A composition for treating kidney disease and a preparation method and application thereof

Abstract

The application provides a composition for treating kidney diseases and a preparation method and application thereof, wherein the efficacy components of the composition are composed of taurine and astragaloside B, and the concentration ratio of the taurine and the astragaloside B is 1:10-10:1; both the taurine and the astragaloside B are safe natural active components, and the combination of the two can realize multi-target anti-fibrosis through synergistically regulating the multi-dimensional inhibition of the'metabolism-DNA methylation-fibrosis' axis, delay the chronic kidney disease process, has the research and development advantages of'multi-target, low risk and short cycle', promotes the medicinal value conversion of the food-cure variety and the combination of the traditional Chinese medicine active components and the modern pharmacological mechanism, and has a remarkable synergistic anti-kidney fibrosis effect.

Description

Technical Field

[0001] This invention relates to a composition, its preparation method, and its application, and more particularly to a composition for treating kidney disease, its preparation method, and its application. Background Technology

[0002] The global incidence of chronic kidney disease (CKD) is rising year by year, with a prevalence of 10.8% among adults in my country. Renal fibrosis is the core pathological mechanism by which CKD progresses to end-stage renal disease (ESRD). At present, there is a lack of highly effective and low-toxicity anti-fibrotic drugs in clinical practice, while imported drugs (such as pirfenidone) are expensive and have hepatotoxic and nephrotoxic effects. Therefore, there is an urgent need to develop new drugs for the treatment of kidney disease. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a composition for treating kidney disease.

[0004] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned composition for treating kidney disease and its application.

[0005] The technical solution adopted in this invention is:

[0006] A composition for treating kidney disease, the active ingredients being taurine and astragaloside A, wherein the concentration ratio of taurine to astragaloside A is 1:10 to 10:1.

[0007] Preferably, in the above-mentioned composition for treating kidney disease, the concentration ratio of taurine to astragaloside A is 0.5 to 10:1.

[0008] Preferably, in the above-mentioned composition for treating kidney disease, the concentration ratio of taurine to astragaloside A is 3:1 to 5:1.

[0009] Preferably, in the above-mentioned composition for treating kidney disease, the concentration ratio of taurine to astragaloside A is 3:1.

[0010] The above-mentioned composition for treating kidney disease is prepared by mixing taurine and astragaloside A according to the concentration ratio.

[0011] The above composition is used in the preparation of drugs for treating kidney disease.

[0012] Preferably, in the above application, the kidney disease is chronic kidney disease.

[0013] Preferably, in the above application, the drug is a drug used to slow the progression of chronic kidney disease.

[0014] Preferably, in the above application, the drug is an anti-fibrotic drug.

[0015] The beneficial effects of this invention are:

[0016] The above-mentioned composition for treating kidney disease contains taurine and astragaloside, both of which are commercially available natural active ingredients with well-established safety profiles. The combined use of the two can achieve multi-target anti-fibrosis by synergistically regulating the "metabolism-DNA methylation-fibrosis" axis and inhibiting multiple dimensions, thus delaying the progression of chronic kidney disease (CKD). It has the advantages of "multi-target, low risk, and short cycle" in research and development, promotes the transformation of medicinal value of food and medicine homology products and the combination of active ingredients of traditional Chinese medicine with modern pharmacological mechanisms, and has a significant synergistic anti-renal fibrosis effect. Detailed Implementation

[0017] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0018] Example 1

[0019] A composition for treating kidney disease, wherein the concentration ratio of taurine to astragaloside A is 3:1, and the two components are mixed to obtain the composition.

[0020] Example 2

[0021] A composition for treating kidney disease, wherein the concentration ratio of taurine to astragaloside A is 5:1, and the preparation method is the same as in Example 1.

[0022] Example 3

[0023] A composition for treating kidney disease, wherein the concentration ratio of taurine to astragaloside A is 1:10, and the preparation method is the same as in Example 1.

[0024] Example 4

[0025] A composition for treating kidney disease, wherein the concentration ratio of taurine to astragaloside A is 10:1, and the preparation method is the same as in Example 1.

[0026] Example 5

[0027] Cell Culture and Processing

[0028] NRK-52E cells from passages 4-10 were selected and cultured in DMEM high-glucose complete medium (10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at a constant temperature of 37°C and 5% CO2. When the cells reached approximately 80% confluence, they were transplanted at a rate of 5 × 10⁻⁶ cells / year. 4 The cells were plated at a density of 10 ng / mL. All groups except the normal group were given 3 mL of 10 ng / mL TGF-β1 stimulant per dish. The normal group was given complete culture medium without the stimulant. The plates were incubated for 24 h. The groups and administration regimens are shown in Table 1.

