A traditional Chinese medicine composition for preventing and / or treating vascular calcification in chronic kidney disease, and its preparation method and use

Abstract

The present invention belongs to the field of Chinese medicine compositions, and in particular to a Chinese medicine composition for preventing and / or treating chronic kidney disease vascular calcification, and its preparation method and use. The Chinese medicine composition is a preparation prepared from the following raw materials in parts by weight: 5-15 parts of Radix Dichroae, 5-20 parts of Eucommia, 5-20 parts of Cistanche, 5-15 parts of Cuscuta, 5-20 parts of Rehmannia, 5-20 parts of Epimedium, 5-15 parts of Rhizoma Drynariae, 5-15 parts of Panax Japonicus, 5-20 parts of Millettia Spatholobi, and 5-20 parts of Semen Persicae. The Chinese medicine composition of the present invention can improve calcium-phosphorus metabolism disorder, protect the cardiovascular system, improve renal function, enhance cardiac function, inhibit vascular calcification, and prevent the transdifferentiation of vascular smooth muscle cells, with "tonifying the kidney, activating blood circulation and unblocking collaterals" as the treatment principle, by regulating the kidney-bone-vascular axis, inhibiting oxidative stress and inflammation, and intervening in calcium-phosphorus metabolism, thereby effectively treating CKD vascular calcification.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine compositions, and in particular relates to a traditional Chinese medicine composition for preventing and / or treating vascular calcification in chronic kidney disease, and a preparation method and use thereof. Background Art

[0002] Chronic kidney disease (CKD) has become a major threat to human health, with nearly 700 million people worldwide affected. It is projected to become the fifth leading cause of death worldwide by 2040. The sixth CKD epidemiological survey report in my country in 2024 showed that the prevalence of CKD in adults reached 8.2%, with the number of adults with the disease reaching 82 million. This incidence continues to rise, posing a serious health threat and a significant socioeconomic burden. Vascular calcification is the most common complication of CKD, occurring in 80%-90% of terminally ill patients. It severely impacts quality of life and is the leading cause of death in these patients. Extensive data confirm that vascular calcification significantly increases mortality in patients with CKD stages 3-5, posing a significant challenge to CKD prevention and treatment in my country.

[0003] Vascular calcification in chronic kidney disease (CKD) refers to the ectopic pathological deposition of calcium and phosphate salts in the form of hydroxyapatite in the vascular media, causing mineralization of the vessel wall, resulting in decreased vascular compliance and increased stiffness. The vascular calcification process involves multiple cellular molecular biological events, including the transdifferentiation of vascular smooth muscle cells to osteoblasts, the production of extracellular matrix proteins, the release of matrix vesicles, and the deposition of calcium hydroxyapatite. The transdifferentiation of vascular smooth muscle cells to osteoblasts is the core pathogenesis of vascular calcification in CKD. During the progression of chronic kidney disease, abnormal metabolic accumulation of inflammatory molecules, reactive oxygen species (ROS), uremic toxins, parathyroid hormone, and harmful substances such as calcium and phosphate induces the transdifferentiation of vascular smooth muscle cells to osteoblasts, leading to the ectopic deposition of calcium and phosphate crystals in the vascular media and promoting the development of vascular calcification in CKD. CKD often affects large arteries such as the thoracic and abdominal aorta, causing elevated blood pressure and increased cardiac afterload, ultimately leading to left ventricular hypertrophy and fatal cardiovascular events, making it the most common complication and cause of death in CKD.

[0004] However, there is currently a lack of clear and effective preventive and therapeutic drugs and intervention methods. Therefore, there is an urgent need to develop effective preventive and therapeutic drugs to prevent and treat vascular calcification in CKD, thereby improving the quality of life of CKD patients and reducing the risk of death. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a traditional Chinese medicine composition for preventing and / or treating vascular calcification in chronic kidney disease, as well as a preparation method and use thereof.

[0006] The present invention provides a traditional Chinese medicine composition for preventing and / or treating vascular calcification in chronic kidney disease. The composition is prepared from the following raw materials in parts by weight: 5-15 parts of Radix Dichroae, 5-20 parts of Eucommia, 5-20 parts of Cistanche, 5-15 parts of Cuscuta, 5-20 parts of Rehmannia, 5-20 parts of Epimedium, 5-15 parts of Drynaria, 5-15 parts of Panax japonicus, 5-20 parts of Millettia reticulata, and 5-20 parts of peach kernel.

