A Chinese medicine composition for treating diabetic nephropathy and its preparation method and application

Abstract

The present invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a kind of membrane kidney prescription for treating membranous nephropathy and its preparation method and application. The Chinese medicine composition is made of the following raw materials: 14-50 parts of Astragalus, 12-35 parts of Rhodiola, 8-25 parts of Radix Pseudostellariae, 8-25 parts of Radix Rehmanniae, 14-50 parts of Fructus Corni, 8-25 parts of Rubia, 8-25 parts of Cuttlebone, 6-22 parts of Herba Lycopodii, 8-25 parts of Semen Cuscutae, 10-35 parts of Smilax glabrae, 5-18 parts of Periostracum Cicadae, and 3-12 parts of Rhubarb. Animal experiments show that the Chinese medicine composition of the present invention has the effect of treating membranous nephropathy in the indicators such as the measurement results of 24h UP and blood BNU of mice. Clinical experiments confirm that the curative effect is definite, and the prescription can significantly reduce the blood sugar and Chinese medicine symptom score of patients with diabetic nephropathy, and has a good therapeutic effect.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a membranous nephropathy-treating membrane kidney prescription and a preparation method and application thereof. Background Art

[0002] In recent years, the prevalence of diabetes has increased significantly worldwide, and diabetic kidney disease (DKD) is one of the major chronic complications of diabetes. At present, DKD has become the leading cause of end-stage renal disease (ESRD) in my country. Although many large-scale clinical studies have shown that intensive glucose-lowering, blood pressure-lowering, and lipid-lowering treatments can significantly reduce urine protein, the residual risk of DKD progression is still as high as 50%-86%. The DKD treatment recommended by the guidelines is mainly anti-renin-angiotensin-aldosterone system treatment, but its benefits have great limitations, and many newly launched DKD treatment drugs, such as renin inhibitors, aldosterone receptor antagonists, endothelin antagonists, etc., have little benefit on renal hard endpoints. Therefore, exploring new pathogenesis of DKD and finding more effective treatment methods (targets) are key issues that urgently need to be addressed.

[0003] Diabetic Kidney Disease (DKD) is a common complication caused by diabetes. It affects the function of the kidneys, causing gradual damage to the kidneys and eventually developing into renal failure. Diabetic nephropathy is usually divided into several stages. The early symptoms may not be obvious, but as the disease worsens, proteinuria (edema, increased foam in the urine, etc.) may occur. If not controlled, it may eventually lead to chronic renal failure or uremia.

[0004] The main treatment measures for diabetic nephropathy include controlling blood sugar, blood pressure, improving renal function and lifestyle intervention, such as changing diet, losing weight, and increasing exercise. PTEC is an important component of the kidney. The potential pathogenesis of DKD involves dysfunction of renal tubules, oxidative stress, inflammation, fibrosis and apoptosis under high glucose environment. Studies have shown that mitochondrial dysfunction and aerobic glycolysis play a key role in the development of DKD. Under high glucose environment, the metabolic flux of glucose can be shifted from oxidative phosphorylation (OXPHOS) in mitochondria to the glycolysis pathway. Therefore, mitochondria and glycolysis pathways are being actively studied as potential biomarkers and therapeutic targets in the kidney. BIM is a key factor mediating the mitochondrial pathway and plays an important role in the maintenance of mitochondrial function. Our previous studies have shown that the mechanism of DKD damage involves early apoptosis mediated by BIM, a key factor in the mitochondrial pathway in PTEC. In DKD, BIM expression is upregulated, and DKD is significantly improved after reducing the expression of BIM in PTEC. When mitochondria are dysfunctional, OXPHOS is inhibited and aerobic glycolysis flux increases. PFKFB3, as one of the key links in the glycolysis pathway, plays an important role in DKD.

[0005] Traditional Chinese medicine has a long history. Shenxiao Yuning Decoction was developed based on previous studies and was derived from Shenqi Dihuang Decoction and the empirical medication cited in the guidelines. It has a good effect in treating proteinuria in early DKD patients in the clinic. Studies have shown that many Chinese herbal compound prescriptions and single herbs can improve proteinuria and renal damage in DKD animal models, improve tubular reabsorption function, and reduce oxidative stress and apoptosis of tubular epithelial cells [72,73]. Based on the above, in order to further explore the mechanism of action of Shenxiao Yuning Decoction, this study focused on BIM-mediated mitochondrial pathway apoptosis and PFKFB3-mediated aerobic glycolysis, and explored the efficacy of Shenxiao Yuning Decoction in the treatment of DKD and the mechanism of tubular damage, in order to provide experimental basis for the treatment of DKD and provide reference for the development of new clinical drugs.

[0006] In my country, the guidelines recommend DKD treatment mainly through anti-renin-angiotensin-aldosterone system and aldosterone receptor antagonists, but their benefits are greatly limited. Many newly launched DKD treatment drugs, such as renin inhibitors and endothelin antagonists, have little benefit on renal hard endpoints. Summary of the invention

[0007] The purpose of the present invention is to provide a Chinese medicine composition for treating diabetic nephropathy according to the deficiencies of the prior art, and a preparation method and application thereof. The Chinese medicine composition is composed of 12 Chinese medicines such as astragalus, rhodiola rosea, and Pseudostellaria heterophylla, which can significantly reduce blood sugar and Chinese medicine symptom scores in patients with diabetic nephropathy, and has a good therapeutic effect.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A traditional Chinese medicine composition for treating diabetic nephropathy is prepared from the following raw materials in parts by weight: 14-50 parts of astragalus, 12-35 parts of rhodiola, 8-25 parts of pseudoginseng, 8-25 parts of raw rehmannia, 14-50 parts of cornus, 8-25 parts of madder, 8-25 parts of cuttlebone, 6-22 parts of zedoaria, 8-25 parts of dodder seeds, 10-35 parts of smilax glabra, 5-18 parts of cicada shells, and 3-12 parts of wine-soaked rhubarb.

[0010] Furthermore, in a preferred embodiment of the present invention, the traditional Chinese medicine composition for treating diabetic nephropathy is made of the following raw materials in parts by weight: 30 parts of Astragalus, 20 parts of Rhodiola, 15 parts of Pseudostellariae Radix, 15 parts of Rehmannia glutinosa, 30 parts of Cornus officinalis, 15 parts of Rubia cordifolia, 15 parts of Cuttlebone, 12 parts of Zedoariae rhizome, 15 parts of Cuscuta australis, 24 parts of Smilax glabra, 9 parts of Periostracum cicadae, and 6 parts of Rhubarb prepared with wine.

[0011] Furthermore, in a preferred embodiment of the present invention, the traditional Chinese medicine composition for treating diabetic nephropathy is made of the following raw materials in parts by weight: 14 parts of Astragalus, 12 parts of Rhodiola, 8 parts of Pseudostellariae Radix, 8 parts of Rehmannia glutinosa, 14 parts of Cornus officinalis, 8 parts of Rubia cordifolia, 8 parts of Cuttlebone, 6 parts of Zedoariae rhizome, 8 parts of Cuscuta australis, 10 parts of Smilax glabra, 5 parts of Periostracum cicadae, and 3 parts of Rhubarb prepared with wine.