[0029] Normal control group: 3 mL DMEM complete culture medium

[0030] Model control group: 3 mL DMEM complete culture medium

[0031] Single-drug group: 3 mL of DMEM complete culture medium containing taurine / astragaloside A

[0032] Combined group: 3 mL of DMEM complete medium containing taurine and astragaloside in different proportions.

[0033] Table 1. Taurine + Astragaloside A groups and dosing regimens in NRK-52E cells

[0034]

[0035] Experimental grouping and drug administration

[0036] Six-week-old male SD rats were randomly divided into four groups after a week of acclimatization: Sham group, 5 / 6 nephrectomy group, taurine group, astragaloside IV group, and taurine:astragaloside IV 3:1 group, with 10 rats in each group. For 5 / 6 nephrectomy, after induction and maintenance anesthesia with isoflurane inhalation, the rats were fixed in a supine position on the operating table. After abdominal preparation and routine disinfection with iodine, a 2-3 cm oblique incision was made 1.5 cm from the left ribcage, extending downwards and outwards (towards the kidney area). The skin and muscle were cut open, and the location of the left kidney was located using the left hand. The left kidney was then lifted from the ventral side towards the incision with the middle finger to fully expose it. The renal capsule was carefully dissected with forceps, and the upper and lower poles of the left kidney were ligated with No. 7 suture. Once the clamped area darkened to a purplish-black color due to ischemia, the upper and lower thirds of the left kidney were quickly removed in an arc shape, primarily removing the cortical portion. Hemostasis was achieved by applying gelatin sponge for a few moments, followed by the application of several drops of gelatin. After administering fibrinogen and thrombin solutions and confirming the absence of active bleeding on the incision surface, the remaining left kidney was repositioned. The muscle and skin were sutured layer by layer, and the surgical sutures were disinfected with povidone-iodine. Once the rats awoke, they were placed in individual cages with their airways kept clear. They were kept fasted for 24 hours post-surgery, but allowed free access to water. The skin incision, mental state, and food and water intake were closely monitored for 3 days post-surgery. Seven days after the first surgery, a second surgery was performed. The rats were anesthetized using the same method, and an incision was made on the right dorsal side to expose the right kidney. The renal capsule was carefully dissected, taking care not to damage the right adrenal gland. The renal pedicle was ligated. Once the kidney became ischemic and darkened, the entire right kidney was removed. The incision was then sutured and disinfected. The sham surgery control group underwent the same procedure, but only the renal capsule was dissected to expose the kidney. The abdomen was closed after exposure, but the renal tissue was not removed.

[0037] After successful modeling, mice in the Sham group and the 5 / 6 nephrectomy group were administered physiological saline by gavage. Mice in the taurine group, astragaloside A group, and taurine:astragaloside A 3:1 group were administered taurine at doses of 120 mg / kg, astragaloside A at doses of 40 mg / kg, and taurine:astragaloside A at doses of 120 + 40 mg / kg, respectively, and were collected after 28 days of continuous gavage.

[0038] Biochemical indicator testing

[0039] Animal serum was isolated, and rat serum creatinine levels were detected using a commercial creatinine assay kit. The creatinine content was calculated by measuring the absorbance at a wavelength of 546 nm.

[0040] Sirius Red Dye

[0041] Rat kidney tissue (from the modeling side or both kidneys) was collected, fixed in 4% paraformaldehyde for 24 h, and then routinely dehydrated in a gradient (70%→80%→90%→95%→100% ethanol), cleared in xylene, and embedded in paraffin to prepare 4 μm thick serial sections, which were then baked at 60℃ for 2 h. The sections were dewaxed in xylene (10 min each, twice), rehydrated in a gradient of ethanol (100%→95%→90%→80%→70% ethanol, 5 min each), and rinsed with distilled water for 5 min. They were then stained with 0.1% Sirius red staining solution (prepared with saturated picric acid solution) at room temperature in the dark for 1 h. After the staining solution was recovered, the sections were differentiated in 0.5% glacial acetic acid aqueous solution for 30 s (to remove non-specific staining), and quickly rinsed with distilled water 3 times. They were then dehydrated again in a gradient of ethanol, cleared in xylene, and mounted with neutral resin. When observed with an optical microscope (equipped with a polarization device), the collagen fibers (fibrotic areas) appear red or orange-red. Under polarized light, a bright red (type I collagen) refractive signal can be seen. ImageJ software is used to quantitatively analyze the percentage of "collagen fiber area / total field of view" to reflect the degree of renal tissue fibrosis.

