Application of lithospermic acid in the preparation of drugs for improving acute kidney injury caused by sepsis

Abstract

The present invention discloses the application of lithospermic acid in the preparation of a drug for improving acute kidney injury caused by sepsis, and belongs to the field of medical technology. The present invention discloses the application of lithospermic acid in the preparation of a drug for improving acute kidney injury caused by sepsis, and the improvement of acute kidney injury caused by sepsis includes inhibiting the polarization and infiltration of M1 macrophages, reducing inflammatory damage, and reducing the release of inflammatory factors in septic mice. The present invention first discovered that lithospermic acid can improve the survival rate of septic mice. Through animal experiments, it was found that lithospermic acid can reduce the 7-day mortality rate of septic mice and reduce kidney damage in septic mice after 24 hours. It has a good application prospect in the preparation of drugs for treating acute kidney injury caused by sepsis, and has opened up a new medical approach for the prevention and treatment of septic complications.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, in particular to application of lithospermic acid in preparing a medicine for improving acute kidney injury caused by sepsis. Background Art

[0002] Acute kidney injury (AKI) is the most common complication of sepsis. About 30% of sepsis patients will experience AKI, which is called sepsis associated with acute kidney injury (SA-SAKI). SA-AKI increases the risk of multiple comorbidities and has a very high mortality rate. It has always occupied an important position in global medical problems. Sepsis includes a series of different types of diseases: since the definition of sepsis and septic shock (Sepsis-3) was proposed in 2016, sepsis-associated acute kidney injury is usually defined as sepsis or septic shock involving the kidneys, leading to progressive decline in renal function. Septic shock is a subset of sepsis, and compared with simple sepsis, it has a higher risk of death in blood circulation, molecular and metabolic abnormalities. As separate syndromes, sepsis and acute kidney injury make the host susceptible to each other, and it is often difficult to determine the exact onset time of these two syndromes clinically.

[0003] Lithospermic acid (LA) is a monomer form isolated from the traditional Chinese medicine Salvia miltiorrhiza, belonging to the salvianolic acid class. Studies have reported the use of lithospermic acid ester compounds in the preparation of drugs for treating malaria, and the use of lithospermic acid in the preparation of anti-influenza virus drugs. So far, there has been no report on the use of lithospermic acid in the preparation of drugs for improving acute kidney injury caused by sepsis. Summary of the invention

[0004] The purpose of the present invention is to provide an application of lithospermic acid in the preparation of a drug for improving acute kidney injury caused by sepsis, so as to solve the problems existing in the prior art. The present invention finds that lithospermic acid can inhibit the polarization of M1 macrophages, inhibit the infiltration of macrophages, reduce the inflammatory response of acute kidney injury caused by sepsis, and inhibit the release of inflammatory factors, thereby reducing acute kidney injury caused by sepsis.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical solution 1: Application of lithospermic acid in the preparation of drugs for improving acute kidney injury caused by sepsis.

[0007] Furthermore, the improvement of acute kidney injury in sepsis includes inhibiting the polarization and infiltration of M1 macrophages, alleviating inflammatory damage and reducing the release of inflammatory factors in septic mice.

[0008] Furthermore, the inflammatory factors of the septic mice include inflammatory factors released by M1 macrophages.

[0009] Furthermore, lithospermic acid inhibits the polarization and infiltration of the M1 macrophages by activating AMPK.

[0010] Technical Solution 2: A drug for improving acute kidney injury caused by sepsis, the active ingredient of which includes lithospermic acid.

[0011] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0012] The present invention discloses the following technical effects:

[0013] Lithospermic acid can effectively improve acute kidney injury caused by sepsis. The present invention first describes that lithospermic acid can alleviate acute kidney injury caused by sepsis, and can not only inhibit the polarization of M1 macrophages, but also inhibit the infiltration of macrophages, alleviate the inflammatory response of acute kidney injury caused by sepsis, and inhibit the release of inflammatory factors, thereby alleviating acute kidney injury caused by sepsis. It has a good application prospect in the preparation of drugs for treating acute kidney injury caused by sepsis, and opens up a new medical approach for the prevention and treatment of complications of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 The survival curves of mice in each group within 7 days are shown in Figure 1. The cecal ligation and puncture technique was used to establish the mouse model of sepsis, with n = 20 in each group;

