Traditional Chinese medicine composition for treating diabetic nephropathy and preparation method thereof

Abstract

The invention provides a traditional Chinese medicine composition for treating diabetic nephropathy and a preparation method thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 30g of astragalus membranaceus, 15g of angelica sinensis, 15g of radix paeoniae rubra, 15g of ligusticum wallichii, 9g of safflower carthamus, 18g of prepared rehmannia root and 15g of Chinese yam. On the basis of the traditional Chinese medicine theory and a transcriptomics sequencing result of a clinical sample, the Yang-tonifying and Yang-recuperating decoction is cut, the improved compound traditional Chinese medicine composition of a new prescription is generated, the pathological changes such as glomerulosclerosis and fibrosis of a diabetic nephropathy animal model can be effectively inhibited, microalbumin in urine, serum creatinine and urea nitrogen are reduced, and the curative effect is good. Compared with an original formula, the traditional Chinese medicine composition has a more remarkable effect on treating diabetic nephropathy.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine and Chinese medicine composition, and in particular to a Chinese medicine composition for treating diabetic nephropathy and a preparation method thereof. Background Art

[0002] Diabetic nephropathy (DKD) is a common microvascular complication of diabetes mellitus (DM) and is the most common cause of end-stage renal disease (ESRD) worldwide. Globally, the number of patients with diabetes is expected to increase from 463 million to 780 million by 2045, and approximately 20%-40% of patients with diabetes will develop DKD. In addition, the risk of death in patients with DKD is 3-12 times higher than that in patients with diabetes alone. DKD poses a serious threat to human health. Currently, the first-line drugs for the treatment of DKD are mainly renin angiotensin system inhibitors or angiotensin receptor blockers, and sodium-glucose cotransporter-2 inhibitors (SGLT2i). Although they can delay the progression of DKD to end-stage renal disease (ESRD), the efficacy is still unsatisfactory. The current control of DKD, despite the optimal management of DKD, including lifestyle changes, strict blood sugar, blood pressure and blood lipid control, still has a considerable residual risk of DKD progression, indicating that it is still necessary to explore new treatments with better effects on DKD.

[0003] Traditional Chinese medicine (TCM) has remarkable properties, including anti-inflammatory and antioxidant activities, which can help treat DKD. The traditional Chinese medicine Buyang Huanwu Decoction comes from "Yi Lin Gaicuo" and is composed of Astragali Radix, Angelicae Sinensis Radix, Paeoniae Radix Rubra, Chuanxiong Rhizoma, Carthami Flos, Prunus persica and Earthworm. It has the effects of replenishing qi, activating blood circulation and unblocking meridians. It is mainly used to treat qi deficiency and blood stasis syndrome of stroke. Hemiplegia, facial paralysis, slurred speech, drooling at the corners of the mouth, frequent urination or incontinence, dark tongue, white fur, and weak pulse. In recent years, it has been found that Buyang Huanwu Decoction can also correct lipid metabolism disorders and reduce urine protein levels in patients with diabetic nephropathy, and has a significant improvement on renal function. The effect of Buyang Huanwu Decoction is still mediocre compared with the Western medicines on the market. Moreover, in clinical practice, when adding or modifying Chinese herbal compound prescriptions according to the symptoms and constitutions of different patients, there is still a lack of modern scientific principles and theoretical support, which hinders the understanding of traditional Chinese medicine from a modern biomedical perspective. Summary of the invention

[0004] In order to address the deficiencies of the prior art, the present invention proposes a Chinese medicine composition for treating diabetic nephropathy and a preparation method thereof. Based on the theory of traditional Chinese medicine and transcriptomics sequencing technology, Buyang Huanwu Decoction is modified to produce a new prescription of an improved compound Chinese medicine composition, which can effectively inhibit diabetic nephropathy fibrosis, inhibit glomerular sclerosis, and improve renal function compared with the original prescription.

