Application of schizandrin B and derivative thereof in preparation of medicine for resisting aristolochic acid-induced renal toxicity diseases and medicine composition for resisting aristolochic acid-induced renal toxicity

Abstract

The invention provides application of schizandrin B and a derivative thereof in preparing a medicine for resisting renal toxicity diseases caused by aristolochic acid, and a pharmaceutical composition containing the schizandrin B and the derivative thereof for resisting renal toxicity caused by aristolochic acid. A mouse suffering from acute renal toxicity injury caused by aristolochic acid is taken as a model, and the protective effect of schisandrin B and the derivative thereof on the mouse suffering from acute renal toxicity caused by aristolochic acid is researched by combining metabonomics and proteomics. It is found that the combined use of schisandrin B and / or schisandrin B derivatives can effectively protect the renal function of mice, and serum creatinine (Cr) and urea nitrogen (BUN) are significantly reduced; the injury of aristolochic acid to the kidney of the mouse is relieved, and the renal tubular necrosis, tubular and renal tubular dilatation and the like are improved; protein expression abnormality and metabolic abnormality of mouse kidneys caused by aristolochic acid are remarkably reversed, and renal function impairment and renal tissue injury caused by aristolochic acid are remarkably reduced. The schisandrin B and the derivative thereof have a remarkable protection effect on acute renal toxicity injury caused by aristolochic acid.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to application of schisandra chinensis alcohol B and its derivatives in preparing medicines for resisting aristolochic acid-induced nephrotoxicity diseases, and a medicine composition for resisting aristolochic acid-induced nephrotoxicity. Background Art

[0002] Aristolochic acids are a class of organic acids found in Aristolochicaceae plants, which are widely found in Aristolochicaceae plants. For centuries, in many parts of the world, Aristolochicaceae plants have often been used as herbal medicines to treat a variety of diseases including hepatitis, urinary tract infections, vaginitis, upper respiratory tract infections, eczema, dysmenorrhea, joint pain, hypertension, bronchitis, pneumonia, heart failure and edema. However, taking plants or extracts containing aristolochic acid can cause severe kidney damage, and its related preparations or products have received safety warnings and have been banned in many countries and regions including the United States and Europe. Due to the abundance of Aristolochicaceae plants in nature and the prevalence of the traditional use of Chinese herbal medicine to treat diseases in Asian countries or regions, the risk of aristolochic acid kidney damage is very high, and cases of aristolochic acid-induced nephrotoxicity are emerging in an endless stream. Kidney damage caused by aristolochic acid is manifested as tubular degeneration and necrosis, renal interstitial fibrosis, and quickly progresses to end-stage renal failure. It is a worldwide disease that poses a great threat to health. Therefore, how to effectively prevent and treat the nephrotoxicity caused by aristolochic acid is a global problem that needs to be solved urgently.

[0003] Schisandrol B is the main active ingredient of the traditional Chinese medicine Schisandra chinensis (including Schisandrasphenanthera Rehd. et Wils. and Schisandra chinensis), and has multiple pharmacological effects, including antioxidant, anti-inflammatory, neuroprotective, anticancer, antihypertensive and hepatoprotective effects. The protective and regenerative effects of Schisandrol B on drug-induced liver injury induced by various factors have been well confirmed, but there are few reports on its renal protective effects. There are no reports on whether Schisandrol B can prevent, inhibit or treat nephrotoxic diseases caused by aristolochic acid. Summary of the invention

[0004] The present invention aims to explore the application of schisandrin B and / or its derivatives in resisting aristolochic acid-induced nephrotoxicity, that is, the application of schisandrin B and its derivatives in preparing drugs for preventing, inhibiting and treating nephrotoxic diseases caused by aristolochic acid, as well as pharmaceutical compositions containing schisandrin B and / or its derivatives for resisting aristolochic acid-induced nephrotoxicity.

[0005] One object of the present invention is to provide the use of schisandrae alcohol B and / or its derivatives in the preparation of drugs for preventing, inhibiting or treating aristolochic acid-induced nephrotoxicity diseases.

