Application of 1-lactulose in preparation of medicine for preventing and treating liver injury or liver fibrosis

Abstract

The invention relates to the technical field of medicines, in particular to application of 1-lactulose in preparation of a medicine for preventing and treating liver injury or liver fibrosis. The traditional Chinese medicine composition has the advantage of remarkable effect of preventing and treating liver injury or liver fibrosis.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of 1-lactulose in preparing medicines for preventing and treating liver damage or liver fibrosis. Background Art

[0002] Liver disease is a common disease in clinical practice. The main causes of liver damage in clinical practice include accidental ingestion of toxic food, excessive drinking, improper medication, viral infection, etc. Chronic liver disease caused by various causes and liver damage caused by various liver-damaging factors may lead to liver cell necrosis, inflammation and secondary fibrosis, and eventually progress to cirrhosis or even liver cancer.

[0003] Recent studies have found that liver fibrosis can be reversed during the course of the disease. Therefore, an effective way to alleviate liver cirrhosis and liver cancer is to prevent and treat the disease at the liver fibrosis stage. Currently, the only effective treatment for liver fibrosis is liver transplantation, but its cost is extremely high. Therefore, there is an urgent need for an effective and low-toxic anti-liver fibrosis drug.

[0004] Liver fibrosis refers to the pathological process of abnormal proliferation of fibrous connective tissue in the liver when liver cells are necrotic and stimulated by inflammation. The basic pathological change is that a large amount of extracellular matrix (ECM) is deposited in the perisinusoidal space and its composition changes, resulting in narrowing of the perisinusoidal space and "capillarization". It is also the wound healing response of the liver to chronic damage caused by different causes. Mild cases are called liver fibrosis, and severe cases lead to the reconstruction of liver lobule structure, pseudolobule and nodule formation, which is called cirrhosis. When the liver is in a state of damage for a long time, it often leads to the occurrence of liver fibrosis, which is mainly manifested by excessive proliferation and abnormal deposition of extracellular matrix components and excessive proliferation of hepatic stellate cells.

[0005] Nowadays, in the research of drugs against liver diseases, the research on the effects of experimental liver injury in animals is constantly deepening. The animal models of chemical liver injury induced by carbon tetrachloride, D-galactosamine, etc., the animal models of alcoholic liver injury induced by ethanol, etc., and the animal models of drug-induced liver injury caused by acetaminophen (APAP), etc. are divided into the animal models.

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

[0007]

[0008] However, there is no report on the use of the novel 1-lactulose in the treatment of liver diseases. Summary of the invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of 1-lactulose in the preparation of a drug for preventing and treating liver damage or liver fibrosis.

[0010] The present invention is achieved through the following technical solutions:

[0011] A use of 1-lactulose in preparing a drug for preventing and treating liver damage or liver fibrosis, wherein the structure of the 1-lactulose is at least one of the following structures:

[0012]

[0013] In some embodiments of the present invention, the liver injury comprises chemical liver injury.

[0014] In some preferred embodiments of the present invention, the chemical liver injury includes carbon tetrachloride-induced liver injury.

[0015] In some embodiments of the present invention, the liver injury comprises alcoholic liver injury.

[0016] In some embodiments of the present invention, the liver injury includes drug-induced liver injury.

[0017] In some preferred embodiments of the present invention, the drug-induced liver injury is acetaminophen-induced liver injury.

[0018] In some embodiments of the present invention, the drug is an oral drug or an injectable drug.

[0019] In some embodiments of the present invention, the method for preparing 1-lactulose comprises the following steps:

[0020] o-Nitrophenol-β-D-galactoside, fructose and PsaGal protease are added to a buffer solution for catalytic reaction, and then the precipitate is removed by centrifugation; the supernatant is extracted, and the extracted solution is freeze-dried to obtain; the amino acid sequence of the PsaGal protease is SEQ ID NO: 2.