[0005] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0006] The invention provides a traditional Chinese medicine composition for treating diabetic nephropathy. The composition is composed of the following raw materials in proportion by weight: 30g of astragalus, 15g of angelica, 15g of red peony root, 15g of ligusticum chuanxiong, 9g of safflower, 18g of rehmannia root and 15g of yam.

[0007] Preferably, the raw material is a decoction piece or a single-drug formula granule equivalent to an equal amount of the decoction piece.

[0008] More preferably, when the raw materials are single-drug formula granules, the Chinese medicine composition is composed of the following raw materials in the following weight proportions: 12g of Astragalus formula granules, 9.99g of Angelica formula granules, 4.5g of Red Peony Root formula granules, 5g of Chuanxiong formula granules, 4.09g of Carthamus tinctorius formula granules, 13.68g of Rehmannia glutinosa formula granules, and 3.75g of Dioscorea opposita formula granules.

[0009] The present invention also provides a Chinese medicine oral preparation for treating diabetic nephropathy, which is prepared using the Chinese medicine composition as described above and a raw material drug containing the active ingredients of the Chinese medicine composition.

[0010] Preferably, the oral Chinese medicine preparation is an aqueous solution of a Chinese medicine decoction or a single-drug formula composition.

[0011] The present invention further provides a method for preparing a Chinese medicine oral preparation for treating diabetic nephropathy, which is prepared using the Chinese medicine composition as described above and a raw material drug containing the active ingredients of the Chinese medicine composition.

[0012] Preferably, the oral Chinese medicine preparation is an aqueous solution of a Chinese medicine decoction or a single-drug formula composition.

[0013] More preferably, the method for preparing the aqueous solution of the single-drug formula composition comprises: mixing the single-drug formula particles of the traditional Chinese medicine composition as described above, adding boiling water, and stirring until fully dissolved to obtain a traditional Chinese medicine solution.

[0014] The beneficial effects of the present invention are:

[0015] The present invention is based on the theory of traditional Chinese medicine and transcriptomic sequencing to modify Buyang Huanwu Decoction to produce a new prescription of improved compound Chinese medicine composition, wherein the monarch drug is Astragalus, which can invigorate qi and raise yang, consolidate and protect the kidney's vital energy, promote diuresis and eliminate dampness, and dissipate kidney stasis and turbidity, with a large amount of strength and special; Angelica is the ministerial drug, which can activate blood circulation and dissipate blood stasis, regulate qi and blood to benefit blood vessels, and clear kidney collaterals to eliminate stasis, and prevent the excessive astringency of Astragalus; the monarch and ministerial drugs are combined to invigorate kidney qi, promote dampness and turbidity, eliminate blood stasis, regulate qi and blood, and invigorate without stagnation; red peony root, Chuanxiong, and safflower are used to activate blood circulation and dissipate blood stasis to clear kidney collaterals; Chinese yam and Rehmannia glutinosa are added to invigorate qi and yin and stop thirst. The whole prescription plays the role of invigorating qi and removing dampness, promoting blood circulation and removing blood stasis, and invigorating without stagnation, so that diabetic kidney can be cured.

[0016] On the one hand, the present invention inherits the advantages of traditional Chinese medicine syndrome differentiation and prescription, and gives play to the characteristics of modern molecular diagnosis and precise treatment. It is convenient for clinical application and has a definite curative effect. Compared with the original Buyang Huanwu Decoction, it has a higher therapeutic efficiency and can significantly improve renal function and reduce weight. On the other hand, the Chinese medicine composition of the present invention uses common medicinal materials as raw materials, has low cost, easy to find medicine sources, effectively reduces the economic burden of patients, can be applied to prepare a variety of different dosage forms, has a simple preparation process, and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the time series cluster analysis of DKD, DM and Control groups.

[0018] Figure 2 Schematic diagram of key gene screening for the improved Buyang Huanwu Decoction, and Venn diagram of the intersection of 5hmC, original Buyang Huanwu Decoction target genes, and the gene set related to DKD in the Genecards database.

[0019] Figure 3 These are the key genes for flavor reduction obtained from 30 genes through the PPI network.