[0012] Furthermore, in a preferred embodiment of the present invention, the Chinese medicine composition for treating diabetic nephropathy is made of the following raw materials in parts by weight: 50 parts of Astragalus, 35 parts of Rhodiola, 25 parts of Pseudostellariae Radix, 25 parts of Rehmannia glutinosa, 50 parts of Cornus officinalis, 25 parts of Rubia cordifolia, 25 parts of Cuttlebone, 22 parts of Zedoariae, 25 parts of Cuscuta, 35 parts of Smilacis Glabrae, 18 parts of Periostracum Cicadae, and 12 parts of Rhubarb prepared with wine.

[0013] Furthermore, the present invention provides a preparation of the Chinese medicine composition, wherein the preparation further comprises a pharmaceutically acceptable excipient and is a clinically acceptable dosage form.

[0014] Furthermore, the preparation is any one of a decoction, granules, tablets, capsules, mixtures, syrups, pills, gels, powders or pastes, preferably a decoction or powders.

[0015] The preparation method of the Chinese medicine composition is as follows: soak each medicine in water for 20-40 minutes, decoct for 1-3 times, the first time for 10-20 minutes, the second time for 15-25 minutes, discard the residue, mix the decoctions from the two times, and obtain a decoction.

[0016] Furthermore, the preparation method of the therapeutic Chinese medicine composition is as follows: soak each medicine in water for 30 minutes, decoct twice, the first time for 15 minutes, the second time for 20 minutes, discard the residue, and mix the decoctions from the two times.

[0017] The decoction is concentrated by a freeze dryer under vacuum and dried to produce a brown powder, which is the powder.

[0018] The prescription of the present invention is derived from the method, and the curative effect is definite. The Chinese medicine composition of the present invention is named Shenxiao Yuning Decoction, which is tailored according to clinical experience, and has the functions of invigorating qi and nourishing yin, removing blood stasis and purging turbidity. The prescription is composed of 12 Chinese medicines, including Astragalus, Rhodiola, Pseudostellaria, Radix Rehmanniae, Cuscuta, Fructus Corni, Cuttlebone, Rubia, Herba Lycopi, Rhubarb, Periostracum Cicadae, and Smilax. In the prescription of Shenxiao Yuning Decoction, Astragalus and Rhodiola are the main medicines. Astragalus is an important medicine for invigorating qi, has a strong power of invigorating qi, is warm in nature and sweet in taste, and is praised as the "longest tonic". Tao Hongjing's "Famous Doctors' Records" records that it has the saying of "replenishing the deficiency of men, and the five labors and thinness". Astragalus has the effects of invigorating the middle qi, raising yang and quenching thirst, and also diuresis and detumescence. Rhodiola tastes sweet and bitter, invigorates qi and strengthens the spleen, activates blood circulation and nourishes blood, and clears yin deficiency and heat. Rhodiola rosea (Tibetan: "Solo Mabu"), known as "highland ginseng", was first recorded in the "Four Medical Classics", which said that it can "cure blood diseases, red sputum diseases, sores, boils and ulcers." "Wen Dao He" records: "Rhodiola rosea tastes sweet, bitter, and astringent, and is cool in nature. It nourishes the lungs, clears away heat, and nourishes the vital energy." Although the specific records in various classics are not exactly the same, they all mention that Rhodiola rosea has two effects, with the effects of "nourishing the vital energy" and "activating blood circulation and unblocking meridians", and is neutral in nature (some say it is cool in nature). Astragalus and Rhodiola rosea are combined, one warm and one cool. Astragalus replenishes qi without restraining evil, and Rhodiola rosea activates blood circulation without hurting the body. On the basis of tonifying deficiency, it also activates blood circulation and eliminates symptoms. They are both monarch drugs and complement each other. Pseudostellaria heterophylla tastes sweet and slightly bitter, is neutral in nature, and enters the spleen and lung meridians. It has the functions of replenishing qi, strengthening the spleen, and promoting fluid and moistening the lungs. "Ben Cao Cong Xin" first recorded the efficacy of Pseudostellariae Radix, believing that it "greatly replenishes the vital energy" and "its power is not inferior to that of Pseudostellariae Radix". "Ben Cao Zai Xin" pointed out that Pseudostellariae Radix "treats qi deficiency and lung dryness, replenishes the spleen and earth, eliminates edema, and resolves phlegm and quenches thirst", believing that Pseudostellariae Radix can treat edema. Shengdihuang is sweet, cold and moist, with a thick smell and a sinking nature. It nourishes the true yin in the kidney. It is compatible with Pseudostellariae Radix, and the ascending and descending are in harmony, replenishing qi and replenishing yin, and nourishing without being greasy. Fructus Corni nourishes the kidney and astringes. One replenishes, one nourishes and one astringes. The three replenish qi and nourish yin, astringe and consolidate. Cuttlebone consolidates the kidney and replenishes essence, and unclogs the blood vessels. Rubia cordifolia promotes blood circulation and unclogs the menstruation. The two are used together in "Siwu Qugu Yi Lu Ru Wan" in "Neijing Su Wen", which replenishes essence and blood, resolves blood stasis and disperses hardness. The above 5 herbs are all ministers. Cuscuta chinensis is used to nourish the liver and kidney, and Zedoariae Radix promotes blood circulation and resolves blood stasis, promotes water circulation and reduces edema. Smilax glabra is sweet and mild in nature. It can clear the meridians and collaterals, and eliminate turbidity and dampness. The guiding drugs are wine rhubarb and cicada slough. Wine rhubarb can promote blood circulation, dissipate blood stasis, eliminate turbidity and promote diuresis. Cicada slough rises, floats, and diffuses, and can regulate the water channels. It is matched with wine rhubarb. Cicada slough raises the clear yang, and wine rhubarb lowers the turbid yin. When the clear rises and the turbid falls, the water will flow. The combination of the two drugs means "rising and falling". Diseases of the kidney collaterals should be nourished and unblocked. The above drugs are used together to play the role of nourishing qi and yin, removing blood stasis and eliminating turbidity, and hit the pathogenesis of DKD "due to deficiency".

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) Shenxiao Yuning Decoction of the present invention has been verified by pharmacological experiments, and the blood glucose content, mouse urine volume, kidney index and renal protein of mice have been measured. In the final therapeutic effect, compared with the comparative example 1-5 groups, the mouse urine volume of the embodiment group at the 6th and 10th weeks was significantly reduced, the blood glucose content was reduced, and there was a significant difference (P<0.01), and the therapeutic effect of embodiment 1 was the best, in the measurement results of 24h UP and blood BNU of mice. Compared with the comparative example groups, the 24h UP and blood BNU of mice in each group of the embodiment were significantly reduced (P<0.01); the mouse renal index between the groups of the embodiment did not have a significant difference (P>0.05). Animal experiments show that the membrane kidney prescription of the present invention reduces the 24h UTP of mice with membranous nephropathy, reduces the BUN level and CRE level, and has the effect of treating membranous nephropathy.

[0021] 2) In the clinical trial of TCM syndrome score evaluation, the main efficacy index, the total effective rate of treatment, was 2 The test showed that the total effective rate of the experimental group was significantly higher than that of the control group, and the difference was statistically significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Comparison of symptom efficacy between the experimental group and the control group 4 weeks after treatment showed that urine ACR and urine α1-MG in the experimental group were lower than those in the control group, and the difference was statistically significant, with P values ​​of 0.044 and 0.037 respectively.

