Application of distention hippocampus submicron powder in preparation of acetaminophen induced liver injury composition

Abstract

The invention discloses an application of distension hippocampus submicron powder in preparation of a composition for acetaminophen-induced liver injury. The application of the superfine powder form of the hippocampus japonicus which can be cultured on a large scale to the APAP liver injury is developed, the effect is equivalent to or superior to that of NAC, the advantages of dose dependence and multi-target protection are achieved, sustainable utilization of marine medicinal materials is promoted, and a novel, safe and effective strategy is provided for clinical prevention and treatment of the liver injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of expanded hippocampal ultrafine powder in the preparation of acetaminophen-induced liver injury compositions. Background Technology

[0002] The liver is the largest internal organ in the human body and a key hub for numerous physiological processes, undertaking important functions such as the metabolism of biological macromolecules, the breakdown of exogenous compounds, and support for the immune system. With changes in modern lifestyles and increased drug use, liver injury has become a common and prevalent clinical disease. Long-term liver injury often progresses to liver fibrosis, cirrhosis, and even liver failure, seriously threatening patients' lives and health. The inducing factors for liver injury are diverse, including drugs, alcohol, chemical toxins, and viral infections. Among these, drug-induced liver injury (DILI) is particularly prominent and has become a major challenge in the global public health field.

[0003] Acetaminophen (APAP), a widely used nonsteroidal anti-inflammatory drug, is commonly used for clinical analgesia and antipyretic treatment and is found in many over-the-counter combination formulations. Due to its easy availability and convenient use, patients often unknowingly overdose, leading to acute liver injury. Studies have shown that APAP is one of the main sources of drug-induced liver injury.

[0004] The mechanism of APAP liver injury primarily involves the production of its metabolite N-acetyl-p-benzoquinone imine (NAPQI). In excess, NAPQI depletes glutathione in hepatocytes, leading to oxidative stress, mitochondrial damage, and cell necrosis, ultimately resulting in widespread liver tissue destruction. Currently, N-acetylcysteine ​​(NAC) is the only approved antidote for APAP liver injury, acting by replenishing glutathione and alleviating oxidative damage. However, the clinical application of NAC has significant limitations. Its therapeutic window is narrow, with optimal efficacy only within 8-10 hours after APAP overdose; beyond this window, its effectiveness declines sharply or even becomes ineffective. This results in many patients missing out on benefits due to delayed diagnosis or treatment, increasing the risk of liver failure. Furthermore, NAC may be associated with allergic reactions or inconvenience with intravenous administration, further limiting its widespread use.

[0005] In current technologies, prevention and treatment strategies for APAP-related liver injury mainly rely on supportive therapies, such as liver transplantation or adjuvant detoxification. However, these methods are costly, resource-limited, and not targeted solutions. In recent years, some studies have explored the potential of natural products in hepatoprotection. For example, certain plant extracts or traditional Chinese medicine components alleviate liver injury through antioxidant and anti-inflammatory pathways. However, these approaches are mostly limited to in vitro experiments or preliminary animal models, lacking comprehensive in vivo validation and clinical translation potential. Meanwhile, marine biological resources have attracted considerable attention as potential drug sources. However, seahorses, as a traditional and valuable medicinal material, are mainly used for kidney tonification and aphrodisiac purposes, and research on their application in the prevention and treatment of liver injury is still lacking. Wild seahorse resources are under national protection, making large-scale utilization difficult. While there are abundant resources of seahorse species that can be farmed on a large scale, current technologies have not reported on the effects of processed forms (such as powder formulations) on regulating APAP-related liver injury.

[0006] In summary, existing technologies suffer from the following main problems: First, the pathogenesis of APAP liver injury is complex, and the therapeutic window of existing specific drugs, such as NAC, is too narrow to meet clinical needs; second, there is a lack of safe and effective preventive or adjuvant treatment options, especially targeted interventions on oxidative stress and cell necrosis mechanisms; and third, the development and utilization of marine medicinal resources are insufficient, failing to fill the gaps in existing hepatoprotective drugs. These shortcomings lead to unsatisfactory prevention and treatment outcomes for APAP liver injury, resulting in poor patient prognosis. There is an urgent need to develop novel, safe, and resource-sustainable protective strategies to improve clinical outcomes and reduce the social burden. Summary of the Invention

[0007] The purpose of this invention is to overcome the defects of the prior art and provide the application of expanded hippocampal ultrafine powder in the preparation of acetaminophen-induced liver injury compositions.

[0008] The technical solution of the present invention is as follows:

[0009] Application of expanded hippocampal ultrafine powder in the preparation of acetaminophen-induced liver injury compositions.

