Application of gold nanocluster in preparation of medicine for treating sepsis liver injury

Abstract

The invention discloses application of a gold nanocluster in preparation of a medicine for treating sepsis liver injury. Animal and cell experiments prove that the gold nanocluster has a remarkable treatment effect on sepsis liver injury (SLI), and the levels of inflammatory factors in serum of model animals and aspartate aminotransferase serving as a liver injury marker can be reduced; in an in-vitro model, the cluster compound can relieve hepatocyte mitochondrial dysfunction caused by cholestasis and bile acid metabolism disorder and promote cell survival, so that the effect of protecting hepatocytes is achieved.

Description

Technical Field

[0001] This invention belongs to the field of liver injury treatment technology, specifically relating to the application of gold nanoclusters in the preparation of drugs for treating sepsis-induced liver injury. Background Technology

[0002] Liver injury refers to damage to the liver caused by various factors, resulting in abnormalities in its structure or function. Common causes of liver injury include viral hepatitis, drugs / toxins, alcohol, metabolic diseases, and other causes such as autoimmune hepatitis (AIH) caused by autoantibodies and septic liver injury (SLI) caused by severe infection.

[0003] Autoimmune hepatitis (AIH) is primarily treated with the nonspecific immunosuppressant prednisone / prednisolone in combination with azathioprine (AZA), or prednisone / prednisolone monotherapy as the first-line standard treatment. Prednisone / prednisolone, as a glucocorticoid, in the early stages of inflammation, increases vascular tone, reduces capillary permeability, and alleviates congestion, while simultaneously inhibiting leukocyte infiltration and phagocytosis, thus reducing exudation and edema. In the later stages of inflammation, glucocorticoids inhibit capillary and fibroblast proliferation, suppress collagen and mucopolysaccharide synthesis, and promote granulation tissue growth, preventing fibrosis and scar formation. Furthermore, glucocorticoids can induce lymphocyte DNA degradation and interfere with their metabolism, exerting an immunosuppressive effect. Through its dual anti-inflammatory and immunosuppressive effects, it can rapidly induce symptom relief. AZA primarily suppresses the immune response by interfering with purine metabolism, thereby inhibiting the synthesis of purine nucleotides. This, in turn, inhibits the synthesis of cellular DNA, RNA, and proteins, exerting an inhibitory effect on T, B, and NK lymphocytes. It takes 6-8 weeks to achieve optimal immunosuppressive effects. Budesonide, as a second-generation glucocorticoid, has fewer systemic adverse reactions and can replace prednisone (or steroids) in the first-line standard treatment regimen. Currently, the first-line standard treatment regimen can significantly improve liver biochemical indicators and prolong survival in most patients with moderate to severe AIH.

[0004] CN117902616A has confirmed that gold nanoclusters exhibit good therapeutic activity in a concanavalin A (Con A)-induced autoimmune hepatitis (AIH) model and regulate macrophage activation, enabling rapid and precise treatment of the hepatitis model. These gold nanoclusters can be used to treat various diseases such as autoimmune hepatitis, viral hepatitis, and fatty liver disease. However, there are no reports in the existing technology regarding the use of these gold nanoclusters for the treatment of septic liver injury (SLI).