[0023] Figure 2 : The total effective rate of TCM syndrome in the experimental group was higher than that in the control group, P=0.005<0.01, and the difference was statistically significant.

[0024] Figure 3 : Random blood glucose was measured at different time points in each group of mice.

[0025] Figure 4 :The urine volume of mice in each group was measured at different time points. The results are shown in the figure.

[0026] Figure 5 : The results of 24h UP were measured on mice in each group at different time points.

[0027] Figure 6 : The renal indexes of mice in the model group, sham operation group and each group of the embodiment were measured at different time points.

[0028] Figure 7 : The blood BNU of mice in each group was measured at different time points. DETAILED DESCRIPTION

[0029] In order to make the purpose and technical solution of the present invention clearer, the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0030] Example 1: A Chinese medicine composition for treating diabetic nephropathy, the dosage and preparation method are as follows:

[0031] Astragalus 30g, Rhodiola rosea 20g, Pseudostellaria pseudoginseng 15g, Rehmannia root 15g, Cornus officinalis 30g, Rubia cordifolia 15g, Cuttlebone 15g, Zedoariae rhizome 12g, Cuscuta chinensis 15g, Smilax glabra 24g, Periostracum cicadae 9g, and Rhubarb prepared with wine 6g.

[0032] The preparation process is:

[0033] The preparation method of the therapeutic Chinese medicine composition is as follows: soak each medicine in water for 30 minutes, decoct twice, the first time for 15 minutes, the second time for 20 minutes, discard the residue, mix the decoctions from the two times, and obtain a decoction.

[0034] Example 2: A Chinese medicine composition for treating diabetic nephropathy, the dosage and preparation method are as follows:

[0035] The Chinese medicine composition for treating diabetic nephropathy is prepared from the following raw materials in parts by weight: 14g of Astragalus, 12g of Rhodiola, 8g of Pseudostellariae Radix, 8g of Rehmannia glutinosa, 14g of Cornus officinalis, 8g of Rubia cordifolia, 8g of Cuttlebone, 6g of Zedoariae Radix, 8g of Cuscuta, 10g of Smilax glabra, 5g of Periostracum cicadae, and 3g of Rhubarb.

[0036] The preparation method of the therapeutic Chinese medicine composition is as follows: soak each medicine in water for 20 minutes, decoct for 3 times, the first time for 20 minutes, the second time for 25 minutes, discard the residue, mix the decoctions from the two times, and obtain a decoction.

[0037] Example 3: A Chinese medicine composition for treating diabetic nephropathy, the dosage and preparation method are as follows:

[0038] 50g of Astragalus, 35g of Rhodiola, 25g of Pseudostellaria, 25g of Rehmannia, 50g of Cornus, 25g of Rubia, 25g of Cuttlebone, 22g of Zedoaria, 25g of Cuscuta, 35g of Smilax, 18g of Periostracum Cicadae, and 12g of Rhubarb.

[0039] The preparation method of the therapeutic Chinese medicine composition is as follows: soak each medicine in water for 40 minutes, decoct for 3 times, the first time for 20 minutes, the second time for 25 minutes, discard the residue, mix the decoctions from the two times, and obtain a decoction.

[0040] Example 4: A Chinese medicine composition for treating diabetic nephropathy, the dosage and preparation method are as follows:

[0041] The decoctions of Examples 1-3 were vacuum concentrated using a freeze dryer and dried to produce brown powder, thereby obtaining the powders of each example.

[0042] Comparative Example 1: A Chinese medicine composition for treating diabetic nephropathy, the dosage and preparation method are as follows:

[0043] Astragalus 30g, Pseudostellaria 15g, Rehmannia glutinosa 15g, Cornus officinalis 30g, Rubia cordifolia 15g, Cuttlebone 15g, Zedoaria 12g, Cuscuta 15g, Smilax glabra 24g, Periostracum cicadae 9g, and Rhubarb prepared with wine 6g.

[0044] The preparation process is the same as that of Example 1.

[0045] Comparative Example 2: A Chinese medicine composition for treating diabetic nephropathy, the dosage and preparation method are as follows:

[0046] Salvia miltiorrhiza 30g, Rhodiola rosea 20g, Pseudostellaria pseudoginseng 15g, Rehmannia root 15g, Cornus officinalis 30g, Rubia cordifolia 15g, Cuttlebone 15g, Eupatorium fortunei 12g, Cuscuta chinensis 15g, Smilax glabra 24g, Periostracum cicadae 9g, and Rhubarb prepared with wine 6g.

[0047] The preparation process is the same as that of Example 1.

[0048] Comparative Example 3: A Chinese medicine composition for treating diabetic nephropathy, the dosage and preparation method are as follows:

[0049] Astragalus 30g, Rhodiola rosea 20g, Pseudostellaria pseudoginseng 15g, Rehmannia root 15g, Cornus officinalis 30g, Eupatorium fortunei 12g, Cuscuta australis 15g, Smilax glabra 24g, Periostracum cicadae 9g, and Rhubarb prepared with wine 6g.

[0050] The preparation process is the same as that of Example 1.

[0051] Comparative Example 4: A Chinese medicine composition for treating diabetic nephropathy, the dosage and preparation method are as follows:

[0052] Astragalus 12g, Rhodiola rosea 38g, Pseudostellaria heterophylla 20g, Rehmannia root 10g, Cornus officinalis 20g, Rubia cordifolia 5g, Cuttlebone 5g, Zedoariae rhizome 30g, Cuscuta seed 130g, Smilax glabra 21g, Periostracum cicadae 2g, Rhubarb with wine 13g.

[0053] The preparation process is the same as that of Example 1.

[0054] Comparative Example 5: A Chinese medicine composition for treating diabetic nephropathy, the dosage and preparation method are as follows:

[0055] Atractylodes macrocephala 30g, saffron 20g, red ginseng 15g, Rehmannia root 15g, Cornus officinalis 30g, Rubia cordifolia 15g, Aconiti Lateralis Preparata 15g, Eupatorium fortunei 12g, Cuscuta australis 15g, Smilax glabra 24g, Periostracum cicadae 9g, and Aloe vera 6g.

[0056] The preparation process is the same as that of Example 1.

[0057] 1. Clinical Experiment of Shenxiaoyuning Decoction, a Chinese Medicine Composition of the Present Invention

[0058] This study actually included 62 patients, 30 in the experimental group and 32 in the control group, which met the sample size requirements.

[0059] 1.2 Random Methods

[0060] This study adopted a completely randomized method, using the "random number generator" to generate random numbers through the RV.Uniform function to generate a random coding table for subjects. Subjects were randomly divided into an experimental group and a control group. Special personnel were responsible for recruiting research participants, generating allocation sequences, and allocating intervention measures.

[0061] 2 Treatment options

[0062] 2.1 Basic Western medicine treatment

[0063] (1) Diabetes education: All study participants were provided with model group dietary guidance, emotional control, smoking and alcohol cessation, and appropriate exercise.

[0064] (2) Blood sugar control: All study participants received routine hypoglycemic treatment with no restrictions on hypoglycemic measures (including oral hypoglycemic drugs, injections, or insulin injections).