[0021] In some preferred embodiments of the present invention, the nucleotide sequence of the gene encoding the PsaGal protease is SEQ ID NO: 1.

[0022] 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.

[0023] The beneficial effects of the present invention are:

[0024] The 1-lactulose of the present invention has the effects of promoting liver cell proliferation, inhibiting inflammatory response and the like, and has a significant therapeutic effect in preventing and treating liver damage and liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 1-lactulose to CCl 4 Dose-effect curve of chemical-induced acute liver injury.

[0026] Figure 2 1-lactulose to CCl 4 Effects of chemical liver injury on serum ALT and AST activities in mice. A: Statistical graph of serum ALT activity in mice. B: Statistical graph of serum AST activity in mice.

[0027] Figure 3 1-lactulose to CCl 4 Effects of chemical liver injury on liver pathology in mice. A: HE staining results of mouse liver, the scales of the four groups are the same. B: Statistical chart of inflammatory cell infiltration area in mouse HE sections.

[0028] Figure 4 This is the survival curve of mice with ethanol-induced alcoholic liver injury model.

[0029] Figure 5 The effect of 1-lactulose on serum ALT and AST activities in mice with ethanol-induced alcoholic liver damage.

[0030] Figure 6 The effect of 1-lactulose on liver pathology in mice with alcoholic liver injury induced by ethanol. A: HE-stained sections of mouse liver, the scales of the four groups are the same. B: Statistical diagram of fat vacuoles in mouse liver sections.

[0031] Figure 7 This is the survival curve of mice with APAP-induced liver injury.

[0032] Figure 8 The effect of 1-lactulose on the activity of ALT and AST in the serum of mice with APAP-induced liver injury. A: Statistical graph of ALT and AST activity in the serum of mice 12 hours after administration. B: Statistical graph of ALT and AST activity in the serum of mice 7 days after administration.

[0033] Fig. 9 The effect of 1-lactulose administration on liver pathology in mice with APAP-induced liver injury 12 hours after administration. A: HE-stained sections of mouse liver 12 hours after administration, with the same scale in each group. B: Statistical chart of necrotic area in mouse liver sections.

[0034] Fig.10The effect of 1-lactulose on liver pathology in mice with APAP-induced liver injury after 7 days of administration. A: HE-stained sections of mouse liver after 7 days of administration, with the same scale in each group. B: Statistical chart of necrotic area in mouse liver sections.

[0035] Fig.11 The effect of 1-lactulose on Ki67 immunohistochemistry in mice with APAP-induced liver injury. A: Ki67 immunohistochemistry results, the scale of each group is the same. B: Statistical chart of Ki67 positive hepatocytes.

[0036] Fig.12 CCl 4 Survival curve of liver fibrosis in mice.

[0037] Fig.13 1-lactulose to CCl 4 Effects of ALT and AST activity in serum of mice with hepatic fibrosis. A: Statistical graph of ALT activity in serum of mice. B: Statistical graph of AST activity in serum of mice.

[0038] Fig.14 CCl 4 HE staining results of livers of mice with hepatic fibrosis. The scale bars in each group are the same.

[0039] Fig.15 CCl 4 Results of Masson staining of livers of mice with hepatic fibrosis. A: Masson staining of mouse livers. The scale bar of each group is the same, 100 μm. B: Statistical diagram of Masson staining positive areas.

[0040] Fig.16 CCl 4 Results of Sirius red staining of mouse livers with hepatic fibrosis. A: Pictures of Sirius red staining of mouse livers, with the same scale for each group. B: Statistical graph of Sirius red staining of collagen in mice.

[0041] Fig.17 1-lactulose to CCl 4 Effect of α-SMA immunohistochemistry on liver fibrosis in mice. A: α-SMA immunohistochemistry results, the scale bar of each group is the same, 100μm. B: Statistical graph of α-SMA positive area.