[0007] Preferably, it is a preparation prepared from the following raw materials in parts by weight: 10 parts of Radix Dichroae, 10 parts of Eucommia, 10 parts of Cistanche, 10 parts of Cuscuta, 15 parts of Rehmannia, 15 parts of Epimedium, 15 parts of Drynaria, 15 parts of Panax notoginseng, 10 parts of Millettia reticulata, and 10 parts of peach kernel.

[0008] The present invention also provides a method for preparing the above-mentioned traditional Chinese medicine composition for preventing and / or treating vascular calcification in chronic kidney disease, comprising the following steps:

[0009] Step 1, weighing the above-mentioned raw material drug in parts by weight;

[0010] Step 2: prepare the drug powder of the raw material drug, the water extract of the raw material drug or the organic solvent extract of the raw material drug, and add the commonly used pharmaceutical excipients to obtain the product.

[0011] Preferably, the water extract of the raw material drug is prepared by adding water to the raw material drug, soaking it, heating it to boiling, extracting it, collecting the supernatant, and concentrating or drying it.

[0012] Preferably, the mass volume ratio of the raw material drug to water is 1: (8-10) g / mL; the number of extractions is 2 to 3 times, and the extraction time is 0.5 to 1 hour each time.

[0013] The present invention also provides use of the above-mentioned traditional Chinese medicine composition in preparing a drug for preventing and / or treating vascular calcification.

[0014] Preferably, the vascular calcification is vascular calcification caused by chronic kidney disease.

[0015] Preferably, the drug can improve calcium and phosphorus metabolism disorders.

[0016] Preferably, the drug can inhibit the transdifferentiation of vascular smooth muscle cells into osteoblasts.

[0017] Preferably, the drug can inhibit the expression level of alkaline phosphatase.

[0018] Explanation of the prescription: Main ingredients (Rehmannia root + Eucommia bark):

[0019] Rehmannia root nourishes yin and tonifies the kidneys, nourishes blood and activates blood circulation, and clears away heat and detoxifies. It is particularly suitable for patients with CKD, improving kidney function and alleviating symptoms of yin deficiency caused by renal insufficiency. Rehmannia root also plays a vital role in kidney repair, regulating calcium and phosphorus metabolism in the body and alleviating the occurrence of vascular calcification.

[0020] Eucommia: In Traditional Chinese Medicine, Eucommia is known to nourish the liver and kidneys, strengthen bones and tendons, and stabilize pregnancy. For patients with CKD, Eucommia can help repair damaged kidney tissue and enhance kidney function. It plays a particularly important role in regulating bone and vascular calcification. Eucommia helps maintain kidney and skeletal health, preventing osteoporosis and vascular calcification.

[0021] Assistant herbs (Dihuang scutellariae, Cistanche deserticola, Cuscuta australis, Epimedium):

[0022] Dihuang herb: Dihuang herb has the properties of dispelling dampness, clearing heat, and strengthening bones and muscles. It can help improve symptoms of chronic kidney disease, such as edema and dampness retention, promote normal excretion function in the kidneys, and enhance the kidneys' detoxification capacity. It can also clear dampness and heat in the body and alleviate pathological changes associated with vascular calcification.

[0023] Cistanche deserticola: Cistanche deserticola nourishes the kidneys and strengthens yang, moistens the intestines and promotes bowel movements, and nourishes and invigorates blood circulation. It is effective in treating constipation and improving blood circulation in CKD. It promotes blood circulation in the kidneys and improves local microcirculation, thereby reducing the risk of vascular calcification.

[0024] Cuscuta: Cuscuta chinensis nourishes the kidneys and improves essence, regulating kidney function and significantly relieving symptoms associated with kidney deficiency. It can enhance kidney function, improve blood circulation, help prevent and alleviate vascular calcification, and enhance the kidneys' ability to repair itself.

[0025] Epimedium: Epimedium nourishes the kidneys and strengthens yang, warms yang and dispels cold. It improves kidney metabolism and enhances kidney function, making it particularly important for patients with chronic kidney disease. It can help restore the kidney's internal environment, reduce vascular calcification, and enhance the body's immune response.

[0026] Auxiliary drugs (Dryonia lactiflora, Millettia reticulata, Peach kernel):

[0027] Drynaria: Drynaria is primarily used to strengthen tendons and bones, and to promote blood circulation and remove blood stasis. It plays a crucial role in patients with chronic kidney disease, especially those with osteoporosis. It can increase bone density, prevent fractures, and promote normal calcium metabolism, reducing the deposition of calcium salts in blood vessel walls and preventing vascular calcification.