[0065] (3) Control blood lipids: Patients whose blood lipids do not meet the standard should receive lipid-lowering treatment.

[0066] (4) Control blood pressure: For patients with blood pressure >140 / 90 mmHg, antihypertensive drugs should be used to control blood pressure. There is no limit to antihypertensive measures (CCBs, ACEI or ARBs, α, β receptor blockers, diuretics, etc.).

[0067] (5) Other symptomatic treatments.

[0068] 2.2 Control group

[0069] Only basic Western medicine treatment is given.

[0070] 2.3 Experimental Group

[0071] In the basic treatment of Western medicine, Shenxiao Yuning Decoction is given. The source of Shenxiao Yuning Decoction is as follows:

[0072] a. All Chinese herbal medicine pieces are from the First Affiliated Hospital of Shandong First Medical University;

[0073] b. The composition of the Chinese medicine prescription is as follows:

[0074] Astragalus 30g, Rhodiola rosea 20g, Pseudostellaria pseudoginseng 15g, Rehmannia root 15g, Cornus officinalis 30g, Rubia cordifolia 15g, Cuttlebone 15g, Zedoaria 12g, Cuscuta 15g, Smilax glabra 24g, Periostracum cicadae 9g, and Rhubarb wine 6g (the prescription of Example 1 of the present invention).

[0075] c. Usage: Soak the above medicine for 30 minutes, one dose per day, decoct twice for each dose, mix the decocted medicine evenly, divide into 2g, 200ml-250ml per gram, and take it warm 30 minutes after breakfast and dinner respectively.

[0076] 2.4 Treatment course

[0077] The course of drug treatment is 4 weeks.

[0078] 3. Diagnostic criteria

[0079] 3.1 Western medicine diagnostic criteria

[0080] 3.1.1 Diagnostic criteria for diabetes

[0081] The T2DM diagnostic criteria proposed in the 2020 "Guidelines for the Prevention and Treatment of Type 2 Diabetes in China" by the Diabetes Branch of the Chinese Medical Association were adopted.

[0082] Table 1 Diagnostic criteria for diabetes

[0083]

[0084] 3.1.2 Diagnostic criteria for diabetic nephropathy

[0085] Refer to the Chinese Medical Association Nephrology Branch's "Chinese Guidelines for Clinical Diagnosis and Treatment of Diabetic Kidney Disease"

[0086] (2021 Study on the efficacy and mechanism of Shenxiao Yuning Decoction in the treatment of diabetic nephropathy. The proposed diagnostic criteria for DKD: meet the diagnostic criteria for DM, have a clear history of DM, and have a causal relationship with changes in urine protein and renal function, and exclude other primary and secondary glomerular diseases and systemic diseases. If one of the following conditions is met, DKD can be diagnosed.

[0087] (1) Random urine albumin-to-creatinine ratio (UACR) ≥ 30 mg / g or urine albumin excretion rate (UAER) ≥ 30 mg / 24 h, and UACR or UAER is repeated within 3 to 6 months, reaching or exceeding the critical value 2 out of 3 times; other interfering factors such as infection are excluded.

[0088] (2) Estimated glomerular filtration rate (eGFR) <60 mL / min / 1.73 m2.

[0089] (3) Renal biopsy showed pathological changes consistent with DKD.

[0090] 3.2. TCM diagnostic criteria

[0091] 3.2.1 Diagnostic criteria for diabetes mellitus

[0092] Table 2 Diagnostic criteria for diabetes mellitus

[0093]

[0094] 3.2.2 Diagnostic criteria for diabetes nephropathy

[0095] Table 3 Diagnostic criteria for diabetes nephropathy

[0096]

[0097] 3.3 TCM syndrome diagnostic criteria

[0098] Referring to the 2002 “Guidelines for Clinical Research of New Chinese Medicines (Trial)” issued by the State Food and Drug Administration and the “Guidelines for the Prevention and Treatment of Diabetic Nephropathy by Traditional Chinese Medicine” issued by the Chinese Association of Traditional Chinese Medicine

[77] (2011 edition), the syndrome differentiation criteria for Qi and Yin Deficiency with Blood Stasis were formulated as follows:

[0099] Main symptoms: dry throat and mouth, fatigue and listlessness.

[0100] Secondary symptoms: eating a lot and getting hungry easily, thirsty and wanting to drink, shortness of breath and laziness to speak, fever in the five parts of the body, palpitations and insomnia, numbness or tingling in the limbs.

[0101] Tongue and pulse: The tongue is red with little moisture, thin or peeled, the pulse is thready, rapid and weak, or thin and stringy; or the tongue is dark with ecchymosis, the pulse is deep and astringent.

[0102] This syndrome can be diagnosed if the patient has two main symptoms, one main symptom and one secondary symptom, or three secondary symptoms, or is accompanied by tongue and pulse.

[0103] 3.4 Inclusion criteria

[0104] (1) Patients who meet the diagnosis of type 2 DKD stage III-IV, with persistent albuminuria and urine ACR>30 mg / g for two consecutive urine tests within 3 months;

[0105] (2) eGFR ≥ 60 mL / min / 1.73 m2 and remained stable during the trial (eGFR decreased by ≤ 25% from enrollment to week 4);

[0106] (3) Patients with diabetes mellitus and kidney disease with deficiency of both qi and yin and blood stasis syndrome;

[0107] (4) Aged 18-80 years, regardless of gender, body mass index (BMI) ≤ 45 kg / m2;

[0108] (5) Glycated hemoglobin (HbA1C) 6-11%, fasting blood glucose (FPG) ≤ 15 mmol / L;

[0109] (6) Systolic / diastolic blood pressure ≤ 160 / 100 mmHg (sitting); Study on the efficacy and mechanism of Shenxiao Yuning Decoction in the treatment of diabetic nephropathy;

[0110] (7) Those with complete medical records and signed informed consent;

[0111] (8) Approved by the Medical Ethics Committee of this hospital.

[0112] 3.5 Exclusion criteria

[0113] (1) Women who are pregnant or breastfeeding;

[0114] (2) Those who have developed acute complications of diabetes such as diabetic ketoacidosis in the past month;

[0115] (3) Patients with concurrent heart failure, myocardial infarction; concurrent infection, immune disease, malignant tumor; concurrent liver disease (ALT, AST or ALP levels exceeding three times the upper limit of normal);

[0116] (4) Serum creatinine (Scr) ≥ 1.50 mg / dL (114.4 μmol / L) for men or ≥ 1.40 mg / dL for women

[0117] (106.8 μmol / L);

[0118] (5) Patients with other kidney or renal vascular diseases, or those with elevated urine protein due to other causes such as excessive exercise or urinary tract infection;

[0119] (6) Those undergoing dialysis or kidney transplantation;

[0120] (7) Drug contraindications: those with allergic constitution or allergic to the drugs used in this study;

[0121] (8) Those who are unable to cooperate due to severe mental illness or mental disorder;

[0122] (9) Those who have not signed the informed consent form.

