Use of aescin in the preparation of a medicament for preventing and / or treating gouty nephropathy

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of aesculin B in preparation of a medicine for preventing and / or treating gouty nephropathy. The aesculin B can reduce a kidney index, reduce contents of serum creatinine, urea nitrogen and uric acid, reduce kidney crystal precipitates and resist renal interstitial fibrosis, so that an effect of preventing and / or treating gouty nephropathy is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Akebia quinata phenylethanoid glycoside B in the preparation of drugs for the prevention and / or treatment of gouty nephropathy. Background Technology

[0002] Gouty nephropathy, or gouty nephropathy for short, is kidney damage caused by hyperuricemia due to excessive uric acid production or reduced excretion. Clinical manifestations of gouty nephropathy may include uric acid stones, low-molecular-weight proteinuria, edema, nocturia, hypertension, elevated blood and urine uric acid levels, and impaired renal tubular function. If uric acid nephropathy is diagnosed early and treated appropriately (controlling hyperuricemia and protecting kidney function), the progression of kidney disease can be reduced or halted.

[0003] Gouty nephropathy is generally treated with oral uric acid-lowering medications, such as febuxostat tablets. Lowering uric acid levels is beneficial for the treatment of gouty nephropathy. If proteinuria is the primary symptom, medications such as enalapril, irbesartan tablets, okra capsules, and baicalin tablets can be taken to reduce proteinuria and lower intraglomerular pressure, thereby protecting kidney function. If serum creatinine is elevated, medications such as uremic toxin-clearing granules, Haikun Shenxi capsules, and Shensuining tablets can be used; long-term use can lower creatinine and delay kidney failure.

[0004] Currently, there are no reports on the use of Akebia quinata phenylethanoid B for the prevention and / or treatment of gouty nephropathy. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of Akebia quinata phenylethanoid glycoside B in the preparation of drugs for the prevention and / or treatment of gouty nephropathy, and this invention provides a new application direction for Akebia quinata phenylethanoid glycoside B.

[0006] This invention provides the use of Akebia quinata phenylethanoid glycoside B in the preparation of a drug for the prevention and / or treatment of gouty nephropathy, wherein the chemical structural formula of Akebia quinata phenylethanoid glycoside B is shown in Formula I.

[0007]

[0008] Preferably, the working concentration of the akebia phenylethanol glycoside B, based on rat administration, is 5–20 mg / kg.

[0009] Preferably, the dosage form of the drug includes an oral dosage form.

[0010] Preferably, the gouty nephropathy is caused by purine accumulation.

[0011] Preferably, the purine includes adenine.

[0012] Preferably, the prevention and / or treatment of gouty nephropathy includes one or more of the following aspects 1) to 4):

[0013] 1) Lower kidney index;

[0014] 2) Reduce the levels of one or more of the following in serum: creatinine, blood urea nitrogen, and uric acid;

[0015] 3) Reduce kidney crystal deposits and / or reduce kidney pathological damage;

[0016] 4) Anti-renal interstitial fibrosis.

[0017] Preferably, the anti-renal interstitial fibrosis includes reducing fibrous tissue and / or inhibiting the expression of renal fibrosis-related proteins.

[0018] Preferably, the fibrous tissue includes fibrous tissue surrounding the glomerulus and / or the renal tubules.

[0019] Preferably, the renal fibrosis-related proteins include one or more of fibronectin, collagen type I, hypoxia-inducible factor 1α, vimentin, and α-smooth muscle actin.

[0020] This invention provides the use of Akebia quinata phenylethanoid glycoside B in the preparation of drugs for the prevention and / or treatment of gouty nephropathy, wherein the chemical structural formula of Akebia quinata phenylethanoid glycoside B is shown in Formula I. It has been verified that Akebia quinata phenylethanoid glycoside B can reduce renal index, decrease serum creatinine, blood urea nitrogen and uric acid levels, reduce renal crystalline deposits and resist renal interstitial fibrosis, thereby achieving the effect of preventing and / or treating gouty nephropathy. Attached Figure Description

[0021] Figure 1 This study indicates the effect of akebia phenylethanoid glycoside B on improving renal fibrosis in rats; where A represents the renal index and blood biochemical indicators of different groups; B represents the serum urea nitrogen content of different groups; C represents the serum creatinine content of different groups; D represents the serum uric acid content of different groups; E represents the HE and Masson scores of rat pathological sections; # represents P<0.05 compared with the normal group; ## represents P<0.01; ### represents P<0.001; * represents P<0.05 compared with the model group; ** represents P<0.01; *** represents P<0.001.