[0028] Millettia reticulata: Millettia reticulata promotes blood circulation, dissipates stasis, nourishes yin and blood, and improves microcirculation, promoting blood flow and enhancing vascular elasticity. By promoting blood circulation and dissipating stasis, Millettia reticulata helps alleviate vascular calcification and improve kidney and vascular health.

[0029] Peach Kernel: Peach kernel promotes blood circulation, removes blood stasis, dredges meridians, and relieves pain. It is particularly effective in improving blood circulation and clearing deposits within blood vessels. Peach kernel can effectively promote blood flow, improve microcirculation, and alleviate blood flow disorders caused by vascular calcification, helping to prevent and treat vascular calcification in patients with CKD.

[0030] Envoy drug (Panax japonicus):

[0031] Bamboo Ginseng: Bamboo Ginseng strengthens the spleen and replenishes Qi, clears away heat and detoxifies, enhances the body's immune function, and regulates the kidneys' Qi and blood. It helps improve the weakness caused by kidney disease, promotes the circulation of Qi and blood throughout the body, and helps prevent the progression of vascular calcification.

[0032] In this formula, all herbs complement each other closely. The main ingredients, Rehmannia root and Eucommia ulmoides, serve as the mainstays, tonifying the kidneys and strengthening bones, while regulating kidney function. Assistant herbs, such as Radix Dichroae, Cistanche deserticola, Cuscuta chinensis, and Epimedium, provide auxiliary support, promoting blood circulation, improving kidney function, and inhibiting vascular calcification. Adjuvant herbs, such as Drynaria fortunei, Millettia reticulata, and Peach kernel, all help activate blood circulation and dissipate stasis, enhancing vascular elasticity and further promoting kidney and vascular health. The guiding ingredient, Panax japonicus, is integrated with the overall formula to maximize its efficacy. The coordinated composition of the entire formula not only enhances kidney tonification but also regulates blood vessels, effectively preventing and treating vascular calcification in chronic kidney disease.

[0033] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0034] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1A traditional Chinese medicine compound improves renal function and reduces mortality in a CKD vascular calcification model mouse model. (A) Model and intervention design: After 2 weeks of adaptive feeding with a standard diet, mice were fed a diet containing 0.2% adenine to induce chronic renal failure (CRF). Vascular calcification was then induced with a diet containing 0.2% adenine and 1.8% phosphorus for 4 weeks. Vascular calcification was then induced with a diet containing 0.2% adenine and 1.8% phosphorus for 16 weeks. Intervention treatment began at the onset of calcification induction. (B) Body weight curves of mice in each group (group settings: control group: standard diet; model group: CKD vascular calcification model; low-dose compound intervention group: CKD calcification model + low-dose compound intervention; high-dose compound intervention group: CKD calcification model + high-dose compound intervention; the animal experimental grouping design in the following figures is consistent with this). (C) Mouse survival curves. (D) Mouse kidney Masson staining results. (E) Mouse renal index. (FH) Mouse renal function, serum creatinine, urea nitrogen, and uric acid levels. p <0.05, *** p <0.001.

[0036] Figure 2 The Chinese herbal compound significantly improved aortic sclerosis and cardiac function in model mice. (A) M-mode echocardiography and abdominal aorta ultrasound of mice; (B) blood flow resistance index; (C) cardiac output; (D) stroke volume; (E) ejection fraction; * p <0.05,** p <0.01,*** p <0.001.

[0037] Figure 3 The Chinese herbal compound significantly inhibited aortic calcification in CKD mice. (A) MicroCT analysis of aortic calcification and ectopic calcium salt deposition in model mice; (B) MicroCT analysis of calcium salt deposition volume in the mouse aorta; (C) Serum alkaline phosphatase (ALP) levels in mice; (D) Serum phosphorus concentration in mice; (E) Serum calcium concentration in mice; ** p <0.01,*** p <0.001.

[0038] Figure 4A traditional Chinese medicine compound inhibits aortic calcium deposition and osteogenic transdifferentiation of vascular smooth muscle cells. (A) Alizarin Red S staining of the entire aorta; (B) Calcium content in mouse aorta; (C) Von Kossa and Alizarin Red staining of aortic sections; (D) Ratio of Von Kossa-positive area in aortic sections; (E) Ratio of Alizarin Red-positive area in aortic sections; (F) Expression of vascular smooth muscle cell marker α-SMA, calcification transcription factor Runx2, and osteocalcin OCN in VSMCs in aorta; (G1) Statistical results of α-SMA, Runx2, and OCN expression in aortic VSMCs;* p <0.05,*** p <0.001.

