Traditional Chinese medicine composition for treating diabetic kidney disease and preparation method thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating diabetic kidney diseases, relates to the field of traditional Chinese medicines, and provides a traditional Chinese medicine composition for treating diabetic kidney diseases, which is prepared from 15 to 30 parts of radix astragali seu hedysari, 10 to 30 parts of radix salviae miltiorrhizae, 3 to 6 parts of leech powder, 3 to 6 parts of raw radix et rhizoma rhei, 15 to 45 parts of rhizoma coptidis and 10 to 30 parts of rhizoma zingiberis recens. The traditional Chinese medicine composition disclosed by the invention can reduce fasting blood-glucose, regulate blood fat, reduce triglyceride and total cholesterol levels, reduce the ratio of urinary albumin to creatinine, inhibit ferroptosis and improve the pathology of the diabetic kidney disease.

Description

Technical Field

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

[0002] Diabetic kidney disease (DKD) is one of the most common complications of diabetes. About 20%-40% of diabetic patients develop DKD, and 30%-50% of DKD patients will progress to end-stage renal disease. The mortality rate of patients with kidney disease has exceeded that of some malignant tumors.

[0003] In current research, the pathological mechanisms and treatment measures for DKD have been extensively explored. Among them, some studies on the role of lipid metabolism disorders and ferroptosis in DKD provide an important theoretical basis for further exploring intervention strategies. Lipid metabolism disorders are a common feature in DKD. Hyperglycemia and insulin resistance cause adipose tissue to release a large amount of free fatty acids, causing dyslipidemia, lipid peroxidation and lipid deposition, and the inflammatory response caused by them aggravates kidney damage. Ferroptosis is a new form of cell death and plays an important role in the development of DKD. In a high glucose environment, iron ions can cause oxidative stress and lipid peroxidation, leading to the death and damage of kidney cells. Multiple cell signaling pathways have been found to play an important role in the lipid metabolism disorders and ferroptosis process in DKD. For example, signaling pathways such as PI3K / AKT and AMPK are associated with lipid metabolism disorders and oxidative stress, affecting the progression of renal lesions. Some studies have explored some potential therapeutic strategies to intervene in the role of lipid metabolism disorders and ferroptosis in DKD. These include antioxidants, regulating lipid metabolism, ironchelating agents, etc. These strategies show certain protective effects, but further research is still needed. Summary of the invention

[0004] In view of the above problems, the present invention aims to provide a Chinese medicine composition for treating diabetic nephropathy and a preparation method thereof.

[0005] The main idea of ​​the technical solution adopted by the present invention: Based on the pathogenesis of diabetic nephropathy of fullness in the middle and internal heat, toxic blood stasis and damage to the collaterals, and qi deficiency and blood stasis, the present invention proposes a prescription for clearing heat, replenishing qi and activating blood circulation, which can effectively control blood sugar, protect kidney function, and reduce urinary protein excretion.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A traditional Chinese medicine composition for treating diabetic nephropathy, characterized in that the traditional Chinese medicine composition is made of the following raw materials in parts by weight: 15-30 parts of astragalus, 10-30 parts of salvia miltiorrhiza, 3-6 parts of leech powder, 3-6 parts of raw rhubarb, 15-45 parts of coptis chinensis, and 10-30 parts of ginger.

[0008] Furthermore, the Chinese medicine composition is made of the following raw materials in parts by weight: 30 parts of astragalus, 15 parts of salvia miltiorrhiza, 3 parts of leech powder, 6 parts of raw rhubarb, 30 parts of coptis chinensis, and 10 parts of ginger.

[0009] The present invention also discloses a method for preparing a Chinese medicine composition for treating diabetic nephropathy, which is characterized by comprising the following steps:

[0010] Step 1: Weigh 3-6 parts of leeches by weight and grind them into powder to obtain Preparation I;

[0011] Step 2: weigh 15-30 parts of astragalus, 10-30 parts of salvia miltiorrhiza, 3-6 parts of raw rhubarb, 15-45 parts of coptis chinensis, and 10-30 parts of ginger in order by weight, add cold water to cover the medicinal materials by 3-5 cm, soak for 30 minutes, bring to a boil over high heat, then continue to simmer over low heat for 30 minutes, filter and separate, and obtain a primary filtrate and a filter residue;

[0012] Step 3: Add 0.9-1L water to the residue, continue to boil over high heat, simmer for 30 minutes over low heat, and filter to obtain the secondary filtrate;

[0013] Step 4: Combine the first filtrate and the second filtrate, add Preparation I to the filtrate, and heat and concentrate to 400ml-500ml.

