Use of 1-lactulose in preparation of drugs for preventing and treating liver injury or liver fibrosis

Abstract

The use of 1-lactulose in the preparation of drugs for preventing and treating liver injury or liver fibrosis has the advantage of significant effects in preventing and treating liver injury or liver fibrosis.

Description

Application of 1-Lactulose in the Preparation of Drugs for the Prevention and Treatment of Liver Injury or Liver Fibrosis Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of 1-lactulose in the preparation of drugs for the prevention and treatment of liver damage or liver fibrosis. Background Technology

[0002] Liver disease is a common clinical condition. Common causes of liver damage include ingestion of toxic food, excessive alcohol consumption, improper medication use, and viral infections. Chronic liver disease caused by various etiologies, as well as liver damage resulting from various hepatotoxic factors, can lead to hepatocyte necrosis, inflammation, and secondary fibrosis, ultimately progressing to cirrhosis or even liver cancer.

[0003] Recent studies have found that liver fibrosis can be reversed during the disease process. Therefore, an effective way to alleviate cirrhosis and liver cancer is to prevent and treat the disease at the fibrotic stage. Currently, the only effective treatment for liver fibrosis is liver transplantation, but it is extremely expensive. Therefore, there is an urgent need for a highly effective and low-toxicity anti-fibrotic drug.

[0004] Liver fibrosis refers to the pathological process of abnormal proliferation of fibrous connective tissue in the liver when hepatocytes undergo necrosis and inflammatory stimulation. Its basic pathological change is the deposition of a large amount of extracellular matrix (ECM) in the perisinusoidal space, with altered composition, leading to narrowing and "capillaryization" of the perisinusoidal space. It is also a wound healing response of the liver to chronic damage caused by various etiologies. Mild cases are called liver fibrosis, while severe cases lead to remodeling of liver lobule structure, formation of pseudolobules and nodules, known as cirrhosis. Long-term liver damage often leads to liver fibrosis, mainly manifested as excessive proliferation and abnormal deposition of extracellular matrix components and excessive proliferation of hepatic stellate cells.

[0005] Currently, research on the effects of drugs on liver diseases, particularly on experimental liver injury in animals, is deepening. Animal models of liver injury caused by liver diseases, including hepatocyte inflammation, increased inflammatory cytokines, lipid peroxidation, and hepatocyte necrosis, can be categorized into chemically induced liver injury animal models (such as those induced by carbon tetrachloride and D-galactosamine); alcoholic liver injury animal models (such as those induced by ethanol); and drug-induced liver injury animal models (such as those induced by acetaminophen (APAP)).

[0006] Chinese Patent Publication No. CN116535448A discloses the antioxidant activity of a novel 1-lactulose, wherein the structural formula of the novel 1-lactulose is any of the following:

[0007] However, there are currently no reports on the use of the aforementioned novel 1-lactulose in the treatment of liver diseases. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of 1-lactulose in the preparation of drugs for the prevention and treatment of liver damage or liver fibrosis.

[0009] This invention is achieved through the following technical solutions:

[0010] The use of 1-lactulose in the preparation of drugs for preventing and treating liver injury or liver fibrosis, wherein the structure of 1-lactulose is at least one of the following:

[0011] In some embodiments of the present invention, the liver injury includes chemically induced liver injury.

[0012] In some preferred embodiments of the present invention, the chemically induced liver injury includes carbon tetrachloride-induced liver injury.

[0013] In some embodiments of the present invention, the liver injury includes alcoholic liver injury.

[0014] In some embodiments of the present invention, the liver injury includes drug-induced liver injury.

[0015] In some preferred embodiments of the present invention, the drug-induced liver injury is acetaminophen-induced liver injury.

[0016] In some embodiments of the present invention, the drug is an oral drug or an injectable drug.

[0017] In some embodiments of the present invention, the method for preparing 1-lactulose includes the following steps:

[0018] The reaction was catalyzed by adding o-nitrophenol-β-D-galactoside, fructose, and PsaGal protease to a buffer solution, followed by centrifugation to remove the precipitate; the supernatant was extracted, and the extract solution was lyophilized to obtain the final product; the amino acid sequence of the PsaGal protease is SEQ ID NO:2.

[0019] In some preferred embodiments of the present invention, the nucleotide sequence of the gene encoding the PsaGal protease is SEQ ID NO:1.

[0020] In some preferred embodiments of the present invention, 10-20 mM o-nitrophenol-β-D-galactoside ONPG, 60-500 mM fructose and 0.1 U / ml PsaGal protease are added to the buffer solution.

[0021] The beneficial effects of this invention are:

[0022] The 1-lactulose of this invention has the effects of promoting hepatocyte proliferation and inhibiting inflammatory response, and has significant therapeutic effects in preventing and treating liver damage and liver fibrosis. Attached Figure Description

[0023] Figure 1 shows the dose-response curve of 1-lactulose on CCl4-induced chemical acute liver injury.

[0024] Figure 2 shows the effect of 1-lactulose on serum ALT and AST activities in mice with CCl4-induced chemical liver injury. A: Statistical graph of serum ALT activity in mice. B: Statistical graph of serum AST activity in mice.