[0039] Figure 5 A traditional Chinese medicine compound-containing serum inhibits vascular smooth muscle cell calcification. (A) Alizarin Red S staining of vascular smooth muscle cells (group settings: control group: normal cell culture medium; model group: culture medium + 3 mmol / L sodium dihydrogen phosphate treatment; compound-containing serum intervention group: cells treated with 3 mmol / L sodium dihydrogen phosphate and treated with low, medium, and high concentrations of the compound-containing serum); (B) Cellular calcium content measurement results; (CE) α-SMA, Runx2, and OCN mRNA expression levels in different experimental groups; (FI) α-SMA, Runx2, and OCN protein expression levels and quantitative results in different experimental groups; * p <0.05, ** p <0.01, *** p <0.001. DETAILED DESCRIPTION

[0040] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available.

[0041] Example 1 Preparation of the Chinese medicinal compound of the present invention

[0042] Prescription: Radix Dichroae 10g, Eucommia ulmoides 10g, Cistanche deserticola 10g, Cuscuta australis 10g, Rehmannia glutinosa 15g, Epimedium 15g, Drynaria fortunei 15g, Panax japonicus 15g, Millettia reticulata 10g, Peach kernel 10g.

[0043] Preparation method of Chinese herbal compound:

[0044] The decoction method was used: 120g of the above-mentioned Chinese medicinal materials were soaked in 10-fold water for 30 minutes, then decocted at 100°C for 1 hour. The supernatant was removed and then added with 8-fold water and decocted at 100°C for 0.5 hour. The two supernatants were combined, filtered, and the mixture was then heated and concentrated in a rotary evaporator (60°C, 50 rpm) to a concentration of 0.9g crude drug / ml. This yielded the Chinese medicinal compound of the present invention.

[0045] The following experimental examples demonstrate the beneficial effects of the Chinese medicine composition of the present invention.

[0046] Experimental Example 1: Traditional Chinese medicine compound can significantly improve vascular calcification in CKD

[0047] 1. Research subjects

[0048] The study included CKD patients who visited the Nephrology Department of the First Affiliated Hospital of Chengdu Medical College between January 2022 and December 2024. Based on the 2021 Kidney Disease Improving Global Outcomes (KDIGO) guidelines, patients with CKD stages 3-5 who were not on dialysis were randomly recruited. CKD stage 3 definition: 30 ≤ eGFR < 60 ml / (min. 1.73 m 2 ), CKD stage 4 definition: 15≤eGFR<30ml / (min.1.73m 2 ), CKD stage 5 definition: eGFR < 15ml / (min.1.73m 2 ).

[0049] 2. Experimental plan

[0050] 2.1 Inclusion criteria

[0051] (1) Meet the diagnostic criteria for CKD stage 3-5: estimated glomerular filtration rate (eGFR) < 60 ml / (min.1.73 m 2 );

[0052] (2) Aged 18-85 years;

[0053] (3) Non-dialysis CKD patients;

[0054] (4) The patient has coronary artery calcification;

[0055] (5) The patients were informed and agreed to participate in this trial.

[0056] 2.2 Exclusion criteria

[0057] (1) Acute kidney injury caused by various reasons;

[0058] (2) Patients with severe primary diseases of the liver and hematopoietic system, mental disorders, or malignant tumors;

[0059] (3) Age <18 or >85 years old;

[0060] (4) Pregnant or breastfeeding women;

[0061] (5) Those who cannot follow the doctor’s orders and have poor compliance.

[0062] 2.3 Grouping and treatment plan

[0063] According to the above inclusion and exclusion criteria, 24 patients with CKD stage 3, 24 patients with CKD stage 4, and 20 patients with CKD stage 5 were recruited. Patients of each CKD stage were randomly divided into a conventional treatment control group and a traditional Chinese medicine compound intervention group in a 1:1 ratio.

[0064] (1) Conventional treatment control group: All patients were treated with chronic kidney disease and its complications. Blood pressure control drugs: According to the patient's condition, calcium channel blockers were used: amlodipine besylate tablets, oral, 2.5 mg / time, once a day; angiotensin II receptor antagonists: irbesartan tablets, oral, 150 mg / time, once a day; beta-blockers: labetalol hydrochloride, oral, 100 mg / time, twice a day. Blood sugar control drugs: According to the condition, metformin: metformin hydrochloride sustained-release tablets, 0.5 g / time, once a day; insulin secretagogue sulfonylureas, gliclazide sustained-release tablets, 60 mg / time, once a day, nateglinide tablets, 60 mg / time, 3 times a day; DDP-4 inhibitors: linagliptin tablets, 5 mg / time, once a day; SGLT-2 inhibitors: dapagliflozin tablets, 10 mg / time, once a day. To control blood lipids, use statins: Atorvastatin calcium tablets, 20 mg once a day. To improve renal function, use Urea Clearing Granules, 10 g three times a day, orally.

