Application of magnesium salvianolate B in preparation of medicine for preventing or treating sepsis and related multi-organ dysfunction thereof

Abstract

The invention discloses an application of magnesium salvianolic acid B (MLB) in preparation of a medicine for preventing or treating sepsis and related multi-organ dysfunction of the sepsis, and particularly discloses an application of magnesium salvianolic acid B (MLB) in preparation of a medicine for preventing or treating the sepsis and the related multi-organ dysfunction of the sepsis and the related multi-organ dysfunction of the sepsis. Experiments prove that in a sepsis animal model, the MLB can significantly improve the survival rate, relieve multi-organ injury, improve hematological abnormality and organ functions, and relieve iron-dependent injury by stabilizing an antioxidant system. And the MLB is natural in source and high in safety, and has a potential application prospect as a candidate drug for preventing and treating sepsis. The invention provides an effective technical means for preventing or treating sepsis and related multi-organ dysfunction thereof.

Description

Technical Field

[0001] This invention relates to a novel use of a natural product in medicine, specifically the use of magnesium lithospermate B (MLB) in the preparation of medicines for the prevention or treatment of sepsis and related multiple organ dysfunction. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, characterized by an imbalance in the body's immune response to infection. This imbalance can lead to impaired tissue perfusion and microcirculatory dysfunction, ultimately resulting in multiple organ dysfunction syndrome (MODS). Clinical data shows that severe sepsis has a high mortality rate, and there are currently no targeted treatments. Current treatments mainly include anti-infection therapy, fluid resuscitation, mechanical ventilation, hemodynamic support, and organ function maintenance. However, these methods are primarily supportive and cannot directly intervene in the pathological process of the disease.

[0003] Existing research has shown that the development of sepsis involves multiple molecular mechanisms, including the release of inflammatory factors, oxidative stress, cellular metabolic disorders, and iron-dependent cell damage. Persistent inflammatory activation leads to cell damage and increased capillary permeability, causing structural and functional abnormalities in multiple organs such as the lungs, liver, kidneys, heart, and brain. Due to the complexity of the pathological process, the overall efficacy of currently used anti-inflammatory or antioxidant drugs is limited, and some drugs also have safety or pharmacokinetic issues. For example, while glucocorticoids can suppress inflammation in the short term, long-term use may lead to immunosuppression and secondary infections; the antioxidant N-acetylcysteine ​​(NAC) has unstable efficacy and dose-related adverse reactions; the mitochondrial-targeting antioxidant MitoQ has certain activity, but its pharmacokinetic properties are unsatisfactory; and inflammatory factor antagonists (such as anti-TNF-α antibodies) may affect normal immune responses in some patients. Therefore, exploring new small molecule compounds related to sepsis treatment is of positive significance for improving patient prognosis.

[0004] Magnesium lithospermate B (MLB) is derived from the traditional Chinese medicine Danshen (Salvia miltiorrhiza). Salvia miltiorrhiza The main water-soluble polyphenolic acid salts isolated from MLB exhibit clear anti-inflammatory and antioxidant activities. The chemical structural formula of MLB is as follows: Figure 1As shown in the figure. Previous studies have shown that MLB has a certain protective effect in models of myocardial ischemia, kidney injury, and liver ischemia-reperfusion, and can improve tissue redox status and reduce inflammatory response. However, to date, there are no literature or patent reports on the application of MLB in the overall pathological process of sepsis, nor has its intervention effect on multiple organ dysfunction been systematically studied.

[0005] Therefore, based on the bioactivity of MLB in various tissue damage models, further exploration of its role and potential value in the treatment of sepsis is of great significance. This invention, focusing on experimental research on sepsis-related multiple organ dysfunction, is the first to discover that MLB can improve survival rates and tissue damage indicators, providing a new research direction for drug intervention in sepsis. Summary of the Invention

[0006] The purpose of this invention is to provide the use of magnesium salvianolic acid B in the preparation of drugs for the treatment of sepsis and related multiple organ dysfunction.

