Application of ginsenoside Rb1 in preparation of medicine for delaying development of acute kidney injury to chronic kidney disease

Abstract

The invention discloses application of ginsenoside Rb1 in preparation of a medicine for delaying development of acute kidney injury to chronic kidney disease. Through the technical scheme of the invention, the ginsenoside Rb1 can improve the renal function and pathological injury in the AKI-CKD period, promote kidney angiogenesis, improve vasodilation, improve the angiogenesis effect and vasodilation function of HUVEC induced by H / R, relieve the pathological injury of kidney and delay the development of acute kidney injury to chronic kidney disease, so that the ginsenoside Rb1 can be used for preparing the medicine for delaying the development of acute kidney injury to chronic kidney disease. Therefore, the progress of AKI to CKD is delayed.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of ginsenoside Rb1 in the preparation of a drug for delaying the progression of acute kidney injury to chronic kidney disease. Background Art

[0002] In the late stages of acute kidney injury (AKI), peritubular capillary rarefaction is a key contributor to renal interstitial fibrosis. Capillary rarefaction can cause renal tissue ischemia and hypoxia, thereby impairing the repair capacity of renal tubular epithelial cells, promoting abnormal proliferation and phenotypic transformation of myofibroblasts, and activating various inflammatory cells. These pathological changes collectively drive the development of renal interstitial fibrosis and the progression of the disease to chronic kidney disease (CKD). Based on this pathophysiological mechanism, improving renal microcirculation by promoting renal angiogenesis may be a novel therapeutic strategy to intervene in the fibrotic process in the late stages of AKI.

[0003] Although AKI is considered a reversible condition, recent studies have shown that a significant proportion of patients do not fully recover renal function, with some even requiring long-term renal replacement therapy, ultimately developing CKD or end-stage renal disease (ESRD). Therefore, the development of novel, safe, and effective treatment strategies to slow its progression is crucial. The search for safe and effective natural compounds that can promote angiogenesis has garnered significant attention.

[0004] Ginseng has been used as a medicinal plant in China for over 5,000 years. Ginsenoside Rb1 is a highly abundant and active component of ginsenoside Rb1. Rb1 can exert multiple pharmacological effects and improve kidney disease. A growing body of research supports its potential role in drug-induced kidney injury and chronic kidney disease (CKD).

[0005] Previous studies have found that Rb1 can alleviate podocyte damage in diabetic kidney disease (DKD) by inhibiting aldose reductase (AR) activity. In addition, it can also alleviate high glucose-induced cell apoptosis and mitochondrial damage, effectively alleviating the progression of DKD.

[0006] Although Rb1 has been shown to have beneficial pharmacological effects in other organ systems, its role in the AKI-CKD transformation process is still unclear, and the efficacy and toxicity of Rb1 in vivo have not yet reached the clinical use standards, limiting its application in clinical practice. Summary of the Invention

[0007] In response to the above problems, the present invention provides a use of ginsenoside Rb1 in the preparation of a drug for delaying the progression of acute kidney injury to chronic kidney disease, which can improve renal function and pathological damage in the AKI-CKD period, promote renal angiogenesis, improve vasodilation, improve the angiogenesis and vasodilation function of HUVECs induced by H / R, reduce pathological damage to the kidneys, and thus delay the progression of AKI to CKD.

[0008] To achieve the above objectives, the present invention provides a use of ginsenoside Rb1 in the preparation of a drug for delaying the progression of acute kidney injury to chronic kidney disease.

[0009] In the above technical solution, preferably, the drug is used to delay the progression of acute kidney injury caused by ischemia-reperfusion to chronic kidney disease.

[0010] In the above technical solution, preferably, the dosage of ginsenoside Rb1 is 40 mg / kg.