[0065] (2) Chinese medicine compound intervention group: Based on conventional treatment, Chinese medicine compound intervention was given for 6 months. The intervention treatment was carried out by taking one set of Chinese medicine per day, decocted and taken three times a day (150 ml each time) in the morning, noon and evening.

[0066] 2.4 Observation indicators: ① Coronary artery calcification score: The Agatston score was used to evaluate the coronary artery calcification score and severity. The specific method was as follows: chest CT scan was performed to detect the calcification of the left main coronary artery, the left anterior descending artery, the circumflex artery, and the right coronary artery. The calcification-positive area was defined as a CT value >130Hu and an area >1mm 2Calcification density scores are also assigned to calcification-positive areas. Higher Hu values ​​are assigned higher scores, with 130-199 Hu being 1, 200-299 Hu being 2, 300-399 Hu being 3, and 400 Hu or greater being 4. Coronary CT scans are performed on each slice, and calcified areas are identified and scored. The sum of all scores for each slice is the patient's coronary calcification score. A coronary calcification score of 10 < 400 indicates mild to moderate calcification, and a coronary calcification score of >400 indicates severe coronary calcification. (2) Blood biochemical indicators: blood urea nitrogen (BUN), serum creatinine (SCr), serum calcium (Ca), serum phosphorus (P), alkaline phosphatase (ALP), and parathyroid hormone (PTH).

[0067] 3. Statistical methods: Normally distributed quantitative data were expressed using t-test, non-normally distributed quantitative data were analyzed using rank sum test, and categorical data were analyzed using chi-square test for univariate analysis. (* P <0.05; ** P <0.01; *** P <0.001)

[0068] 3. Treatment effect

[0069] As shown in Table 1: After treatment with the Chinese herbal compound, patients with CKD stage 3 effectively improved renal function, improved calcium and phosphorus metabolism disorders, reduced blood phosphorus and parathyroid hormone, reduced coronary artery calcification scores, and significantly inhibited the occurrence and development of CKD vascular calcification compared with before treatment and conventional treatment plans.

[0070] Table 1 Renal function, calcium and phosphorus metabolism, and coronary artery calcification scores in patients with chronic kidney disease stage 3

[0071]

[0072] Note: * Compared with before treatment P <0.05, **compared with before treatment P <0.01, ***compared with before treatment P <0.001, #compared with conventional treatment group P <0.01.

[0073] As shown in Table 2: After treatment with the Chinese herbal compound, patients with CKD stage 4 effectively improved renal function, improved calcium and phosphorus metabolism disorders, reduced blood phosphorus and parathyroid hormone, reduced coronary artery calcification scores, and significantly inhibited the occurrence and development of CKD vascular calcification compared with before treatment and conventional treatment plans.

[0074] Table 2 Renal function, calcium and phosphorus metabolism and coronary artery calcification scores in patients with chronic kidney disease CKD stage 4

[0075]

[0076] Note: * Compared with before treatment P <0.05, **compared with before treatment P <0.01, ***compared with before treatment P <0.001, #compared with conventional treatment group P <0.01.

[0077] As shown in Table 3: After treatment with the Chinese herbal compound, patients with CKD stage 5 effectively improved renal function, improved calcium and phosphorus metabolism disorders, reduced blood phosphorus and parathyroid hormone, reduced coronary artery calcification scores, and significantly inhibited the occurrence and development of CKD vascular calcification compared with before treatment and conventional treatment plans.

[0078] Table 3 Renal function, calcium and phosphorus metabolism and coronary artery calcification scores in patients with chronic kidney disease CKD stage 5

[0079]

[0080] Note: *Compared with before treatment P <0.05, **compared with before treatment P <0.01, ***compared with before treatment P <0.001, #compared with conventional treatment group P <0.01.

[0081] Experimental Example 2: A Chinese herbal formula significantly improved renal function and reduced mortality in mice with CKD and vascular calcification

[0082] 1. In vivo experimental methods in mice

[0083] 1.1 Establishment of CKD vascular calcification model: 8-week-old male C57BL / 6J mice were fed with a normal diet for 2 weeks, then fed with a diet containing 0.2% adenine for 4 weeks, and then fed with a diet containing 1.8% high phosphorus + 0.2% adenine to induce calcification until 16 weeks. At the same time, a control group and a Chinese medicine compound intervention group ( Figure 1 A). The body weight and general physiological / pathological conditions of the mice were measured weekly. At week 16, cardiac function and aortic calcification were assessed using ultrasound.