[0014] The beneficial effects of the present invention are as follows: compared with the prior art, the present invention has the following improvements:

[0015] The Chinese medicine composition of the present application can lower fasting blood sugar, regulate blood lipids, reduce triglycerides, total cholesterol levels, reduce urine albumin-creatinine ratio, inhibit ferroptosis, and improve pathological changes in diabetic nephropathy.

[0016] Astragalus was originally called "Huangqi". The name of Huangqi is explained in Compendium of Materia Medica as follows: "Qi means long. Huangqi is yellow in color and is the best tonic." It is sweet and warm in nature. It has the effects of replenishing qi and raising yang, consolidating the exterior and stopping sweating, supporting sores and promoting tissue regeneration, and promoting diuresis and reducing swelling. It is often used in clinical practice for qi deficiency or sinking of the middle qi, sores and ulcers that do not ulcerate or ulcers that do not heal for a long time; edema, beriberi, facial edema, etc. Professor Tong Xiaolin uses Huangqi to treat diabetic nephropathy, mainly for its function of replenishing deficiency and promoting diuresis. Leeches, commonly known as leeches, live in paddy fields and ditches and feed on the blood of humans and animals. They taste salty and bitter, and are neutral in nature. They have the effects of removing blood stasis, promoting menstruation, and eliminating symptoms. The Compendium of Materia Medica says: "Leech is a medicine for expelling bad blood and blood stasis." It is often used in clinical practice to treat blood stasis, amenorrhea, lumps, and pain caused by falls. According to the idea that "long-term illness enters the collaterals", "long-term illness must cause blood stasis" and "the treatment of diabetes should be full of collaterals", leeches are often used to enhance the ability to remove blood stasis. Rhubarb is bitter and cold in nature. It has the effects of purging and attacking accumulation, clearing away heat and purging fire, cooling blood and detoxifying, removing blood stasis and promoting menstruation, and removing dampness and jaundice. It is often used in clinical practice to treat constipation caused by real heat stagnation, vomiting and bleeding due to blood heat, red eyes and swollen throat, abdominal pain due to intestinal carbuncle, amenorrhea caused by blood stasis, traumatic injuries, damp-heat dysentery, jaundice and red urine, stranguria, edema, burns and scalds, etc. Rhubarb is a powerful medicine. Don't be afraid of its strong purgative effect. It can both relieve constipation and relieve turbidity. It is especially suitable for the stage of diabetic nephropathy and the accumulation of pathological products such as phlegm, turbidity, blood stasis and toxins. Salvia miltiorrhiza, commonly known as red ginseng, grows on hillsides and forest edges. It is famous for promoting blood circulation and removing blood stasis, promoting menstruation and relieving pain. It tastes bitter and slightly cold. It enters the heart and liver meridians. It has the effects of promoting blood circulation and removing blood stasis, clearing the heart and removing troubles, and cooling blood and reducing swelling. "Compendium of Materia Medica" records: "Danshen is an important medicine for promoting blood circulation, dredge menstruation, dispersing blood stasis and relieving pain". In clinical practice, Danshen is often used to treat symptoms such as angina pectoris, irregular menstruation, and blood stasis pain. Danshen nourishes blood and promotes blood circulation, "mainly for accumulation, breaking up tumors and removing masses", and is a target drug for dredging large blood vessels. When used with Astragalus, it can protect the myocardium and resist renal fibrosis to improve renal function and circulation. Motherwort, also known as Yimu and Kuncao, grows in fields and roadsides. It tastes sweet and bitter, is slightly cold in nature, and belongs to the liver, heart, and bladder meridians. It has the effects of promoting blood circulation and regulating menstruation, promoting diuresis and reducing swelling, and clearing away heat and detoxification. It is often used in clinical practice to treat irregular menstruation, amenorrhea, dysmenorrhea, edema, oliguria, and hot stranguria. The blood circulation and diuresis effects of motherwort help improve kidney blood circulation, reduce edema, and promote the discharge of metabolic waste. Epimedium, also known as Xianlingpi, grows in the damp land of mountains and forests. It tastes spicy and sweet, is warm in nature, and enters the kidney and liver meridians. It has the effects of tonifying the kidney and strengthening yang, strengthening tendons and bones, and removing rheumatism. It is often used clinically to treat symptoms such as kidney yang deficiency, soreness of waist and knees, rheumatic pain, impotence and infertility. If the patient's yang qi is obviously depleted, epimedium can also be used in combination. First, it can protect yang and restrict the bitter and cold nature of rhubarb. Second, it can warm the kidney and help the kidney restore the function of gasification and drainage, and improve the symptoms of kidney deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the effect of the Chinese medicine composition of the present invention on the renal function of HFD / STZ mice.