[0123] 3.6 Suspension / Exit Criteria

[0124] (1) The researcher considers it necessary for the research participant to terminate the trial from a medical perspective;

[0125] (2) Pregnancy;

[0126] (3) The research participants themselves request to stop the trial;

[0127] (4) Those whose compliance was determined by the researchers to be very poor;

[0128] (5) Study participant lost to follow-up: The study participant could not be contacted after three recorded phone calls, faxes, or emails. All situations should be recorded in the study participant's medical record;

[0129] (6) Incorrect inclusion, i.e., the research participants do not meet the necessary inclusion / exclusion criteria specified in the research.

[0130] 3.7 Safety indicators

[0131] (1) Basic physical examination, such as body temperature, heart rate, blood pressure, respiration, etc.

[0132] (2) Routine blood test, liver and kidney function test, and electrolytes.

[0133] (3) Adverse events: Observe whether there are any discomfort symptoms. If relevant adverse events occur, record the time, symptoms, severity and whether symptomatic treatment has been given, and record the time when the adverse reaction disappears.

[0134] 3.8 Efficacy Observation Indicators

[0135] (1) Main evaluation index: treatment effectiveness.

[0136] (2) Secondary evaluation indicators:

[0137] ① Urine indicators (mid-morning urine): urine ACR, urine α1-microglobulin (urine α1-MG), urine immunoglobulin G (urine IgG), urine microalbumin (urine mALB), and urine transferrin.

[0138] ② Renal function indicators: serum creatinine (Scr), blood urea nitrogen (BUN), serum uric acid (UA), cystatin C (Cys-C), retinol binding protein, and β2-microglobulin.

[0139] ③ Blood sugar and blood lipid related indicators: fasting blood glucose (FPG), glycosylated hemoglobin (HbA1c), fasting insulin, fasting C-peptide, blood lipids (cholesterol, triglycerides, HDL, LDL), serum albumin (ALB) and other indicators.

[0140] ④ TCM syndrome scores: According to the TCM syndrome quantitative score table, observe, record and analyze the changes in the TCM syndrome scores of the experimental group and the control group.

[0141] 4. Efficacy and safety evaluation criteria

[0142] 4.1 Criteria for determining disease efficacy

[0143] Table 4 Symptom efficacy evaluation criteria

[0144]

[0145] (1) Inter-group comparison: Measurement data that conformed to normal distribution were expressed as mean ± standard deviation (X ± SD). If the data conformed to normal distribution and variance was homogeneous, the two groups were compared using independent sample t-test; if the data conformed to normal distribution but did not conform to variance homogeneity, the adjusted t-test was used.

[0146] (2) Intra-group comparison: Based on the normality of the differences in measurement data before and after treatment, paired sample t-test or Wilcoxon signed rank test of paired design was used.

[0147] 4.2 Safety indicators

[0148] Safety was evaluated according to the above safety evaluation criteria.

[0149] Blood routine, liver function, and electrolytes were examined twice before the study and 4 weeks after treatment. No abnormalities in liver and kidney function, blood routine, or electrolytes were found in the experimental group before treatment; two patients in the control group had a mild increase in alanine aminotransferase before treatment, with ALT of 59.6 and 56.2 U / L, respectively. Four weeks after treatment, the two patients still had a mild increase in ALT, which was 61.4 and 58.8 U / L, respectively. One patient in the experimental group had a mild increase in alanine aminotransferase 4 weeks after treatment, and no abnormalities in blood routine and electrolytes were found in either group after treatment. The chi-square test showed no significant difference in the incidence of abnormal liver function before and after treatment between the two groups, P>0.05.

[0150] 4.3 Efficacy observation indicators

[0151] The main evaluation indicators include: treatment effectiveness; secondary evaluation indicators include: urine ACR, urine α1-MG, TCM syndrome score, etc.

[0152] 5 Results Analysis

[0153] 5.1 Symptom efficacy evaluation criteria test results

[0154] After testing, the symptom efficacy judgment criteria of the two groups after 4 weeks of treatment were significantly different, P < 0.05, and the urine ACR and urine α1-MG of the experimental group were lower than those of the control group, and the differences were statistically significant, with P values ​​of 0.044 and 0.037, respectively. Figure 1 shown.

[0155] 5.2 Comparison of TCM syndrome scores

[0156] The TCM syndrome scores of the two groups of patients were evaluated before treatment and 4 weeks after treatment. After analysis, the TCM syndrome scores met the normal distribution and variance homogeneity, and the independent sample t test was used. There was no significant difference in the TCM syndrome scores of the two groups of patients before treatment. 4 weeks after treatment, the TCM syndrome scores of the experimental group were significantly lower than those of the control group. P = 0.027 < 0.05, the difference was statistically significant.

[0157] Table 5 TCM syndrome score evaluation results of the two groups of patients before treatment and 4 weeks after treatment

[0158]

[0159] 4.5 Overall efficacy

[0160] Compared with the control group, the experimental group was superior to the control group in reducing urine protein.

[0161] The overall efficacy was evaluated based on the above-mentioned disease efficacy assessment criteria and symptom efficacy assessment criteria.

[0162] The efficacy of disease and TCM syndrome was divided into three categories: markedly effective, effective and ineffective. Total effective rate = (number of markedly effective cases + number of effective cases) / total number of cases. According to the chi-square test, the total effective rate of TCM syndrome in the experimental group was higher than that in the control group, P = 0.005 < 0.01, and the difference was statistically significant, as shown in Table 6 and Figure 2 .

[0163] Table 6 Comparison of TCM syndrome efficacy between the two groups (n, %)

[0164]

[0165] For the main efficacy index, the total effective rate of treatment was 2 The test showed that the total effective rate of the experimental group was significantly higher than that of the control group, P=0.003<0.01, and the difference was statistically significant. See Table 7 for details.

[0166] Table 7 Comparison of therapeutic efficacy between the two groups (n, %)

[0167]

[0168] 2. Pharmacological effects of the composition of the present invention in diabetic nephropathy mouse model

[0169] 2 Research Materials

[0170] 2.1 Experimental animals

[0171] Several male C57BL / 6J mice aged 6-8 weeks and weighing 16-22g were used. They were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and the animal production license number is SCXK (Beijing) 2021-0006. They were raised in the specific pathogen-free (SPF) animal room of the First Affiliated Hospital of Shandong First Medical University, with room temperature (25±1)℃, relative humidity (50±10)%, alternating light and dark (12h / 12h), free drinking water, and normal food intake. All operations in this experiment were carried out in strict accordance with relevant animal ethics regulations.

[0172] 2.2 Main medicines and reagents and consumables

[0173] (1) 1.5 mL and 10 mL sterile centrifuge tubes: purchased from Anhui Biosharp Biotechnology Co., Ltd.