[0084] 1.2 Preparation of Chinese herbal compound: Take Radix Dipterocarpa 10g, Eucommia ulmoides 10g, Cistanche deserticola 10g, Cuscuta australis 10g, Rehmannia glutinosa 15g, Epimedium 15g, Drynaria fortunei 15g, Panax japonicus 15g, Millettia reticulata 10g, and Peach kernel 10g.

[0085] For Chinese herbal medicine slices, soak the slices in 10-fold water for 30 minutes, boil at 100°C for 1 hour, remove the supernatant, add 8-fold water, and boil at 100°C for 0.5 hour. Combine the two supernatants, filter the mixture, and heat in a rotary evaporator (60°C, 50 rpm) to evaporate and concentrate to produce an extract. Store in a refrigerator until ready for use.

[0086] 1.3 Mouse experimental grouping:

[0087] Control group: fed with ordinary diet until 16 weeks (n=10);

[0088] Model group: The CKD vascular calcification model of mice was established according to the above vascular calcification model experiment (n=10);

[0089] Model + low-dose compound group: During the modeling process, mice were given 1.8% high phosphorus + 0.2% adenine induced calcification feed for 4 weeks, and then given a low-dose compound intervention by gavage (n=10)

[0090] Model + high-dose compound group: During the modeling process, mice were given 1.8% high phosphorus + 0.2% adenine induced calcification feed for 4 weeks, and then given high-dose compound intervention by gavage (n=10).

[0091] 1.4 Compound gavage intervention method

[0092] To model CKD vascular calcification in mice, after four weeks of calcification-inducing diet supplemented with 1.8% high phosphorus and 0.2% adenine, a traditional Chinese medicine compound was administered by gavage once daily for 16 weeks. The gavage volume was calculated based on mouse body weight, with a dose of 0.1 ml / 10 g administered. The dosage of the traditional Chinese medicine compound was prepared according to the "Experiments in Traditional Chinese Medicine Pharmacology" (edited by Ma Shiping, Southeast University Press, 2015). The body surface area conversion method was used to calculate the human-grade crude drug dosage in mice. In the intervention group, low-dose (10 mg / g / day) and high-dose (40 mg / g / day) of the compound were administered by gavage, respectively.

[0093] 1.5 Analysis of mouse histopathological characteristics

[0094] At the end of the experiment, mice were anesthetized by intraperitoneal injection of sodium pentobarbital and killed by cervical dislocation. The aorta and kidney tissues of the mice were isolated, embedded in paraffin, and sections were prepared. The kidney and vascular tissues were observed by HE staining.

[0095] Morphology and Masson staining were used to observe renal fibrosis, and Alizarin Red S staining and Von Kassa staining were used to detect calcification deposition; immunohistochemistry was used to detect the expression of calcification marker molecules a-SMA, Runx2, and OCN in blood vessels.

[0096] 2. Experimental results

[0097] The experimental results showed that the Chinese herbal compound could significantly inhibit the weight loss of model mice and reduce the mortality rate of mice ( Figure 1 B, C); The results of Masson staining of the kidneys showed that the renal tissue structure of the control group was normal, the model group showed obvious fibrosis, and the degree of fibrosis in the TCM compound treatment groups (low dose and high dose) was reduced, indicating that the TCM compound can inhibit renal fibrosis in a dose-dependent manner ( Figure 1 D); The compound can improve the renal index of model mice and reduce their blood creatinine, urea nitrogen and uric acid levels ( Figure 1 EH); The results showed that the Chinese herbal compound can significantly improve the renal function of mice with chronic kidney disease and vascular calcification and reduce pathological damage to the kidneys.

[0098] Experimental Example 3: A Traditional Chinese Medicine Compound Significantly Improves Aortic Sclerosis and Heart Function in CKD Mice with Vascular Calcification

[0099] 1. Experimental methods

[0100] A CKD vascular calcification mouse model was established according to Experimental Example 2, and the M-mode echocardiography and abdominal aorta ultrasound images, as well as blood flow resistance index, cardiac output (CO), stroke volume (SV), and ejection fraction (EF) of each group of mice were detected.

[0101] 2. Experimental results

[0102] CKD vascular calcification reduces vascular compliance, increases vascular resistance and cardiac afterload, and ultimately leads to left ventricular hypertrophy and heart failure. Experimental results show that the Chinese herbal compound can significantly improve the compliance of the abdominal aorta of mice and reduce the blood flow resistance index ( Figure 2 A, B), and can also improve the cardiac function of model mice: cardiac output (CO), stroke volume (SV) and ejection fraction (EF) ( Figure 2 CE). The results showed that the Chinese herbal compound could significantly improve aortic sclerosis and cardiac function in CKD mice with vascular calcification, and the effect was dose-dependent.

