats in the Qingre Yishen Xiaozheng Recipe group was significantly increased, while the expression of ASCL4 was significantly decreased (P < 0.05). There were no significant differences in the expression of GPX4 and ACSL4 proteins in the kidneys of rats in the dapagliflozin group compared with the model group. Figure 13 PCR results of DE showed that the expression level of GPX4 mRNA in the kidneys of rats in the model group was significantly decreased compared with the sham-operated group (P<0.001), while the expression level of ACSL4 mRNA was significantly increased (P<0.001). The expression level of GPX4 mRNA in the kidneys of rats in the Qingre Yishen Xiaozheng prescription group was significantly increased compared with the model group (P<0.001), while the expression level of ACSL4 mRNA was significantly decreased (P<0.001). The expression level of GPX4 mRNA in the kidneys of rats in the dapagliflozin group was significantly increased compared with the model group (P<0.05), while the expression level of ACSL4 mRNA showed a downward trend, but the difference was not statistically significant.

[0158] WB experiments showed (see Figure 14 AC), compared with the sham operation group, the expression of FTH1 in the kidney of the rats in the model group was significantly decreased (P<0.01), and the expression level of NCOA4 was significantly increased (P<0.01). The expression of FTH1 in the kidney of the rats in the Qingre Yishen Xiaozheng prescription group was significantly increased compared with the model group, and the expression of NCOA4 was significantly decreased, and the differences were statistically significant (P<0.05); compared with the model group, there was no significant statistical difference in the expression of FTH1 protein in the kidney of the rats in the dapagliflozin group, and the expression of NCOA4 was significantly decreased (P<0.05). PCR results showed (see Figure 14 Compared with the sham-operated group, the model group showed a significant decrease in FTH1 mRNA expression in the kidneys of rats (P<0.01), while the NCOA4 mRNA expression level was significantly increased (P<0.001). Compared with the model group, the Qingre Yishen Xiaozheng formula group showed a significant increase in FTH1 mRNA expression in the kidneys of rats (P<0.05), while the NCOA4 mRNA expression level was significantly decreased (P<0.01). There were no statistically significant differences in FTH1 and NCOA4 mRNA expression in the dapagliflozin group compared with the model group.

[0159] 7.11 Qingre Yishen Xiaozheng Recipe Inhibits the HIF-1a / HO-1 Signaling Pathway

[0160] WB experiments showed (see Figure 15 AC), compared with the sham operation group, the protein expressions of HIF-1a (P<0.05) and HO-1 (P<0.001) in the kidneys of rats in the model group were significantly increased. The protein expressions of HIF-1a (P<0.05) and HO-1 (P<0.001) in the kidneys of rats in the Qingre Yishen Xiaozheng prescription group and the dapagliflozin group were significantly decreased compared with the model group. PCR results further showed that ( Figure 15 In the model group, the mRNA expression levels of HIF-1a and HO-1 in the kidneys of rats in the sham operation group were significantly higher than those in the model group (P < 0.001). The mRNA expression levels of HIF-1a and HO-1 in the kidneys of rats in the Qingre Yishen Xiaozheng recipe and dapagliflozin groups were significantly lower than those in the model group (P < 0.001). This suggests that the Qingre Yishen Xiaozheng recipe may exert its effects by inhibiting the HIF-1a / HO-1 signaling pathway.

[0161] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A granule for clearing away heat, benefiting the kidney and eliminating symptoms, characterized in that: The medicine comprises the following raw materials: 30g of raw astragalus, 10g of pseudoginseng, 10g of angelica, 15g of raw rehmannia, 15g of dodder seed, 10g of forsythia, 15g of burdock fruit, 10g of scutellaria, 3g of coptis root, 10g of turtle shell, 3g of leech and 10g of seaweed.

2. The heat-clearing, kidney-tonifying and symptom-reducing granules according to claim 1, wherein Prepared by the following method: Weigh all raw materials according to the formula, mix them, extract with water, filter and concentrate to obtain extract, dry and grind to obtain dry powder, add dextrin and granulate to obtain the extract.

3. The heat-clearing, kidney-tonifying and symptom-reducing granules according to claim 2, wherein: The extraction with water is performed by adding 5 times the weight of water and boiling for 2 hours, extracting twice, and combining the decoctions.

4. The heat-clearing, kidney-tonifying and symptom-reducing granules according to claim 2, wherein: The weight ratio of the dry powder to the dextrin is 1:

4.

5. Use of the Qingre Yishen Xiaozheng granules according to claim 1 in preparing a medicament for treating diabetic nephropathy.

6. A drug for treating diabetic nephropathy, characterized in that: The invention comprises the heat-clearing, kidney-tonifying and symptom-eliminating granules according to claim 1.

7. The drug according to claim 6, characterized in that The Qingre Yishen Xiaozheng granules play a role in treating diabetic nephropathy by resisting oxidative stress.

8. The drug according to claim 6, wherein The Qingre Yishen Xiaozheng granules play a role in treating diabetic nephropathy by inhibiting iron overload and reducing the degree of ferroptosis.

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