[0042] Fig.18 1-lactulose to CCl 4 Effect of Collagen I immunohistochemistry on liver fibrosis in mice. A: Results of Collagen I immunohistochemistry, the scale bar of each group is the same, 100 μm. B: Statistical graph of Collagen I positive area. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0044] 1-Lactulose was prepared according to the method disclosed in CN116535448A.

[0045] The nucleotide sequence of the DNA fragment for recombinantly expressing glycosidic transferase (PsaGal) is SEQ ID NO: 1, and the amino acid sequence of the glycosidic transferase (PsaGal) encoded by the fragment is SEQ ID NO: 2.

[0046] The preparation of 1-lactulose was carried out in 20mM phosphate buffer (pH, 7.5, PB buffer system), which included 20mM o-nitrophenol-β-D-galactoside ONPG, 400mM fructose and 0.1U / ml recombinant expressed PsaGal enzyme. The above system was reacted at 25℃ for 4h, heated at 100℃ for 10min to terminate the reaction, and then centrifuged at 10000g / min for 10min to remove the precipitate. Chloroform was used to mix the supernatant in a volume ratio of 1:1 and then extracted. The extraction process was repeated 3 times to remove ONPG and residual protein in the reaction solution. The extracted solution was freeze-dried to obtain 1-lactulose.

[0047] The structure of 1-lactulose of the present invention is at least one of the following structures:

[0048]

[0049] The acetal or ketal structure in sugar compounds is unstable, especially the hemiacetal structure at the terminal reducing end, which is even more unstable. It exists in two forms: open chain and cyclic. The cyclic structure has two forms: pyranose (six-membered ring) and furose (five-membered ring) according to the size of the ring. Therefore, the above three configurations are interconvertible and can be considered to be the same sugar.

[0050] Example 11 - Lactulose on carbon tetrachloride (CCl 4 Study on the therapeutic effect and mechanism of chemical liver injury induced by ) in mice

[0051] 1.1 Experimental methods

[0052] 1.1.1 Animal Experiment Design

[0053] In this experiment, SPF male Kunming mice, 6 weeks old, weighing about 20-25 g, were selected and kept in an animal room with a constant temperature (22±2°C), constant humidity (50±10%), and a day-night cycle of light (12 / 12h light / darkness). The mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0054] After 7 days of adaptive feeding, the mice were randomly divided into 4 groups, with 3 mice in each group. The animals were divided into the control group, 1-lactulose (3.125 mg / kg / day) group, 1-lactulose (6.25 mg / kg / day) group, and 1-lactulose (12.5 mg / kg / day) group, and the liver weight ratio test was performed to determine the dosage of 1-lactulose.

[0055] Liver weight ratio test experiment: Each group of mice was intraperitoneally injected with CCl 4 (1ml / kg, 1:3 corn oil), 24h after intraperitoneal injection, the drug treatment began. The mice in the 1-lactulose group were intraperitoneally injected with 1-lactulose 3.125mg / kg / day, 6.25mg / kg / day and 12.5mg / kg / day, respectively, and the mice in the control group were intraperitoneally injected with 0.9% saline once a day for 7 consecutive days. After the last administration, fasting but not water was performed for 16h. After the mice were anesthetized, blood was collected from the eye sockets. After the mice were killed, the livers were dissected and weighed.

[0056] After 7 days of adaptive feeding, the mice were randomly divided into 4 groups, with 3 mice in each group. The animals were divided into a normal group, a control group, a positive control group (recorded as bifendate), and a 1-lactulose group, and then subsequent experiments were carried out.

[0057] The normal group of mice was intraperitoneally injected with the same amount of corn oil as the other groups, and the other groups of mice were intraperitoneally injected with CCl 4 (1ml / kg, 1:3 corn oil), 24h after intraperitoneal injection, the drug treatment began. The mice in the positive control group were intraperitoneally injected with bifendate (10mg / kg / day), and the mice in the 1-lactulose group were intraperitoneally injected with 1-lactulose (10mg / kg / day), once a day, for 7 consecutive days. After the last administration, fasting but not water for 16h, the mice were anesthetized, blood was collected from the eye sockets, and the mice were killed and the liver was dissected and weighed.