[0025] Figure 3 shows the effect of 1-lactulose on the pathological characteristics of mouse livers with CCl4-induced chemical liver injury. A: HE staining results of mouse livers, with the same scale bar for all four groups. B: Statistical graph of inflammatory cell infiltration area in mouse HE sections.

[0026] Figure 4 shows the survival curves of mice in the ethanol-induced alcoholic liver injury model.

[0027] Figure 5 shows the effect of 1-lactulose on serum ALT and AST activities in mice with ethanol-induced alcoholic liver injury.

[0028] Figure 6 shows the effect of 1-lactulose on the pathological characteristics of mouse livers in mice with ethanol-induced alcoholic liver injury. A: HE-stained sections of mouse livers, with the same scale bar for all four groups. B: Statistical graph of fat vacuoles in mouse liver sections.

[0029] Figure 7 shows the survival curves of mice with APAP-induced liver injury.

[0030] Figure 8 shows the effect of 1-lactulose on serum ALT and AST activities in mice with APAP-induced liver injury. A: Statistical graph of serum ALT and AST activities in mice 12 hours after administration. B: Statistical graph of serum ALT and AST activities in mice 7 days after administration.

[0031] Figure 9 shows the effect of 1-lactulose administration 12 hours after administration on the liver pathology of mice with APAP-induced liver injury. A: HE-stained sections of mouse liver 12 hours after administration; scale bars are the same for all groups. B: Statistical graph of necrotic area in mouse liver sections.

[0032] Figure 10 shows the effect of 1-lactulose administration for 7 days on the liver pathology of mice with APAP-induced liver injury. A: HE-stained sections of mouse livers 7 days after administration, with the same scale bar for each group. B: Statistical graph of necrotic area in mouse liver sections.

[0033] Figure 11 shows the effect of 1-lactulose on Ki67 immunohistochemistry in mice with APAP-induced liver injury. A: Ki67 immunohistochemical results, with the same scale bar for each group. B: Statistical graph of Ki67-positive hepatocytes.

[0034] Figure 12 shows the survival curve of CCl4-induced liver fibrosis in mice.

[0035] Figure 13 shows the effect of 1-lactulose on serum ALT and AST activities in CCl4-induced liver fibrosis mice. A: Statistical graph of serum ALT activity in mice. B: Statistical graph of serum AST activity in mice.

[0036] Figure 14 shows the HE staining results of the livers of mice with CCl4-induced liver fibrosis. The scale bars are the same for all groups.

[0037] Figure 15 shows the Masson staining results of the livers of mice with CCl4-induced liver fibrosis. A: Masson staining images of mouse livers; the scale bar is the same for all groups, 100 μm. B: Statistical graph of Masson staining positive areas.

[0038] Figure 16 shows the results of Sirius red staining in the livers of mice with CCl4-induced liver fibrosis. A: Images of Sirius red staining in mouse livers; scale bars are the same for all groups. B: Statistical graph of collagen stained with Sirius red in mice.

[0039] Figure 17 shows the effect of 1-lactulose on the immunohistochemical staining of α-SMA in CCl4-induced liver fibrosis mice. A: Immunohistochemical results of α-SMA, with the same scale bar of 100 μm for each group. B: Statistical graph of the positive area of ​​α-SMA.

[0040] Figure 18 shows the effect of 1-lactulose on the immunohistochemical activity of Collagen I in CCl4-induced liver fibrosis mice. A: Immunohistochemical results of Collagen I, with the same scale bar of 100 μm for each group. B: Statistical graph of Collagen I positive area. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. 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.

[0042] 1-Lactulose was prepared according to the method disclosed in CN116535448A.

[0043] The nucleotide sequence of the recombinant DNA fragment expressing glycosyltransferase (PsaGal) is SEQ ID NO:1, and the amino acid sequence of the glycosyltransferase (PsaGal) it encodes is SEQ ID NO:2.

[0044] The preparation of 1-lactulose was carried out in 20 mM phosphate-buffered saline (pH 7.5, PB buffer system), which included 20 mM o-nitrophenol-β-D-galactoside (ONPG), 400 mM fructose, and 0.1 U / ml of recombinantly expressed PsaGal enzyme. The reaction system was incubated at 25 °C for 4 h, then heated at 100 °C for 10 min to terminate the reaction, followed by centrifugation at 10000 g / min for 10 min to remove the precipitate. The supernatant was extracted with chloroform at a 1:1 volume ratio, and the extraction process was repeated three times to remove ONPG and residual protein from the reaction solution. The extracted solution was lyophilized to obtain 1-lactulose.

[0045] The structure of 1-lactulose of the present invention is at least one of the following structures:

[0046] Acetal or ketal structures in carbohydrates are unstable, especially the hemiacetal structure at the terminal reducing end, which is even more unstable. They exist in both open-chain and cyclic forms. Cyclic structures, depending on the ring size, can be further divided into pyran-type (six-membered ring) and furan-type (five-membered ring). Therefore, these three configurations are interconvertible and can be considered as the same sugar.

[0047] Example 1: Therapeutic effect and mechanism of 1-lactulose on carbon tetrachloride (CCl4)-induced chemical liver injury in mice.