[0042] ELISA testing

[0043] For animal kidney tissue samples, the tissue was first weighed and then homogenized with pre-cooled physiological saline or homogenization buffer at a mass-to-volume ratio of 1:9. The homogenate was then collected under ice bath conditions. After centrifugation at low temperature, the supernatant was used as the test sample. For cell samples, cells were collected, washed with PBS, and then an appropriate amount of cell lysis buffer was added. The cells were lysed under ice bath conditions, and the supernatant was collected as the test sample after centrifugation. Kidney tissue and cell samples were pretreated, and the total protein concentration in the supernatant was determined using a BCA protein quantification kit. The protein concentration was adjusted to 1 mg / mL, and the samples were aliquoted and stored at -80℃ for later analysis. ROS, CTGF, and 5-methylcytosine enzyme-linked immunosorbent assay (ELISA) kits were used. Standards, test samples, and blank controls (standard dilution) were added to the corresponding wells of the ELISA plate, respectively. After incubation and washing, biotin-labeled detection antibodies were added to each well, followed by incubation and washing. Horseradish peroxidase (HRP)-labeled streptavidin was added to each well, followed by incubation and washing. Substrate solution (TMB) was added to each well, and the plates were incubated in the dark. Stop solution was added quickly, and the absorbance (OD value) of each well was measured at 450 nm using an ELISA reader within 10 min. The ROS content (U / mg), CTGF concentration (pg / mL), and 5-methylcytosine level (%) in the samples were calculated using a standard curve.

[0044] Statistical processing

[0045] The experimental results were statistically analyzed using Graphpad Prism 9.5.1 software. Quantitative data were expressed as mean ± standard deviation (SD). The mean ± SD indicates that one-way ANOVA was used for comparisons among multiple groups, and the significance level was set at ± SD. p <0.05.

[0046] result

[0047] (1) Taurine and astragaloside synergistically inhibit ROS accumulation, inhibit DNA methylation, reduce CTGF secretion, and alleviate renal tubular cell fibrosis damage (Table 2).

[0048] Table 2. Indicators related to TGF-β1-induced rat renal tubular epithelial cells

[0049]

[0050] Note: Compared with the normal group ### p <0.001; compared with the model group, *** p <0.001

[0051] (2) A rat model of 5 / 6 nephrectomy was established. The area of ​​renal collagen deposition was reduced by 66% in the combined drug group (≤45% for single drug) and serum creatinine was reduced by 60%, verifying the in vivo effectiveness of the cross-regulation of "metabolism-DNA methylation-fibrosis" (Table 3).

[0052] Table 3. Relevant Indicators in Rats with 5 / 6 Nephrectomy

[0053]

[0054] Note: Compared with the sham surgery group. ### p <0.001, ## p <0.01; compared with the 5 / 6 nephrectomy group, *** p <0.001, ** p <0.01, * p <0.05

[0055] (3) According to the dosing regimen in Table 1, the 13 ratio tests of taurine and astragaloside A showed that the optimal ratio range was 3:1 to 5:1, of which 3:1 was the optimal ratio (Table 4).

[0056] Table 4. Tests on 13 ratios of taurine and astragaloside A

[0057]

[0058] Note: Compared with the normal group ### p <0.001; compared with the model group, *** p <0.001, ** p <0.01, * p <0.05

[0059] In summary, taurine and astragaloside A, as naturally occurring active ingredients with well-established safety profiles, have been found to exert a synergistic effect through a cross-regulatory pathway of metabolism-epigenetics-fibrosis. This significantly inhibits the accumulation of reactive oxygen species (ROS) and DNA methylation, thereby reducing the secretion of connective tissue growth factor (CTGF) and mechanistically improving renal fibrosis. In a 5 / 6 nephrectomy rat model, this combination demonstrated significant efficacy, reducing renal collagen deposition area by 66% and serum creatinine by 60%, far exceeding the effects of monotherapy. Furthermore, a taurine:astragaloside A concentration ratio ranging from 3:1 to 5:1 (with 3:1 being optimal) ensures the maximization of the synergistic effect and the stability of the therapeutic efficacy, fully demonstrating the outstanding substantive features and significant advancements of this invention in the treatment of kidney disease.

[0060] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A composition for treating kidney disease, characterized in that: The active ingredients consist of taurine and astragaloside A, wherein the concentration ratio of taurine to astragaloside A is 3:1 to 5:

1.

2. The composition for treating kidney disease according to claim 1, characterized in that: The concentration ratio of taurine to astragaloside A is 3:

1.

3. A method for preparing the composition for treating kidney disease according to claim 1 or 2, characterized in that: Taurine and astragaloside A are mixed according to the specified concentration ratio to obtain the final product.

4. Use of the composition of claim 1 or 2 in the preparation of a medicament for treating renal fibrosis.

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