[0005] Sepsis is a multi-organ dysfunction caused by dysregulation of the inflammatory response in the infected host. Septic liver injury (SLI) is liver damage and liver dysfunction that occurs in sepsis patients. Therefore, the treatment of septic liver injury includes two aspects: one is basic treatment to improve the overall condition and maintain vital signs, using antibiotics to control infection, adequate fluid resuscitation to avoid organ hypoperfusion and septic shock, oxygen therapy and ventilation support to improve blood oxygen saturation, and insulin to prevent stress hyperglycemia and hepatocellular dysfunction; the other is drug treatment to protect the liver. Commonly used drugs include reducing agents such as bicyclol, thioproline, glutathione or their precursor N-acetylcysteine ​​to scavenge free radicals and improve antioxidant capacity, glycyrrhizic acid preparations to inhibit inflammatory factors, polyene phosphatidylcholine and silymarin to stabilize and repair cell membranes, ursodeoxycholic acid to reduce cholesterol saturation in bile, and choleretic drugs such as S-adenosylmethionine to increase hepatocyte membrane fluidity and facilitate bile excretion. In clinical practice, in addition to the basic treatments of anti-infection and maintenance of vital signs mentioned above, patients with septic liver injury typically receive one or two of the following three classes of drugs for liver protection: antioxidants, hepatocyte membrane stabilizers, and inhibitors of inflammatory factors. If elevated levels of indicators suggesting biliary obstruction and cholestasis, such as direct bilirubin, gamma-glutamyl transferase, and alkaline phosphatase, are present, choleretic hepatoprotective drugs are used in combination. Glucocorticoids and immunosuppressants are generally not used in the treatment of septic liver injury. Hydrocortisone is usually only used in adult patients who develop septic shock and whose target mean arterial pressure level has not been achieved after administration of vasopressors.

[0006] Based on the above analysis, it is clear that AIH and SLI have different causes and mechanisms, and therefore different clinical treatment plans. The treatment plan for AIH includes anti-inflammatory and immunosuppressive glucocorticoids and AZA (anti-inflammatory drugs), while the treatment plan for SLI involves basic treatment to maintain vital signs and a comprehensive treatment plan that protects liver function through multiple mechanisms such as anti-oxidation, stabilizing hepatocyte membranes, inhibiting inflammatory factors, and choleretic effects. Glucocorticoids are not routinely used in the treatment plan for AIH, and AZA is usually not used. Summary of the Invention

[0007] This invention provides the application of gold nanoclusters in the preparation of drugs for treating sepsis-induced liver injury.

[0008] This invention provides the application of gold nanoclusters in the preparation of drugs for reducing serum levels of pro-inflammatory cytokines.

[0009] This invention provides the application of gold nanoclusters in the preparation of drugs for reducing serum aspartate aminotransferase levels.

[0010] This invention provides the application of gold nanoclusters in the preparation of drugs for reducing the cytotoxicity of lithocholic acid to the hepatocyte line AML12.

[0011] This invention provides the application of gold nanoclusters in the preparation of drugs for improving the mitochondrial membrane potential of hepatocytes.

[0012] This invention provides the application of gold nanoclusters in the preparation of drugs for reducing intracellular reactive oxygen species levels in hepatocytes.

[0013] This invention provides a product for treating sepsis-induced liver injury, comprising gold nanoclusters.

[0014] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of gold nanoclusters, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier.

[0015] The gold nanoclusters described in this invention have the molecular composition (Au)x(S)y, where x = 3-144, y = 1-140, and S represents a biocompatible ligand molecule containing a thiol group.

[0016] The thiol-containing biocompatible ligand molecule includes polypeptides or proteins, and is not limited to any type; further, it may contain cysteine ​​or cysteine-containing proteins.

[0017] The hydrated particle size of the gold nanoclusters is between 1 and 5 nm.

[0018] The preparation of the gold nanoclusters includes the following steps: mixing a gold salt solution with a thiol-containing ligand to form a mixture; adding a reducing agent under certain temperature and pH conditions to cause a reduction reaction in the mixture system; and forming the (Au)x(S)y cluster by reacting with the thiol group of the ligand.

[0019] The gold salt is a trivalent gold compound, such as chloroauric acid, wherein the trivalent gold is reduced to gold atoms and / or monovalent gold.

[0020] The reducing agent is sodium borohydride (NaBH4), sodium hydroxide (NaOH), vitamin C, carbon monoxide (CO), or trisodium citrate dihydrate, etc., and the preferred method is selected based on the actual synthesis.