[0058] 1.1.2 Determination of mouse liver weight ratio

[0059] Before killing the mice, weigh the weight of mice in each group. After anesthesia, kill all mice and remove the liver to weigh the weight. After collecting the data, calculate the liver weight ratio of mice in each group.

[0060] Liver weight ratio = mouse liver weight (g) / mouse body weight (g) × 100%

[0061] 1.1.3 Determination of liver function-related enzyme activities in mouse serum

[0062] The mouse blood obtained by orbital blood sampling was placed in a 1.5 ml EP tube at room temperature for 2 hours, and then centrifuged at 4°C and 2000 rpm for 20 minutes, and the supernatant was placed in a 1.5 ml EP tube. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were determined using the corresponding kit according to the instructions.

[0063] 1.1.4 HE staining of mouse liver

[0064] The liver tissue sections were stained with hematoxylin and eosin (H&E) staining. The liver tissue fixed in formalin solution was removed and the sections were prepared through dehydration, transparency, paraffin embedding, sectioning, dewaxing, staining, washing, dehydration, fixation and sealing. The physiological and pathological states of the sections were observed using an optical microscope.

[0065] 1.2 Experimental results:

[0066] 1.2.11-Lactulose on CCl 4 Effect of chemical liver injury model on liver-to-weight ratio in mice

[0067] Liver damage induced by various reasons will increase the weight of the mouse liver and increase the percentage of the mouse's total body weight. Therefore, the liver weight ratio is a simple and important indicator to measure the degree of liver damage in mice. The calculation results of the mouse liver weight ratio are shown in Table 1.

[0068] Table 11 - Lactulose to CCl 4 Effect of chemical liver injury model on liver-to-weight ratio in mice

[0069]

[0070] Compared with the control group, *p<0.05, **p<0.01; n=3.

[0071] The experimental results showed that compared with the control group, the liver weight ratio of mice in each dose group of 1-lactulose decreased in a dose-dependent manner, and there was a significant difference (p<0.05) when the 1-lactulose concentration was 12.5 mg / kg compared with the control group, indicating that 1-lactulose can alleviate the increase in liver weight caused by acute liver damage.

[0072] At the same time, the relationship between 1-lactulose dose and efficacy was plotted to determine the ED of 1-lactulose. 50 Calculate the result as Figure 1 As shown, ED 50 =6.218mg / kg, therefore, the subsequent selection is slightly higher than ED 50The subsequent experiments were carried out with 1-lactulose (10 mg / kg).

[0073] 1.2.21-Lactulose on CCl 4 Effect of chemical liver injury model mice serum ALT and AST activities

[0074] Alanine aminotransferase (ALT) is mainly present in the cytoplasm of liver cells. Under normal circumstances, the ALT content in the blood is low. When the liver is damaged and liver cells are necrotized, ALT will be released into the blood in large quantities, indicating that the liver is damaged. Aspartate aminotransferase (AST) is mainly distributed in the mitochondria of cells in tissues such as the myocardium, liver and kidneys. Under normal circumstances, the AST content in serum is low. When the liver is damaged, the permeability of the liver cell membrane increases, allowing AST to be released into the blood. When the AST content in the serum increases, it indicates that the above-mentioned tissues and organs are damaged. Since the levels of ALT and AST in serum are positively correlated with the degree of liver damage, the levels of ALT and AST in serum are important indicators for assessing the degree of liver damage.

[0075] The experimental results are as follows Figure 2 As shown: 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 increased significantly (p<0.05), indicating that the liver of the control group mice was damaged. Compared with the control group, after 1-lactulose treatment, ALT and AST indicators decreased (decline rate 55.4% and 35.7%). This shows that 1-lactulose has an effect on CCl 4 There was no significant difference between the 1-lactulose group and the bifendate group (p>0.05), indicating that the effect of 1-lactulose in reducing ALT and AST activities was similar to that of bifendate.

