Application of chiral palladium nano-cluster in preparation of medicine for treating multiple sclerosis

Abstract

The invention discloses an application of a chiral palladium nano-cluster in preparation of a medicine for treating multiple sclerosis. The chiral palladium nano-cluster with good biocompatibility is prepared by using L-tartaric acid as a stabilizer through a specific synthesis method. The chiral palladium nano-cluster can relieve inflammatory infiltration and demyelinating lesion of a central nervous system of an EAE mouse, regulate the proportion of immune cells in the spleen and reduce the content of various inflammatory cytokines in serum, so that the disease state is comprehensively improved. Compared with the existing therapeutic drugs, the chiral palladium nano-cluster provided by the invention can relieve inflammatory infiltration and demyelinating lesion of the central nervous system of an EAE mouse, deeply regulate the proportion of immune cells in the spleen of the mouse, and remarkably reduce the level of various inflammatory factors in serum, thereby providing a new thought and method for treating multiple sclerosis, and having broad application prospects. Wide application prospects and important clinical significance are realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to the application of chiral palladium nanoclusters in the preparation of drugs for treating multiple sclerosis. Background Art

[0002] Multiple sclerosis (MS) is a complex autoimmune disease, the main feature of which is inflammatory demyelination of the central nervous system (CNS). This disease is common in young adults aged 20 to 40, and the incidence rate in women is higher than that in men. The pathogenesis of MS is complex, involving multiple factors such as abnormal immune system, environmental factors and genetic factors, resulting in demyelination and axonal injury, and triggering a series of neurological dysfunctions.

[0003] Experimental autoimmune encephalomyelitis (EAE), as a classical animal model of MS, shows similar pathological features of inflammatory demyelination of the central nervous system to MS by simulating the autoimmune response of MS patients. In the EAE model, the strong immune activation of myelin antigen-specific CD4 + T cells is a key link in the occurrence of the disease. Therefore, by intervening in the disease process of the EAE model, valuable clues can be provided for the development of new drugs for treating MS.

[0004] Although there are currently various immunomodulatory and immunosuppressive drugs used to treat MS, these drugs are often accompanied by certain side effects, and the curative effects vary from person to person. Therefore, the development of new, highly effective and low-side-effect drugs for treating MS has become an urgent need in current research.

[0005] In recent years, the application of nanotechnology in the field of biomedicine has provided new ideas and methods for disease treatment. Chiral nanomaterials, especially chiral palladium nanoclusters, have shown great application potential in the field of biomedicine due to their unique physical and chemical properties, good biocompatibility and potential optical activity. However, the application of chiral palladium nanoclusters in the treatment of multiple sclerosis and its animal model EAE has not been fully explored. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides the application of chiral palladium nanoclusters in the preparation of drugs for treating multiple sclerosis.

[0007] In one embodiment of the present invention, the chiral palladium nanoclusters are further prepared into drugs for treating experimental autoimmune encephalomyelitis (EAE).

[0008] In one embodiment of the present invention, the drug is administered by dissolving dry chiral palladium nanoclusters in physiological saline to form a drug solution, and the drug solution is administered to EAE model mice by tail vein injection at a dose of 0.2 - 0.8 mg / kg body weight;

[0009] Further, a dose of 0.8 mg / kg body weight is selected as the administration dose.

[0010] In one embodiment of the present invention, the drug solution, as one type of drug dosage form, includes but is not limited to dosage forms suitable for intravenous administration such as aqueous solutions, suspensions, emulsions, etc.

[0011] In one embodiment of the present invention, the drug can reduce the degree of inflammatory infiltration in the central nervous system of EAE mice and reduce the occurrence of demyelination lesions.

[0012] In one embodiment of the present invention, the drug can regulate the relative proportion of immune cells in the spleen of mice.