[0174] (2) 15 mL and 50 mL centrifuge tubes: purchased from Guangzhou Jiete Biofiltration Co., Ltd.;

[0175] (3) Streptozotocin STZ: Product No. S8050, purchased from Beijing Solaibao Technology Co., Ltd.;

[0176] (4) Isoflurane: Product No. R510-22-10, purchased from Shenzhen Ruiwode Life Science Technology Co., Ltd.;

[0177] (5) Erythromycin ointment: purchased from Fuyuan Pharmaceutical Co., Ltd.;

[0178] (6) Sodium citrate buffer: Product No. C1013, purchased from Beijing Solebow Technology Co., Ltd.;

[0179] (7) Paraformaldehyde fixative: Product No. G1101, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0180] (8) Bayer blood glucose test strips: purchased from Bayer Healthcare, Inc., USA;

[0181] (9) Urine protein quantitative test kit: Product No. C035-2-1, purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.;

[0182] (10) Serum creatinine (SCr) assay kit: Product No. C011-2-1, purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.;

[0183] (11) Urea nitrogen (BUN) test kit: Product No. C013-2-1, purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.;

[0184] (12) Citric acid: Product No. 10007118, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0185] (13) Trisodium citrate: Product No. 10019408, purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0186] (14) Xylene: Product No. 10023418, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0187] (15) Anhydrous ethanol: Product No. 10009265, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0188] (16) PBS buffer (dry powder): Product No. G0002, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0189] (17) SABC-POD (mouse / rabbit IgG) kit: Catalog No. SA1020, purchased from Wuhan Boster Biotechnology Co., Ltd.;

[0190] (18) DAB colorimetric kit (brown yellow): Catalog No. AR1022, purchased from Wuhan Boster Biotechnology Co., Ltd.;

[0191] (19) PAS stain: Product No. G1008, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0192] (20) Hematoxylin stain: Product No. G1004, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0193] (21) Bovine serum albumin (BSA): Catalog No. A8020, purchased from Beijing Solebow Technology Co., Ltd.;

[0194] (22) DAPI staining solution: Product No. C1005, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0195] (23) Anti-fluorescence quenching mounting solution: Product No. P0126, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0196] (24) RIPA lysis buffer: Catalog number PC101, purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.;

[0197] (25) Protease inhibitor (PMSF): Catalog No. P0100, purchased from Beijing Solebao Technology Co., Ltd.;

[0198] (26) BCA protein concentration assay kit: catalog number P0010S, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0199] (27) PAGE gel rapid preparation kit: Catalog number PG112, purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.;

[0200] (28) Prestained protein ladder: Catalog No. 26616, purchased from ThermoScientific, USA;

[0201] (29) Electrophoresis buffer (dry powder): Product No. G2018, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0202] (30) PVDF membrane: Product No. ISEQ00010, purchased from Millipore Corporation, USA;

[0203] (31) Transfer buffer (dry powder): Product No. G2017, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0204] (32) TBS buffer (dry powder): Product No. G0001, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0205] (33) Tween 20: Catalog No. GC204002, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0206] (34) Skimmed milk powder: Product No. D8340, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0207] (35) WB primary antibody diluent: Product No. PS114, purchased from Shanghai Yamei Biopharmaceutical Technology Co., Ltd.;

[0208] (36) ECL luminescent liquid: Product No. WBKLS0500, purchased from Millipore Corporation, USA;

[0209] (37) RNA extraction FreeZol Reagent: Product No. R711-01, purchased from Nanjing Novogene Biotechnology Co., Ltd.;

[0210] (38) Isopropanol: Product No. 30157428, purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0211] (39) DEPC water: Product No. R0021, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0212] (40) cDNA synthesis premix reagent: Catalog number KR118-02, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.;

[0213] (41) Quantitative PCR detection kit: Catalog No. Q711-02, purchased from Nanjing Novogene Biotechnology Co., Ltd.;

[0214] (42) TUNEL apoptosis detection kit (green fluorescence): Catalog number C1086, purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0215] (43) Lactic acid (LD) test kit (colorimetric method): Product No. A019-2-1, purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.;

[0216] (44) Absorbable surgical suture (4-0): purchased from Shanghai Pudong Jinhuan Medical Supplies Co., Ltd.;

[0217] (45) Small animal surgery related instruments: purchased from the Surgical Instrument Factory of Shanghai Medical Instrument (Group) Co., Ltd.;

[0218] (46) Insulin glargine injection (Changxiulin): purchased from Ganli Pharmaceutical Co., Ltd.;

[0219] (47) Electron microscopy fixative: Product No. G1102, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0220] 2.3 Administration

[0221] The Chinese medicine compositions of Examples 1-3 and Comparative Examples 1-5 of the present invention were freeze-dried in a freeze dryer to obtain 28 g of powder. The estimated dosage for mice was: 28 g / 60 kg×12.33=5.8 g / kg. Each mouse was gavaged with about 0.2 mL of the extract.

[0222] 2.4 Experimental Antibodies

[0223] (1) KIM-1: Catalog No. BA3537, purchased from Wuhan Boster Biotechnology Co., Ltd.;

[0224] (2) BIM: catalog number ab32158, purchased from Abcam, UK;

[0225] (3) PFKFB3: Catalog number ab181861, purchased from Abcam, UK;

[0226] (4) DPP4; Catalog number ab187048, purchased from Abcam, UK;

[0227] (5) β-actin: Catalog number TA-09, purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.;

[0228] (6) Horseradish enzyme-labeled goat anti-rabbit IgG (H+L): Product No. ZB-2301, purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.;

[0229] (7) Horseradish enzyme-labeled goat anti-mouse IgG (H+L): Product No. ZB-2305, purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.;

[0230] (8) Donkey anti-rabbit AlexaFluor546: Catalog No. A10040, purchased from Invitrogen, USA;

[0231] (9) Goat anti-mouse FITC: Catalog No. ab6785, purchased from Abcam, UK;

[0232] (10) Goat anti-rabbit AlexaFluor488: Catalog number ab150077, purchased from Abcam, UK.

[0233] 2.5 Experimental Equipment

[0234] Optical microscope Olympus Japan, confocal microscope Nikon Japan, 4℃ refrigerator Changhong Meiling Co., Ltd., -20℃ refrigerator Changhong Meiling Co., Ltd., -80℃ refrigerator Changhong Meiling Co., Ltd., ice maker Ningbo Xinzhi Biotechnology Co., Ltd., normal temperature centrifuge Beijing Baiyang Medical Equipment Co., Ltd., 4℃ low temperature centrifuge German Eppendorf, micropipette gun German Eppendorf, constant temperature metal bath German Eppendorf, constant temperature box German MemmertINE, magnetic stirrer Guangzhou RH-KT / C, vortex oscillator Guangzhou IKA, automatic double pure water distiller American Cascada

[0235] Electronic balance Beijing Sartorius Instrument Co., Ltd., electrophoresis instrument Bio-Rad, USA, ultrasonic crusher Xiaomei Ultrasonic Instrument (Kunshan) Co., Ltd., grinder Wuhan Sevier Biotechnology Co., Ltd., multifunctional imager GE, USA, RNA concentration meter ThermoScientific, USA, ordinary PCR amplifier ThermoScientific, USA, real-time fluorescence quantitative PCR instrument Bio-Rad, USA, ultrathin slicer Lcica, Germany, case slice drying machine Changzhou Zhongwei Electronic Instrument Co., Ltd., microplate reader ThermoScientific, USA, small animal anesthetic Shenzhen Ruiwode Life Science Technology Co., Ltd., mouse metabolic cage Columbia, USA, fingertip blood glucose meter Bayer Pharmaceuticals and Healthcare Co., Ltd., freeze dryer Shanghai Bilang Instrument Manufacturing Co., Ltd.