[0048] 1.1 Experimental Methods

[0049] 1.1.1 Animal Experiment Design

[0050] This experiment used SPF-grade male Kunming mice, 6 weeks old, weighing approximately 20-25g. The animal housing environment was kept at a constant temperature (22±2℃), constant humidity (50±10%), and with a diurnal light cycle (12 / 12h light / dark). The mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0051] After 7 days of acclimatization, the mice were randomly divided into 4 groups of 3 mice each. The animals were divided into a control group, a 1-lactulose (3.125 mg / kg / day) group, a 1-lactulose (6.25 mg / kg / day) group, and a 1-lactulose (12.5 mg / kg / day) group. The liver weight ratio was tested to determine the dosage of 1-lactulose.

[0052] Liver weight ratio test experiment: Mice in each group were intraperitoneally injected with CCl4 (1 ml / kg, 1:3 corn oil). Treatment began 24 hours after intraperitoneal injection. Mice in the 1-lactulose group were intraperitoneally injected with 3.125 mg / kg / day, 6.25 mg / kg / day, and 12.5 mg / kg / day, respectively, while mice in the control group were intraperitoneally injected with 0.9% saline once daily for 7 consecutive days. After the last administration, mice were fasted for 16 hours but allowed free access to water. After anesthesia, blood was collected from the orbital region, and the mice were euthanized. The livers were then dissected and weighed.

[0053] After 7 days of acclimatization, the mice were randomly divided into 4 groups of 3 mice each. The animals were divided into a normal group, a control group, a positive control group (denoted as biphenyl diester), and a 1-lactulose group, and then subsequent experiments were carried out.

[0054] Mice in the normal control group received the same amount of corn oil intraperitoneally as the other groups, while mice in the other groups received CCl4 (1 ml / kg, 1:3 corn oil) intraperitoneally. Treatment began 24 hours after intraperitoneal injection. Mice in the positive control group received bifendate (10 mg / kg / day) intraperitoneally, and mice in the 1-lactulose group received 1-lactulose (10 mg / kg / day) intraperitoneally, once daily for 7 consecutive days. After the last administration, mice were fasted for 16 hours but allowed free access to water. After anesthesia, blood was collected from the orbital region, and the mice were euthanized. The livers were then dissected and weighed.

[0055] 1.1.2 Determination of liver weight ratio in mice

[0056] Before sacrifice, the mice in each group were weighed. After anesthesia, all mice were sacrificed, and their livers were removed and weighed. Data were collected, and the liver weight ratio of each group was calculated. Liver weight ratio = (Mice liver weight (g) / Mice body weight (g)) × 100%

[0057] 1.1.3 Determination of liver function-related enzyme activity in mouse serum

[0058] Mouse blood obtained by orbital sampling was placed in a 1.5 ml EP tube and allowed to stand at room temperature for 2 hours. After centrifugation at 2000 rpm for 20 minutes at 4°C, the supernatant was collected and placed in a 1.5 ml EP tube. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were measured using the corresponding kits according to the manufacturer's instructions.

[0059] 1.1.4 HE staining of mouse liver

[0060] Hematoxylin and eosin (H&E) staining was used to stain liver tissue sections. Liver tissue fixed in formalin solution was removed and sectioned sequentially through the following steps: dehydration, clearing, paraffin embedding, sectioning, dewaxing, staining, washing, dehydration, fixation, and mounting. The physiological and pathological conditions of the sections were observed using an optical microscope.

[0061] 1.2 Experimental Results:

[0062] 1.2.1 Effect of 1-Lactulose on Liver Weight Ratio in a Mice Model of CCl4-Induced Liver Injury

[0063] Liver injury induced by various factors will increase the weight of the liver in mice, and the percentage of the liver weight to the total body weight of the mouse will increase. Therefore, the liver weight ratio is a simple yet important indicator for measuring the degree of liver injury in mice. The results of the calculation of the liver weight ratio in mice are shown in Table 1.

[0064] Table 1. Effects of 1-Lactulose on Liver Weight Ratio in a Mice Model of CCl4-Induced Liver Injury

[0065] Compared with the control group, *p<0.05, **p<0.01; n=3.

[0066] The experimental results showed that, compared with the control group, the liver weight ratio of mice in all 1-lactulose dosage groups decreased in a dose-dependent manner, and the difference was statistically significant at a 12.5 mg / kg concentration compared with the control group (p<0.05). This indicates that 1-lactulose can alleviate the increase in liver weight caused by acute liver injury.

[0067] Simultaneously, a curve showing the relationship between 1-lactulose dosage and efficacy was plotted to investigate the effect of 1-lactulose on ED. 50 The calculation was performed, and the results are shown in Figure 1. ED 50 =6.218 mg / kg, therefore, a slightly higher ED was subsequently selected. 50 The dosage of 1-lactulose (10 mg / kg) was used for subsequent experiments.

[0068] 1.2.2 Effects of 1-Lactulose on ALT and AST Activities in Serum of Mice with CCl4-Induced Liver Injury Model

[0069] Alanine aminotransferase (ALT) is mainly found in the cytoplasm of hepatocytes, and its levels in the blood are normally low. When the liver is damaged, leading to hepatocyte necrosis, a large amount of ALT is released into the bloodstream, indicating liver damage. Aspartate aminotransferase (AST) is mainly distributed in the mitochondria of cells in tissues such as the myocardium, liver, and kidneys, and its levels in serum are normally low. When the liver is damaged, the permeability of the hepatocyte membrane increases, causing AST to be released into the bloodstream. An increase in serum AST levels indicates damage to these tissues and organs. Since the levels of serum ALT and AST are positively correlated with the degree of liver damage, their levels are important indicators for assessing the extent of liver damage.