[0013] In one embodiment of the present invention, the drug improves the disease state of EAE mice by reducing the contents of cellular inflammatory factors such as interleukin - 2, interleukin - 4, interleukin - 10, interleukin - 17, and tumor necrosis factor - γ in the serum of EAE mice.

[0014] Traditional drugs for the treatment of multiple sclerosis mostly rely on immune regulation and immunosuppression mechanisms. Although they can relieve the condition to a certain extent, they are often accompanied by non - negligible side effects such as decreased immune function and increased infection risk, and the curative effect is limited for some patients. In addition, the mechanism of action of these drugs is relatively single and it is difficult to comprehensively deal with the complex pathogenesis of MS.

[0015] Compared with the prior art, it has the following advantages:

[0016] As a new treatment means, the chiral palladium nanoclusters of the present invention have the following advantages: First, they have good biocompatibility, and using L - tartaric acid as a stabilizer reduces side effects; Second, through a specific synthesis method, the operation is simple, chiral palladium nanoclusters are successfully prepared, and their significant curative effect in treating EAE mouse models is confirmed; while effectively reducing the inflammatory infiltration and demyelination lesions in the central nervous system of EAE mice, it can also deeply regulate the proportion of immune cells in the spleen of mice and significantly reduce the levels of various inflammatory factors in the serum, thereby comprehensively improving the disease condition of EAE mice; Third, the administration method is simple and safe. After being dissolved in physiological saline, it is administered to EAE model mice by tail vein injection, realizing the rapid and efficient delivery of the drug; improving bioavailability and reducing the inconvenience of medication. In short, the present invention provides a new idea for the treatment of MS and has broad prospects and clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Neurological dysfunction scores for mice with EAE model, mice treated with 0.2 mg / kg chiral palladium nanoclusters, mice treated with 0.5 mg / kg chiral palladium nanoclusters, and mice treated with 0.8 mg / kg chiral palladium nanoclusters.

[0018] Figure 2 Hematoxylin-eosin (HE) staining images of the spinal cords of two groups of mice, scale bar: 200 μm.

[0019] Figure 3 Detection results of macrophages, neutrophils, and lymphocytes in the spleens of two groups of mice.

[0020] Figure 4 Determination results of the contents of serum inflammatory cytokines in two groups of mice. Detailed implementation manners

[0021] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the specific embodiments cited are not intended to limit the present invention.

[0022] Example 1

[0023] Dissolve 1.4 g of L-tartaric acid in 100 mL of benzyl alcohol, place it in a water bath at 55 °C, and stir for 60 minutes under a nitrogen atmosphere to ensure that the tartaric acid is completely dissolved and a homogeneous 0.1 M mixture is formed. While continuously stirring, slowly add 10 mL of 0.01 M chloropalladic acid (H 2 PdCl 4 ) solution dropwise to 100 mL of the tartaric acid-benzyl alcohol mixture. Subsequently, quickly add 1 mL of 0.2 M potassium borohydride (KBH 4 ) solution, and continue to stir for 60 minutes at 55 °C under a nitrogen atmosphere. After the reaction is completed, let the reaction system cool naturally to room temperature. Centrifuge at 12,000 rpm for 15 minutes to collect the palladium nanocluster precipitate. Wash it with anhydrous ethanol 3 - 5 times, and centrifuge again after each wash to completely remove the unreacted chloropalladic acid, potassium borohydride and its decomposition products, as well as the residual benzyl alcohol and tartaric acid. Finally, place the washed chiral palladium nanocluster precipitate in a vacuum drying oven and dry it at 42 °C for several hours to obtain dry chiral palladium nanocluster powder.