[0007] To achieve the above objectives, the present invention employs the following technical means: In a cecal ligation and puncture (CLP)-induced sepsis mouse model, tail vein injection of MLB (10–30 mg / kg) significantly improved the 5-day survival rate of the animals. Histological examination showed that MLB significantly alleviated pathological changes such as alveolar structural damage, renal tubular injury, and hippocampal neuronal degeneration, and reduced the wet-to-dryness ratio of lung tissue and Evans Blue exudate, suggesting that it can effectively improve tissue permeability and organ edema. Serum biochemical tests showed that MLB could reduce tissue damage-related enzymatic indicators such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and alanine aminotransferase (ALT), while significantly improving serum creatinine (Cr) and blood urea nitrogen (BUN) levels, thereby alleviating liver and kidney dysfunction.

[0008] Blood routine tests further confirmed that MLB can correct peripheral blood abnormalities caused by sepsis. Compared with the CLP group, MLB treatment significantly reduced the number of white blood cells (WBC) and granulocytes (GRA), significantly increased the number of lymphocytes (LYM), and restored platelet (PLT) to near control levels, while red blood cell (RBC) showed no significant change, suggesting that MLB can restore inflammation-related hematological balance.

[0009] To explore its potential mechanism of action, this invention further employed molecular docking, molecular dynamics simulations, and biophysical experiments to verify the binding ability of MLB to glutathione peroxidase 4 (GPX4) protein. The results showed that MLB and GPX4 can form a stable hydrogen bond network, and the complex remained stable in a 100 ns molecular dynamics simulation. CETSA experiments demonstrated that MLB treatment significantly improved the thermal stability of GPX4 protein; surface plasmon resonance (SPR) analysis showed that the equilibrium dissociation constant (K_D) of the two proteins was approximately 25.94 μM, proving a direct binding between MLB and GPX4. Transmission electron microscopy and Western blot results further showed that MLB could alleviate mitochondrial structural damage in CLP mice and upregulate the expression levels of GPX4 and SLC7A11, suggesting that it can alleviate iron-dependent cell damage by maintaining the stability of the antioxidant defense system.

[0010] Based on the above research, this invention proposes the use of magnesium lithospermate B (MLB) in the preparation of drugs for the treatment or prevention of sepsis and related multiple organ dysfunction.

[0011] Preferably, the magnesium tanshinone B can significantly reduce the structural damage and edema of lung tissue caused by sepsis, and improve hepatocyte degeneration, renal tubular necrosis and neuronal damage in the hippocampus.

[0012] Preferably, the magnesium tanshinone B can reduce serum levels of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and alanine aminotransferase (ALT), significantly reducing liver damage; at the same time, it reduces creatinine (Cr) and blood urea nitrogen (BUN) levels, improving renal function.

[0013] Preferably, the drug is an injectable drug.

[0014] Preferably, the purity of magnesium tanshinone B in the drug is not less than 98%.

[0015] Preferably, the drug is prepared using sterile PBS, 5% w / w glucose solution, or 0.9% w / w sodium chloride injection as a solvent.

[0016] Preferably, the drug also contains 0.1% w / w ascorbic acid to prevent oxidation.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention proposes for the first time a novel use of magnesium salvianolic acid B (MLB) in the preparation of drugs for the treatment or prevention of sepsis and related multiple organ dysfunction. MLB significantly improves survival rates, reduces multi-organ damage, improves hematological abnormalities and function in animal models of sepsis, and alleviates iron-dependent damage by stabilizing the antioxidant system. MLB is naturally derived, has a well-defined chemical structure, favorable pharmacokinetic properties, and high safety profile, demonstrating its potential application value as a candidate drug for the prevention and treatment of sepsis and multiple organ dysfunction. Attached Figure Description