[0076] 1.2.3 Effect of 1-lactulose on liver pathological changes in mice with CCl4-induced liver injury model

[0077] like Figure 3 As shown: compared with the control group, *p<0.05, **p<0.01; n=3. The liver lobe structure of mice in the normal group was intact, and the liver cell structure was clear and tightly arranged; the mice in the control group were intraperitoneally injected with CCl 4 After treatment, obvious liver damage occurred in the liver, with inflammatory cell infiltration and large-area cell necrosis near the central vein of the liver, hepatocyte expansion, blurred cell boundaries, and atrophy of the cell nucleus; after treatment with bifendate, liver cell necrosis decreased and inflammation was alleviated; after treatment with 1-lactulose, liver cells returned to normal morphology. The results showed that 1-lactulose can improve CCl 4The results showed that the treatment of acute liver injury in mice was better than that of bifendate. Example 21 - Study on the therapeutic effect and mechanism of lactulose on ethanol-induced alcoholic liver injury in mice 2.1 Experimental methods

[0078] 2.1.1 Animal Experiment Design

[0079] This experiment used SPF grade male Kunming mice, 6 weeks old, weighing about 20-25g, and the animal room was kept in a constant temperature (22±2℃), constant humidity (50±10%), and a day and night cycle of light (12 / 12h light / darkness). The mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. After 7 days of adaptive feeding, the mice were randomly divided into 4 groups, 3 in each group. The animals were divided into a normal group, a control group, a positive control group (recorded as silymarin), and a 1-lactulose group, and then subsequent experiments were carried out.

[0080] The mice in the normal group were gavaged with the same dose of 0.9% saline, the mice in other groups were gavaged with 56% Beijing Erguotou (14ml / kg / day) at 9 am every day, the mice in the positive control group were intraperitoneally injected with silymarin (10mg / kg / day) at 3 pm every day, and the mice in the 1-lactulose group were intraperitoneally injected with 1-lactulose (10mg / kg / day) once a day for 7 consecutive days. After the last administration, the mice were fasted but not watered for 16 hours, and the mice were anesthetized, blood was collected from the eye sockets, and the livers were dissected and weighed after the mice were killed.

[0081] 2.1.2 Determination of mouse liver weight ratio

[0082] Before killing the mice, weigh the weight of mice in each group. After anesthesia, kill all mice and remove the liver to weigh the weight. After collecting the data, calculate the liver weight ratio of mice in each group.

[0083] Liver weight ratio = mouse liver weight (g) / mouse body weight (g) × 100%

[0084] 2.1.3 Determination of liver function-related enzyme activities in mouse serum

[0085] The mouse blood obtained by orbital blood sampling was placed in a 1.5 ml EP tube at room temperature for 2 hours, and then centrifuged at 4°C and 2000 rpm for 20 minutes, and the supernatant was placed in a 1.5 ml EP tube. The activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum was determined using the corresponding kit according to the instructions.

[0086] 2.1.4 HE staining of mouse liver

[0087] The liver tissue sections were stained with hematoxylin and eosin (H&E) staining. The liver tissue fixed in formalin solution was removed and the sections were prepared through dehydration, transparency, paraffin embedding, sectioning, dewaxing, staining, washing, dehydration, fixation and sealing. The physiological and pathological states of the sections were observed using an optical microscope.

[0088] 2.2 Experimental results:

[0089] 2.2.11-Effect of lactulose on liver weight ratio in mice with ethanol-induced alcoholic liver injury model

[0090] As shown in Table 2, the experimental results show that compared with the normal group, the liver weight ratio of the control group increased significantly (p<0.01), indicating that the liver of the mice in the control group was damaged. Compared with the control group, the liver weight ratio of the mice in the 1-lactulose group decreased significantly (p<0.01) and was concentration-dependent. This shows that 1-lactulose can alleviate the increase in liver weight caused by ethanol-induced acute alcoholic liver injury.