[0006] Schisandrin B derivative refers to the sodium salt or potassium salt of Schisandrin B.

[0007] Another object of the present invention is to provide a pharmaceutical composition for resisting aristolochic acid-induced nephrotoxicity.

[0008] The above-mentioned pharmaceutical composition comprises schisandra chinensis alcohol B and / or its derivatives.

[0009] The pharmaceutical composition comprises pharmaceutically acceptable excipients; the pharmaceutical excipients include any one of fillers, disintegrants, binders, surfactants, glidants, preservatives, or flavoring agents, or a combination of at least two of them.

[0010] Among the above-mentioned pharmaceutical excipients, the filler is starch, dextrin, cellulose, mannitol and / or lactose; the disintegrant is microcrystalline cellulose, calcium carbonate and / or sodium bicarbonate; the binder is ethanol, dextrin, and / or sodium hydroxymethylcellulose; the surfactant is polysorbate, polyethylene glycol, and / or sorbitan trioleate; the glidant is talc, micropowdered silica gel, and / or magnesium stearate; the preservative is propylene glycol, glycerol, and / or benzyl alcohol; the flavoring agent is sucrose, monosaccharide syrup and / or trehalose.

[0011] Another object of the present invention is to provide a pharmaceutical composition for resisting aristolochic acid-induced nephrotoxicity for use in preparing drugs for preventing, inhibiting or treating aristolochic acid-induced nephrotoxicity diseases.

[0012] The dosage form of the above-mentioned medicine is tablet, capsule, granule, powder, solution, emulsion, suspension, suppository, powder or lyophilized powder injection.

[0013] The beneficial effects of the present invention are as follows: using a model of acute renal injury in mice caused by intraperitoneal injection of aristolochic acid for 4 consecutive days, metabolomics and proteomics are combined to comprehensively evaluate the protective effect of Schisandrin B and its derivatives on mice with acute renal toxicity caused by aristolochic acid. The combined use of Schisandrin B and / or Schisandrin B derivatives can effectively protect the renal function of mice, significantly reduce blood creatinine (Cr) and urea nitrogen (BUN); reduce the damage of aristolochic acid to the kidneys of mice, including improving tubular necrosis, casts and tubular dilatation; significantly reverse the abnormal protein expression and metabolic abnormalities in the kidneys of mice caused by aristolochic acid, and significantly reduce the renal function damage and renal tissue damage caused by aristolochic acid. Schisandrin B and its derivatives have a significant protective effect on acute renal toxicity caused by aristolochic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This is the result of the effect of Schisandrin B on the renal tissue damage of mice caused by aristolochic acid. A is a 40-fold microscopic image of a kidney tissue section of mice in the blank control group, and A' is a 200-fold microscopic image of a kidney tissue section of mice in the blank control group; B is a 40-fold microscope image of the renal tissue section of mice in the aristolochic acid model group, and B' is a 200-fold microscope image of the renal tissue section of mice in the aristolochic acid model group; C is a 40-fold microscopic image of the renal tissue section of mice in the low-dose schisandrae alcohol B plus aristolochic acid model group, and C' is a 200-fold microscopic image of the renal tissue section of mice in the low-dose schisandrae alcohol B plus aristolochic acid model group; D is a 40-fold microscopic image of the renal tissue section of mice in the medium-dose schisandrae alcohol B plus aristolochic acid model group, and D' is a 200-fold microscopic image of the renal tissue section of mice in the medium-dose schisandrae alcohol B plus aristolochic acid model group; E is a 40-fold microscopic image of the renal tissue section of mice in the high-dose schisandrae alcohol B plus aristolochic acid model group, and E' is a 200-fold microscopic image of the renal tissue section of mice in the high-dose schisandrae alcohol B plus aristolochic acid model group; Figure 2a This is the result of the effect of Schisandrin B on serum creatinine in mice with renal damage induced by aristolochic acid; Figure 2b This is the result diagram of the effect of Schisandrin B on serum urea nitrogen in mice with renal damage induced by aristolochic acid; Figure 3 This is a volcano plot of the effects of Schisandrin B on the metabolomics of renal tissue in mice with aristolochic acid-induced renal injury. F is a comparison chart between the aristolochic acid model group and the blank control group; G is a comparison chart of the high-dose schisandrae alcohol B plus aristolochic acid model group and the blank control group: Figure 4 This is a volcano diagram of the effects of Schisandrin B on the renal tissue proteomics of mice with aristolochic acid-induced renal injury. H is a comparison chart between the aristolochic acid model group and the blank control group; J is a comparison chart between the high-dose schisandrae alcohol B plus aristolochic acid model group and the blank control group. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with specific examples, but the examples of the present invention should never be construed as any limitation of the present invention. Unless otherwise specified, the reagents and methods involved in the examples are all commonly used reagents and methods in the art.