[0024] Example 2

[0025] The method for establishing an experimental autoimmune encephalomyelitis (EAE) mouse model used in the present invention is as follows: Female C57BL / 6 mice aged 8 - 10 weeks are randomly divided into an EAE model group and a treatment group, with 5 mice in each group. In the EAE model group, 0.2 mL of a mixture of MOG35 - 55 and Freund's complete adjuvant is subcutaneously injected into the back. 100 μL of pertussis toxin solution (1 g / L) is intraperitoneally injected 2 h and 24 h after immunization to induce EAE lesions in the mice. In the treatment group, a suspension of MOG35 - 55 and Freund's complete adjuvant is injected into the back and an equal amount of pertussis toxin solution is intraperitoneally injected. The dried chiral palladium nanoclusters are dissolved in physiological saline (ultrasonic acceleration for dissolution), and the palladium nanocluster solution is injected into the tail vein at a dose of 0.2 - 0.8 mg / kg (body weight). The dose groups of the treatment group are further divided into: 0.2 mg / kg, 0.5 mg / kg, and 0.8 mg / kg (body weight).

[0026] After the EAE model is constructed, the degree of lesion change in the mice is evaluated every 3 days, and the neurological deficit score is recorded. The neurological deficit score of the mouse EAE model is based on the following criteria: 0, no obvious abnormality in motor function; 0.5, the tail begins to be weak; 1, the tail is completely soft and weak; 1.5, the mouse's tail is completely weak and the hind limbs show abnormalities; 2, bilateral hind limb dragging; 2.5, unilateral hind limb paralysis; 3, all hind limbs of the mouse are paralyzed; 3.5, the forelimbs become weak; 4, partial paralysis of the forelimbs; 4.5, complete paralysis of all four limbs; 5, on the verge of death.

[0027] As Figure 1 shown, after the EAE model is established in the mice, the neurological deficit scores of all groups are the highest and the tails are paralyzed, indicating successful model establishment. In the model group, as time goes by, the mice gradually show symptoms such as reduced appetite, listlessness, hair loss, unsteady gait, and varying degrees of tail paralysis and limb paralysis. In the chiral palladium nanocluster treatment group, the onset symptoms of the mice gradually improve with treatment, and are related to the dose of chiral palladium nanoclusters. The mental state and neurological deficit score of the mice are improved, indicating that chiral palladium nanoclusters can effectively improve the development of MS. In the present invention, the dose of 0.8 mg / kg with the best treatment effect is selected as the experimental dose for the subsequent examples.

[0028] Example 3

[0029] To further evaluate chiral palladium nanoclusters at the histopathological level, the mice are sacrificed on the 15th day after treatment. The spinal cord tissues of the mice are fixed in 4% paraformaldehyde, dehydrated with gradient alcohol, embedded in paraffin, sectioned, stained with hematoxylin - eosin (HE), dehydrated again with gradient alcohol, and sealed with neutral gum. Observation is carried out under an optical microscope, and the target area is selected for photographing.

[0030] The HE staining results show ( Figure 2) In the spinal cord tissues of the mice in the EAE model group, a large number of inflammatory cells were infiltrated, and a large amount of nerve myelin was lost. However, in the spinal cord tissues of the mice in the chiral palladium nanocluster treatment group, the degree of inflammatory infiltration and demyelination was significantly lower than that of the mice in the model group, indicating that the chiral palladium nanocluster prepared by the present invention has a significant alleviating effect on experimental autoimmune encephalomyelitis (EAE). This finding not only verifies the therapeutic potential of chiral palladium nanoclusters, but also provides strong histological evidence for the further development of chiral palladium nanoclusters as a candidate drug for the treatment of multiple sclerosis. As a novel therapeutic means, chiral palladium nanoclusters have shown significant effects of reducing inflammatory responses, protecting nerve myelin, and improving neuropathological structures in the experimental autoimmune encephalomyelitis model.