[0010] In a preferred embodiment of the present invention, the preparation method of the bloated seahorse ultrafine powder includes: washing, draining, and drying fresh bloated seahorses in a hot circulating oven, and then subjecting them to coarse pulverization, fine pulverization and low-temperature ultrafine pulverization in sequence to obtain bloated seahorse ultrafine powder.

[0011] More preferably, the coarse grinding is performed to a particle size of 8-11 mm.

[0012] More preferably, the fine grinding is performed by grinding to pass through a 70-90 mesh sieve.

[0013] In a further preferred embodiment, the temperature of the low-temperature ultrafine pulverization is -11 to -9°C, and the time is 45-55 minutes.

[0014] In a preferred embodiment of the present invention, the effective dose of the expanded seahorse ultrafine powder for humans is 0.05-0.23 g / kg.

[0015] More preferably, the effective dose of the expanded seahorse ultrafine powder for humans is 0.21-0.23 g / kg.

[0016] The beneficial effects of this invention are:

[0017] 1. This invention utilizes the scalable cultured seahorse resources to develop its application in ultrafine powder form for APAP liver injury. The effect is equivalent to or better than NAC, with dose-dependent and multi-target protection advantages, promoting the sustainable use of marine medicinal materials and providing a new, safe and effective strategy for the prevention and treatment of clinical liver injury.

[0018] 2. This invention can significantly reduce the increase in liver index induced by APAP: Experimental results showed that the liver index of mice in the APAP treatment group was significantly increased, while the low, medium and high dose groups of the expanded hippocampus ultrafine powder (0.65g / kg, 1.3g / kg and 2.6g / kg) could significantly reduce the index, indicating that it effectively inhibits hepatomegaly and organ lesions.

[0019] 3. This invention can effectively improve liver function biochemical indicators: The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the APAP-induced group of mice were significantly increased, indicating liver parenchymal damage; all dose groups of the distended hippocampus ultrafine powder significantly reduced AST and ALT levels in a dose-dependent manner. Among them, the high-dose group showed better results than the positive control drug N-acetylcysteine ​​(NAC, 100 mg / kg), demonstrating stronger liver function protective potential.

[0020] 4. This invention can alleviate pathological changes in liver tissue: HE staining showed that the liver tissue of mice in the APAP group showed obvious cell necrosis and erythrocyte filling, while the liver cell structure in the group treated with bloated hippocampus ultrafine powder tended to be normal and the necrotic area was reduced. Only a small amount of edema was seen in the high-dose group, which proved that it significantly reduced tissue necrosis.

[0021] 5. This invention can inhibit hepatocyte death: TUNEL staining results showed that the number of dead cells around the central vein in the liver tissue of the APAP group increased, while the number of dead cells in the bloated hippocampus ultrafine powder group was significantly reduced, confirming that it protects hepatocytes by inhibiting apoptosis or necrosis.

[0022] 6. This invention can reduce the level of reactive oxygen species (ROS) in liver tissue: APAP induces H2O2, NO and O2. - Significantly elevated ROS levels in the group with bloated hippocampus ultrafine powder led to oxidative stress; however, significantly lower ROS levels in the group with bloated hippocampus ultrafine powder suggested that its antioxidant mechanism is key to liver protection.

[0023] 7. This invention can improve the survival rate of mice under lethal doses of APAP: Under treatment with 450 mg / kg APAP, the NAC group could not improve the survival rate, while the high-dose abdominal distended hippocampus ultrafine powder group significantly improved the survival rate (p<0.05), showing a survival rate superior to existing specific drugs. Attached Figure Description

[0024] Figure 1 This invention demonstrates that, in Example 1, the distended hippocampal ultrafine powder significantly inhibited the liver index in APAP-treated mice (CON: control group; APAP: APAP-treated group; APAP+NAC: APAP+positive control NAC group; APAP+L: APAP+low-dose hippocampal ultrafine powder treatment group; APAP+M: APAP+medium-dose hippocampal ultrafine powder treatment group; APAP+H: APAP+high-dose hippocampal ultrafine powder treatment group; ##p<0.01: compared with the control group; **p<0.01: compared with the APAP-treated group).

[0025] Figure 2 The results of Example 1 of this invention show that the abdominal distending hippocampal ultrafine powder significantly reduced APAP-induced AST and ALT levels (CON: control group; APAP: APAP treatment group; APAP+NAC: APAP+positive drug NAC group; APAP+L: APAP+low-dose hippocampal ultrafine powder treatment group; APAP+M: APAP+medium-dose hippocampal ultrafine powder treatment group; APAP+H: APAP+high-dose hippocampal ultrafine powder treatment group; ##p<0.01: compared with the control group; **p<0.01: compared with the APAP treatment group).