[0091] Table 21 - Effect of lactulose on liver weight ratio in mice with ethanol-induced alcoholic liver injury model

[0092]

[0093] Compared with the normal group, *p<0.05, **p<0.01; compared with the control group, # p<0.05, ## p<0.01; n=3.

[0094] 2.2.21-Effect of lactulose on the survival of mice with ethanol-induced alcoholic liver injury model

[0095] like Figure 4 As shown, 1-lactulose treatment can improve the mortality of mice with ethanol-induced alcoholic liver damage and reduce the number of mouse deaths, and its effect is better than silymarin.

[0096] 2.2.3 Effect of 1-lactulose on the activities of ALT and AST in the serum of mice with ethanol-induced alcoholic liver injury model

[0097] The experimental results are as follows Figure 5As shown: 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 increased significantly (p<0.05), indicating that the liver of the control group mice was damaged. Compared with the control group, after 1-lactulose treatment, ALT and AST indicators decreased (the decrease rate was 30.48% and 22.46%). It shows that 1-lactulose has a significant alleviating effect on ethanol-induced alcoholic liver damage, which is similar to the effect of silymarin in the positive control group, and there is no significant difference between the two groups (p>0.05).

[0098] 2.2.4 Effect of 1-lactulose on liver pathological changes in mice with ethanol-induced alcoholic liver injury model

[0099] like Figure 6 As shown: compared with the control group, *p<0.05, **p<0.01; n=3. The liver cells of the mice in the normal group were regular and neatly arranged, with clear nuclei and cell boundaries; after the mice in the control group were fed with alcohol, the liver of the mice was obviously damaged, fat vacuoles appeared in the liver, fat droplets of different sizes accumulated in the cytoplasm, the nuclei shifted to the periphery of the cells, and inflammatory cell infiltration occurred. After silymarin treatment in the positive control group, the number of vacuoles and inflammatory cell infiltration decreased; after 1-lactulose treatment, the integrity of the liver cell boundaries was restored and the appearance tended to be normal. This shows that 1-lactulose is better than silymarin in reducing inflammation and fatty degeneration in mice with alcoholic liver damage.

[0100] Example 31 - Study on the therapeutic effect and mechanism of lactulose acetaminophen (APAP) on drug-induced liver injury in mice

[0101] 3.1 Experimental methods

[0102] 3.1.1 Animal Experiment Design

[0103] This experiment used SPF grade male Kunming mice, 8 weeks old, weighing about 20-25g, and the animal room was kept in a constant temperature (22±2℃), constant humidity (50±10%), and a day and night cycle of light (12 / 12h light / darkness). The mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. After 7 days of adaptive feeding, the mice were randomly divided into 4 groups, normal group n=3, control group n=6, 1-lactulose group and positive control group n=7. The animals were divided into normal group, control group, positive control group (recorded as N-acetylcysteine), 1-lactulose group, and then subsequent experiments were carried out.

[0104] The mice in the normal group were intraperitoneally injected with 0.9% saline, and the mice in the other groups were intraperitoneally injected with APAP (400 mg / kg). 12 hours after the intraperitoneal injection, the drug treatment was started. The mice in the positive control group were intraperitoneally injected with N-acetylcysteine ​​(NAC) (10 mg / kg / day), and the mice in the 1-lactulose group were intraperitoneally injected with 1-lactulose (10 mg / kg / day), once a day, for 7 consecutive days. After the last administration, the mice were fasted but not watered for 16 hours. After the mice were anesthetized, blood was collected from the eye sockets, and the livers were dissected and weighed after the mice were killed.

[0105] 3.1.2 Determination of mouse liver weight ratio

[0106] Before killing the mice, weigh the weight of mice in each group. After anesthesia, kill all mice and remove the liver to weigh the weight. After collecting the data, calculate the liver weight ratio of mice in each group.