[0070] The experimental results are shown in Figure 2: Compared with the control group, *p<0.05, **p<0.01; n=3. Compared with the normal group, the activity levels of ALT and AST in the control group mice were significantly increased (p<0.05), indicating that the liver of the control group mice was damaged. Compared with the control group, after treatment with 1-lactulose, the ALT and AST levels decreased (reduction rates of 55.4% and 35.7%, respectively). This indicates that 1-lactulose has a significant alleviating effect on CCl4-induced acute liver injury. There was no significant difference between the 1-lactulose group and the biphenyl diester group (p>0.05), indicating that the effect of 1-lactulose in reducing ALT and AST activity is similar to that of biphenyl diester.

[0071] 1.2.3 Effects of 1-Lactulose on Liver Pathological Changes in a Mouse Model of CCl4-Induced Liver Injury

[0072] As shown in Figure 3: Compared with the control group, *p<0.05, **p<0.01; n=3. In the normal group, the liver lobe structure of mice was intact, and the liver cells were clearly structured and tightly arranged. In the control group, mice injected intraperitoneally with CCl4 showed significant liver damage, with inflammatory cell infiltration and extensive cell necrosis near the central hepatic vein. Hepatocytes were swollen with blurred cell boundaries and atrophied nuclei. After treatment with biphenyl diester, liver cell necrosis decreased and inflammation lessened. After treatment with 1-lactulose, liver cells returned to normal morphology. The results indicate that 1-lactulose can improve the pathological condition of CCl4-induced acute liver injury in mice, and its effect is superior to that of biphenyl diester.

[0073] Example 2: Study on the therapeutic effect and mechanism of 1-lactulose on ethanol-induced alcoholic liver injury in mice.

[0074] 2.1 Experimental Methods

[0075] 2.1.1 Animal Experiment Design

[0076] This experiment used SPF-grade male Kunming mice, 6 weeks old, weighing approximately 20-25g. The animal housing environment was maintained at a constant temperature (22±2℃), constant humidity (50±10%), and with a 12 / 12h light / dark cycle. The mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. After 7 days of acclimatization, the mice were randomly divided into 4 groups of 3 mice each. The animals were divided into a normal group, a control group, a positive control group (denoted as silymarin), and a 1-lactulose group, and then subsequent experiments were conducted.

[0077] Mice in the normal control group were administered the same dose of 0.9% saline by gavage. Mice in other groups were administered 56% Beijing Erguotou (14 ml / kg / day) by gavage at 9:00 AM daily. Mice in the positive control group were injected intraperitoneally with silymarin (10 mg / kg / day) at 3:00 PM daily. Mice in the 1-lactulose group were injected intraperitoneally with 1-lactulose (10 mg / kg / day), once daily for 7 consecutive days. After the last administration, mice were fasted for 16 hours but allowed free access to water. After anesthesia, blood was collected from the orbital sinus, and the mice were euthanized. The livers were then dissected and weighed.

[0078] 2.1.2 Determination of liver weight ratio in mice

[0079] Before sacrifice, the mice in each group were weighed. After anesthesia, all mice were sacrificed, and their livers were removed and weighed. Data were collected, and the liver weight ratio of each group was calculated. Liver weight ratio = (Mice liver weight (g) / Mice body weight (g)) × 100%

[0080] 2.1.3 Determination of liver function-related enzyme activity in mouse serum

[0081] Mouse blood obtained by orbital sampling was placed in a 1.5 ml EP tube and allowed to stand at room temperature for 2 hours. After centrifugation at 4°C and 2000 rpm for 20 minutes, the supernatant was collected and placed in a 1.5 ml EP tube. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were measured using the corresponding kits according to the manufacturer's instructions.

[0082] 2.1.4 HE staining of mouse liver

[0083] Hematoxylin and eosin (H&E) staining was used to stain liver tissue sections. Liver tissue fixed in formalin solution was removed and sectioned sequentially through the following steps: dehydration, clearing, paraffin embedding, sectioning, dewaxing, staining, washing, dehydration, fixation, and mounting. The physiological and pathological conditions of the sections were observed using an optical microscope.

[0084] 2.2 Experimental Results:

[0085] 2.2.1 Effect of 1-Lactulose on Liver Weight Ratio in Ethanol-Induced Alcoholic Liver Injury Model Mice

[0086] As shown in Table 2, the experimental results indicate that the liver weight ratio in the control group was significantly increased compared to the normal group (p<0.01), indicating that the livers of the control group mice were damaged. In contrast, the liver weight ratio in the 1-lactulose group was significantly decreased compared to the control group (p<0.01), and this decrease was concentration-dependent. This suggests that 1-lactulose can alleviate the increase in liver weight caused by ethanol-induced alcoholic acute liver injury.

[0087] Table 2. Effects of 1-Lactulose on Liver Weight Ratio in Ethanol-Induced Alcoholic Liver Injury Model Mice

[0088] Compared with the normal group, *p<0.05, **p<0.01; compared with the control group, # p<0.05, ## p<0.01; n=3.