[0026] Figure 3 The results of Example 1 of this invention show that the expanded hippocampal ultrafine powder significantly reduced APAP-induced liver necrosis in mice (CON: control group; APAP: APAP-treated group; APAP+NAC: APAP+positive drug NAC group; APAP+L: APAP+low-dose hippocampal ultrafine powder treatment group; APAP+M: APAP+medium-dose hippocampal ultrafine powder treatment group; APAP+H: APAP+high-dose hippocampal ultrafine powder treatment group; ##p<0.01: compared with the control group; **p<0.01: compared with the APAP-treated group).

[0027] Figure 4The results of Example 1 of this invention show that the expanded hippocampal ultrafine powder significantly inhibited APAP-induced hepatocyte death in mice (CON: control group; APAP: APAP-treated group; APAP+NAC: APAP+positive control NAC group; APAP+L: APAP+low-dose hippocampal ultrafine powder treatment group; APAP+M: APAP+medium-dose hippocampal ultrafine powder treatment group; APAP+H: APAP+high-dose hippocampal ultrafine powder treatment group; ##p<0.01: compared with the control group; **p<0.01: compared with the APAP-treated group).

[0028] Figure 5 This invention demonstrates how the distended hippocampus ultrafine powder inhibited APAP-induced levels of reactive oxygen species (ROS) in the liver in Example 1 (CON: control group; APAP: APAP-treated group; APAP+NAC: APAP+positive control NAC group; APAP+L: APAP+low-dose hippocampus ultrafine powder treatment group; APAP+M: APAP+medium-dose hippocampus ultrafine powder treatment group; APAP+H: APAP+high-dose hippocampus ultrafine powder treatment group; ##p<0.01: compared with the control group; **p<0.01: compared with the APAP-treated group).

[0029] Figure 6 This invention demonstrates that the abdominal-expanding hippocampal ultrafine powder in Example 1 significantly improved the survival rate of mice treated with lethal doses of APAP (NC: control group; MC: APAP treatment group; PC: APAP + positive control drug NAC treatment group; L: APAP + low-dose hippocampal ultrafine powder treatment group; M: APAP + medium-dose hippocampal ultrafine powder treatment group; H: APAP + high-dose hippocampal ultrafine powder treatment group; *p<0.05: compared with the APAP treatment group). Detailed Implementation

[0030] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0031] Example 1

[0032] I. Materials and Methods:

[0033] (1) Preparation of bloated seahorse ultrafine powder: Fresh bloated seahorses were washed, drained, and dried in a hot circulating oven. They were then crushed into small particles with a particle size of 8-11 mm using a coarse pulverizer, then crushed into small particles with a particle size of 80-11 mm using a fine pulverizer and passed through an 80-mesh sieve. Finally, they were crushed into bloated seahorse ultrafine powder at -10℃ for 50 min using a low-temperature ultrafine pulverizer. The particle size distribution is shown in Table 1 below. The powder was stored in a -20℃ refrigerator for later use.

[0034] Table 1. Particle size distribution of the expanded seahorse powder

[0035]

[0036] Note: D10: Particle size at which the cumulative distribution is 10%; D50: Particle size at which the cumulative distribution is 50%; D90: Particle size at which the cumulative distribution is 90%; span: Particle span.

[0037] (2) Establishment of APAP-induced acute liver injury mouse model and drug intervention protocol: After 7 days of acclimatization, mice were randomly divided into a normal group (n=10) and a model group (n=50). The model group was further divided into a model group, a positive drug group (NAC, 100 mg / kg), and low (0.65 g / kg), medium (1.3 g / kg), and high (2.6 g / kg) dose groups of distended hippocampus ultrafine powder, with 10 mice in each group. Except for the normal group and the model group, the other mice were administered the corresponding drugs by gavage for 7 days. One hour after the last administration, except for the blank group, each group was injected intraperitoneally with APAP solution 250 mg / kg. After the injection, the mice were fasted but allowed to drink water. The survival status of the mice was observed, and samples were collected 24 hours later.