[0018] Among them, A is a schematic diagram of the procedure for STZ / HFD-induced diabetic mice;

[0019] B is the fasting blood glucose (FBG) values ​​of the mouse groups treated at different times (n=6);

[0020] C is the body weight of mice groups treated at different time periods;

[0021] D is mouse kidney weight / body weight;

[0022] E is the urine albumin / creatinine ratio (UACR) of mice (n=6);

[0023] FL is the blood renal function and lipid-related indicators of each group of mice at the time of sampling, including serum creatinine (SCr, F), blood urea nitrogen (BUN, G), uric acid (UA, H), total cholesterol (TC, I), triglycerides (TG, J), high-density lipoprotein cholesterol (HDL-C, K) and low-density lipoprotein cholesterol (LDL-C, L), n = 6.

[0024] Figure 2 This is a diagram showing that the Chinese medicine composition of the present invention improves the renal pathological changes in HFD / STZ mice.

[0025] Among them, A is the pathological staining results of morphological examination of renal changes by hematoxylin-eosin (HE) staining and PAS staining (n=3 in each group);

[0026] B is the pathological staining results of morphological examination of kidney changes by MASSON staining and Oil Red O staining (n = 3 in each group);

[0027] C is the PAS-positive area;

[0028] D is the MASSON-positive area;

[0029] E is the Oil Red O positive area;

[0030] G is a transmission electron microscopy photograph; the glomerular ultrastructure was observed by transmission electron microscopy, with the original magnification of 10000× (n=5 per group);

[0031] F, H, I are quantitative analysis by transmission electron microscopy; glomerular basement membrane (GBM) thickness (F), podocyte width (H) and the number of podocytes per micrometer of GBM (podocyte number / GBM length, I), (3 images were randomly selected for each sample, n = 5 per group). Data are expressed as mean ± SEM. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001vs.Con; p<0.05, **p<0.01, ***p<0.001, ****p<0.0001vs.Mod.

[0032] Figure 3 It is the principal component analysis (PCA) diagram of the traditional Chinese medicine composition of the present invention.

[0033] Figure 4 It is a volcano plot of the differential lipids of the traditional Chinese medicine composition of the present invention.

[0034] Figure 5 This is a diagram of the ChemRICH enrichment results of the present invention.

[0035] Figure 6 This is a diagram of the enrichment results of LION terms in the present invention.

[0036] Figure 7 This is a percentage diagram of the significantly enriched lipid species detected in the comparison between the model group and the control group of the present invention.

[0037] Figure 8 It is a heat map of phospholipid alcoholamines (PEs) contained in the polyunsaturated fatty acid chains (PUFA) of the present invention.

[0038] Fig. 9 This is a diagram showing the effect of the Chinese medicine composition of the present invention on ferroptosis-related indicators.

[0039] Among them, AC are RT–PCR analysis of GPX4 (A), FTH1 (B), and ACSL4 (C) (n = 4 per group);

[0040] D is the average density of GHPX4 immunofluorescence quantitative analysis;

[0041] EH are representative Western blots (E, n = 4 blots in total) and quantitative analysis of ACSL4 (F), FTH1 (G), and GPX4 (H);

[0042] I is immunofluorescence of GPX4 (n = 4 per group);

[0043] JL are the levels of MDA (J), 4HNE (K), and GSH (L) in different groups (n = 4 in each group);

[0044] M is the iron ion in the kidney of each group (n=4 per group), and the data are expressed as mean±SEM. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 vs.Con; p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs.Mod.

[0045] Fig.10 This is a diagram of the PI3K / AKT / GSK3β signaling pathway in HFD / STZ mice regulated by the traditional Chinese medicine composition of the present invention.