[0022] Figure 2 HE staining results of kidney pathological sections of rats in each group treated with phenylethanoid B of Akebia quinata;

[0023] Figure 3 Masson staining results of kidney pathological sections of rats in each group treated with adenine-induced rat model by phenylethanoid B;

[0024] Figure 4The effects of administration of Akebia trifoliata phenylethanoid glycoside B on the expression of marker proteins of renal fibrosis in rat kidneys are shown. A: Western blotting detection of marker protein expression bands in the kidneys; B: Semi-quantitative analysis of Fibronectin protein expression; C: Semi-quantitative analysis of Collagen I protein expression; D: Semi-quantitative analysis of HIF-1α protein expression; E: Semi-quantitative analysis of Vimentin protein expression; F: Semi-quantitative analysis of α-SMA protein expression. # Compared with the normal group, # This represents P < 0.05; ## This represents P < 0.01; ### This represents P < 0.001;

[0025] Figure 5 This indicates the effect of administration of Akebia trifoliata phenylethanoid B on the survival rate of NRK-52E cells;

[0026] Figure 6 The study investigated the effects of administration of Akebia trifoliata phenylethanoid B on the expression of renal fibrosis marker proteins in NRK-52E cells. Specifically, A: Western blotting analysis of renal fibrosis marker protein expression bands; B: Semi-quantitative analysis of Fibronectin protein expression; C: Semi-quantitative analysis of Vimentin protein expression; and D: Semi-quantitative analysis of α-SMA protein expression. # Compared with the normal group, # This represents P < 0.05; ## This represents P < 0.01; ### * indicates P < 0.001; * indicates the difference from the model group, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001). Detailed Implementation

[0027] This invention provides the use of Akebia quinata phenylethanoid glycoside B in the preparation of a drug for the prevention and / or treatment of gouty nephropathy, wherein the chemical structural formula of Akebia quinata phenylethanoid glycoside B is shown in Formula I.

[0028]

[0029] In this invention, the chemical formula of the akebia phenylethanol glycoside B is preferably C. 23 H 26 O 11 ;CAS Number: 105471-98-5.

[0030] This invention does not have any special restrictions on the source of Akebia quinata phenylethanoid B; commercially available products are sufficient. In the specific implementation of this invention, the Akebia quinata phenylethanoid B was purchased from Baoji Chenguang Biotechnology Co., Ltd.

[0031] In the present invention, based on the administration to rats, the working concentration of lignstroside B is preferably 5 - 20 mg / kg, more preferably 10 mg / kg.

[0032] In the present invention, the dosage form of the drug preferably includes an oral dosage form.

[0033] In the present invention, the gouty nephropathy is preferably caused by purine accumulation; the purine preferably includes adenine.

[0034] In the present invention, the prevention and / or treatment of gouty nephropathy preferably includes one or more of the following aspects 1) - 4):

[0035] 1) Reducing the kidney index;

[0036] 2) Reducing the content of one or more of serum creatinine, urea nitrogen, and uric acid;

[0037] 3) Reducing kidney crystal precipitates and / or reducing kidney pathological damage;

[0038] 4) Anti - renal interstitial fibrosis.

[0039] In the present invention, the anti - renal interstitial fibrosis preferably includes reducing fibrous tissue and / or inhibiting the expression of renal fibrosis - related proteins. [[ID=2…]]

[0040] In the present invention, the fibrous tissue preferably includes the fibrous tissue around glomeruli and / or around renal tubules.

[0041] In the present invention, the renal fibrosis - related proteins preferably include one or more of fibronectin, type I collagen, hypoxia - inducible factor 1α, vimentin, and α - smooth muscle actin.

[0042] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the examples in the present invention.