[0103] Experimental Example 4: Traditional Chinese Medicine Compound Inhibits Aortic Vascular Calcification in CKD Vascular Calcification Mice

[0104] 1. Experimental methods

[0105] A CKD mouse model of vascular calcification was established according to Experimental Example 2. MicroCT was used to visualize vascular calcification and ectopic calcium salt deposition in the aortic wall of each group of mice, and the volume of calcium salt deposition in the aorta of the mice was analyzed. The ALP enzyme level, phosphorus concentration, and calcium concentration in the serum of the mice were measured.

[0106] 2. Experimental results

[0107] MicroCT examination revealed severe calcification of the thoracic and abdominal aorta wall in the model mice (marked by the red dotted box), while no obvious calcification was observed in the Chinese medicine compound treatment groups (low and high doses). In addition, multiple ectopic calcium salt deposits were observed in the model group mice (the red arrows indicated severe calcium salt deposition in the subcutaneous tissue of the chest). Figure 3 A, B); The serum ALP and blood phosphorus levels of mice in the model group increased significantly, while the blood calcium level decreased significantly. After intervention with the Chinese herbal compound, the calcium and phosphorus metabolism disorder was significantly improved and the blood ALP level was suppressed ( Figure 3 CE). The results showed that the Chinese herbal compound can significantly inhibit the occurrence and development of vascular calcification in CKD.

[0108] Experimental Example 5: Traditional Chinese Medicine Compound Inhibits Aortic Calcium Deposition and Osteogenic Transdifferentiation of Vascular Smooth Muscle Cells

[0109] 1. Experimental methods

[0110] A CKD vascular calcification mouse model was established according to Experimental Example 2 and grouped. The mouse aorta was isolated, and calcium salt deposition in the entire aorta was analyzed by Alizarin Red S staining and Von Kossa silver staining, and the calcium content in vascular tissue was detected. Immunohistochemistry was used to analyze the expression levels of calcification-related molecules in vascular smooth muscle cells in the aortic tunica media.

[0111] 2. Experimental results

[0112] The results showed that the Chinese herbal compound significantly inhibited calcium salt deposition in the aorta of model mice ( Figure 4 A) and reduced the calcium content in the aorta tissue of the model mice ( Figure 4 B). Aortic sections were stained with Alizarin Red S and Von Kossa silver respectively. The results showed that the Chinese herbal compound significantly inhibited aortic calcification ( Figure 4 CE). Immunohistochemistry was further used to analyze the expression levels of calcification-related molecules in the aortic media of vascular smooth muscle cells. The results showed that the expression of α-SMA, a marker molecule of vascular smooth muscle cells in the aorta of mice in the calcification model group, was significantly reduced, while the expression of Runx2 and OCN, indicators of bone transdifferentiation, was significantly increased; intervention with a Chinese herbal compound significantly inhibited the calcification indicator molecules Runx2 and OCN, while increasing the expression of α-SMA ( Figure 4 FI). The above results indicate that the Chinese herbal compound can inhibit the transdifferentiation of VSMC into osteocytes and prevent the occurrence and development of vascular calcification.

[0113] Experimental Example 6: Chinese herbal compound-containing serum inhibits calcification of vascular smooth muscle cells

[0114] 1. Experimental methods

[0115] 1.1 Cell calcification model: Mouse aortic vascular smooth muscle cell line (MOVAS) was cultured in DMEM complete medium and treated with inorganic phosphate NaH2PO4 (Pi, 3 mmol / L) for 7 days to induce cell calcification. Alizarin red S staining was used to identify cell calcification. Real-time PCR and Western blotting were performed.

[0116] -blotting was used to detect the expression of vascular calcification-related indicator molecules.

[0117] 1.2 Preparation of serum containing Chinese herbal compound: 8-week-old healthy male Wistar rats (200-220g) were selected to prepare low-, medium-, and high-dose serum containing Chinese herbal compound. The low-dose (10mg / g / d), medium-dose (20mg / g / d), and high-dose (40mg / g / d) of the compound were calculated and administered by gavage. The drug was administered by gavage at 10 times the above-calculated dose. After 7 consecutive days of administration with the same volume of blood, blood was collected from the abdominal aorta, and the serum was separated, complement inactivated, filtered through a 0.22μm filter, packaged, and stored at -80°C for later use.