[0046] Among them, A is the immunofluorescence of GSK3β (n=4 per group);

[0047] B is the relative fluorescence intensity of GSK3β;

[0048] CF are representative Western blots (E, n=4 blots in total) and quantitative analysis of P-PI3K / PI3K (C), P-AKT / AKT (D), P-GSK3β / GSK3β (F). DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0050] Example 1

[0051] The present application discloses a Chinese medicine composition for treating diabetic nephropathy, comprising astragalus, salvia miltiorrhiza, raw rhubarb, leech powder, coptis chinensis, and ginger, wherein the dosage of each component is: 30g of astragalus, 15g of salvia miltiorrhiza, 3g of leech powder, 6g of raw rhubarb, 30g of coptis chinensis, and 10g of ginger.

[0052] Example 2

[0053] The present application discloses a Chinese medicine composition for treating diabetic nephropathy, comprising astragalus, salvia miltiorrhiza, raw rhubarb, leech powder, coptis chinensis, and ginger, wherein the dosage of each component is: 20g of astragalus, 12g of salvia miltiorrhiza, 5g of leech powder, 4g of raw rhubarb, 21g of coptis chinensis, and 23g of ginger.

[0054] Example 3

[0055] The present application discloses a Chinese medicine composition for treating diabetic kidney disease, comprising astragalus, salvia miltiorrhiza, raw rhubarb, leech powder, coptis chinensis, and ginger, wherein the dosage of each component is: 15g of astragalus, 10g of salvia miltiorrhiza, 3g of leech powder, 3g of raw rhubarb, 15g of coptis chinensis, and 10g of ginger.

[0056] Examples 1-3 are all prepared by the following method, and Example 1 is used as an example for specific description:

[0057] Step 1: weigh 3 g of leech and grind it into powder to obtain Preparation I;

[0058] Step 2: Weigh 30g of Astragalus, 15g of Salvia miltiorrhiza, 6g of Rhubarb, 30g of Coptis chinensis, and 10g of ginger in sequence, put them into a casserole, add cold water to cover the medicinal materials by 5cm, soak for 30 minutes, bring to a boil over high heat, then continue to simmer over low heat for 30 minutes, and filter to obtain the primary filtrate;

[0059] Step 3: Add 1L of warm water to the casserole, continue to boil over high heat, simmer for 30 minutes over low heat, and filter to obtain the secondary filtrate;

[0060] Step 4: Combine the first filtrate and the second filtrate, add Preparation I to the filtrate, and heat and concentrate to 400 ml.

[0061] Usage and dosage of the medicine of the present invention: decoction in water, take warm twice a day.

[0062] It should be noted that the Chinese medicine composition of the present invention is hereinafter referred to as the heat-clearing, qi-invigorating and blood-activating prescription for explanation.

[0063] The following are clinical cases for the Chinese medicine composition of this application

[0064] Clinical Case 1:

[0065] Meng, male, 47 years old, weight 100kg, BMI: 32.28kg / m 2 . First visit on February 27, 2023. Chief complaint: Blood sugar has been elevated for half a year. Irregularly take metformin 0.25g 3 times a day to control blood sugar. History of coronary heart disease for 4 years. Symptoms: dry mouth, bitter taste, dizziness, fatigue, back pain, irritability. Appetite is OK, sleep is shallow and easy to wake up, and there are many dreams. There is foam in the urine, and there is occasional nocturia. The stool is sticky and smelly, 1 to 2 times a day. Physical examination: blood pressure: 125 / 85mm Hg, heart rate: 73 times per minute. The tongue is fat and dark red, with fine cracks on it, the bottom of the tongue is dark red and stagnant, the root of the moss is light yellow and greasy, and the pulse is deep and stringy. Auxiliary examination: glycosylated hemoglobin 6.8%, fasting blood sugar 8.51mmol / L. Alanine aminotransferase 43U / L, aspartate aminotransferase 33U / L, blood uric acid 625μmol / L, blood creatinine 68.3μmol / L, urine protein (+), urine microalbumin 243mg / L, urine microalbumin creatinine ratio (ACR) 270.48mg / g. Total cholesterol 3.69mmol / L, triglyceride 3.67mmol / L, high-density lipoprotein 0.99mmol / L, low-density lipoprotein 1.81mmol / L, anti-glutamic acid decarboxylase antibody (GAD-Ab), anti-islet cell antibody (ICA) and insulin antibody (IAA) are all negative. Western medicine diagnosis: type 2 diabetes, type 2 diabetes diabetic nephropathy stage III. Traditional Chinese medicine diagnosis: turbid urine with phlegm-heat, qi deficiency and blood stasis syndrome. Treatment is to clear away heat and resolve phlegm, invigorate qi, remove blood stasis and dredge collaterals. The prescription is modified from the prescription of clearing heat, invigorating qi and activating blood circulation. The prescription composition is: 30g of Astragalus, 6g of Hirudo, 15g of Salvia miltiorrhiza, 6g of Raw Rhubarb, 15g of Coptis chinensis, 15g of Pinellia ternata, 30g of Trichosanthes, 45g of Clematidis, 15g of Artemisia capillaris, 45g of Raw Hawthorn, 9g of Ginger, and 9g of Jujube. 56 doses, decocted in water, 1 dose per day, divided into morning and evening. Advise to control diet, exercise appropriately to lose weight, monitor blood sugar, and recheck biochemistry, glycosylated hemoglobin, 24h urine protein quantitative, and ACR.