[0236] 2.6 Preparation of main reagents

[0237] (1) PBS buffer: 1 bag of PBS powder, 2000 mL of ddH2O (mix well and sterilize under high pressure for later use)

[0238] (2) Electrophoresis buffer: 1 bag of electrophoresis buffer powder, 1000 mL of ddH2O

[0239] (3) Transfer buffer: 1 bag of transfer buffer powder, 200 mL of methanol, 800 mL of ddH2O

[0240] (4) TBST buffer: 1 bag of TBST powder, 2 mL of Tween-20, 2000 mL of ddH2O

[0241] (5) 5% skim milk: 1 g skim milk powder, 20 mL TBST buffer

[0242] 3 Experimental methods

[0243] 3.1 Grouping and construction of diabetic nephropathy mouse model

[0244] 3.1.1 Grouping

[0245] C57BL / 6J mice aged 6-8 weeks and weighing 18-22 g were selected and, after adaptive feeding for 1 week, were randomly divided into a sham operation group, a model group, an embodiment 1 group, an embodiment 2 group, an embodiment 3 group, a comparative example 1 group, a comparative example 2 group, a comparative example 3 group, a comparative example 4 group and a comparative example 5 group.

[0246] 3.1.2 Construction of diabetic nephropathy mouse model

[0247] Experimental mice were fasted for 12h before surgery but were not restricted to drinking water to ensure fasting state. Mask inhalation of isoflurane anesthetized them, and surgery can be performed after mice were in an unconscious state. Ear tags were recorded for each group of mice, conventional hair removal, iodine cotton balls were disinfected in the surgical area and surrounding skin 2 times, and transverse incisions were made with ophthalmic scissors along the line connecting the ear tip and the tail and the intersection of the lower edge of the left rib arch, about 1cm long. Skin, mucosa and muscle three layers were separated layer by layer, and the sham operation group sutured the muscle and mucosa layer continuously immediately, and the skin layer adopted simple interrupted suture to close the incision. After cutting each layer of tissue, each embodiment group exposed perirenal fat and performed blunt separation, and the left kidney of mice was pulled up with hemostatic forceps to fully expose, and the perirenal fat and adrenal tissue were carefully peeled off with tweezers, and the left renal hilar blood vessels were ligated with surgical sutures and then the left kidney of mice was removed with ophthalmic scissors close to the kidney tissue side. After checking that there was no bleeding, the muscle and mucosa layer were sutured continuously, and the skin layer adopted simple interrupted suture. After the mice woke up from the anesthesia, they were put back into the cage. The incision was disinfected with iodine every day until it healed. One week after surgery, the mice in each group of the example were fasted but not watered for 12 hours, and streptozocin (STZ) powder was dissolved in sodium citrate buffer (prepared and used in dark conditions), and 1% STZ (10 mg / mL) was injected intraperitoneally into the mice at a dose of 65 mg / kg.

[0248] The mice were not restricted from eating feed and drinking water within 12 hours after the injection, and then they were fasted for 12 hours, but not water, and then injected with STZ again for 5 consecutive days. 72 hours after all the injections, the mice were fixed with a mouse fixator, and the tail tip was pierced with a blood collection needle to collect blood. The random blood sugar was measured with a blood glucose meter. If the results of the two measurements were higher than 16.7mmol / L, the model was considered to be successfully established, and the mice with a level lower than 16.7mmol / L were eliminated from the experiment.

[0249] 3.1.3 Administration

[0250] After the model was established, the mice continued to be fed with ordinary feed and free drinking water for 2 weeks. After 2 weeks, 24h urine was collected, and each group was gavaged with 5.8g / kg of freeze-dried powder, and the sham operation group was gavaged with the same dose of distilled water, once a day for 8 consecutive weeks. Each example group was injected with 2U / kg of glargine insulin to maintain blood sugar stability, once a day for 8 consecutive weeks.

[0251] 3.2 Collection and processing of mouse urine, blood and tissue specimens

[0252] (1) Random blood sugar and weight testing

[0253] In order to detect the body weight and random blood glucose values ​​of mice, blood was collected from the tail tip of the mice at 2, 6, and 10 weeks after model establishment.

[0254] (2) Urine volume and urine protein detection: 24-hour urine of mice was collected in metabolic cages (without water or food deprivation) at 2, 6, and 10 weeks after model establishment, and the total urine volume was recorded.

[0255] After mixing, draw 1 mL of urine into a 1.5 mL centrifuge tube and place it in a 4°C centrifuge for centrifugation (speed 3000 rpm, time 10 min). The supernatant urine is tested by urine protein quantitative test kit (CCB method), and the remaining urine is placed in a -80°C ultra-low temperature refrigerator for future use.

[0256] (3) Blood and specimen collection and processing

[0257] After collecting urine from mice at the end of the 10th week of the experiment (the time of model establishment was 0 week), isoflurane was inhaled through a mask.

[0258] After the mouse loses consciousness and enters a completely anesthetized state, trim the beard with scissors to prevent hemolytic reaction. Fix the mouse to expose the eyeball with congestion, clamp the eyeball with elbow forceps, and collect blood into a 1.5mL centrifuge tube. Then fix the mouse on the operating table with its face up in a supine position, shave the hair on the chest and abdomen of the mouse with a shaver, and disinfect it twice with iodine cotton balls. Use ophthalmic scissors to expose the chest and abdominal cavities of the mouse, use a 20mL syringe to absorb normal saline, puncture from the apex to the left ventricle, cut the right auricle of the mouse, perfuse an appropriate amount of normal saline, and stop perfusion after observing that the mouse liver changes from red to yellow-white. Separate the right kidney of the mouse to avoid breaking it, cut off the renal pedicle and ureter, carefully remove the perirenal capsule and fat, and weigh it. Fix the kidney with forceps, cut the kidney in two along the coronal plane, take half of the kidney tissue to separate the renal cortex, put it into a 1.5mL centrifuge tube, and place it in a -80℃ ultra-low temperature refrigerator for storage. The other half of the kidney tissue was fixed in 4% paraformaldehyde and made into paraffin sections for subsequent histopathological staining and other related experiments. The collected blood was placed at room temperature for 2 hours and then kept at 4°C overnight. After observing the blood coagulation and clot shrinkage, it was placed in a 4°C centrifuge and centrifuged (speed 4000rpm, time 10min). Use a pipette to carefully draw the upper serum into a new 1.5mL centrifuge tube and place it in a -80°C ultra-low temperature refrigerator for storage.

[0259] (4) Detection of protein concentration

[0260] Preparation of protein standard: Take 1.2mL of protein standard solution and add it to the protein standard (30mg BSA)

[0261] After fully dissolving, prepare a 25 mg / mL protein standard; take an appropriate amount of the above protein standard and dilute it with PBS buffer to a final concentration of 0.5 mg / mL; prepare BCA working solution: reagent A and reagent B are prepared in a ratio of 50:1; protein concentration detection: ① add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to a 96-well plate, and then add PBS to make up to 20 μL / well, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL respectively; ② use a pipette to draw an appropriate volume of test sample into a 96-well plate; ③ then add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min; ④ use an enzyme marker to measure the absorbance of the wavelength of A562; ⑤ calculate the standard curve and calculate the protein concentration of each sample according to the sample volume.