[0011] Compared with the existing technology, the beneficial effects of the present invention are: ginsenoside Rb1 can improve renal function and pathological damage in the AKI-CKD period, promote renal angiogenesis, improve vasodilation, improve the angiogenesis and vasodilation function of HUVECs induced by H / R, reduce pathological damage to the kidneys, and thus delay the progression of AKI to CKD. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A schematic diagram of the experimental grouping and timeline arrangement of ginsenoside Rb1 in improving renal function and pathological damage in the AKI-CKD stage disclosed in one embodiment of the present invention; Figure 2 This is a schematic diagram of the experimental results of ginsenoside Rb1 improving the renal function of AKI-CKD mice disclosed in one embodiment of the present invention; Figure 3 This is a schematic diagram of the experimental results of ginsenoside Rb1 alleviating pathological damage in mice during the AKI-CKD period disclosed in one embodiment of the present invention; Figure 4 This is a schematic diagram of the experimental results of ginsenoside Rb1 alleviating renal fibrosis in mice during AKI-CKD period disclosed in one embodiment of the present invention; Figure 5 This is a schematic diagram of the experimental results of ginsenoside Rb1 promoting angiogenesis in AKI-CKD kidneys disclosed in one embodiment of the present invention; Figure 6 This is a schematic diagram of the experimental results of ginsenoside Rb1 improving the vascular function of AKI-CKD kidneys disclosed in one embodiment of the present invention; Figure 7This is a schematic diagram of the experimental results of using CCK-8 to measure the effects of Rb1 treatment at different concentrations and times on HUVECs viability disclosed in one embodiment of the present invention; Figure 8 This is a schematic diagram of the experimental results of the effect of ginsenoside Rb1 on the tube-forming ability of HUVECs disclosed in one embodiment of the present invention; Figure 9 This is a schematic diagram of the experimental results of ginsenoside Rb1 improving the diastolic function of HUVECs disclosed in one embodiment of the present invention. DETAILED DESCRIPTION

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0014] The present invention is described in further detail below with reference to the accompanying drawings: The present invention provides a use of ginsenoside Rb1 in the preparation of a drug for delaying the progression of acute kidney injury to chronic kidney disease.

[0015] In the above embodiment, preferably, the drug is used to delay the progression of acute kidney injury caused by ischemia-reperfusion to chronic kidney disease.

[0016] In the above embodiment, preferably, the dosage of ginsenoside Rb1 is 40 mg / kg.

[0017] According to the use of ginsenoside Rb1 disclosed in the above embodiment in the preparation of a drug for delaying the progression of acute kidney injury to chronic kidney disease, its application and effects are illustrated through the following examples.

[0018] 1.1 Animal model construction and animal grouping Twenty male C57 / 6J mice, 6-8 weeks old, were selected and maintained in the specific pathogen-free facility of the Animal Center of the Chinese People's Liberation Army General Hospital.

[0019] Mice underwent unilateral renal ischemia-reperfusion surgery. After anesthesia with pentobarbital (50 mg / kg, ip), the left renal pedicle was clamped for 28 minutes. The clamp was then released to induce ischemia. The animal body temperature was maintained at 37°C during the surgery. Sham-operated mice underwent the same surgery, but the renal pedicle was not clamped.

[0020] All mice were randomly divided into four groups, with five mice in each group: sham operation group (Sham), sham operation + ginsenoside Rb1 group (Sham+Rb1), unilateral ischemia-reperfusion group (14 days uIRI), and unilateral ischemia-reperfusion group (14 days uIRI + Rb1). The treatment conditions for each group are shown in the table below.

[0021] Animal grouping and treatment:

[0022] like Figure 1 As shown, the dose of ginsenoside Rb1 (catalog number: 112127, supplier: J&K Bailingwei Technology Co., Ltd., Beijing) was 40 mg / kg, and the solvent was prepared with normal saline. It was administered by gavage for 7 consecutive days starting from the 7th day after surgery.

[0023] 1.2 Cell culture methods and cell grouping Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC). HUVECs were cultured in RPMI medium 1640 basic 1X (gibco, 6123092, China) supplemented with 10% bovine serum and 1% penicillin and streptomycin.

[0024] All cells were cultured in a humidified incubator at 37°C with 5% CO₂ and 95% air. A hypoxia / reoxygenation (H / R) model was established. Briefly, HUVECs were exposed to hypoxic conditions (37°C, 1% O₂, 94% N₂, and 5% CO₂) for 12 hours in a glucose- and serum-free culture medium to induce hypoxic injury. Subsequently, the culture medium was replaced and the cells were reoxygenated under normal conditions (37°C, 95% air and 5% CO₂) for 12 hours according to the experimental design. A control group was cultured under conventional conditions (5% CO₂ and 95% air).

[0025] The cell groups and treatment methods are as follows:

[0026] 1.3 Biochemical function index detection The right healthy kidney of the mouse was removed the day before sampling. GFR was measured before sampling. The mouse's back hair was removed before the experiment, and a Dielectric Lighthouse transcutaneous micro GFR monitor was attached to the depilated skin. After the monitor was fixed for 5 minutes, fluorescein isothiocyanate (FITC)-inosine (70 mg / kg body weight) was injected via the tail vein. The mouse was caged, and the fluorescence signal in the skin was monitored for 2 hours. The microarray data was then read. Data were analyzed using MB Studio software.

[0027] Blood and kidneys were collected from mice. After standing at room temperature for 2 hours, the blood samples were centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected as serum. Blood urea nitrogen (BUN) and serum creatinine (Scr) levels were then determined according to the manufacturer's instructions.