[0089] 2.2.2 Effect of 1-Lactulose on Survival in Mice with Ethanol-Induced Alcoholic Liver Injury Model

[0090] As shown in Figure 4, 1-lactulose treatment can improve the mortality rate of mice with ethanol-induced alcoholic liver injury, reduce the number of mice that die, and the effect is better than that of silymarin.

[0091] 2.2.3 Effects of 1-Lactulose on ALT and AST Activities in Serum of Mice with Ethanol-Induced Alcoholic Liver Injury Model

[0092] The experimental results are shown in Figure 5: Compared with the control group, *p<0.05, **p<0.01; n=3. Compared with the normal group, the activity levels of ALT and AST in the control group mice were significantly increased (p<0.05), indicating that the liver of the control group mice was damaged. Compared with the control group, after treatment with 1-lactulose, the ALT and AST levels decreased (the decrease rates were 30.48% and 22.46%, respectively). This indicates that 1-lactulose has a significant alleviating effect on ethanol-induced alcoholic liver injury, similar to the effect of silymarin in the positive control group, and there was no significant difference between the two groups (p>0.05).

[0093] 2.2.4 Effects of 1-Lactulose on Liver Pathological Changes in a Mouse Model of Ethanol-Induced Alcoholic Liver Injury

[0094] As shown in Figure 6: Compared with the control group, *p<0.05, **p<0.01; n=3. In the normal group, mouse liver cells were morphologically regular and neatly arranged, with clear nuclei and cell boundaries. In the control group, after being fed alcohol, the mice showed significant liver damage, with fat vacuoles appearing in the liver, fat droplets of varying sizes accumulating in the cytoplasm, cell nuclei shifting to the periphery, and inflammatory cell infiltration. In the positive control group, after treatment with silymarin, the number of vacuoles and inflammatory cell infiltration decreased; after treatment with 1-lactulose, the liver cell boundaries regained integrity, and the appearance tended to be normal. This indicates that 1-lactulose is more effective than silymarin in reducing inflammation and steatosis in mice with alcoholic liver injury.

[0095] Example 3: Therapeutic effect and mechanism of 1-lactulose-induced drug-induced liver injury in mice induced by acetaminophen (APAP).

[0096] 3.1 Experimental Methods

[0097] 3.1.1 Animal Experiment Design

[0098] This experiment used SPF-grade male Kunming mice, 8 weeks old, weighing approximately 20-25g. The animal housing environment was maintained at a constant temperature (22±2℃), constant humidity (50±10%), and with a 12 / 12h light / dark cycle. Mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. After 7 days of acclimatization, the mice were randomly divided into 4 groups: a normal group (n=3), a control group (n=6), a 1-lactulose group, and a positive control group (n=7). The animals were then divided into the normal group, control group, positive control group (denoted as N-acetylcysteine), and 1-lactulose group for subsequent experiments.

[0099] Mice in the normal control group were injected intraperitoneally with 0.9% saline, while mice in other groups were injected intraperitoneally with APAP (400 mg / kg). Treatment began 12 hours after the intraperitoneal injection. Mice in the positive control group were injected intraperitoneally with N-acetylcysteine ​​(NAC) (10 mg / kg / day), and mice in the 1-lactulose group were injected intraperitoneally with 1-lactulose (10 mg / kg / day), once daily for 7 consecutive days. After the last administration, mice were fasted for 16 hours but allowed free access to water. After anesthesia, blood was collected from the orbital region, and the mice were euthanized. The livers were then dissected and weighed.

[0100] 3.1.2 Determination of liver weight ratio in mice

[0101] Before sacrifice, the mice in each group were weighed. After anesthesia, all mice were sacrificed, and their livers were removed and weighed. Data were collected, and the liver weight ratio of each group was calculated. Liver weight ratio = (Mice liver weight (g) / Mice body weight (g)) × 100%

[0102] 3.1.3 Determination of liver function-related enzyme activity in mouse serum

[0103] Mouse blood obtained by orbital sampling was placed in a 1.5 ml EP tube and allowed to stand at room temperature for 2 hours. After centrifugation at 2000 rpm for 20 minutes at 4°C, the supernatant was collected and placed in a 1.5 ml EP tube. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were measured using the corresponding kits according to the manufacturer's instructions.

[0104] 3.1.4 Immunohistochemical staining of mouse liver

[0105] Immunohistochemical staining of liver tissue was performed. Liver specimens fixed in formalin were embedded in paraffin blocks. For immunohistochemical analysis, liver sections were separated, rehydrated, and then incubated sequentially with rabbit Ki67 primary antibody and goat anti-rabbit IgG secondary antibody. The area of ​​positive staining was measured in a high-power field on each slide and quantified using ImageJ.

[0106] 3.1.5 HE staining of mouse liver

[0107] Hematoxylin and eosin (H&E) staining was used to stain liver tissue sections. Liver tissue fixed in formalin solution was removed and sectioned sequentially through the following steps: dehydration, clearing, paraffin embedding, sectioning, dewaxing, staining, washing, dehydration, fixation, and mounting. The physiological and pathological conditions of the sections were observed using an optical microscope.