[0066] Second visit on April 24, 2023, BMI: 30.67kg / m 2 , dry mouth and bitter taste are better than before, dizziness and fatigue are better than before, waist pain is better than before, and the mood is irritable. Appetite is good, and sleep is poor as before. Urine is foamy, and stool is 1 to 2 times / day, unformed and sticky. Auxiliary examination: glycosylated hemoglobin 6.3%, fasting blood sugar 7.16mm ol / L, blood uric acid 610μmol / L, blood creatinine 67.9μmol / L, total cholesterol 2.73mmol / L, triglyceride 2.08mmol / L, high-density lipoprotein 0.99mmol / L, low-density lipoprotein 1.35mmol / L. Urine microalbumin 117mg / L, ACR 104.75mg / g, prescription: add 15g of Clematis chinensis and raw hawthorn to the above prescription, add 30g of Smilax glabra, 30g of Chuanxiong, and 15g of Polygonum cuspidatum. 30 doses, decocted in water, 1 dose per day, divided into morning and evening.

[0067] On June 27, 2023, the third visit showed that the dry mouth and bitter taste were better than before, the dizziness, fatigue, and waist pain were better than before, and the mood was calmer than before. The appetite and sleep were good, the urine was foamy, and the stool was once a day, which was sticky. Auxiliary examination: glycosylated hemoglobin 6%, urine microalbumin 44mg / L, ACR 31.67mg / g. Keep the front, 56 doses, decocted in water, 1 dose per day, divided into morning and evening.

[0068] Clinical Case 2:

[0069] Wang, male, 52 years old, first visit on June 6, 2019. Height: 183cm, weight: 63kg, BMI: 18.81kg / m 2. Chief complaint: elevated blood sugar for more than 3 years, accompanied by foam in urine for more than 1 year. Current medical history: In 2016, the patient went to a local hospital for treatment due to symptoms such as dry mouth, weight loss, and foam in urine. Fasting blood glucose (FPG) was 12.5mmol / L, glycosylated hemoglobin (HbA1C) was 8.4%, and he was diagnosed with type 2 diabetes. He was treated with oral glimepiride and metformin, and his blood sugar was under control. In 2017, he did not pay attention to treatment, so his condition worsened. In 2019, the FPG was 8.6mmol / L and the urine protein was 2+. He was then hospitalized in the Affiliated Hospital of Qingdao University and diagnosed with diabetic nephropathy. For further treatment, he came to the outpatient clinic today. Current symptoms: chest tightness and shortness of breath, occasional palpitations, bowel sounds in the abdomen, excessive flatulence, numbness and coldness of the hands and feet, blurred vision, good appetite, poor sleep, constipation, 1 line every 2 days, large amount of urine with foam, and 1 to 2 nocturia. The tongue coating is pale yellow and thick, the bottom of the tongue is congested, and the pulse is stringy, hard and rapid. Current medication: repaglinide 1mg, 3 times / d, acarbose 50mg, 3 times / d, vildagliptin 50mg, 2 times / d, valsartan 80mg, 1 time / d, Bailing capsule 2 capsules, 3 times / d, rosuvastatin 5mg, 1 time / d. Past history: hypertension for more than half a year. Personal history: smoking for 36 years, 20 cigarettes / d, drinking for 30 years. Family history: twin brother has diabetes, denies allergy history. Auxiliary examination: (May 29, 2019) 24-hour urine protein quantification (24h UTP) 2.32g / 24h (normal value <0.15g / 24h). Western medicine diagnosis: stage IV of type 2 diabetic nephropathy; Chinese medicine diagnosis: diabetes (liver and stomach stagnation, qi deficiency and blood stasis). Prescription Qingre Yiqi Huoxue Tongluo Fang plus or minus. Prescription composition: 30g of Astragalus, 6g of processed leech powder, 15g of Salvia miltiorrhiza, 6g of raw rhubarb (single bag), 15g of Coptis chinensis, 9g of Bupleurum, 15g of Scutellaria baicalensis, 9g of Pinellia ternata, 30g of White Peony Root, 9g of Curcuma Atractylodes, 9g of Panax Notoginseng, 9g of Fritillaria thunbergii, 15g of ginger (added later), 9g of Jujube, decocted in water, 1 dose / d, once in the morning and evening, and repaglinide and acarbose were stopped. The patient was re-examined after taking the medicine for 1 month, and the chest tightness and shortness of breath were reduced by 60%, the bowel sounds and flatulence in the abdomen basically disappeared, the numbness and coldness of the hands and feet were the same as before, the blurred vision was aggravated, dizziness, poor appetite, poor sleep, 4-5 nocturia, normal bowel movements, reduced urine foam, reddish and dry tongue, thin yellow fur, and stringy and hard pulse. Current medication: vildagliptin 50mg, 2 times / d, valsartan 80mg, 1 time / d, Bailing capsule 2 capsules, 3 times / d, rosuvastatin 5mg, 1 time / d, dipyridamole 1 tablet, 3 times / d, canagliflozin 100mg, 1 time / d. Auxiliary examination: (July 3, 2019, Jiaozhou People's Hospital) HbA1C5.6%, 24hUTP 0.71g / 24h, urine protein±, blood pressure: 133 / 71mmHg (1mmHg≈0.133kPa). On the basis of the original prescription, add Millettia reticulata 30g, red peony root 15g, raw rehmannia root 30g, reduce white peony root, reduce coptis root to 6g, add Panax notoginseng to 12g, and add Fritillaria thunbergii to 15g.The patient came back for a follow-up visit one month after taking the medication. His blood sugar level was stable, and his urine protein continued to decrease. He continued to receive blood sugar-lowering and complication treatment.