[0020] Figure 4 This is a table of traditional Chinese medicines that target key genes in the original formula of Buyang Huanwu Decoction.

[0021] Figure 5 The following are the key genes for flavoring obtained from 477 genes through machine learning screening. The following is the ROC plot of the accuracy of the 10 key genes in distinguishing the DKD and DM groups.

[0022] Figure 6 The following are the key genes for flavoring obtained from 477 genes screened by machine learning. The following is a heat map of the accuracy of the 10 key genes in distinguishing the DKD and DM groups.

[0023] Figure 7 It is a potential "key target-ingredient-traditional Chinese medicine network".

[0024] Figure 8 The animal experiments in Example 1 verified the changes in body weight and biochemical indicators of mice after administration of the original and improved prescriptions.

[0025] Fig. 9 This is a schematic diagram of verifying the staining and immunofluorescence results of mouse kidney tissue sections through animal experiments. DETAILED DESCRIPTION

[0026] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0027] Example

[0028] The original formula of Buyang Huanwu Decoction replenishes qi, activates blood circulation, and dredges the collaterals. It is mainly used to treat stroke syndrome caused by qi deficiency and blood stasis. Raw Astragalus is used as the main medicine to replenish vital energy, which is intended to make qi strong and blood flow, remove blood stasis and dredge the collaterals, so as to remove blood stasis without hurting the body. Angelica is good at activating blood circulation and nourishing blood, removing blood stasis without hurting blood, and is the ministerial medicine. It is used together with Astragalus to form "Danggui Buxue Decoction", which can replenish qi and produce blood, not only make up for the blood deficiency caused by blood stasis in the meridians, but also activate blood circulation and dredge the collaterals without hurting the body. Chuanxiong and red peony root activate blood circulation and nourish the body; peach kernel and safflower activate blood circulation and remove blood stasis; earthworm is good at running around, dredges the meridians and activates the collaterals, and travels all over the body to promote the efficacy of the medicine, and they are all adjuvants.

[0029] Through clinical studies on diabetic nephropathy patients, diabetic patients and healthy people, the key genes for the progression of diabetic nephropathy and 5hmC differential genes were jointly analyzed to determine the key differential genes related to treatment. The intersection of the target of Buyang Huanwu Decoction database and the 5hmC differential genes revealed 30 differential genes related to drugs and 477 differential genes outside the intersection (such as Figure 1 , Figure 2 ); PPI network analysis of the 30 differentially expressed genes in the intersection revealed five key genes associated with diabetic nephropathy, including IL6, TLR4, LPL, LTF, and SREBF1 (e.g. Figure 3 ); For the intersections, the clinical use of key 5hmC differential genes related to efficacy was used for temporal cluster analysis, and key targets other than drug intersections, such as PSMA6 and RAD51B (such as Figure 5 , Figure 6 ), and these targets are closely related to diabetic nephropathy.

[0030] Based on the in-depth exploration of key genes and the search for related Chinese medicine, PSMA6 and RAD51B were significantly downregulated in patients with diabetic nephropathy (such as Figure 5 , Figure 6), the lack of regulatory ability of the original formula of Buyang Huanwu Decoction on related targets may be a defect of the original formula when used in the treatment of diabetic nephropathy. Through network pharmacology analysis, it was deduced that the targets of PSMA6 and RAD51B corresponded to Chinese herbs such as yam and Rehmannia glutinosa (such as Figure 7 ); Through the joint analysis of 5hmC and the Buyang Huanwu Decoction target database, it was inferred that yam and Rehmannia glutinosa together with the traditional Buyang Huanwu Decoction targets constitute a new prescription to improve treatment.

[0031] Based on the in-depth exploration of key genes, the flavor of Buyang Huanwu Decoction was reduced. The intersection of the Buyang Huanwu Decoction database target and the 5hmC differentially expressed genes revealed 30 drug-related differentially expressed genes. Through PPI network analysis, five key genes related to diabetic nephropathy, including IL6, TR4, LPL, LTF, and SREBF1 (such as Figure 3 The above key genes were searched through the ETCM database. The genes acting on peach kernel and earthworm in the original prescription (astragalus, angelica, red peony root, chuanxiong, safflower, peach kernel and earthworm) did not contain the above key genes (such as Figure 4 ), which may be poorly associated with the disease.