[0108] 3.2 Experimental Results

[0109] 3.2.1 Effect of 1-Lactulose on Liver Weight Ratio in Mice with APAP-Induced Liver Injury

[0110] As shown in Table 3, the experimental results indicate that the liver weight ratio of the control group was significantly increased compared with the normal group (p<0.05), indicating that the livers of the control group mice were damaged. Compared with the control group, the liver weight ratio of the 1-lactulose group was significantly decreased (p<0.05), and even lower than that of the positive control group mice. This suggests that 1-lactulose can alleviate the increase in liver weight caused by APAP drug-induced liver injury, and its effect is superior to that of NAC.

[0111] Table 3. Effects of 1-Lactulose on Liver Weight Ratio in APAP-Induced Liver Injury Model Mice

[0112] Compared with the normal group, *p<0.05, **p<0.01; compared with the control group, #p<0.05, ##p<0.01; normal group n=3, control group n=6, 1-lactulose group and positive control group n=7.

[0113] 3.2.2 Effect of 1-Lactulose on Survival in Mice with APAP-Induced Liver Injury

[0114] As shown in Figure 7, 1-lactulose treatment can improve the mortality of mice with APAP-induced drug-induced liver injury, reduce the number of mice that die, and the effect is better than that of NAC.

[0115] 3.2.3 Effects of 1-Lactulose on Serum ALT and AST Activities in Mice with APAP-Induced Liver Injury

[0116] NAC, a positive control drug, is currently the only FDA-approved antidote for APAP. NAC is a prodrug of glutathione. After entering the body, it is first converted to cysteine ​​through deacetylation. Then, in hepatocytes, it is converted to glutamate by gamma-glutamylcysteine ​​synthase. Subsequently, glutamate is converted to glutathione by glycine through glutathione synthase. NAC replenishes the amount of intracellular glutathione, reduces the covalent binding of NAPQI to cellular proteins, and thus reduces hepatocyte necrosis. However, NAC has disadvantages such as respiratory and gastrointestinal side effects and a narrow therapeutic window; its therapeutic effect is significantly reduced after 8 hours of APAP poisoning. In this invention, mice were administered 1-lactulose and NAC 12 hours after modeling, and were sacrificed 12 hours later. Serum and liver samples were collected for testing.

[0117] The experimental results are shown in Figure 8: Compared with the control group, *p<0.05, **p<0.01; n=3. Compared with the normal group, the activity levels of ALT and AST in the control group mice were significantly increased (p<0.05), indicating that the liver of the control group mice was damaged. Compared with the control group, NAC did not show a significant difference in reducing AST activity (p>0.05). Compared with the control group, after treatment with 1-lactulose, both ALT and AST levels decreased significantly (the decrease rates were 49.7% and 50.9%, respectively). This indicates that administration of 1-lactulose 12 hours after APAP poisoning has a significant therapeutic effect and is superior to NAC.

[0118] 3.2.4 Effects of 1-Lactulose on Liver Pathological Changes in Mice with APAP-Induced Liver Injury

[0119] As shown in Figure 9: Compared with the control group, *p<0.05, **p<0.01; n=3. In the control group, mice injected with APAP for 24 hours showed congestion around the central vein and hepatocyte necrosis, with a large number of inflammatory cells in the necrotic area. The positive control group (NAC) showed similar results, with congestion and cell necrosis around the central vein. However, after treatment with 1-lactulose, the 1-lactulose group showed only mild congestion and a small amount of cell necrosis in the liver. This indicates that 1-lactulose can alleviate congestion and cell necrosis caused by APAP-induced liver injury, and its therapeutic efficacy remained good even 12 hours after model establishment, superior to NAC.

[0120] As shown in Figure 10: Compared with the control group, *p<0.05, **p<0.01; n=3. On day 7 after administration, the control group still had a large number of hepatocyte necrosis areas. However, the inflammation and necrosis of liver tissue in the 1-lactulose group were improved, and the effect was better than that of NAC.

[0121] 3.2.5 Effects of 1-Lactulose on Hepatocyte Regeneration-Related Indicators in Mice with APAP-Induced Liver Injury

[0122] Based on the HE results, it is hypothesized that 1-lactulose reduces cell necrosis caused by APAP-induced liver injury by promoting hepatocyte regeneration. Therefore, this invention uses immunohistochemistry to detect Ki67 expression in liver tissue.

[0123] As shown in Figure 11: Compared with the control group, *p<0.05, **p<0.01; compared with the normal group, #p<0.05, ##p<0.01; n=3. Compared with the normal group of mice, the Ki67 expression in the control group was decreased. However, after treatment with 1-lactulose, the Ki67 expression in the liver tissue of mice was significantly increased (by 215.6% compared with the control group), indicating that 1-lactulose can promote the proliferation of hepatocytes in mice with liver injury.

[0124] in conclusion:

[0125] 1. Several common acute liver injury models were established using CCl4, ethanol, and APAP to investigate the therapeutic effect of 1-lactulose on acute liver injury. 1-Lactulose significantly reduced serum AST and ALT levels in model mice, indicating that 1-lactulose can significantly improve the condition of several types of acute liver injury.

[0126] 2. HE staining of the livers of mice in three acute liver injury models revealed that 1-lactulose could reduce the inflammatory response in the liver of mice, thereby improving the symptoms of acute liver injury.

[0127] 3. After measuring the hepatocyte regeneration-related indicators in the liver of mice with drug-induced acute liver injury, it was found that 1-lactulose can increase the expression of Ki67, promote hepatocyte regeneration, and thus improve the large-area hepatocyte necrosis caused by acute drug-induced liver injury.