[0018] Figure 1 The chemical structural formula of MLB; Figure 2 MLB improves the survival rate of CLP mice and reduces multi-organ tissue damage; Where A represents the 5-day survival rate of mice in each group; B represents the HE staining results of lung, kidney, and brain tissues of mice in each group; C and D represent the decrease in pathological scores of lung and kidney tissues of mice in each group; F represents the wet-to-dry ratio of lungs of mice in each group; G represents the Evans Blue content of mice in each group; and E and H represent the serum ALT, ALP, LDH, BUN, and Cr levels of mice in each group. Figure 3 MLB improves abnormal blood counts in CLP mice; The results for AE represent the white blood cell (WBC), lymphocyte (LYM), granulocyte (GRA), platelet (PLT), and red blood cell (RBC) counts, respectively. Figure 4A- E and Figure 4F- I represents the binding of MLB to GPX4 and the inhibition of iron-dependent cell damage; In the figure, A is the simulation result of MLB and GPX4 molecule docking; B is the RMSD result of MLB and GPX4 molecule binding; C is the RMSF result of MLB and GPX4 molecule binding; D and E are the stability results of GPX4 protein after cell lysate is treated with different temperature gradients; F and G are the binding affinity of MLB and GPX4 determined by surface plasmon resonance analysis (SPR); H is the transmission electron microscopy result of mouse lung tissue mitochondria; and I is the Western blot detection result of GPX4 and SLC7A11. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0020] Example 1: Protective effect of MLB on a CLP-induced sepsis mouse model method: 1. Establishment of experimental animals and models The experimental animals were SPF-grade male C57BL / 6J mice (20 ± 2 g), purchased from the Beijing Vital River Laboratory Animal Center. They were housed under constant temperature and humidity conditions and a 12-hour light-dark cycle, with free access to food and water.

[0021] A sepsis model was established using cecal ligation and puncture (CLP). The procedure involved anesthetizing mice, making a midline abdominal incision to expose the cecum, ligating it approximately halfway down the cecum, and puncturing it twice with a 22G needle. After gently squeezing out a small amount of feces, the cecum was returned to its original position and the abdominal wall was sutured. In the Sham group, only an abdominal incision was performed without ligation or puncture.

[0022] 2. Grouping and Dosing Regimen (n = 6 / group) Sham group: An equal volume of normal saline was injected via tail vein; CLP group: After modeling, physiological saline was injected into the tail vein; CLP + MLB-L group: MLB (15 mg / kg) was injected via tail vein 24 h before and 2 h after surgery. CLP + MLB-H group: MLB (30 mg / kg) was injected via the tail vein 24 hours before and 2 hours after surgery.

[0023] 3. Detection indicators Survival rate: Survival curves were plotted after 5 consecutive days of postoperative observation; Histological examination: Lung, kidney, and brain tissues were taken, fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (HE). Lung permeability and edema: Detection of lung wet-to-dry ratio and Evans Blue exudate; Serum biochemical indicators: The levels of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), alanine aminotransferase (ALT), creatinine (Cr), and blood urea nitrogen (BUN) were measured. Multi-organ injury scoring: Pathological scoring of lung and kidney tissues in mice of each group.

[0024] Experimental results: The results are as follows Figure 2 As shown, MLB significantly improved the 5-day survival rate of CLP mice. Figure 2 A). HE staining showed that MLB treatment could reduce alveolar structural damage, renal tubular necrosis, and neuronal degeneration in the hippocampus. Figure 2 B). MLB treatment reduced lung and kidney tissue pathological scores in mice. Figure 2 C, D), lung wet-to-dry ratio and Evans Blue content were significantly reduced ( Figure 2F, G), serum ALT, ALP, LDH, BUN and Cr levels all decreased ( Figure 2 E, H), indicating that MLB effectively reduces pathological damage to multiple organs and improves functional indicators.