[0028] 1.4 Renal pathology experiments Fresh kidney tissue was washed with pre-chilled saline, dried with filter paper, and fixed overnight with 4% paraformaldehyde. The tissue was then dehydrated, cleared, embedded in paraffin, and sectioned at a thickness of 2–5 μm. Staining was performed according to the manufacturer's instructions for periodic acid-Schiff (PAS), Masson's, and Sirius red stains.

[0029] 1.5 Western blot experiment For Western blot experiments, kidney tissue was lysed in RIPA buffer (Solarbio, Shanghai, China) at 4°C for 30 minutes. Subsequently, the sample was centrifuged at 15,000 rpm at 4°C for 30 minutes, and the supernatant was collected. Protein concentration was determined using a BCA protein assay kit (Genstar, Beijing, China) according to the manufacturer's instructions. Finally, the sample was boiled at 95°C for 15 minutes in 2x SDS sample buffer. The resulting protein sample was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Darmstadt, Germany). After blocking with 5% BSA for 2 hours, the PVDF membrane was incubated with the primary antibody overnight at 4°C and then with an enzyme-conjugated secondary antibody for 2 hours at room temperature.

[0030] 1.6 Immunohistochemistry For immunohistochemistry, paraffin sections were routinely deparaffinized and hydrated, followed by peroxidase blocking with 3% H₂O₂ for 30 minutes. Sections were then acid- or alkaline-fixed and microwave-fixed for 10 minutes, blocked with 5% BSA for 1 hour, and incubated with the primary antibody overnight at 4°C. Secondary antibodies were added, and the sections were incubated at room temperature for 2 hours. DAB staining was monitored microscopically, and sections were counterstained with hematoxylin and mounted with gum. For immunofluorescence, frozen sections were washed in PBS for 15 minutes, blocked with 5% BSA for 1 hour, and incubated with the primary antibody overnight at 4°C. Cy5 secondary antibodies were incubated at room temperature for 2 hours, followed by DAB staining. Sections were then stained with hematoxylin, deblued, antiblued, and cleared, and then mounted with nail polish. ImageJ software was used to analyze the percentage of positive area (positive area / total area).

[0031] 1.7 Immunofluorescence experiments In the immunofluorescence experiment, the frozen sections were washed with PSB and blocked with 1×Casein for 60 minutes. The blocking solution was discarded and Casein was prepared as the primary antibody diluent at a ratio of 1:50. After the primary antibody was prepared, it was incubated at 4°C overnight. The primary antibody was discarded and the sections were washed with PBS for 10 minutes × 3 times. The sections were incubated with secondary antibodies at room temperature for 2 hours in the dark. The sections were washed with PBS for 10 minutes × 3 times, washed with PBS, and washed with DAPI. The sections were sealed with resin in the dark and observed under a microscope.

[0032] 1.8 NO detection Mouse serum was collected and nitric oxide (NO) concentration was quantified using a nitric oxide (NO) detection kit (Nanjing Jiancheng, A012-1-2) according to the manufacturer's instructions.

[0033] HUVEC cell supernatants were collected. Nitric oxide (NO) concentrations were quantified using a nitric oxide (NO) detection kit (Nanjing Jiancheng, A012-1-2) according to the manufacturer's instructions. Western blot assays were performed as previously described.

[0034] 1.9 Cytotoxicity and proliferation assays HUVEC cell suspensions were prepared and counted, with triplicates performed for each sample. Experimental groups were incubated with different concentrations of Rb1 (0, 0.1, 1, 10, and 100 μM). Control wells were incubated with culture medium containing 0.1% DMSO. Cell-free culture medium served as blank wells. The plates were incubated in a hypoxic, three-gas incubator and a normal, two-gas incubator for 12 and 24 hours, respectively. 10 μL of CCK-8 reagent was added to each well, and absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0035] 2.1 Cell tube formation experiment The angiogenesis-promoting effect of ginsenoside Rb1 was investigated using a tube formation experiment. The experiment was divided into three groups: control group, H / R group, and H / R+Rb1 group. HUVECs (2-4×10 4 Tubes were cultured in 96-well plates (Corning) coated with 50 µL of Matrigel basement membrane matrix (Corning, 356230, USA) and incubated at 37°C for 4-6 hours. Tubes were observed and measured under a microscope. Tube formation was quantitatively analyzed using Image J software. Image-J (National Institute of Health, USA) was used to analyze the ability of tubes to form connections, meshwork, branching, and length.

[0036] According to the experimental process disclosed in the above embodiment, the analysis and verification of the embodiment are described as follows.