[0128] 4. Based on all the above results, this invention suggests that 1-lactulose exerts its anti-inflammatory effect on different types of acute liver injury by reducing the inflammatory response and promoting hepatocyte proliferation. The primary reason for this effect is the direct promotion of hepatocyte proliferation, which differs from other clinically used anti-inflammatory drugs that treat acute liver injury by reducing the inflammatory response and thus promoting hepatocyte repair.

[0129] Example 4: Study on the effect and mechanism of 1-lactulose against CCl4-induced liver fibrosis in mice

[0130] 4.1 Experimental Methods

[0131] 4.1.1 Animal Experiment Design

[0132] This experiment used SPF-grade male Kunming mice, 8 weeks old, weighing approximately 20-25g. The animal housing environment was maintained at a constant temperature (22±2℃), constant humidity (50±10%), and with a 12 / 12h light / dark cycle. The mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. After 7 days of acclimatization, the mice were randomly divided into 4 groups: a normal group (n=3), a control group, a 1-lactulose group, and a positive control group (n=7). The animals were then divided into the normal group, control group, positive control group (denoted as silymarin), and 1-lactulose group for subsequent experiments.

[0133] Mice in the normal control group were intraperitoneally injected with the same dose of corn oil as other groups, while mice in other groups were intraperitoneally injected with a 25% CCl4 corn oil solution (1 ml / kg) three times a week, ensuring consistent dosing intervals for 6 weeks. Starting from week 5, mice in the positive control group were intraperitoneally injected with silymarin (10 mg / kg / day), and mice in the 1-lactulose group were intraperitoneally injected with 1-lactulose (10 mg / kg / day) once daily for 14 consecutive days. Two days after the last CCl4 injection, the mice were fasted for 16 hours but allowed free access to water. After anesthesia, blood was collected from the orbital sinus, the mice were euthanized, and their livers were dissected and weighed.

[0134] 4.1.2 Determination of liver weight ratio in mice

[0135] Before sacrifice, the mice in each group were weighed. After anesthesia, all mice were sacrificed, and their livers were removed and weighed. Data were collected, and the liver weight ratio of each group was calculated. Liver weight ratio = (Mice liver weight (g) / Mice body weight (g)) × 100%

[0136] 4.1.3 Determination of liver function-related enzyme activity in mouse serum

[0137] Mouse blood obtained by orbital sampling was placed in a 1.5 ml EP tube and allowed to stand at room temperature for 2 hours. After centrifugation at 2000 rpm for 20 minutes at 4°C, the supernatant was collected and placed in a 1.5 ml EP tube. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were measured using the corresponding kits according to the manufacturer's instructions.

[0138] 4.1.4 Immunohistochemical staining of mouse liver

[0139] Immunohistochemical staining of liver tissue was performed. Liver specimens fixed in formalin were embedded in paraffin blocks. For immunohistochemical analysis, liver sections were separated, rehydrated, and then incubated sequentially with rabbit α-SMA and Collagen I primary antibody and goat anti-rabbit IgG secondary antibody. The area of ​​positive staining was measured in a high-power field on each slide and quantified using ImageJ.

[0140] 4.1.5 Mouse liver HE staining, Masson staining, and Sirius red staining

[0141] Liver tissue sections were stained using hematoxylin and eosin (H&E) staining, Masson staining, and Sirius red staining. Liver tissue fixed in formalin solution was removed and sectioned sequentially through dehydration, clearing, paraffin embedding, sectioning, dewaxing, staining, washing, dehydration, fixation, and mounting. The physiological and pathological conditions of the sections were observed using an optical microscope.

[0142] 4.2 Experimental Results

[0143] 4.2.1 Effect of 1-Lactulose on Liver Weight Ratio in CCl4-Induced Liver Fibrosis Mice

[0144] As shown in Table 4, the experimental results indicate that the liver weight ratio of the control group was significantly increased compared to the normal group (p<0.05), indicating that the livers of the control group mice were damaged. Compared to the control group, the liver weight ratio of the 1-lactulose group was significantly decreased (p<0.05), and even lower than that of the positive control group mice. This suggests that 1-lactulose can alleviate the increase in liver weight in mice caused by CCl4-induced liver fibrosis, and its effect is superior to that of silymarin.

[0145] Table 4. Effects of 1-Lactulose on Liver Weight Ratio in CCl4-Induced Liver Fibrosis Mice

[0146] Compared with the normal group, *p<0.05, **p<0.01; compared with the control group, #p<0.05, ##p<0.01; n=3 for the normal group, and n=7 for the control group, 1-lactulose group and positive control group.

[0147] 4.2.2 Effect of 1-Lactulose on Survival in Mice with CCl4-Induced Liver Fibrosis

[0148] As shown in Figure 12, 1-lactulose treatment can improve mortality in mice with CCl4-induced liver fibrosis and reduce the number of deaths.

[0149] 4.2.3 Effects of 1-Lactulose on ALT and AST Activities in Serum of CCl4-Induced Liver Fibrosis Mice

[0150] The experimental results are shown in Figure 13: Compared with the control group, *p<0.05, **p<0.01; n=3. Compared with the normal group, the activity levels of ALT and AST in the control group mice were significantly increased (p<0.05), indicating that the liver of the control group mice was damaged. Compared with the control group, after treatment with 1-lactulose, the ALT and AST levels decreased significantly (the decrease rates were 56.93% and 36.61%, respectively).