[0016] Figure 2 HE staining of kidneys of mice with sepsis 24 hours later, n=5 per group; the scale bar at 4X magnification is 1000μm, and the scale bar at 20X magnification is 200μm;

[0017] Figure 3 A in the middle is immunofluorescence double-labeling staining of kidneys of septic mice 24 hours after treatment, n=5 in each group; the scale bar for 4X magnification is 1000μm, and the scale bar for 20X magnification is 200μm; Figures B, C, and D respectively show that lithospermic acid inhibits the release of inflammatory factors MCP-1, TNF-α, and IL-6 in septic mice; *P<0.05, vs.Con; #P<0.05, vs.LPS; n=10;

[0018] Figure 4Lithospermic acid inhibited the polarization of M1 macrophages. A is the expression and quantification results of iNOS and CD206 in RAW264.7 cells detected by Western blot; B is the expression and quantification results of iNOS and CD206 in THP-1 cells detected by Western blot; *P<0.05, vs.Con; **P<0.05, vs.LPS; ***P<0.05, vs.LPS; n=4;

[0019] Figure 5 Lithospermic acid inhibited the release of inflammatory factors in macrophages; ELISA was used to detect the expression levels of TNF-a and IL-6 in macrophages; *P<0.05, vs.Con; **P<0.05, vs.LPS; ***P<0.05, vs.LPS; n=4;

[0020] Figure 6 The molecular structure of lithospermic acid;

[0021] Figure 7 A three-dimensional ribbon model of the lithospermic acid-AMPK complex, showing the amino acid residues interacting with lithospermic acid and the length of hydrogen bonds;

[0022] Figure 8 It is a surface simulation model of the lithospermic acid-AMPK complex;

[0023] Fig. 9 Lithospermic acid increases the phosphorylation level of AMPK; A is the expression and quantification results of p-AMPK in RAW264.7 cells detected by Western blot; B is the expression and quantification results of p-AMPK in THP-1 cells detected by Western blot; #P<0.05, vs.LPS; **P<0.05, vs.LPS; n=4;

[0024] Fig.10 Knocking down the expression of AMPK can significantly block the inhibitory effects of lithospermic acid on M1 macrophage polarization and inflammation; A is the Western blot detection of iNOS and CD206 expression in RAW264.7 cells and the quantification results; B is the Western blot detection of iNOS and CD206 expression in THP-1 cells and the quantification results; C and D are the inhibition of the release of macrophage inflammatory factors TNF-α and IL-6 by lithospermic acid after knocking down AMPK; *P<0.05, vs. NC-si; **P<0.05, vs. LPS+NC-si; n=3. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Experimental animals: SPF male C57BL / 6 mice were purchased from Guangdong Experimental Animal Center, 5-6 weeks old, 20-22g. All experimental animals were humanely treated in accordance with the Guidelines for the Management and Use of Experimental Animals. The experimental protocol was approved by the Experimental Animal Ethics Committee of Guangzhou Medical University. The disease model was established after 8-10 weeks of age and 22-25g. Before the experiment, they were free to forage for food, drink water, and be exposed to natural light. Cecal ligation and puncture (CLP) was used to induce a sepsis model in mice: 1.5% sodium pentobarbital was injected intraperitoneally at a dose of 2.5ml / kg according to the weight of the mouse for anesthesia, the mouse was fixed and depilated, and sterilized with iodine tincture twice and covered with a sterile drape. Then the skin and muscle layer were cut along the midline of the mouse abdomen, the cecum was carefully turned out, the feces in the intestine were squeezed to the blind end, one-third of the cecum was ligated, and then a 10mL syringe was used to puncture the blind end to squeeze out a small amount of feces. Finally, the cecum was returned and the skin and muscular layer were sutured, and the animals were kept warm and resuscitated on a 37°C warming blanket. Compared with the model group, the sham operation group had the same anesthesia method and surgical laparotomy steps as the model group. After turning out the cecum, the intestine was directly returned to the abdomen and the skin and muscular layer were sutured.