[0032] The Buyang Huanwu Decoction was modified to construct a new Chinese medicine composition based on the theory of traditional Chinese medicine and network pharmacology. Among them, the monarch drug is Astragalus membranaceus, which can invigorate qi and raise yang, consolidate the kidney's vital energy, promote diuresis and eliminate dampness, and dissipate kidney stasis and turbidity. The amount is strong and specific. Angelica sinensis is the minister drug, which can promote blood circulation and remove blood stasis, regulate qi and blood to promote blood vessels, and clear kidney collaterals to eliminate stasis and stagnation, and prevent the excessive astringency of Astragalus membranaceus; the monarch and minister are combined to invigorate kidney qi, promote dampness and turbidity, eliminate blood stasis, regulate qi and blood, and invigorate without stagnation. Red peony root, Chuanxiong, and safflower are used to promote blood circulation and remove blood stasis to clear kidney collaterals; Chinese yam and Rehmannia glutinosa are added to invigorate both qi and yin and stop thirst. The whole prescription has the functions of invigorating qi and removing dampness, promoting blood circulation and removing blood stasis, and invigorating without stagnation, so diabetic kidney can be cured.

[0033] 1. Sequencing of patients with diabetic nephropathy and network pharmacology study on treatment with the improved Buyang Huanwu Decoction Chinese medicine composition of the present invention

[0034] 5hmC-Seal sequencing technology was used to detect changes in epigenetic markers in plasma cfDNA of diabetic nephropathy patients, diabetic patients and healthy people, to discover significant markers of the development and progression of diabetic nephropathy and differences in marker levels after treatment, and to screen potential drug targets behind the markers. Based on network pharmacology screening, tailoring was performed to construct a new Chinese medicine formula for anti-diabetic nephropathy (hereinafter referred to as the new formula), and to provide support for the efficacy of the improved Buyang Huanwu Decoction for diabetic nephropathy.

[0035] 1.1 Sample collection

[0036] Plasma was collected from diabetic patients (diabetes group, diabetic nephropathy, healthy subjects) from Peking University Third Hospital. The inclusion criteria were as follows: the diagnosis of diabetes was based on the diabetes medical care standards published by the American Diabetes Association in 2020, with a fasting blood glucose value ≥126 mg / dL (7.0 mmol / L) and / or a glucose load test of 75 g of anhydrous glucose dissolved in water orally for at least 8 hours, and a blood glucose value ≥200 mg / dL (11.1 mmol / L) 2 hours later. The diagnostic criteria for DKD were based on the KDIGO 2020 clinical practice guidelines and the 2014 consensus on the prevention and treatment of diabetic nephropathy by the Chinese Diabetes Association, mainly including urine albumin / creatinine ratio (UACR) ≥30 mg / g and / or estimated glomerular filtration rate (eGFR) <60 mL·min －1 ·(1.73m 2 ) －1 , and lasted for more than three months. Exclusion criteria: Evidence of other diseases; concurrently receiving experimental treatments in other clinical studies.

[0037] Blood samples from 15 patients with diabetic nephropathy and 14 patients with diabetes were collected from Peking University Third Hospital as the DKD group and DM group; blood samples from 12 healthy volunteers in the same period were collected as the control group. The patient bed information was checked, the sample information sheet was sorted, whole blood was collected in a blood collection tube, transported to the laboratory, and the sample information was checked after receipt, and plasma was separated.

[0038] The sample collection procedures were in compliance with the patient informed consent and medical ethics approval requirements.

[0039] 1.2 Plasma separation

[0040] Take 8 ml of the patient's peripheral blood, centrifuge it at 1350×g, 4℃ for 12 minutes, and take the supernatant; centrifuge it again under the same conditions, and take the supernatant; finally centrifuge it at 13500×g, 4℃ for 5 minutes, take the supernatant, and obtain plasma.