[0031] Example 4

[0032] Take the spleens of the mice in the EAE model group and the chiral palladium nanocluster treatment group, grind them, and prepare 200 μL of single-cell suspension. Pipette 50 μL from each of the two groups of cell suspensions into a flow cytometry sample tube, wash twice with an appropriate amount of PBS, add a mouse-specific antibody suspension (CD4-PE-Cy7, CD8-V500, CD11b-FITC, GR-1-V450, F4 / 80-Percp-Cy5.5), incubate at room temperature in the dark for 15 min, then wash, resuspend with an appropriate amount of PBS, and detect the relative proportions of immune cells in the mouse spleens by flow cytometry. Analyze the results with Flowjo software.

[0033] Macrophages play an important role in the inflammatory response. They can phagocytose pathogens and release pro-inflammatory factors, thus exacerbating the inflammatory process. Compared with the EAE model group, the macrophages (CD11b + , F4 / 80 + ) in the spleens of the mice in the chiral palladium nanocluster treatment group decreased ( Figure 3 A), and the treatment with chiral palladium nanoclusters reduced the inflammatory response in the spleen and decreased the intensity of autoimmune attack. Neutrophils are another important type of inflammatory cells. They can rapidly migrate to the site of infection or injury, release a variety of inflammatory mediators, and participate in the acute inflammatory response. The decrease in the percentage of neutrophils (CD11b + , GR-1 + ) also indicates that this treatment helps to reduce the inflammatory activity in the spleen and possibly in the central nervous system ( Figure 3 B). It is worth noting that the percentage of CD4 + T cells increased ( Figure 3 C), further confirming that the treatment with chiral palladium nanoclusters can significantly change the relative proportions of immune cells in the spleens of EAE mice.

[0034] Example 5

[0035] Cell inflammatory factors, such as interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-17 (IL-17) and tumor necrosis factor-γ (TFN-γ), play crucial roles in the pathogenesis of multiple sclerosis (MS) and its animal model EAE. Their abnormal expression is often closely related to the progression and severity of the disease. To further explore the effect of chiral palladium nanocluster treatment on the content of cell inflammatory factors in the serum of EAE mice, we extracted the serum of mice from the tail vein and detected the content of cytokines in the serum by flow cytometry. As Figure 4 shown, compared with the mice in the EAE model group, the contents of IL-2, IL-4, IL-10, IL-17, and TFN-γ in the serum of EAE mice treated with chiral palladium nanoclusters showed a decreasing trend. Among them, the content of IL-4 decreased most significantly after treatment with chiral palladium nanoclusters, nearly halving compared with the control group. The above results indicate that chiral palladium nanocluster treatment can effectively reduce the content of serum inflammatory cytokines in EAE mice and has an effective improvement effect on the disease state of EAE mice.

[0036] The embodiments described above are only a part of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but only represents the selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

Claims

1. Application of chiral palladium nanoclusters in the preparation of drugs for treating multiple sclerosis.

2. The use according to claim 1, characterized in that: The chiral palladium nanoclusters are specifically used for preparing drugs for treating experimental autoimmune encephalomyelitis (EAE).

3. The use according to claim 2, characterized in that: The drug is administered by dissolving the dried palladium nanoclusters in physiological saline to form a drug solution, and then injecting the drug solution into EAE model mice via tail vein at a dose of 0.2 to 0.8 mg / kg body weight.

4. The use according to claim 3, characterized in that: The administration dose is preferably 0.8 mg / kg.

5. The use according to claim 2, characterized in that: The drug solution, as a kind of drug dosage form, includes but is not limited to aqueous solution, suspension, emulsion and other dosage forms suitable for intravenous administration.

6. The use according to claim 2, characterized in that: The drug can reduce the degree of inflammatory infiltration in the central nervous system of EAE mice and reduce the occurrence of demyelinating lesions.

7. The use according to claim 2, characterized in that: The drug can regulate the relative proportion of immune cells in the spleen of mice.

8. The use according to claim 2, characterized in that: The drug improves the disease state of EAE mice by reducing the content of interleukin-2, interleukin-4, interleukin-10, interleukin-17, tumor necrosis factor-γ and other cellular inflammatory factors in the serum of EAE mice.

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