[0031] Example 1

[0032] 1. Experimental Materials

[0033] Lithospermic acid was purchased from Chengdu Profa Pharmaceuticals Co., Ltd.; LPS was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; DMEM medium was purchased from Gibco, USA; 1640 medium was purchased from Gibco, USA; fetal bovine serum was purchased from Wuhan Pronosai Life Science Co., Ltd.; ELISA kit was purchased from Wuhan Fine Biotechnology Co., Ltd.; BCA kit was purchased from Thermo Fisher Scientific, USA; iNOS, CD206, AMPK and other antibodies were purchased from CST, USA;

[0034] 2. Experimental cell culture and model establishment

[0035] RAW264.7 and human peripheral blood mononuclear cells (THP-1) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences.

[0036] The cells were cultured in a medium containing 95% air and 5% CO 2 When the cells were in good growth condition and the density reached 80%, they were divided into 4 groups according to the following treatments:

[0037] (1) Control group: control group;

[0038] (2) LPS alone group: LPS (500 ng / mL);

[0039] (3) LPS+50LA group: LPS(500ng / ml)+LA(50μM);

[0040] (4) LPS+75LA group: LPS (500 ng / ml) + LA (75 μM).

[0041] 3. Experimental methods

[0042] (1) 7-day mortality of septic mice and staining of kidney sections of septic mice 24 hours later

[0043] 60 mice were equally divided into three groups, namely Sham group (sham operation group), CLP group (cecal ligation and puncture) and LA+CLP group (cecal ligation and puncture were performed after intraperitoneal injection of lithospermic acid 50 mg / kg 24 hours before surgery). The survival status of mice in each group within 7 days was observed and recorded, and the survival curve of mice in each group within 7 days was drawn.

[0044] Another batch of 15 mice were divided into the above three groups. The kidneys of septic mice in each group were taken 24 hours after surgery for kidney HE staining and immunofluorescence double labeling staining.

[0045] (2) Western blot was used to detect the protein expression of iNOS and CD206, and ELISA was used to detect the expression of inflammatory factors

[0046] RAW264.7 and THP-1 cells were cultured with DMEM and 1640 medium containing 10% serum, respectively. When the cell density reached 80%, RAW264.7 and THP-1 were plated in 6-well plates and treated according to the above grouping for 24 hours. The cell supernatant was collected and centrifuged at 4°C, 2500 rpm for 5 minutes. The supernatant was taken out and set aside. Cell lysis buffer was added to the 6-well plate, cells were lysed on ice, and centrifuged at 4°C, 12000g for 15 minutes. The supernatant was transferred to a new EP tube, which was the protein sample stock solution. The protein concentration was determined using a BCA kit. 5× loading Buffer and protein sample were mixed in a ratio of 1:4, and placed in a 100°C water bath for 10 minutes; cooled on ice for 1 minute for later use; proteins were separated by 10% SDS-PAGE gel electrophoresis, and then transferred to the membrane, blocked, and incubated with the relevant primary antibody overnight. The next day, after washing the membrane and incubating with the secondary antibody, the PVDF membrane was placed in a chemiluminescence instrument for image acquisition, and quantitative analysis was performed using ImageJ software, with β-actin as the internal reference. The cell supernatant was tested for the expression of the corresponding inflammatory factors using the ELISA kit of Wuhan Fine Biotechnology Co., Ltd. according to the experimental steps, and the data obtained were converted using the calculation software provided by the company to obtain the target concentration of the sample for subsequent analysis.

[0047] 4. Statistical analysis

[0048] SPSS 21.0 software was used to perform statistical analysis on the data. Each group of experiments was repeated at least 3 times, and all data were expressed as mean ± standard error. Indicates. The Mann-Whitney U test was used in the nonparametric test for the significant difference between two groups or two subgroups with unequal variances, and the Kruskal-Wallis H test was used in the nonparametric test for the significant difference between multiple groups or multiple subgroups with unequal variances. The independent sample t test was used for the comparison between two groups with equal variances. P<0.05 was considered statistically significant.