[0041] 1.3 Extraction of plasma free DNA

[0042] Using the Quick-CfDNA serum and plasma kit (ZYMO kit), 8-10 ng of plasma CfDNA was extracted from the plasma samples prepared in step 1.1.

[0043] 1.4 End-filling of CfDNA and ligation with sequencing adapters

[0044] According to the instructions of the KAPA HyperPlus library preparation kit, a reaction mixture containing 20 μL cfDNA, 2.8 μL end repair & A-tailing buffer and 1.2 μL end repair & A-tailing enzyme mixture (total volume of 24 μL) was prepared, incubated at 20°C for 30 minutes, and then incubated at 65°C for 30 minutes. The following ligation reaction mixture was prepared in a 0.5 mL low-adsorption EP tube: 2 μL nuclease-free water, 12 μL ligation buffer and 4 μL DNA ligase. Add 2 μL of sequencing adapter to 18 μL ligation reaction mixture, mix, add to 24 μL reaction sample, and heat at 20°C for 4 hours. The reaction product was purified using a DNA CLean&Concentrator 5 (ZYMO) purification kit and eluted with 20 μL elution buffer to obtain the final DNA ligation sample.

[0045] 1.5 5hmC labeling

[0046] Prepare a total volume of 4 μL of labeling reaction mixture: 1 μL 50uM UDP-N3-Glu, 1 μL BGT enzyme (NEB) and 2 μL HEPES buffer (pH 8.0, final concentration 50mM), and add 4 μL of labeling mixture to 20 μL DNA connection sample. Incubate the mixture in a 37°C water bath for 2 hours. Take out the mixture, purify the reaction product using a DNA CLean&Concentrator 5 (ZYMO) purification kit to obtain 30 μL of purified DNA. Then add 1 μL DBCOPEG4-biotin (Click Chemistry Tools) to the above-purified 30 μL DNA, incubate in a 37°C water bath for 1 hour, and purify the reaction product using a DNA Clean&Concentrator 5 (ZYMO) purification kit to obtain 30 μL of purified labeling product.

[0047] 1.6 5hmC enrichment

[0048] First, prepare the binding magnetic beads according to the following steps: take out 2.5 μL Dynabeads (Invitrogen) and add 100 μL washing buffer (5mM Tris (DH7.5) 1M NaCl and 0.02% Tween 20), mix well by blowing, place on a 1.5 mL magnetic stand, repeat washing the binding magnetic beads with 100 μL washing buffer 3 times, and finally add 32 μL binding buffer (10mM Tris (pH7.5), 2M NaCl and 0.04% Tween 20) and mix well. Then, add the purified labeled product obtained in the above steps to the magnetic bead mixture, and mix it in a rotating mixer for 30 minutes to fully bind. Finally, wash the magnetic beads 5 times with 100 μL washing solution (5mM Tris (pH7.5), 1M NaCl and 0.02% Tween 20), centrifuge to remove the supernatant, and add 23.8 μL RNase-free water.

[0049] 1.7 PCR amplification

[0050] Add 25 μL 2×PCR master mix and 1.25 μL PCR primers (total volume 50 μL) to the final system of the above step, and amplify according to the temperature and conditions of the PCR reaction cycle: purify the amplified product with AmpureXP beads to obtain the final 5hmC library, and determine the library concentration with Qubit 3.0.

[0051] 1.8 High-throughput sequencing after quality control of 5hmC library

[0052] The obtained 5hmC library was quality controlled by Fragment Analyzer TN fully automatic capillary electrophoresis system to determine the size of DNA fragments in the library and whether it contained impurities; the concentration was determined by qPCR. The sequencing libraries that passed the quality inspection were mixed at the same concentration and sequenced using Illumina NextSeg500. The sequencing kit used was High Output Kitv2 (75 cycles). The sequencing throughput of each sample was 1.5Gb, and the sequencing band size was 75bp.