[0021] In the mixed solution, the concentration of the thiol-containing ligand is 0.1 μM-10 M, and the concentration of the gold salt is 0.01 M-10 M. The preferred molar ratio of ligand to gold salt is 1:0.5-1:5, which is selected based on the actual synthesis.

[0022] The mixture undergoes a reduction reaction at a temperature of 20-80 °C and a pH condition that is weakly acidic, neutral, or alkaline, which can be adjusted and optimized as needed.

[0023] The mixture undergoes a reduction reaction and is stirred at 20-80℃ in the dark for 1-10 h. The solution color changes from colorless to light yellow and then changes color again. AuS clusters of different molecular weights are obtained by purification methods such as ultrafiltration and dialysis. The hydrated particle size of the AuS clusters is generally less than 5 nm.

[0024] Compared with the prior art, the beneficial effects achieved by the present invention are: This invention demonstrates through animal and cell experiments that gold nanoclusters have a significant therapeutic effect on septic liver injury (SLI), reducing the levels of inflammatory factors and liver injury marker aspartate aminotransferase in the serum of model animals. In in vitro models, the cluster compound not only has no cytotoxicity, but also alleviates hepatocyte mitochondrial dysfunction caused by cholestasis and bile acid metabolism disorders, promotes hepatocyte survival, and thus plays a protective role for hepatocytes. Attached Figure Description

[0025] Figure 1 The ratio of the weight of the heart, liver, spleen, lungs, and kidneys to the body weight of each group of animals in Example 2 of this invention.

[0026] Figure 2 The AuH content in the livers of each group of animals in Example 2 of this invention (expressed as the ratio of gold atoms to body weight).

[0027] Figure 3 The content of pro-inflammatory cytokine IL-6 in the peripheral blood of each group of animals in Example 3 of the present invention.

[0028] Figure 4 The content of pro-inflammatory cytokine IL-1β in the peripheral blood of each group of animals in Example 3 of the present invention.

[0029] Figure 5 The content of AST, a marker of liver injury, in the peripheral blood of each group of animals in Example 3 of this invention.

[0030] Figure 6 These are representative images of H&E staining of livers from each group of animals in Example 3 of this invention, along with their histopathological scores.

[0031] Figure 7 The effects of different concentrations of AuH on the proliferation activity of AML12 cells in Example 4 of this invention (columns 1-5) and the effect of different concentrations of AuH on reducing the cytotoxicity of LCA to AML12 cells (columns 6-10).

[0032] Figure 8 The relative abundance of LCA in the serum of each group of animals in Example 4 of this invention was detected by non-targeted metabolomics.

[0033] Figure 9These are representative images of TMRE staining of cells in each group in Example 5 of this invention, used to characterize changes in mitochondrial membrane potential.

[0034] Figure 10 These are representative images of cells from each group in Example 5 of this invention, showing the intracellular ROS levels characterized by DCFH-DA staining. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.

[0038] Example 1: Preparation of AuH clusters The preparation steps were performed according to CN117902616A, as follows: 1) At a certain temperature (preferably 37 ℃ in this example), 0.02 g of human serum albumin HSA powder is dissolved in 2 mL of ultrapure water, and a stir bar is added and stirred on a magnetic stirrer; 2) Add 2 mL of chloroauric acid solution (4.3 mM) dropwise to the above solution and stir for 2 min; 3) Immediately add 500 μL of NaOH (0.5 M) solution dropwise to adjust the pH of the solution to alkaline, and continue stirring for 12 h; The stir bar was removed using a magnetic rod, yielding gold clusters emitting orange fluorescence. The clusters were purified by adding 30 mL of ethanol to 10 mL of the synthesized clusters. Free metal ions and unreacted ligands were removed using an ultrafiltration tube with a molecular weight cutoff of 3000. The synthesized clusters appeared brownish-yellow under visible light and orange under ultraviolet light.

[0039] The tests in the following embodiments all used the AuH cluster compound obtained in Example 1.