[0043] Example 1

[0044] 1 Experimental materials

[0045] 1.1 Experimental animals

[0046] 72 male SPF - grade Sprague Dawley (SD) rats, purchased from Shanghai Slack Experimental Animal Co., Ltd., with a body weight of 160 - 180 g. Production license number: SCXK(Shanghai)2022 - 0004. The animal experiment was approved by the Animal Ethics Committee of Zhejiang Chinese Medical University, approval number: IACUC - 20220829 - 12. Before the start of the experiment, the animals were housed in an air - conditioned room at 23 - 25°C, with a 12 - hour alternating cycle of darkness / light. During the experiment, sufficient food and water were provided to the animals.

[0047] 1.2 Drug Preparation

[0048] (1) Weigh out the standard powder of Akebia quinata phenylethanol glycoside B, dissolve it in 0.5% CMC-Na solution to prepare a solution with a concentration of 2 mg / ml.

[0049] (2) Modeling drug: Adenine powder is dissolved in 0.5% CMC-Na solution, mixed and shaken to prepare a drug solution with a concentration of 150mg / ml, and stored in a refrigerator at 4℃.

[0050] (3) Positive drug: Dissolve losartan potassium powder in 0.5% CMC-Na solution, mix and shake well to prepare a drug solution with a concentration of 5mg / ml, and store in a refrigerator at 4℃.

[0051] 2 Experimental Methods

[0052] 2.1 Establishment of an animal model of gouty nephropathy and grouping for drug administration

[0053] Male SD rats were randomly divided into 6 groups: normal group, model group, positive drug group, and low-, medium-, and high-dose groups of Akebia trifoliata phenylethanoid B (5, 10, and 20 mg / kg, respectively), with 12 rats in each group. After one week of acclimatization, except for the normal group, all other groups were administered 150 mg / kg adenine via gavage to induce a model (once daily) for 28 days. The drug-treated groups received interventional administration, administering the drug while initiating the model, 4–5 hours after model initiation via gavage (once daily) for 28 days. Specific treatment methods for each group are shown in Table 1. During the experiment, the rats were weighed and their weight recorded daily, and they were provided with sufficient normal food and water.

[0054] Table 1 Animal grouping and drug treatment

[0055]

[0056] 2.2 Serum biochemical index detection

[0057] The levels of creatinine (CREA), blood urea nitrogen (BUN), and uric acid (UA) in serum were detected using a fully automated biochemical analyzer.

[0058] 2.3 Kidney tissue staining

[0059] 2.3.1 Fixation and embedding sections

[0060] (1) Fix the kidney tissue sample with 10% formalin for one week; (2) Remove the kidney tissue from the fixative, cut a section of kidney tissue about 4 mm thick, place it in the embedding box, and rinse with running tap water for more than 2 hours; (3) 40% ethanol - 20 min; (4) 70% ethanol - 15 min; (5) 80% ethanol - 10 min; (6) 95% ethanol I 10 min; (7) 95% ethanol II 10 min; (8) 100% ethanol I 10 min; (9) 100% ethanol II 10 min; (10) 100% ethanol III 10 min; (11) Stearic acid: paraffin (1:1; 60℃) 30 min; (12) Paraffin (60℃) 40 min; (13) Embedding: Take out the tissue, place it at the bottom of the iron box, carefully pour in the hot paraffin solution, being careful not to shake the tissue, place the embedded paraffin block on the freezing stage, and remove the embedding groove after solidification. (14) Sectioning: Before slicing, freeze the paraffin block containing the kidney tissue sample in a -20℃ freezer for more than 40 minutes and set the slicing thickness to 4μm. Place the tissue slices in a water bath at 37℃ until the tissue slices are flattened, then attach them to a glass slide, and then place them in an oven at 60℃ for 6 hours until the paraffin on the glass slide melts. Store them for later use.