[0053] 1.9 Alignment of raw sequence data

[0054] Each raw sequencing FASTQ data was first trimmed for low-quality data using Trimmomatic software, and then aligned to the human genome hg19 using Bowtie2 software. MACS software was used to identify read peaks containing 5hmC, with the following parameters: effective genome size = 2.72e+09; tag size = 38: oandwidth = 100; model fold = 10; P value cutoff = 1.00e-05. The peak information of plasma CFDNA from patients with effective treatment and patients with ineffective treatment was filtered, with the condition that each peak appeared in at least 10 samples, so as to call peaks and generate counts files; DEse2 software was used to compare the 5hmc peak regions with reads greater than 50 found in counts files from different samples, and the differential biomarkers of 5hmC up and down regulation were screened according to log2FoldChange>=1 and p value <0.01. Further time series cluster analysis was performed using the Shanghai Tengyun mapping system (hiplot) to screen genes that were continuously upregulated or downregulated from the healthy state, diabetic state to diabetic nephropathy state, thereby identifying potential drug targets.

[0055] 1.10 Statistical analysis

[0056] The difference analysis between the two groups of data was performed using RStudio software, Sangerbox website (http: / / www.sangerbox.com / login.html) and GraphPed Prism 8 software, and p < 0.05 proved to be statistically significant. The measurement data in the clinical sample information were expressed as mean ± standard deviation express.

[0057] 2. Construction of improved Buyang Huanwu Decoction based on target and network pharmacology

[0058] Based on the clinical cohort, key targets and TCM theories were screened, and Buyang Huanwu Decoction was modified by combining network pharmacology technology. Based on the TCMSP and ETCM databases, Chinese medicines that act on key sites of diabetic nephropathy were retrieved and added.

[0059] Firstly, the key target genes in Buyang Huanwu Decoction were searched through the ETCM database, and the traditional Chinese medicines peach kernel and earthworm that did not target the key genes were removed.

[0060] The potential key target genes were searched through the TCM Bank database to obtain potential Chinese medicine components, and the potential Chinese medicine components were further searched to obtain potential Chinese medicines.

[0061] 3. Effectiveness verification of modified Buyang Huanwu decoction based on network pharmacology

[0062] The improved Buyang Huanwu Decoction was screened out based on 5hmC-Seal sequencing, and the "traditional Chinese medicine-active ingredients-target" regulatory network of the improved Buyang Huanwu Decoction was obtained using Cytoscape3.7.2 software to verify the effectiveness of the new prescription.

[0063] The Chinese medicine composition of the present invention is used to treat diabetic nephropathy and inhibit diabetic nephropathy fibrosis, and the efficacy of the Chinese medicine composition is verified by a randomized controlled experiment on an animal model.

[0064] Test Example 1

[0065] 1. Animal model grouping, modeling and drug administration

[0066] The study used 8-week-old male SPF C57BKS-DB(- / -) mice (db / db) to establish a DKD mouse model. The db / db mouse has a point mutation in the Leptin receptor, which leads to a disorder in the leptin signaling pathway, resulting in obesity, insulin resistance, hyperglycemia, fatty liver and other symptoms in the mouse. db / db mice can show obvious obesity and increased fasting blood sugar 6 weeks after birth, and increase water intake and urine output, which is most obvious at 8-12 weeks, and complications such as diabetic nephropathy may occur. db / db mice are usually used as the control group of the experiment with db / m mice.

[0067] All animals were placed under standard 12-hour light / 12-hour dark cycle conditions and fed a standard diet. After 1 week of adaptive feeding, random blood glucose >16.7mmol / L db / db and urine microalbumin >30mg / 24h were determined as DKD models.