[0016] The experiment in the embodiment of the present invention uses acute kidney injury in mice induced by intraperitoneal injection of aristolochic acid for 4 consecutive days as a model, and combines metabolomics and proteomics to comprehensively evaluate the protective effect of schisandrae alcohol B on mice with aristolochic acid acute nephrotoxicity from a holistic perspective.

[0017] 1. Experimental Materials Experimental animals: SPF-grade C57BL / 6 mice, 5-6 weeks old, provided by Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., with experimental animal production license number SCXK (Zhejiang) 2019-0001. They were fed normally in the barrier environment animal room in the southern area of ​​the Drug Evaluation Center of Guangzhou Boji Pharmaceutical Biotechnology Co., Ltd.

[0018] Drugs and reagents: Schisandrin B (≥99%, Shanghai Ronghe Pharmaceutical Technology Co., Ltd.), Aristolochic acid І (>99%, Sigma-Aldrich Co.).

[0019] Experimental instruments: electronic balance (model: BP211D, Germany / sartorius), fully automatic biochemical analyzer (Dimension Xpand Plus, USA / Siemens), desktop low-speed centrifuge: (TDZ5-WS, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), UPLC-MS / MS high-resolution liquid chromatography-mass spectrometry analyzer (instrument model: Vanquish, manufacturer: Thermo Fisher Scientific, USA).

[0020] 2. Experimental Methods and Dosing Scheme 1. Preparation of Schisandrin B intragastric solution: Take an appropriate amount of Schisandrin B, add an appropriate amount of 0.5% CMC-Na solution, and ultrasonicate for 10 minutes to prepare Schisandrin B intragastric solutions with concentrations of 0.625, 2.5, and 10 mg / mL, respectively (the intragastric administration volume is 0.2 mL / 10 g, and the doses are 12.5, 50, and 200 mg / kg, respectively).

[0021] 2. Preparation of aristolochic acid solution: Take an appropriate amount of aristolochic acid, weigh it accurately, and dissolve it in 1% sodium bicarbonate solution to prepare a mouse acute kidney injury modeling solution with a concentration of 1 mg / mL (intraperitoneal injection volume is 0.1 mL / 10 g, and the dose is 10 mg / kg).

[0022] 3. Experimental process: Mice were randomly divided into 8 groups (n=5), including normal control group (Control), Schisandrin B control group (CY, 12.5, 50, 200 mg / kg), aristolochic acid model group (AA, intraperitoneal injection of 10 mg / kg), and Schisandrin B (12.5, 50, 200 mg / kg) + aristolochic acid group (CY / AA). The mice were weighed before the experiment. The mice in the CY control group and CY / AA group were continuously gavaged with Schisandrin B solution (0.2 mL / 10g, v / w) for 3 days, and the mice in the Control group and AA model group were continuously gavaged with the same volume of 0.5% CMC-Na solution for 3 days, once a day. On days 4-7, 30 minutes after gavage with 0.5% CMC-Na or Schisandrin B, 1 mg / mL aristolochic acid solution (dose of 10 mg / kg) or the corresponding volume of 1% NaHCO 3 On the 8th day (i.e., 24 h after intraperitoneal injection of AA), the mice were anesthetized with ulose and then killed, and blood samples and kidneys were collected for the determination of relevant biochemical indices, the left kidney for renal tissue morphology observation, and the right kidney for proteomics and metabolomics detection.