[0061] 2.3.2 HE (Hematoxylin-eosin) staining

[0062] (1) Before staining, place the tissue sections in an oven at 60℃ to melt the paraffin for 2 hours; (2) While still hot, immerse them in xylene solution for 20 minutes; (3) immerse in 100% ethanol I for 5 minutes; (4) immerse in 100% ethanol II for 5 minutes; (5) immerse in 95% ethanol for 5 minutes; (6) immerse in 90% ethanol for 5 minutes; (7) immerse in 85% ethanol for 5 minutes; (8) rinse with tap water under a gentle stream for 3 minutes; (9) incubate with hematoxylin for 10 minutes; (10) rinse with running water for 8 minutes; (11) stain with eosin for 2 minutes; (12) rinse with running water for 5 minutes; (13) immerse in 75% ethanol for 10 seconds; (14) immerse in 95% ethanol for 1 minute.

[0063] (15) 100% ethanol for 10s; (16) xylene for 10min; (17) neutral resin for mounting and observation under an optical microscope.

[0064] 2.3.3 Masson staining

[0065] The sections were dewaxed and stained according to the kit instructions, following the sequence of Weigert iron hematoxylin staining, acid ethanol differentiation, Masson blue solution for blue reversion, Ponceau S and fuchsin staining, washing with weak acid working solution, phosphomolybdic acid washing, aniline blue staining, dehydration, xylene clearing, and mounting with neutral resin. The sections were then observed under an optical microscope.

[0066] 2.4 MTT assay for the effect of phenylethanoid glycoside B on the cell viability of NRK-52E cells.

[0067] NRK-52E cells in logarithmic growth phase were digested with trypsin and then added to complete culture medium to adjust the cell density to 2.5 × 10⁻⁶. 4 Cells were seeded at a density of 1 / ml into 96-well plates, with 200 μl of cell suspension added to each well. Cells were then incubated in a cell culture incubator. Cell status was observed after 6–8 h of culture. Once cell adhesion was stable, 100 μl of incomplete culture medium (without fetal bovine serum) was added to the normal group cells. Eight concentrations of akebia quinata phenylethanoid B were prepared: 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM, with 100 μl of the corresponding solution added to each well. Five replicates were performed for each concentration. 24 h after drug administration, 10 μl of MTT solution was added to each well. Cells were incubated for 3–5 h for color development. OD values ​​were measured at 490 nm using a multi-mode microplate reader. The OD value of the control group was used as a reference. Cell viability was calculated using the following formula.

[0068]

[0069] Note: OD drug administration group: absorbance of wells containing cells, MTT solution and drug solution; OD blank group: absorbance of wells containing cells and MTT solution but without drug solution.

[0070] 2.5TGF-β1 stimulation assay for the anti-renal fibrosis activity of Akebia trifoliata phenylethanoid glycosides in cells

[0071] NRK-52E cells in logarithmic growth phase were seeded in 6-well plates (density 2 × 10⁻⁶). 5 Cells were divided into 6 groups (3 replicates per well), as shown in Table 2. After the cells were stably adhered, the serum-free medium was replaced. Except for the blank group, all other groups were induced with TGF-β1 (10 ng / ml) for 24 h, and then cultured in the corresponding serum-free medium containing the drug for another 24 h.

[0072] Table 2 Cell drug administration groups

[0073]

[0074]

[0075] 2.6 Western blot detection

[0076] 2.6.1 Detection of tissue protein expression

[0077] Total protein extraction from tissue: Weigh 20 mg of renal cortex tissue, add 100 μl of lysis buffer (RIPA:PMSF = 100:1) per 10 mg of tissue, add magnetic beads, and homogenize the tissue using a pre-cooled cell disruptor until no obvious tissue fragments remain. Centrifuge at 15000 rpm for 10 min and collect the supernatant. Add 5× Loading buffer (sample:Loading buffer = 4:1) to the protein sample, vortex to mix, place in a 100℃ metal bath to denature the protein, and then store in a -80℃ freezer. After determining the protein concentration of the sample using BCA, perform SDS-PAGE electrophoresis, transfer to a membrane, block, incubate with primary antibody, incubate with secondary antibody, develop, and detect the expression of the target protein.