[0040] Example 2: Biosafety and liver targeting of AuH in rats To demonstrate that the cluster compound synthesized in Example 1 could alleviate sepsis-induced liver injury in animals, a cecal ligation-puncture (CLP) induced sepsis rat model was used. The weight of each animal was measured before modeling. Sham rats served as normal controls (sham group, n=6), and CLP rats served as the experimental group (n=12, considering the potential for animal mortality). Two additional administration groups were established: sham and CLP rats were administered the drug by gavage once one day before and immediately after modeling, respectively, at a dose of 10 mg / kg, namely the sham+AuH group (n=6) and the CLP+AuH group (n=12). Rats were sacrificed 24 h after modeling, and peripheral blood and internal organs such as the heart, liver, spleen, lungs, and kidneys were collected.

[0041] First, each organ of the animal was weighed, and the ratio of a single organ to body weight (i.e., organ coefficient) was calculated. The sham group and the sham+AuH group were compared. The results showed that there was no significant difference in the organ coefficients of the two groups of animals, indicating that the cluster compound synthesized in Example 1 did not cause organ toxicity such as edema, congestion, or damage. Figure 1 A suitable amount of rat liver tissue was taken, and the Au content in the tissue was detected by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that the Au content in the sham+AuH group was similar to that in the CLP+AuH group. Figure 2 This study confirmed that the nanoclusters could reach the liver of animals via oral administration and blood circulation in both control and sepsis models, laying the foundation for their biological effects.

[0042] Example 3: AuH alleviates CLP-induced septic liver injury in rats The main characteristic of sepsis is a significant increase in pro-inflammatory cytokines in peripheral blood. This invention prepares serum from peripheral blood samples collected in the above experiments, and first assesses the levels of pro-inflammatory cytokines IL-6 and IL-1β in the serum after modeling / sham surgery using enzyme-linked immunosorbent assay (ELISA). Figure 3 , Figure 4 The results indicated that the aforementioned cytokines were significantly higher in the CLP group than in the sham group. Subsequently, the level of serum aspartate aminotransferase (AST), a marker of liver injury, was measured. Figure 5 The results indicated that the AST level in the CLP group was significantly elevated, and the animals in this group developed septic liver damage.

[0043] To investigate the effects of AuH in animal models, we further analyzed the aforementioned indicators in the CLP+AuH group. The results showed that the levels of pro-inflammatory cytokines in the CLP+AuH group were significantly lower than those in the CLP group, suggesting that this nanocluster can reduce systemic inflammation levels in rats with septic liver injury. More importantly, the AST level in the CLP+AuH group was significantly lower than that in the CLP group, indicating that this nanocluster can alleviate septic liver injury.

[0044] At the histological level in animals, liver sections stained with hematoxylin and eosin (H&E) showed hepatocellular congestion (red arrow), hepatocellular degeneration (white arrow), and inflammatory cell infiltration (black arrow) in the CLP group; indicating that the cluster compound synthesized in Example 1 can alleviate the above-mentioned pathological changes. Figure 6 Histopathological scores also demonstrated the protective effect of this gold nanocluster against septic liver injury. These experimental results indicate that AuH is a potential candidate drug for the treatment of septic liver injury.

[0045] Example 4: Detection of AuH cytotoxicity against hepatocyte cell line AML12 at the cellular level AML12 cells were spaced at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μM into 96-well plates and incubated at 37 °C for 24 h. Cells were then treated with 20, 50, 100, and 200 µM gold clusters for 24 h each. Further, 10 μL of CCK-8 reagent diluted with 100 μL of culture medium was added to each well, and the cells were incubated for 60 min; absorbance at 450 nm was measured using a microplate reader.

[0046] Figure 7 Statistical analysis was conducted to investigate the potential effects of this gold cluster compound on the proliferation activity of AML12 cells. The results of comparing columns 1 with columns 2 to 5 showed that the gold cluster compound did not significantly reduce cell proliferation activity in the range of 20-200 µM, suggesting that the gold cluster compound has no obvious cytotoxicity to AML12 cells.