[0023] 3. Detection of Renal Tissue and Serum Biochemical Indexes Renal tissue sections and HE staining were completed by Wuhan Saiweier Biotechnology Co., Ltd. The results of mouse kidney tissue sections are as follows Figure 1 Serum creatinine (Cr) and urea nitrogen (BUN) were detected using the American Dimension Xpand Plus automatic biochemical analyzer, and the results are shown in Figure 2.

[0024] 4. Metabolomics and proteomics testing UPLC-QE-MS technology combined with principal component analysis (PCA) and OPLS-DA analysis was used to investigate the effect of schisandrin B on the renal metabolomics of mice with aristolochic acid nephrotoxicity. Figure 3 The effect of Schisandrin B on renal proteomics in mice with aristolochic acid nephrotoxicity was investigated by proteomics combined with TMT labeling and LC-MS / MS technology. The results are shown in Figure 4 shown.

[0025] 5. Statistics The experimental data of each group were expressed as mean ± SEM and statistically analyzed using GraphPad Prism 5 software. The unpaired Student's t test was used for comparison between the two groups, and P < 0.05 was considered to be significantly different.

[0026] 6. Experimental Results 1. Effect of Schisandrin B on the degree of renal tissue damage in mice induced by aristolochic acid like Figure 1 As shown in the figure, compared with the blank control group, a large number of renal tubular necrosis occurred at the junction of the renal cortex and medulla in the aristolochic acid modeling group, and most of the cortical renal tubules were involved. The nuclei of epithelial cells shrank and dissolved, the eosinophilic cytoplasm increased, and detached from the basement membrane (black arrow). In the cortex and medulla, more renal tubular dilatation, thinning of the tubular wall, and enlargement of the tubular lumen were seen (red arrow), and more protein casts were seen, and the lumen was filled with eosinophilic protein-like fluid (yellow arrow). It can be seen that continuous administration of aristolochic acid for 4 days also caused serious damage to the morphology of mouse renal tissue. The combined use of different doses of schisandrae alcohol B can reduce the damage of aristolochic acid to the renal tissue of mice to varying degrees, including improving renal tubular necrosis, casts and renal tubular dilatation. The protective effect of schisandrae alcohol B on renal tissue damage caused by aristolochic acid is dose-dependent. The higher the dose of schisandrae alcohol B, the stronger its effect on improving renal tissue damage caused by aristolochic acid.

[0027] 2. Effect of Schisandrin B on Serum Cr and BUN in Mice with Aristolochic Acid-Induced Renal Injury As shown in Figure 2, compared with the blank control group, continuous intraperitoneal injection of aristolochic acid for 4 days significantly increased the serum creatinine (Cr) and urea nitrogen (BUN) of mice. The combined use of different doses of Schisandrin B can reduce the trend of the increase of serum creatinine and urea nitrogen in mice caused by aristolochic acid, among which high dose of Schisandrin B can improve the trend of the increase of serum creatinine and urea nitrogen caused by aristolochic acid the most significantly, indicating that high dose of Schisandrin B can significantly resist the renal function damage caused by AA.

[0028] 3. Effect of Schisandrin B on renal tissue metabolomics in mice with aristolochic acid-induced renal injury like Figure 3 As shown in the figure, differential metabolites were screened according to VIP greater than 1, Fold Change greater than 2 or less than 0.5, and P-value less than 0.05. Each point in the volcano plot represents a metabolite, the horizontal axis represents the fold change of the group compared with each substance (taking the logarithm with base 2), the vertical axis represents the P value of the Student's t-test (taking the negative number of the logarithm with base 10), and the size of the scatter point represents the VIP value of the OPLS-DA model. The larger the scatter point, the greater the VIP value. The color of the scatter point represents the final screening result. The significantly upregulated metabolites are represented in red, the significantly downregulated metabolites are represented in blue, and the metabolites with non-significant differences are gray. Compared with the blank control group, a total of 2,680 differential metabolites were screened in the aristolochic acid model group, while the high-dose schisandrae alcohol B + aristolochic acid group had much fewer differential metabolites. It can be seen that the combined use of schisandrae alcohol B can significantly reverse the metabolic abnormalities of the kidneys of mice caused by aristolochic acid.