[0068] Twenty-four db / db mice were randomly divided into four groups: the model group (db / db) was gavaged with normal saline (0.1 mL / 10 g), the positive drug group (db / db+EM) was gavaged with empagliflozin (1.3 mg / kg, 0.1 mL / 10 g), the original Buyang Huanwu Decoction group (db / db+BYHW) was gavaged with the original Buyang Huanwu Decoction (14.94 g / kg, 0.1 mL / 10 g), and the new Buyang Huanwu Decoction group (db / db+New) was gavaged with the new Buyang Huanwu Decoction (14.94 g / kg, 1 mL / 10 g). Another group of normal controls (db / m) was gavaged with normal saline (0.1 mL / 10 g).

[0069] 2. Preparation of Chinese Herbal Decoction

[0070] Buyang Huanwu Decoction Group: Astragalus 30g, Angelica 15g, Red Peony 15g, Chuanxiong 15g, Peach Kernel 15g, Safflower 15g, Pheretima 10g.

[0071] That is the Buyang Huanwu Decoction group: Astragalus formula granules 12g, Angelica formula granules 9.99g, Red Peony root formula granules 4.5g, Chuanxiong formula granules 5g, Peach kernel formula granules 2.49g, Safflower formula granules 6.82g, and Pheretima formula granules 2.5g.

[0072] New prescription group: Astragalus 30g, Angelica 15g, Red Peony Root 15g, Chuanxiong 15g, Carthamus tinctorius 9g, Rehmannia glutinosa 18g, and Dioscorea opposita 15g.

[0073] That is, the new prescription group: Astragalus formula granules 12g, Angelica formula granules 9.99g, Red Peony root formula granules 4.5g, Chuanxiong formula granules 5g, Safflower formula granules 4.09g, Rehmannia root formula granules 13.68g, and Chinese yam formula granules 3.75g.

[0074] The above-mentioned single-drug formula granules were all purchased from Guangdong Yifang Pharmaceutical Co., Ltd.

[0075] The dose for mice was converted to 9.1 times the daily dose for adults. The drug was administered for a total of 8 weeks, and the urine microalbumin, blood creatinine and urea nitrogen of the mice were measured at the end of the experiment.

[0076] 3. Statistical Methods

[0077] The data of each group were compared by one-way analysis of variance, and the data related to the animal experiments were visualized by GraphPad Prism8 software.

[0078] IV. Experimental Results

[0079] like Figure 8 As shown, A is the change of mouse body weight during the administration period, it can be seen that compared with the model group, the body weight of the Buyang Huanwu Decoction original prescription group and the new prescription group was reduced; B is the change of random blood glucose of mice during the administration period; C is the improvement of renal atrophy of mice after administration, it can be seen that compared with the model group, the renal atrophy of mice in the new prescription group was significantly improved; D is the change of urinary microalbumin in mice after administration, it can be seen that compared with the model group, the urinary microalbumin in the new prescription group was significantly reduced; E is the change of blood urea nitrogen in mice after administration, it can be seen that compared with the model group, the blood urea nitrogen in the new prescription group was significantly reduced; F is the change of blood creatinine in mice after administration, it can be seen that compared with the model group, the blood creatinine in the new prescription group was significantly reduced.

[0080] In summary, the new prescription has the effect of reducing weight and improving renal function.

[0081] Test Example 2

[0082] 1. Staining of Kidney Tissue Sections

[0083] After the experiment, some kidney tissues were fixed in 4% PFA for 12 hours (4°C), dehydrated with graded ethanol, and embedded in paraffin. 5 μm kidney cross sections were prepared according to the manufacturer's instructions and used for H&E, PAS, and Masson staining.

[0084] 2. Immunofluorescence Method

[0085] Kidney tissues were fixed with 4% formalin and blocked with 5% BSA. Following standard de-affinity and hydration protocols, sections were incubated with appropriate primary antibodies overnight at 4°C. The following day, they were incubated with matching secondary antibodies for one hour. After incubation, cells were washed and nuclei were re-stained. Fluorescent images of tissues and cells were analyzed using confocal laser microscopy.

[0086] 3. Statistical Methods

[0087] The data of each group were compared by one-way analysis of variance, and the data related to the animal experiments were visualized by GraphPad Prism8 software.