[0070] The following are the clinical experimental results of the Chinese medicine composition prepared in the embodiment of the present invention:

[0071] 1. QRF (Qingre Yiqi Huoxue Fang) improves renal function and pathological damage in C57 mice induced by high fat and STZ (streptozotocin)

[0072] Hyperglycemia is the initiating factor and important promoting factor of the pathological process of DKD. To evaluate the protective effect of QRF on renal function of HFD (high-fat diet) / STZ-induced DKD (diabetic kidney disease) mice, 20-week-old C57BL / 6J mice were gavaged with QRF or took dapagliflozin (Dap) for 16 weeks. The above operation process is as follows Figure 1 As shown in A.

[0073] pass Figure 1 B shows that the blood glucose level of mice induced by HFD / STZ was significantly higher than that of the control group. After 4 weeks of treatment with dapagliflozin and QRF, blood glucose had significantly decreased compared with the model group. During the subsequent administration, the FBG (fasting blood glucose) level of the QRF group gradually decreased. Figure 1 C shows that due to the effects of high fat and STZ injection on the weight of mice, there is no statistical difference in the weight of mice induced by HFD / STZ and the control group. It still showed a downward trend at 4 weeks. It then gradually increased, but it was not statistically significant. Figure 1 D shows that there was no significant change in the body weight of mice in the QRF group, but the kidney weight to body weight ratio was significantly reduced. Figure 1 E shows that urine microalbumin creatinine can reflect renal function. The urine test collected in the metabolic cage showed that QRF could significantly reduce the UACR (urine microalbumin creatinine ratio) level at 16 weeks, which was comparable to the level of dapagliflozin.

[0074] In order to further evaluate the efficacy of QRF on renal function, the present application detected the levels of Cr (creatinine), BUN (urea nitrogen), and UA (uric acid) in the blood, and the results are shown in Figure 1 The results showed that QRF could significantly reduce the elevated BUN and UA in DKD, and the elevated Cr level also tended to decrease.

[0075] Blood lipids are also an important cause of DKD. This application tested the levels of TC (total cholesterol) and TG (triglycerides) in the blood, and the results are shown in Figure 1IL. The results showed that QRF could significantly reduce TC and TG levels. However, the effect on HDL (high-density lipoprotein) and LDL (low-density lipoprotein) levels was not obvious. Dapagliflozin also had a similar effect, which is basically consistent with the reported literature. These results indicate that QRF has a renal protective effect on HFD / STZ-induced DKD mice.