[0118] 1.3 Cell experiment grouping:

[0119] Control group: cultured in DMEM complete medium and treated with rat serum without Chinese herbal compound ingredients.

[0120] Model group (high phosphorus): cells were cultured in DMEM complete medium and calcified according to the cell calcification model method.

[0121] Model + low-dose compound intervention group: Starting from the third day of the cell model, low-dose compound (10 mg / g / d) drug-containing serum was added to the DMEM complete medium culture for intervention until the seventh day.

[0122] Model + medium-dose compound intervention group: Starting from the third day of the cell model, low-dose compound (20 mg / g / d) drug-containing serum was added to the DMEM complete medium culture medium for intervention until the seventh day.

[0123] Model + high-dose compound intervention group: Starting from the third day of the cell model, low-dose compound (40 mg / g / d) drug-containing serum was added to the DMEM complete medium culture medium for intervention until the seventh day.

[0124] 2. Experimental results

[0125] The results showed that the Chinese herbal compound could significantly inhibit calcium salt deposition in vascular smooth muscle cells, and showed a dose-effect relationship ( Figure 5 A); while significantly inhibiting the calcium content in cells ( Figure 5B); PCR results showed that the Chinese herbal compound significantly inhibited the expression of Runx2 and OCN molecules in calcification model cells and reversed the expression of α-SMA, a marker of vascular smooth muscle cells ( Figure 5 CE); Western-blotting results also showed that the Chinese herbal compound significantly inhibited the expression of Runx2 and OCN molecules in calcification model cells and reversed the expression of α-SMA, a marker molecule of vascular smooth muscle cells ( Figure 5 FI). The above results show that the serum containing the Chinese herbal compound can significantly inhibit the occurrence of vascular smooth muscle cell calcification.

[0126] In summary, the traditional Chinese medicine formula of the present invention effectively treats vascular calcification in chronic kidney disease (CKD) by improving calcium and phosphorus metabolism disorders, protecting the cardiovascular system, enhancing renal and cardiac function, inhibiting vascular calcification, and preventing the transdifferentiation of vascular smooth muscle cells. This formula leverages the kidney-tonifying and bone-strengthening effects of the main herbs Rehmannia glutinosa and Eucommia ulmoides, combined with the synergistic effects of the auxiliary and adjuvant herbs, to effectively address chronic kidney disease (CKD) and the accompanying vascular calcification. Each herb in the formula has its own unique pharmacological effects, working in synergy to achieve the therapeutic goals of tonifying the kidney, activating blood circulation, unblocking meridians, and preventing and treating vascular calcification.

Claims

1. A Chinese medicine composition for treating vascular calcification in chronic kidney disease, characterized by: The preparation is prepared from the following raw materials in parts by weight: 5-15 parts of Radix Dichroae, 5-20 parts of Eucommia ulmoides, 5-20 parts of Cistanche deserticola, 5-15 parts of Cuscuta australis, 5-20 parts of Rehmannia glutinosa, 5-20 parts of Epimedium, 5-15 parts of Drynaria rhizome, 5-15 parts of Panax japonicus, 5-20 parts of Millettia reticulata, and 5-20 parts of peach kernel.

2. The Chinese medicine composition according to claim 1, characterized in that: The preparation is prepared from the following raw materials in parts by weight: 10 parts of Radix Dichroae, 10 parts of Eucommia ulmoides, 10 parts of Cistanche deserticola, 10 parts of Cuscuta australis, 15 parts of Rehmannia glutinosa, 15 parts of Epimedium, 15 parts of Drynaria rhizome, 15 parts of Panax japonicus, 10 parts of Millettia reticulata, and 10 parts of peach kernel.

3. The method for preparing the traditional Chinese medicine composition for treating vascular calcification in chronic kidney disease according to claim 1 or 2, characterized in that: The following steps are involved: Step 1, weighing the raw material drug according to claim 1 or 2; Step 2, preparing the water extract of the raw material drug, adding the commonly used pharmaceutical excipients, and obtaining the product.

4. The preparation method according to claim 3, characterized in that: The preparation method of the water extract of the raw material medicine is as follows: adding water to the raw material medicine to soak, heating to boiling, extracting, collecting the supernatant, and concentrating or drying to obtain the water extract.

5. The preparation method according to claim 4, characterized in that: The mass volume ratio of the raw material drug to water is 1:8-10 g / mL; the number of extractions is 2-3 times, and the extraction time is 0.5-1 hour each time.

6. Use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of a medicament for treating vascular calcification; the vascular calcification is vascular calcification caused by chronic kidney disease.

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