[0151] 4.2.4 Effects of 1-Lactulose on Liver Pathological Changes in CCl4-Induced Liver Fibrosis Mice

[0152] As shown in Figure 14, after continuous injection of CCl4 for 6 weeks, the control group mice exhibited significant hepatocyte necrosis, with a large number of inflammatory cells appearing in the necrotic areas. However, after treatment with 1-lactulose, the hepatocyte necrosis in the 1-lactulose group was significantly improved. This indicates that 1-lactulose can alleviate CCl4-induced liver necrosis in mice.

[0153] 4.2.5 Effect of 1-Lactulose on the Degree of Liver Fibrosis in CCl4-Induced Liver Fibrosis Mice

[0154] As shown in Figure 15: Compared with the control group, *p<0.05, **p<0.01; n=3. The control group mice showed significant collagen fiber proliferation, with fibrotic mice at grade S2, indicating successful establishment of the liver fibrosis model. However, after treatment with 1-lactulose, collagen fibers in the mouse liver tissue decreased, and 1-lactulose could alleviate the degree of liver fibrosis in mice, with a reduction rate of 58.73%.

[0155] As shown in Figure 16: Compared with the control group, *p<0.05, **p<0.01; compared with the normal group, #p<0.05, ##p<0.01; n=3. Compared with the normal group, the synthesis of both type I and type III collagen in the control group was significantly increased, and liver fibrosis occurred in the control group mice. However, after treatment with 1-lactulose, the collagen synthesis in the liver of mice in the 1-lactulose group was relatively reduced (the decrease rate was 43.75%), indicating that 1-lactulose can alleviate the degree of liver fibrosis in mice.

[0156] 4.2.6 Effects of 1-Lactulose on fibrosis markers α-SMA and Collagen I in CCl4-induced fibrotic mice

[0157] As shown in Figure 17: Compared with the control group, *p<0.05, **p<0.01; compared with the normal group, #p<0.05, ##p<0.01; n=3. Compared with the normal group, the expression of α-SMA in both the control group and the drug-treated group was significantly increased, indicating an increased degree of liver fibrosis. Compared with the control group, the expression of α-SMA in both the 1-lactulose group and the silymarin group was decreased, with a decrease rate of 44.75% in the 1-lactulose group.

[0158] As shown in Figure 18: Compared with the control group, *p<0.05, **p<0.01; compared with the normal group, #p<0.05, ##p<0.01; n=3. Compared with the normal group, the expression of Collagen I in both the control group and the drug-treated group was significantly increased, indicating an increased degree of liver fibrosis. Compared with the control group, the expression of Collagen I in both the 1-lactulose group and the silymarin group was decreased, with a decrease rate of 41.07% in the 1-lactulose group.

[0159] in conclusion:

[0160] 1. By detecting the activity of ALT and AST in mouse serum and performing HE staining, Masson staining, and Sirius red staining on liver tissue, this invention successfully constructed a CCl4-induced liver fibrosis mouse model.

[0161] 2. Masson staining and Sirius red staining results showed that the fibrotic mice in the control group were at a moderate S2 level of fibrosis. After treatment with 1-lactulose, the degree of fibrosis in the mice was reduced.

[0162] 3. Further immunohistochemical analysis of mouse liver revealed that 1-lactulose reduced the expression of α-SMA and Collagen I. This indicates that 1-lactulose treatment resulted in less fibrosis in mice compared to the control group, suggesting that 1-lactulose can inhibit the progression of liver fibrosis in mice.

[0163] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. 1-The application of lactulose in the preparation of drugs for the prevention and treatment of liver injury or liver fibrosis, characterized in that, The structure of 1-lactulose is at least one of the following structures:

2. The application according to claim 1, characterized in that, The liver injury mentioned includes chemical liver injury.

3. The application according to claim 2, characterized in that, The chemical liver injury mentioned includes liver injury induced by carbon tetrachloride.

4. The application according to claim 1, characterized in that, The liver injury mentioned includes alcoholic liver injury.

5. The application according to claim 1, characterized in that, The liver injury mentioned includes drug-induced liver injury.

6. The application according to claim 5, characterized in that, The drug-induced liver injury was acetaminophen-induced liver injury.

7. The application according to claim 1, characterized in that, The drug is an oral or injectable drug.

8. The application according to claim 1, characterized in that, The preparation method of the 1-lactulose includes the following steps: The reaction was catalyzed by adding o-nitrophenol-β-D-galactoside, fructose, and PsaGal protease to a buffer solution, followed by centrifugation to remove the precipitate; the supernatant was extracted, and the extract solution was lyophilized to obtain the final product; the amino acid sequence of the PsaGal protease is SEQ ID NO:

2.

9. The application according to claim 8, characterized in that, The nucleotide sequence of the gene encoding the PsaGal protease is SEQ ID NO:

1.

10. The application according to claim 8, characterized in that, The buffer solution contains 10-20 mM o-nitrophenol-β-D-galactoside ONPG, 60-500 mM fructose, and 0.1 U / ml PsaGal protease.

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