[0076] Figure 2 F-I shows transmission electron microscopy photos compared with the control group, where G is an electron microscopy photo, and F, H, and I are the results of electron microscopy quantitative analysis. The basement membrane of DKD mice is unevenly thickened, the foot processes are widened, and there is fusion, while QRF can reduce the width of the basement membrane, reduce the width of the foot processes, and reduce the number of foot processes per micron of the basement membrane. Figure 2 A shows that under HE staining (hematoxylin-eosin staining), the renal tissue structure of group M (model group) was severely abnormal, with a large number of renal tubular degeneration, vacuoles in the cytoplasm of epithelial cells, and unclear tubular structure; a large number of inflammatory cells infiltrated the interstitium; glomerular hypertrophy, and obvious thickening of the basement membrane; after QRF treatment, the renal tissue structure was basically normal, with no obvious damage. Under PAS staining (peroxy acid Schiff staining), the basement membrane of group M was thickened as a whole, with more purple-red positive areas, and the basement membrane of group QRF was significantly thinner than that of group M, with a lighter purple-red color. Figure 2 In B, under Masson staining, large-area fibrosis was observed in group M, and the overall blue fibers were significantly increased compared with group C (blank control group). The renal fibrosis in group QRF was significantly reduced compared with group M. Under oil red staining, a large number of fat vacuoles were observed in group M, and the lipid droplets in group QRF were significantly reduced compared with group M. The results of quantitative analysis are shown in Figure 2 CE.

[0077] 2. Lipidomics explains the mechanism of QRF in treating DKD

[0078] In order to investigate the systemic changes in lipid metabolism in the HFD / STZ-induced DKD model induced by QRF treatment, the present invention carried out lipidomics analysis of mouse kidney tissues by UPLC-Orbitrap MS / MS (ultra-performance liquid chromatography-orbitrap mass spectrometry / tandem mass spectrometry, LC-MS). Figure 3 The tightly clustered QC (quality control) samples in the principal component analysis score plot indicated that the stability of the LC-MS system was ensured. PLSDA (partial least squares discriminant) analysis in positive and negative ion modes showed that DKD significantly affected the lipid composition of kidney tissue compared with the control group, and lipid homeostasis could be restored after QRF treatment.

[0079] Volcano map ( Figure 4 ) analysis was used to screen lipid biomarker candidates based on FC criteria ≥2 or ≤0.5 and P value ≥1, and 1089 differential lipid molecules were identified between the model group and the control group ( Figure 4Left), 593 differential lipid molecules were identified in the QRFvs Model group ( Figure 4 Right). The top 10 significant lipid molecules are labeled in the figure.

[0080] Cluster analysis was performed on the lipid molecules identified in Model vs Control, QRF vs Model. Figure 5 The top 10 terms filtered by up- and down-regulation FDR q-value (FDR-corrected q-value) in the two group comparisons shown in the figure. The node size indicates how many lipid species are expected to be associated with the term in the identified lipid subset. The analysis results showed that in the DKD mouse model, alkyldiacylglycerols, glycerolipids, headgroup with neutral charge, lipid droplet, lipid storage, triradylglycerols, significantly decreased above average bilayer thickness, diacylglycerophosphocholines [GP0101], diacylglycerophosphoglycerophosphodiradylglycerols, glycerophospholipids (glycerophospholipids) ( Figure 5 Left), and showed an opposite trend after QRF treatment ( Figure 5 right).

[0081] Figure 6 Shown in the figure are clusters with significant differences identified based on the Kolmogorov-Smirnov test, where the x-axis represents the median XlogP of each cluster and the y-axis represents statistical significance. The node size represents the number of lipids in each cluster, with red indicating an increase and blue indicating a decrease. ChemRICH analysis showed that the levels of TG, AC (acetate), SM (sphingolipids), PE (phosphatidylethanolamine), S-PC (sulfated phosphatidylcholine), and S-DG (sulfated diacylglycerol) were significantly increased in the DKD mouse model ( Figure 6 Left), while QRF intervention led to an increase in TG, Acyl-CoA (acyl-CoA), But (butyrate), SM, PE, S-PC, S-DG and other lipid subclasses ( Figure 6 right).