[0037] Example 1: Rb1 improves renal function and alleviates pathological damage in AKI-CKD mice like Figure 2 As shown in A, B, and C, the experimental results showed that compared with the sham group and the sham+Rb1 group, the SCr and BUN levels of the uIRI group increased, and the GFR decreased. In the uIRI+Rb1 group, the SCr and BUN levels of the mice decreased, and the GFR increased significantly. The PAS results showed that compared with the uIRI group, the renal damage of the mice in the uIRI+Rb1 group was alleviated, and the ATN score was significantly decreased ( Figure 3 A, B). Masson staining and picrosirius red staining showed that a large amount of collagen fibers were deposited in the interstitial region of the kidneys in the uIRI group. After Rb1 treatment, the area of ​​collagen fibers in the interstitial region of the mice was reduced compared with that in the uIRI group ( Figure 3 C, D, E, F). Figure 4 The immunohistochemical results of AF showed that in the uIRI group, the expression levels of Vimentin and Collagen I were significantly increased, while after Rb1 treatment, the expression levels decreased. The results of semi-quantitative statistical analysis showed that the differences were statistically significant.

[0038] Example 2: Rb1 promotes renal angiogenesis in AKI-CKD, improves vascular function, and reduces renal fibrosis Figure 5 The experimental results showed that in order to evaluate the density of peritubular capillaries, the expression levels of Endo and CD31 were detected. The results showed that there was no significant difference in vascular density between the sham+Rb1 group and the sham group. The density of peritubular capillaries in the uIRI group decreased, while the density of peritubular capillaries in the uIRI+Rb1 group increased, which was statistically significant compared with the uIRI group, suggesting that Rb1 can promote the formation of peritubular capillaries ( Figure 5 A, B, C, D). Figure 6 Western blot results in A and B demonstrated that the expression level of eNOS in uIRI mice was significantly decreased, while that in mice treated with Rb1 was increased. In addition, the results of serum NO level test showed that the NO level in uIRI mice was significantly decreased, while that in mice treated with uIRI+Rb1 was significantly increased ( Figure 6 C).

[0039] Example 3: Rb1 treatment increases HUVECs viability, promotes hypoxia-induced tube formation, and improves vasodilation Figure 7The results of experiments A and B showed that under normal conditions, Rb1 at concentrations of 10µM and 1µM had no toxic effect on cells, while high concentrations of Rb1 inhibited cell growth. After 12 hours of hypoxia / reoxygenation, 1µM concentration of Rb1 significantly increased the viability of HUVECs. The tube formation experiment of HUVECs showed that under H / R conditions, the tube formation ability of HUVECs was significantly weakened, and Rb1 improved the tube formation ability of cells. Figure 8 The results showed that the tubular connections in the H / R group were relatively sparse and small, while those in the H / R+Rb1 group were thicker and tighter. The number of junctions, number of meshes, number of branches, and total length of tubules in the H / R+Rb1 group were all higher than those in the H / R group. These results suggest that Rb1 can promote the formation of tubular structures in HUVECs. Figure 8 A, B). Western Blot results showed that the expression level of eNOS in HUVECs induced by H / R was significantly decreased, while Rb1 effectively increased the expression of eNOS protein ( Figure 9 A, B). NO detection showed that H / R reduced the NO level in the cell supernatant, and after Rb1 treatment, the NO level increased ( Figure 9 C).

[0040] Based on the above examples, it was found and confirmed that Rb1 has a therapeutic effect on AKI-CKD, and can alleviate the pathological damage of the kidney by promoting angiogenesis and improving vascular function, thereby delaying the progression of AKI to CKD.

[0041] Therefore, by studying the activity of ginsenoside Rb1 in treating the progression of AKI-CKD caused by ischemia-reperfusion, it was found that this small molecule compound can effectively improve kidney tissue and kidney pathological damage, promote angiogenesis, and reduce the accumulation of renal collagen fibers. It can be used as a drug for treating kidney disease or renal injury and as a lead compound for the development and preparation of drugs for treating kidney disease or renal injury. According to the research results disclosed in the present invention, ginsenoside Rb1 can be used as a pharmaceutical preparation for treating the progression of AKI-CKD.

[0042] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Use of ginsenoside Rb1 in the preparation of a drug for delaying the progression of acute kidney injury to chronic kidney disease.

2. The use according to claim 1, characterized in that The drug is used to delay the progression of acute kidney injury caused by ischemia-reperfusion to chronic kidney disease.

3. The use according to claim 1, characterized in that The dosage of the ginsenoside Rb1 is 40 mg / kg.

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