[0038] Based on the body surface area (BSA) normalization method calculated from drug dosage, the standard formula for converting the mouse doses of the above-mentioned low, medium, and high dose groups of the abdominal distended hippocampus ultrafine powder to the human equivalent dose (HED) is: HED (g / kg) = mouse dose (g / kg) × (mouse Km / human Km), where the mouse Km value is 3 and the human Km value is 37 (based on an average adult weight of 60 kg). Therefore, the conversion factor is approximately 3 / 37 ≈ 0.081;

[0039] Based on this, the conversion results are as follows:

[0040] Low dose: The human equivalent dose is approximately 0.0527 g / kg (assuming an adult weight of 60 kg, the total daily dose is approximately 3.16 g);

[0041] Medium dose: The human equivalent dose is approximately 0.1054 g / kg (assuming an adult weight of 60 kg, the total daily dose is approximately 6.32 g);

[0042] High dose: The human equivalent dose is approximately 0.2108 g / kg (assuming an adult weight of 60 kg, the total daily dose is approximately 12.65 g);

[0043] (3) Histochemical staining:

[0044] A. Tissue embedding: Take a fresh tissue block of appropriate size, immerse it in neutral formaldehyde fixative for 24 hours, and then pass it sequentially through PBS for 2 hours, 50% alcohol for 1 hour, 70% alcohol for 50 minutes (this step can be left for a longer period), 85% alcohol for 40 minutes, 95% alcohol for 30 minutes, anhydrous ethanol I for 30 minutes, and anhydrous ethanol II for 20 minutes for dehydration; then pass it through a mixture of equal parts anhydrous ethanol and xylene for 30 minutes, xylene I for 20 minutes, and xylene II for 20 minutes for clearing; place the tissue block in a 65℃ electric thermostatic drying oven in a mixture of equal parts xylene and paraffin for 30 minutes, and then place it in paraffin I and paraffin II for 1 hour each for paraffin penetration, and then embed it in a paraffin block for later use.

[0045] B. Tissue Sectioning: Trim the embedded tissue blocks and section them using a paraffin microtome. The section thickness should be 6-7 μm. Use glass slides treated with poly-L-lysine adhesive to collect the sections. After the sections have air-dried, place them in a 65℃ drying oven for 30 min to 1 h, and store at room temperature. C. After dewaxing the paraffin sections with xylene I and II for 5 min each, immerse them sequentially in 100% ethanol I and 100% ethanol II for 5 min each, then in 95%, 70%, and 50% ethanol solutions for 3 min each, and finally in distilled water for 3 min for dewaxing and rehydration. Stain the tissue sections with hematoxylin and eosin, and perform microscopic examination during staining to determine the staining time.

[0046] (4) TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) staining: During apoptosis or programmed cell death, endogenous endonucleases are activated, cutting DNA to produce a large number of fragmented double-stranded or single-stranded DNA, exposing the 3'-OH ends. TUNEL is a molecular biology technique used to detect programmed cell death, identifying dead cells by labeling the 3'-hydroxyl ends (3'-OH) of DNA breaks. A commercially available TUNEL assay kit (Beyotime; product number: C1091) was used to detect cell death in mouse liver tissue.

[0047] II. Experimental Results:

[0048] (1) Abdominal-expanding hippocampal ultrafine powder reduces liver index in APAP-induced acute liver injury mice: Organ index is a commonly used indicator to assess the health or disease severity of organs, reflecting organ enlargement, atrophy, or disease status. Mice were weighed before the endpoint experiment, and the isolated livers were weighed during the endpoint experiment. The liver organ coefficient (liver weight / mouse body weight) of each group of mice was calculated. The results are as follows: Figure 1As shown in the figure, compared with the blank control group, the liver organ index of mice in the APAP treatment group was significantly increased; compared with the APAP group, the liver index of mice in the NAC and low, medium and high doses of distended hippocampus treatment groups was significantly decreased. These results indicate that the distended hippocampus ultrafine powder prepared in this embodiment inhibits the APAP-induced increase in mouse liver index.

[0049] (2) Distended hippocampus reduces serum AST / ALT levels in mice with APAP-induced acute liver injury: AST and ALT are biochemical indicators for evaluating the degree of liver lesions and jointly reflect liver function. Elevated AST and ALT levels indicate extensive liver parenchymal damage, and this method is widely used in clinical testing. In this example, AST and ALT levels in mouse serum were detected using a biochemical reagent kit, and the results are as follows: Figure 2 As shown, compared with the blank control group, the APAP group mice had significantly higher AST and ALT levels, indicating that APAP caused liver damage in mice. Compared with the APAP group mice, the NAC and low, medium, and high doses of distended hippocampus treatment groups had significantly lower AST and ALT levels, and the distended hippocampus treatment groups showed a dose-dependent effect, with the high-dose distended hippocampus group showing better improvement than the NAC group. These results indicate that the distended hippocampus ultrafine powder prepared in this embodiment can alleviate APAP-induced liver function damage in mice, thereby exerting a hepatoprotective effect.