[0049] 5. Results Analysis

[0050] (1) Lithospermic acid can reduce the 7-day mortality of septic mice and alleviate kidney damage in septic mice 24 hours later

[0051] Observe and record the survival status of each group of mice within 7 days, and draw the survival curve of each group of mice within 7 days, such as Figure 1 As shown, within the observation period of 7 days, lithospermic acid can reduce the 7-day mortality of septic mice.

[0052] Kidneys of septic mice in each group were obtained 24 hours after surgery and fixed and stained. Figure 2 and 3 As shown, lithospermic acid can alleviate sepsis-induced renal injury.

[0053] (2) Lithospermic acid can inhibit the polarization of monocytes into M1 macrophages and the release of inflammatory factors

[0054] RAW264.7 and THP-1 were plated in 6-well plates and treated according to the above groups for 24 hours before collecting cell proteins and supernatants. The expression and quantification of iNOS, CD206 protein and inflammatory factors were detected by Western blot and ELISA. Figure 4 and 5 As shown, lithospermic acid can inhibit the polarization of monocytes (RAW264.7, THP-1) to M1 macrophages, and inhibit the release of M1 macrophage inflammatory factors MCP-1 and TNF-α. Fig. 9 It can be seen that lithospermic acid increases the phosphorylation level of AMPK; Fig.10 It can be seen that knocking down the expression of AMPK can significantly block the effects of lithospermic acid in inhibiting M1 macrophage polarization and inhibiting inflammation.

[0055] (3) Molecular docking and molecular dynamics simulation results of lithospermic acid and AMPK

[0056] Molecular docking software Autodock Vina and molecular dynamics software YASARA were used for molecular docking and molecular dynamics simulation. Observing the binding affinity of ligand and receptor As long as the molecular docking is used, the amino acid residues that are worthy of attention in the interaction can reflect the interaction details between ligand and receptor. According to the analysis, the binding energy of AMPK-lithospermic acid complex is -9.07kcal / mol. Figure 6 This is the molecular structure of lithospermic acid; Figure 7 The three-dimensional ribbon model of the lithospermic acid-AMPK complex, as well as the amino acid residues and hydrogen bond lengths of the lithospermic acid interaction, is shown in the figure. As shown in the figure, four hydrogen bonds are formed between lithospermic acid and AMPK, and the hydrogen bond lengths are Figure 8 It is the surface simulation model of the lithospermic acid-AMPK complex at 0 ns and 100 ns.

[0057] The above results indicate that the binding of AMPK and lithospermic acid is stable. Molecular docking, molecular dynamics simulation and surface plasmon resonance experiments confirm that lithospermic acid is a potential AMPK activator and can directly bind to AMPK.

[0058] The above experimental results prove that lithospermic acid can inhibit the polarization and infiltration of M1 macrophages by activating AMPK, reduce the transcription of inflammation, reduce the release of inflammatory factors, and alleviate inflammatory damage.

[0059] In acute kidney injury caused by sepsis, uncontrollable inflammatory response is an important cause of its pathogenesis. Acute kidney injury caused by sepsis can be alleviated by reducing inflammatory response. In the development of inflammation, macrophages are the earliest and most common immune cells throughout the entire process, and macrophages have polarization characteristics. Lithospermic acid can inhibit the polarization of M1 type, thereby inhibiting its pro-inflammatory effect and alleviating acute kidney injury caused by sepsis. Therefore, the drug provided by the present invention has a good application prospect in the treatment of acute kidney injury caused by sepsis, and opens up a new medical approach for the prevention and treatment of acute kidney injury caused by sepsis.

[0060] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of lithospermic acid in the preparation of drugs for improving acute kidney injury caused by sepsis.

2. The use according to claim 1, characterized in that The improvement of acute kidney injury induced by sepsis includes inhibiting the polarization and infiltration of M1 macrophages, alleviating inflammatory damage and reducing the release of inflammatory factors in septic mice.

3. The use according to claim 2, characterized in that: The inflammatory factors of sepsis mice include inflammatory factors released by M1 macrophages.

4. The use according to claim 2, characterized in that: Lithospermic acid inhibits the polarization and infiltration of M1 macrophages by activating AMPK.

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