[0078] 2.6.2 Detection of cellular protein expression

[0079] Total cellular protein extraction: Discard the culture medium, wash three times with pre-chilled PBS along the cell wall, add 1 ml of PBS solution to each well, collect the cell suspension from each group, centrifuge at 5000 rpm for 5 min at 4°C, and discard the supernatant PBS solution. Add 30 μl of cell lysis buffer to the cell pellet and place on ice, vortex once every 10 min, repeat 3 times, then centrifuge at 15000 rpm for 15 min at 4°C, and transfer the supernatant to a new EP tube for later use. Take a small amount to determine the concentration, and aliquot the rest and store at -80°C to avoid repeated freeze-thaw cycles that could cause protein degradation. After determining the BCA protein concentration in the sample, Western blot was used to detect the expression of the target protein.

[0080] 3 Results

[0081] 3.1 Effects of Akebia trifoliata phenylethanoid glycoside B on serum biochemical indicators in rats with gouty nephropathy

[0082] The normal group of rats exhibited good appetite, agile movement, and normal urination and defecation. After adenine gavage, the rats began to show lethargy, sluggishness, frequent curling up, weight loss, increased thirst, increased urine output, and soft, wet stools that easily led to damp bedding after the second week. Treatment with Akebia quinata phenylethanoid B improved the rats' activity level, coat luster, and urine output. At the start of the experiment, after SPSS analysis, the rats were randomly divided into 9 groups according to their initial body weight, ensuring no significant difference in initial body weight among the groups.

[0083] like Figure 1 As shown in A and Table 3, there were significant differences in the renal index between the control group and the model group. ### P<0.001). The renal index of the positive control group (losartan potassium, 5 mg / kg) and the high-dose group of akebia phenethyl glycoside B were significantly lower than those of the model group (* represents P<0.05; ** represents P<0.01; *** represents P<0.001). Other groups showed no significant difference from the model group. Blood urea nitrogen in rat serum was measured. Figure 1 B in Table 4), creatinine ( Figure 1 (C in Table 5), uric acid ( Figure 1 Compared with the normal group, the serum creatinine, blood urea nitrogen, and uric acid levels in the model group rats were significantly higher (###P<0.001). The high-dose administration of the positive control drug losartan potassium and akebia phenylethanoid B significantly inhibited the upward trend of serum creatinine and blood urea nitrogen, showing a significant difference compared with the model group; the positive control drug losartan potassium had no significant regulatory effect on uric acid, while the high-dose administration of akebia phenylethanoid B significantly inhibited the upward trend of serum uric acid, showing a significant difference compared with the model group (* represents P<0.05; ** represents P<0.01; *** represents P<0.001). The scoring criteria for rat pathological sections are shown in Table 7, and the HE and Masson comprehensive scores for rat pathological sections are shown in (…). Figure 1 (See E in Table 8). Typical HE staining for each group is shown in Table 8. Figure 2 For typical Masson staining, see [link to Masson staining]. Figure 3 It can be seen that HE staining ( Figure 2 In the normal group, the kidneys of rats showed clear and tightly packed structures, with no abnormalities observed in the renal tubules, glomeruli, or interstitium. In the model group, the kidney tissue structure was disordered, with vacuolar formation, abundant brownish-yellow 2,8-dihydroxyadenine deposits in the renal tubules, a reduced number of glomeruli with atrophy, and dilated and irregularly arranged renal tubules. In the group treated with akebia quinata phenylethanoid B, the amount of crystalline precipitate decreased, and other diseased structures showed improvement. Based on Masson staining results (… Figure 3 As shown in the figure, there is a small amount of fibrous tissue around the glomeruli in the normal group, while there is blue-stained collagen deposits around the glomeruli and renal tubules in the model group, forming renal interstitial fibrosis. The fibrous tissue in the drug-treated group is significantly reduced.