[0088] IV. Experimental Results

[0089] like Fig. 9 As shown in the figure, A is H&E staining, PAS staining and Masson staining of kidney tissue sections. From the results of H&E staining, it can be seen that the size and morphology of the glomeruli of the normal control group mice are normal, the boundaries are clear, and the glomerular capsule cavity is obvious. The glomeruli of the model group mice are deformed, enlarged, filled with blood cells, and the glomerular capillaries are dilated; the glomerular capsule cavity is getting smaller and smaller, and even adheres to the glomerulus; the renal tubular epithelial cells are degenerated and necrotic. Compared with the model group, the renal tissue damage of the positive drug group mice, the Buyang Huanwu Decoction original group mice and the Buyang Huanwu Decoction new group mice were improved to varying degrees, among which the Buyang Huanwu Decoction new group was closest to the renal morphology of the normal group mice. The PAS staining results showed that compared with the normal group, the glomerular basement membrane of the model group mice was diffusely thickened, and the glomerular mesangial matrix was diffusely increased. Compared with the model group, the Buyang Huanwu Decoction new group was closest to the renal morphology of the normal group mice. The results of Masson staining showed that compared with the normal group, the mice in the model group showed obvious tissue fibrosis, while the Buyang Huanwu Decoction new prescription group was closest to the kidney morphology of the mice in the normal group. B is the immunofluorescence result of renal tissue fibrosis proteins E-caderin, α-SMA and Fibronectin. The red-stained part in the figure is the target protein, and the blue-stained part is the cell nucleus. The results showed that compared with the normal group, the expression of E-caderin in the model group was downregulated, and the expression of α-SMA and Fibronectin was upregulated, indicating that the model group had obvious fibrosis. The positive drug group, the original Buyang Huanwu Decoction group and the new prescription group all showed different degrees of improvement compared with the model group, among which the new prescription group had the best effect.

[0090] The above results indicate that the new formula can significantly improve diabetic nephropathy fibrosis.

[0091] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A Chinese medicine composition for treating diabetic nephropathy, comprising the following raw materials in the following weight proportions: Astragalus 30g, Angelica 15g, Red Peony Root 15g, Chuanxiong 15g, Safflower 9g, Rehmannia Glutinosa 18g, and Chinese Yam 15g.

2. The Chinese medicine composition according to claim 1, characterized in that: The raw materials are medicinal pieces or single-drug formula granules equivalent to the same amount of medicinal pieces.

3. The Chinese medicine composition according to claim 2, characterized in that: When the raw materials are single-drug formula granules, the Chinese medicine composition is composed of the following raw materials in the following weight proportions: 12g of Astragalus formula granules, 9.99g of Angelica formula granules, 4.5g of Red Peony Root formula granules, 5g of Chuanxiong formula granules, 4.09g of Carthamus tinctorius formula granules, 13.68g of Rehmannia glutinosa formula granules, and 3.75g of Dioscorea opposita formula granules.

4. An oral Chinese medicine preparation for treating diabetic nephropathy, prepared using the Chinese medicine composition according to any one of claims 1 to 3 and a raw material drug containing the active ingredient of the Chinese medicine composition.

5. The oral Chinese medicine preparation according to claim 4, characterized in that: The Chinese medicine oral preparation is a Chinese medicine decoction or an aqueous solution of a single medicine formula composition.

6. A method for preparing an oral Chinese medicine preparation for treating diabetic nephropathy, comprising preparing the preparation using the Chinese medicine composition according to any one of claims 1 to 3 and a raw material drug containing the active ingredient of the Chinese medicine composition.

7. The preparation method according to claim 6, characterized in that: The Chinese medicine oral preparation is a Chinese medicine decoction or an aqueous solution of a single medicine formula composition.

8. The preparation method according to claim 7, characterized in that: The method for preparing the aqueous solution of the single-drug formula composition comprises: mixing the single-drug formula particles of the traditional Chinese medicine composition, adding boiling water, and stirring until fully dissolved to obtain a traditional Chinese medicine liquid.

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