[0029] 4. Effect of Schisandrin B on renal tissue proteomics in mice with aristolochic acid-induced renal injury Differentially expressed proteins were screened according to the criteria of P value less than 0.05 and Fold Change < 0.67 or Fold Change > 1.5. The experimental results were shown in Figure 4 Volcano plot. Volcano plot can be used to show the distribution of differences in gene expression levels between two groups of samples. Each point in the figure represents a detected protein, and the horizontal axis represents the fold change of each protein in the group (taking the logarithm with base 2). The proteins with greater differences are distributed at both ends of the X-axis. The positive and negative values ​​of the Log-Fold Change value can be used to determine the up- and down-regulation of the protein. The vertical axis represents the P-value of the Student's T test (taking the negative number of the logarithm with base 10). The smaller the P value, the larger the value after taking the logarithm, and the higher it goes in the vertical axis direction, the more significant the difference. The color of the scatter points represents the final screening results. The significantly upregulated differentially expressed proteins are represented in red, the significantly downregulated differentially expressed proteins are represented in blue, and the proteins with non-significant differences are represented in gray. Figure 4 The results showed that compared with the blank control group, a total of 488 proteins in the aristolochic acid model group underwent significant changes in expression, of which 260 proteins were upregulated and 228 proteins were downregulated; while in the high-dose schisandrin B + aristolochic acid group, only 171 proteins underwent significant changes, of which 74 proteins were upregulated and 97 proteins were downregulated; it can be seen that the combined use of schisandrin B can significantly reverse the abnormal protein expression in the mouse kidneys caused by aristolochic acid.

[0030] The above experiments and tests were repeated using the sodium or potassium salt of Schisandrin B, or a mixture of Schisandrin B and Schisandrin B sodium salt, instead of Schisandrin B in the above experiments, and the results obtained were basically consistent, indicating that Schisandrin B and / or its derivatives have a protective effect on mice with acute nephrotoxicity caused by aristolochic acid.

[0031] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of schisandrae alcohol B and / or its derivatives in the preparation of drugs for preventing aristolochic acid-induced nephrotoxicity.

2. Use of schisandrae alcohol B and / or its derivatives in the preparation of drugs for inhibiting aristolochic acid-induced nephrotoxicity.

3. Use of schisandrae alcohol B and / or its derivatives in the preparation of drugs for treating aristolochic acid-induced nephrotoxicity diseases.

4. The use according to any one of claims 1 to 3, characterized in that: The derivative is the sodium salt or potassium salt of schisandra chinensis alcohol B.

5. A pharmaceutical composition for resisting aristolochic acid-induced nephrotoxicity, characterized in that: The pharmaceutical composition comprises schisandra chinensis alcohol B and / or its derivatives.

6. The pharmaceutical composition according to claim 5, characterized in that The derivative is a pharmaceutically acceptable sodium salt or potassium salt of Schisandrin B.

7. The pharmaceutical composition according to claim 5, characterized in that It also contains pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical excipients include any one of fillers, disintegrants, binders, surfactants, glidants, preservatives, or flavoring agents, or a combination of at least two of them.

9. Use of the pharmaceutical composition according to claim 5 in the preparation of drugs for preventing, inhibiting or treating aristolochic acid-induced nephrotoxicity diseases.

10. The drug according to claim 9, characterized in that The dosage form of the drug is tablet, capsule, granule, powder, solution, emulsion, suspension, suppository, powder or lyophilized powder injection.

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