[0084] Table 3. Rats' kidney index (bilateral kidney weight / body weight %)

[0085]

[0086] Table 4. Serum urea nitrogen levels in rats (mmol / L)

[0087]

[0088]

[0089] Table 5. Serum creatinine levels in rats (μmol / L)

[0090]

[0091] Table 6. Serum uric acid levels in rats (μmol / L)

[0092]

[0093] Table 7 Scoring criteria for rat pathological sections

[0094]

[0095] Table 8. Rat pathological section scores

[0096]

[0097] 3.2 Effects of Akebia trifoliata phenylethanoid glycoside B on renal morphology and renal histopathology in rats with gouty nephropathy

[0098] Kidney pathology sections stained with HE as follows Figure 2 As shown, the normal group rats had clear and tightly arranged kidney structures, with no abnormalities observed in the renal tubules, glomeruli, or interstitium. The model group showed disordered kidney tissue structure with vacuolar formation, abundant brownish-yellow 2,8-dihydroxyadenine deposits in the renal tubules, a reduced number of glomeruli with atrophy, and dilated and irregularly arranged renal tubules. After administration of Akebia trifoliata phenylethanoid B, the amount of crystalline precipitate decreased, and other diseased structures showed improvement. Based on Masson staining results (… Figure 3 As shown in the figure, there is a small amount of fibrous tissue around the glomeruli in the normal group, while there is obvious blue-stained collagen deposition around the glomeruli and renal tubules in the model group, forming renal interstitial fibrosis. The fibrous tissue decreased after administration of Akebia quinata phenylethanol glycoside B.

[0099] 3.3 Western Blot analysis of the effect of Akebia trifoliata phenylethanoid glycoside B on the expression of related proteins in rats with gouty nephropathy

[0100] The efficacy of Akebia trifoliata phenylethanoid glycoside B in preventing renal fibrosis in rats with gouty nephropathy induced by adenine accumulation was verified through statistical analysis. (See attached table for results.) Figure 4 And Table 9, by Figure 4 As shown in Table 9, the expression of renal fibrosis marker proteins—fibronectin, collagen I, hypoxia-inducible factor 1α (HIF-1α), vimentin, and α-smooth muscle actin (α-SMA)—was significantly increased in the model group (##P<0.01). The high-dose treatment group of akebia phenylethanoid glycoside B significantly downregulated the expression of all five proteins. Therefore, this verifies that akebia phenylethanoid glycoside B from Plantago asiatica does indeed have an ameliorative effect on gouty nephropathy in rats induced by adenine accumulation.

[0101] Table 9. Western blot results of rat kidneys

[0102]

[0103]

[0104] 2.1 Effect of Akebia trifoliata phenylethanoid B on NRK-52E cell viability

[0105] Cell viability of Akebia trifoliata phenylethanoid B at eight concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 μM) was detected using the MTT assay. The results are as follows: Figure 5 As shown in Table 10, when the concentration of Akebia trifoliata phenylethanoid B reached 200 μM, there was no significant change in cell viability after 24 hours of administration, and the cell morphology remained normal under a microscope. Therefore, administration of Akebia trifoliata phenylethanoid B at a concentration not exceeding 200 μM for 24 hours does not affect the cell viability of NRK-52E cells.

[0106] Table 10. Cytotoxicity assay of Akebia trifoliata phenylethanoid B against NRK-52E cells (cell viability %)

[0107]

[0108]

[0109] 2.2 Western Blot detection of renal fibrosis-related protein expression in NRK-52E cells after intervention with Akebia trifoliata phenylethanoid B

[0110] To verify the anti-renal fibrosis effect of Akebia trifoliata phenylethanoid glycoside B in NRK-52E cells. Results are shown below. Figure 6 And Table 11, by Figure 6 As shown in Table 11, the marker proteins of renal fibrosis—fibronectin, vimentin, and α-smooth muscle actin (α-SMA)—showed a significant increase in TGF-β1 levels. ## P<0.01). Akebia quinata phenylethanoid glycoside B significantly downregulated the expression of all three proteins. Cell-level pharmacodynamic verification experiments showed that Akebia quinata phenylethanoid glycoside B had an anti-renal fibrosis effect on NRK-52E cells.

[0111] Table 11 Western blot results of NRK-52E Akebia trifoliata phenylethanoid B administration

[0112]

[0113] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of Akebia benzene ethanol glycoside B in the preparation of a medicine for preventing and / or treating gouty nephropathy, wherein the chemical structural formula of the Akebia benzene ethanol glycoside B is shown as formula I; and the Akebia benzene ethanol glycoside B is the only active ingredient of the medicine.

2. Use according to claim 1, characterized in that, The dosage form of the medicine is an oral dosage form.

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