[0262] 4 Statistical methods

[0263] All samples were repeated more than 3 times for statistical analysis. Experimental data were expressed as mean plus or minus standard error (X ± SEM). GraphPad Prism9.0 software was used for statistical analysis and mapping of all data. The t test was used for comparison between two groups, and one-way ANOVA was used for comparison between three or more groups. The statistical significance of the difference was set at: P < 0.05.

[0264] 5 Results Analysis

[0265] Figure 3-5 The meanings of @#$%& are as follows:

[0266] @: There is a significant difference compared with Comparative Example 1 (P<0.05, or P<0.01);

[0267] #: There is a significant difference compared with comparative example 2 (P<0.05, or P<0.01);

[0268] $: Significant difference compared with comparative example 3 (P<0.05, or P<0.01);

[0269] %: There is a significant difference compared with Comparative Example 4 (P<0.05, or P<0.01);

[0270] &: There is a significant difference compared with Comparative Example 5 (P<0.05, or P<0.01);

[0271] Blood glucose analysis details see Figure 3 :The random blood glucose of mice in each group was measured at different time points. At the second week of modeling, the random blood glucose of mice in the sham operation group was significantly higher than that in the model group (P<0.01); compared with the control group, there was no statistical difference in the random blood glucose of mice in the embodiment groups (P>0.05). At the end of the 10th week, the random blood glucose of mice in the embodiment groups was significantly higher than that in the model group (P<0.01); compared with the control group, there was a statistical difference (P<0.05).

[0272] The urine volume of mice in the model group, sham operation group and each group of the embodiment was measured at different time points. Figure 4 As shown, when the model was established in the second week, the urine volume of mice in the sham operation group increased significantly compared with the model group (P<0.01); compared with the groups in the examples, there was no statistical difference in the urine volume of mice in each group (P>0.05). At the end of the sixth week, compared with the control group, the urine volume of mice in the example group was significantly reduced (P<0.01); at the end of the tenth week, the difference in the urine volume of mice was significantly increased, and compared with the groups in the examples, the urine volume of mice in the example 1 group decreased most significantly, but there was no statistical difference between the groups in the examples (P>0.05).

[0273] The UP of mice in the model group, sham operation group and each group of the examples was measured at different time points at 24h. The results are as follows Figure 5 The results are shown in Table 2. Figure 5 As shown, at the second week of model formation. Compared with the model group, the 24h UP of the model group mice in the sham operation group was significantly increased (P<0.01); compared with the model group, there was no statistical difference in the 24h UP of the mice in the control group and the embodiment groups (P>0.05). At the end of the 6th and 10th week, compared with the control group, the 24h UP of the model group mice in the embodiment groups was significantly increased (P<0.01); compared with the control group, the 24h UP of the mice in the embodiment 1-3 groups was significantly reduced (P<0.01).

[0274] The right kidney / body weight × 100% of mice in the model group, sham operation group and each group of the examples were measured at different time points. The results are as follows Figure 6 The renal index (right kidney / body weight × 100%) was measured. Compared with the sham-operated mice, the renal index of the model group mice increased significantly (P<0.01), indicating that the model was successfully established; compared with the control groups, the renal index of the mice in the example groups decreased significantly (P<0.01); there was no significant difference in the renal index of the mice in the example groups (P>0.05).

[0275] The blood BNU of mice in the model group, sham operation group and each group of the embodiment was measured at different time points. Figure 7 As shown, blood BNU was measured at the end of the 10th week. Compared with the sham-operated mice, the blood BNU of the model group mice increased (P<0.01); compared with the control groups, the blood BNU of the mice in the example groups was significantly reduced (P<0.01); there was no significant difference in the renal index of the mice in the example groups (P>0.05), indicating that the therapeutic effects of the examples were similar.

Claims

1. A Chinese medicine composition for treating diabetic nephropathy, characterized in that: The Chinese medicine composition for treating diabetic nephropathy is prepared from the following raw materials in parts by weight: 14-50 parts of astragalus, 12-35 parts of rhodiola, 8-25 parts of pseudoginseng, 8-25 parts of raw rehmannia, 14-50 parts of cornus, 8-25 parts of madder, 8-25 parts of cuttlebone, 6-22 parts of zedoaria, 8-25 parts of dodder seeds, 10-35 parts of smilax glabra, 5-18 parts of cicada shells, and 3-12 parts of wine rhubarb.

2. The Chinese medicine composition for treating diabetic nephropathy according to claim 1, characterized in that: The Chinese medicine composition for treating diabetic nephropathy is prepared from the following raw materials in parts by weight: 30 parts of astragalus, 20 parts of rhodiola, 15 parts of pseudoginseng, 15 parts of raw rehmannia, 30 parts of cornus, 15 parts of madder, 15 parts of cuttlebone, 12 parts of zedoaria, 15 parts of dodder seeds, 24 parts of smilax glabra, 9 parts of cicada shells, and 6 parts of wine-soaked rhubarb.

3. The Chinese medicine composition for treating diabetic nephropathy according to claim 1, characterized in that: The Chinese medicine composition for treating diabetic nephropathy is prepared from the following raw materials in parts by weight: 14 parts of astragalus, 12 parts of rhodiola, 8 parts of pseudoginseng, 8 parts of raw rehmannia, 14 parts of cornus, 8 parts of madder, 8 parts of cuttlebone, 6 parts of zedoaria, 8 parts of dodder seeds, 10 parts of smilax glabra, 5 parts of cicada shells, and 3 parts of wine-soaked rhubarb.

4. The Chinese medicine composition for treating diabetic nephropathy according to claim 1, characterized in that: The Chinese medicine composition for treating diabetic nephropathy is prepared from the following raw materials in parts by weight: 50 parts of astragalus, 35 parts of rhodiola, 25 parts of pseudoginseng, 25 parts of raw rehmannia, 50 parts of cornus, 25 parts of madder, 25 parts of cuttlebone, 22 parts of zedoaria, 25 parts of dodder seeds, 35 parts of smilax glabra, 18 parts of cicada shells, and 12 parts of wine-soaked rhubarb.

5. A preparation comprising the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The preparation also includes pharmaceutically acceptable excipients.

6. The preparation for treating diabetic nephropathy according to claim 5, characterized in that: The preparation is a clinically acceptable dosage form for administration.

7. The preparation for treating diabetic nephropathy according to claim 6, characterized in that: The dosage form is any one of decoction, granules, tablets, capsules, mixtures, syrups, pills, gels, powders or pastes, preferably decoction or powders.

8. The Chinese medicine composition for treating diabetic nephropathy according to any one of claims 1 or 2, characterized in that: The preparation method of the decoction is as follows: soak each herb in water for 20-40 minutes, decoct for 1-3 times, the first time for 10-20 minutes, the second time for 15-25 minutes, discard the herb residue, and mix the decoctions from the two times.

9. The Chinese medicine composition for treating diabetic nephropathy according to claim 7, characterized in that: The preparation method of the decoction is as follows: soak each herb in water for 30 minutes, decoct twice, the first time for 15 minutes and the second time for 20 minutes, discard the residue, mix the decoctions from the two times, vacuum concentrate the decoction with a freeze dryer, and dry to produce brown powder.

10. The diabetic nephropathy according to any one of claims 1 to 4 is diabetic nephropathy of qi and yin deficiency with blood stasis syndrome.

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