[0050] (3) Effects of distended hippocampus on pathological changes in liver tissue of mice with APAP-induced acute liver injury: To investigate the degree of APAP-induced liver tissue damage in mice, HE staining was performed on mouse liver tissue. The results are as follows: Figure 3 As shown, in the blank control group, the liver tissue cells of mice had round and well-shaped nuclei, and the hepatocyte cords were arranged radially with uniformly sized gaps, showing no obvious pathological changes. In the APAP group, obvious hepatocyte necrosis was observed in the liver tissue, with the necrotic areas filled with red blood cells. Compared to the APAP group, the cellular structure of the liver tissue of mice treated with NAC and expanded hippocampus ultrafine powder was significantly more normal, especially in the high-dose expanded hippocampus group, where the hepatocytes were arranged more neatly, with only a small number of hepatocytes showing edema. These results indicate that the expanded hippocampus ultrafine powder prepared in this embodiment can significantly alleviate liver tissue necrosis caused by APAP-induced acute liver injury in mice.

[0051] (4) Inhibition of APAP-induced hepatocyte death in mice by ultrafine powder of distended hippocampus: To investigate the effect of ultrafine powder of distended hippocampus on APAP-induced hepatocyte death in mice, this example used the TUNEL assay to detect hepatocyte death in mice. The results showed that, compared with normal mice (control group), a large number of hepatocytes died around the central vein in the liver tissue of mice treated with APAP. Figure 4Compared to the APAP-treated group (model group), the number of dead hepatocytes in the liver tissue of mice treated with hippocampal ultrafine powder was significantly reduced. Figure 4 The results indicate that the expanded hippocampal ultrafine powder prepared in this embodiment significantly inhibited APAP-induced hepatocyte death in mice.

[0052] (5) Inhibition of APAP-induced Reactive Oxygen Species (ROS) Levels in the Liver by Abdominal Distension Hippocampus Ultrafine Powder: Extensive studies have shown that APAP treatment induces the production of reactive oxygen species (ROS) in the liver, and strong oxidative stress can lead to hepatocyte death, ultimately resulting in liver tissue damage. The results of this study show that APAP treatment does indeed lead to the production of three types of reactive oxygen species (H2O2, NO, O2) in liver tissue. - The levels of the three reactive oxygen species were significantly increased in the liver tissue of mice treated with hippocampal ultrafine powder, while the levels of the three reactive oxygen species in the liver tissue of mice treated with hippocampal ultrafine powder were significantly decreased compared with those treated with APAP. Figure 5 These results indicate that the expanded hippocampal ultrafine powder prepared in this embodiment may improve APAP-induced liver injury in mice by inhibiting APAP-induced levels of reactive oxygen species (ROS) in the liver.

[0053] (6) Abdominal-expanding hippocampal ultrafine powder significantly improved the survival rate of mice treated with lethal doses of APAP: To investigate the effect of abdominal-expanding hippocampal ultrafine powder on APAP-induced mortality in mice, this study treated mice with a lethal dose of APAP (450 mg / kg) and then monitored the effect of abdominal-expanding hippocampal ultrafine powder on APAP-induced mouse mortality. The study showed that the positive control drug NAC did not improve the survival rate of mice treated with a lethal dose of APAP (450 mg / kg), but the high-dose hippocampal ultrafine powder treatment group significantly improved the survival rate of mice (see...). Figure 6 The results indicate that the expanded hippocampal ultrafine powder prepared in this embodiment significantly improved the survival rate of mice treated with lethal doses of APAP.

[0054] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. Application of expanded hippocampal ultrafine powder in the preparation of acetaminophen-induced liver injury compositions.

2. The application as described in claim 1, characterized in that: The preparation method of the bloated seahorse ultrafine powder includes: washing, draining, and drying fresh bloated seahorses in a hot circulating oven, and then coarsely crushing, finely crushing and low-temperature ultrafine crushing to obtain bloated seahorse ultrafine powder.

3. The application as described in claim 2, characterized in that: The coarse grinding refers to grinding to a particle size of 8-11 mm.

4. The application as described in claim 3, characterized in that: The fine grinding refers to grinding to pass through a 70-90 mesh sieve.

5. The application as described in claim 4, characterized in that: The temperature for the low-temperature ultrafine pulverization is -11 to -9℃, and the time is 45-55 minutes.

6. The application as described in any one of claims 1 to 5, characterized in that: The effective dose of the expanded seahorse ultrafine powder for humans is 0.05-0.23 g / kg.

7. The application as described in claim 6, characterized in that: The effective dose of the expanded seahorse ultrafine powder for humans is 0.21-0.23 g / kg.

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