[0047] A key characteristic of sepsis-related liver injury is cholestasis, including the accumulation of bile acid metabolites such as lithocholic acid (LCA). Previous studies have commonly used LCA treatment as a cell model of cholestasis. In a mouse sepsis model previously constructed using CLP, non-targeted metabolomics analysis based on chromatography-mass spectrometry revealed that the abundance of LCA in the CLP group was significantly higher than that in the sham group (…). Figure 8 The study confirmed that LCA levels were significantly elevated in SLI in vivo.

[0048] To investigate the potential role of AuH in septic liver injury at the cellular level, AML12 cells were treated with 100 µM LCA for 24 h to simulate septic liver injury in vitro. The results showed that LCA reduced the proliferation activity of the hepatocyte line AML12 to approximately 40% of the control group. Figure 7(Columns 1 and 6). AML12 cells were co-cultured with 20, 50, 100, and 200 µM AuH for 2 h as a therapeutic intervention, followed by the addition of 100 µM LCA. Cell proliferation activity was then assessed after 24 h of co-treatment. The results showed that 50, 100, and 200 µM AuH significantly restored cell proliferation activity, suggesting that this gold nanocluster can reduce the cytotoxicity of LCA and alleviate sepsis-related liver injury in vitro.

[0049] Example 5: AuH-mediated reduction of mitochondrial dysfunction in hepatocytes in a cell model of sepsis-induced liver injury. Mitochondrial dysfunction in hepatocytes is an important mechanism of sepsis-induced liver injury, which is usually manifested as a decrease in mitochondrial membrane potential and accumulation of intracellular reactive oxygen species (ROS).

[0050] A sepsis-induced liver injury cell model was constructed using the cell treatment method described in Example 3, and 50 and 100 µM AuH were used as therapeutic interventions. Tetramethylrhodamine ethyl ester (TMRE) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were used as probes to detect mitochondrial membrane potential and intracellular ROS in hepatocytes.

[0051] In TMRE staining experiments, carbonyl cyanochlorophenylhydrazone (CCCP), an inhibitor of mitochondrial oxidative phosphorylation, was used as a positive control. The results showed that both CCCP and 100 µM LCA significantly reduced mitochondrial membrane potential, indicating mitochondrial functional impairment. Figure 9 AuH can significantly restore mitochondrial membrane potential.

[0052] In the DCFH-DA staining experiment, 100 µM LCA significantly increased intracellular ROS levels ( Figure 10 AuH can reduce the accumulation of ROS in hepatocytes, suggesting that hepatocyte mitochondrial damage is reduced.

[0053] The above results demonstrate that the gold cluster compound can exert a protective effect on mitochondria in a cell model of septic liver injury, which is a potential mechanism for its treatment of septic liver injury.

[0054] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of gold nanoclusters in the preparation of drugs for treating sepsis-induced liver injury.

2. Application of gold nanoclusters in the preparation of drugs for reducing serum pro-inflammatory cytokine levels.

3. Application of gold nanoclusters in the preparation of drugs for reducing serum aspartate aminotransferase levels.

4. Application of gold nanoclusters in the preparation of drugs to reduce the cytotoxicity of lithocholic acid to the hepatocyte cell line AML12.

5. Application of gold nanoclusters in the preparation of drugs for improving mitochondrial membrane potential in hepatocytes.

6. Application of gold nanoclusters in the preparation of drugs for reducing intracellular reactive oxygen species levels in hepatocytes.

7. A product for treating sepsis-induced liver injury, characterized in that, Including gold nanoclusters.

8. A pharmaceutical composition for treating sepsis-induced liver injury, characterized in that, This includes therapeutically effective amounts of gold nanoclusters, or pharmaceutically acceptable salts, solvates, or prodrugs thereof, and pharmaceutically acceptable carriers.

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