[0025] Example 2: The ameliorative effect of MLB on hematological abnormalities 1. Experimental Methods In the CLP model established in Example 1, peripheral blood was collected 24 hours post-surgery, and key hematological parameters, including white blood cell (WBC), lymphocyte (LYM), granulocyte (GRA), platelet (PLT), and red blood cell (RBC) counts, were detected using a fully automated hematology analyzer. The grouping and administration regimens were the same as in Example 1.

[0026] 2. Experimental Results The results are as follows Figure 3 As shown in the AE, compared with the Sham group, the CLP group mice exhibited significant inflammation-related hematological abnormalities: increased WBC and GRA, significantly decreased LYM, and decreased PLT. After MLB treatment, all of the above indicators were significantly improved: WBC and GRA decreased, LYM recovered, and PLT significantly increased, while RBC showed no significant change.

[0027] The results showed that MLB can effectively correct peripheral blood abnormalities caused by sepsis and improve systemic inflammatory status.

[0028] Example 3: MLB binds to GPX4 and inhibits iron-dependent cell damage 1. Protein-protein interaction analysis Molecular docking simulations revealed (Figure 4A) that MLB can form a stable hydrogen bond network with glutathione peroxidase 4 (GPX4). Molecular dynamics (MD) simulations showed that the composite structure remained stable within 100 ns (RMSD fluctuation less than 0.8 nm) (Figures 4B and 4C).

[0029] 2. Protein stability assay (CETSA) Cell lysates were treated with different temperature gradients, and the stability of GPX4 protein was then assessed. The results showed that the GPX4 signal in the MLB-treated group decreased more slowly with increasing temperature, indicating that MLB can enhance the thermal stability of GPX4 (Figures 4D and E).

[0030] 3. Bond strength test (SPR) The binding affinity between MLB and GPX4 was determined by surface plasmon resonance (SPR) analysis, and the equilibrium dissociation constant (K_D) was calculated to be approximately 25.94 μM, indicating that the two are directly bound together (see Figures 4F and G).

[0031] 4. In vivo validation Transmission electron microscopy revealed swollen and fractured mitochondria in the lung tissue of CLP-treated mice, while MLB treatment significantly restored mitochondrial morphology (see Figure 4H). Western blot analysis showed that MLB upregulated the expression of GPX4 and SLC7A11, suggesting that it can maintain cell morphology. Cellular antioxidant defense capacity, reducing iron-dependent damage (see...) Figure 4I ).

[0032] The results of the above examples show that: MLB can significantly improve the survival rate of septic animals, reduce histological damage to multiple organs such as the lungs, liver, kidneys, and brain, improve hematological abnormalities and organ function indicators, and maintain cell homeostasis by binding to the antioxidant protein GPX4.

[0033] The experimental results fully verify the protective effect of MLB in sepsis and its related multiple organ dysfunction, proving that the technical solution described in this invention is feasible and has application value.

Claims

1. Use of magnesium lithospermate B (MLB) in the preparation of drugs for the prevention or treatment of sepsis and related multiple organ dysfunction.

2. The use as described in claim 1, characterized in that, The aforementioned magnesium tanshinone B can significantly reduce the structural damage and edema of lung tissue caused by sepsis, and improve hepatocyte degeneration, renal tubular necrosis and neuronal damage in the hippocampus.

3. The use as described in claim 1, characterized in that, The aforementioned magnesium tanshinone B can reduce serum levels of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and alanine aminotransferase (ALT), significantly reducing liver damage; at the same time, it reduces creatinine (Cr) and blood urea nitrogen (BUN) levels, improving renal function.

4. The use as described in claim 1, characterized in that, The drug in question is an injectable drug.

5. The use as described in claim 4, characterized in that, The purity of magnesium tanshinone B in the drug is not less than 98%.

6. The use as described in claim 5, characterized in that, The drug is prepared using sterile PBS, 5% w / w glucose solution, or 0.9% w / w sodium chloride injection as a solvent.

7. The use as described in claim 6, characterized in that, The drug also contains 0.1% w / w ascorbic acid to prevent oxidation.

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