[0082] Figure 7 It is shown in the data that a total of 26 lipid subclasses with significant differences were identified in the DKD mouse model. In addition to the high proportion of TG content, PE is also one of the subclasses with relatively high content. PE has been shown to be the main substrate for peroxidation in ferroptosis; PEs undergo peroxidation through iron-catalyzed enzymes and Fenton reactions (non-enzyme catalysis) to generate phospholipid hydroperoxides, which ultimately leads to the occurrence of ferroptosis.

[0083] exist Figure 8 According to the number of double bonds, 20 PE lipid molecules containing PUFA (unsaturated fatty acid chains) that were significantly upregulated in the model group were further analyzed by lipidomics. The results showed that QRF could downregulate lipid molecules such as PE (16:0e18:2)-H, PE (18:2e20:4)-H, PE (20:2e16:1)+Na, PE (16:0p18:2)+H, PE (18:1p18:2)+H.

[0084] 3. QRF protects renal function by inhibiting ferroptosis

[0085] GO enrichment analysis showed that 18 genes had regulatory effects, involving oxidative reactions and ferroptosis. Therefore, this application speculates that the inhibitory effect of QRF may be related to the recovery of renal function.

[0086] Fig. 9 Compared with the model group, the mRNA expression levels of GPX4 (glutathione peroxidase 4) and FTH1 (iron regulatory protein 1) were increased, while the mRNA expression of ASCL4 (acyl-CoA synthetase long-chain family member 4) was decreased in the QRF group in AB. Fig. 9 C). Immunofluorescence showed that GPX4 expression was decreased in the model group, but QRF could increase GPX4 expression ( Fig. 9 D, I). WB (Western Blot) results also showed similar trends in the expression levels of GPX4, ASCL4, and FTH1 (Figure EH). In the QRF group, compared with the model group, the levels of MDA (malondialdehyde) and 4HNE (4-hydroxynonenal) were decreased, and GSH (glutathione) was increased. In addition, the iron concentration in the QRF group was decreased. These findings suggest that the changes associated with ferroptosis in the model group were reversed by QRF ( Fig. 9 JM), suggesting that it may help improve kidney function.

[0087] 3.1QRF exerts renal protective effects by inhibiting the PI3K / AKT / GSK3β signaling pathway. The results of network pharmacology showed that among the significantly enriched pathways, the PI3K / AKT / GSK3β (Phosphatidylinositol 3-kinase / ProteinKinase B / Glycogen Synthase Kinase 3beta) signaling pathway is the most likely pathway for QRF to treat DKD. In the analysis of ferroptosis, GSK3β is also an important target. Immunofluorescence showed that GSK3β increased after QRF treatment ( Fig.10 A, B). Therefore, this application verifies the expression of this signaling pathway. Western blot results showed that the relative expression of P-PI3K / PI3K, P-AKT / AKT, and P-GSK3β / GSK3β decreased in the Mod group, and after QRF intervention, the relative expression increased significantly. These results indicate that QRF may play a renal protective role by inhibiting the PI3K / AKT / GSK3β signaling pathway ( Fig.10 CF).

[0088] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A Chinese medicine composition for treating diabetic nephropathy, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-30 parts of astragalus, 10-30 parts of salvia miltiorrhiza, 3-6 parts of leech powder, 3-6 parts of raw rhubarb, 15-45 parts of coptis chinensis and 10-30 parts of ginger.

2. A Chinese medicine composition for treating diabetic nephropathy according to claim 1, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 30 parts of astragalus, 15 parts of salvia miltiorrhiza, 3 parts of leech powder, 6 parts of raw rhubarb, 30 parts of coptis chinensis and 10 parts of ginger.

3. A method for preparing a Chinese medicine composition for treating diabetic nephropathy according to claim 1 or 2, characterized in that: The following steps are involved: Step 1: Weigh 3-6 parts of leeches by weight and grind them into powder to obtain Preparation I; Step 2: weigh 15-30 parts of astragalus, 10-30 parts of salvia miltiorrhiza, 3-6 parts of raw rhubarb, 15-45 parts of coptis chinensis, and 10-30 parts of ginger in order by weight, add cold water to cover the medicinal materials by 3-5 cm, soak for 30 minutes, bring to a boil over high heat, then continue to simmer over low heat for 30 minutes, filter and separate, and obtain a primary filtrate and a filter residue; Step 3: Add 0.9-1L water to the residue, continue to boil over high heat, simmer for 30 minutes over low heat, and filter to obtain the secondary filtrate; Step 4: Combine the first filtrate and the second filtrate, add Preparation I to the filtrate, and heat and concentrate to 400ml-500ml.

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