[0107] Liver weight ratio = mouse liver weight (g) / mouse body weight (g) × 100%

[0108] 3.1.3 Determination of liver function-related enzyme activities in mouse serum

[0109] The mouse blood obtained by orbital blood sampling was placed in a 1.5 ml EP tube at room temperature for 2 hours, and then centrifuged at 4°C and 2000 rpm for 20 minutes, and the supernatant was placed in a 1.5 ml EP tube. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were determined using the corresponding kit according to the instructions.

[0110] 3.1.4 Immunohistochemical staining of mouse liver

[0111] Immunohistochemical staining of liver tissues. Liver specimens fixed in formalin were embedded in paraffin blocks. For immunohistochemical analysis, liver sections were separated, rehydrated, and then incubated with rabbit Ki67 primary antibody and goat anti-rabbit IgG secondary antibody. The positive staining area was measured in the high-power field of each slide and quantified using ImageJ.

[0112] 3.1.5 HE staining of mouse liver

[0113] The liver tissue sections were stained with hematoxylin and eosin (H&E) staining. The liver tissue fixed in formalin solution was removed and the sections were prepared through dehydration, transparency, paraffin embedding, sectioning, dewaxing, staining, washing, dehydration, fixation and sealing. The physiological and pathological states of the sections were observed using an optical microscope.

[0114] 3.2 Experimental Results

[0115] 3.2.11-Effect of lactulose on liver-to-weight ratio in mice with APAP-induced liver injury

[0116] As shown in Table 3, the experimental results show that compared with the normal group, the liver weight ratio of the control group increased significantly (p<0.05), indicating that the liver of the mice in the control group was damaged. Compared with the control group, the liver weight ratio of the mice in the 1-lactulose group decreased significantly (p<0.05), and was lower than the liver weight ratio of the mice in the positive control group. This shows that 1-lactulose can alleviate the increase in liver weight caused by APAP drug-induced liver injury, and the effect is better than NAC.

[0117] Table 31-Effect of lactulose on liver weight ratio in APAP drug-induced liver injury model mice

[0118]

[0119] 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.

[0120] 3.2.21-Effect of lactulose on the survival of mice with APAP-induced liver injury

[0121] like Figure 7 As shown, 1-lactulose treatment can improve the mortality of mice with APAP-induced drug-induced liver injury and reduce the number of mouse deaths, and its effect is better than NAC.

[0122] 3.2.31-Effect of lactulose on serum ALT and AST activities in mice with APAP-induced liver injury

[0123] Positive drug NAC is the only APAP antidote approved by the FDA. NAC is a prodrug of glutathione. After entering the body, it is first converted into cysteine ​​by deacetylation, and then combined with glutamate in liver cells through the action of glutamylcysteine ​​synthase to convert into glutamylcysteine. Afterwards, glutamylcysteine ​​combines with glycine through the activity of glutathione synthetase and is finally converted into glutathione. NAC supplements the amount of glutathione in cells, reduces the covalent binding of NAPQI to cell proteins, and thus reduces liver cell necrosis. However, the disadvantage of NAC is that it has side effects related to the respiratory tract and gastrointestinal tract, and the treatment window is narrow. For APAP poisoning for more than 8 hours, the therapeutic effect is significantly reduced. The present invention selects to administer 1-lactulose and NAC 12h after modeling, and kills mice 12h after administration, and takes serum and liver for detection.

[0124] The experimental results are as follows Figure 8As shown: 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 increased significantly (p<0.05), indicating that the liver of the control group mice was damaged. Compared with the control group, NAC had no significant difference in reducing AST activity (p>0.05). Compared with the control group, after 1-lactulose treatment, ALT and AST indicators decreased, and there was a significant difference (the decrease rate was 49.7% and 50.9%). It shows that 1-lactulose has a significant therapeutic effect when administered 12h after APAP poisoning, and is better than NAC.

[0125] 3.2.41-Effect of lactulose on liver pathological changes in mice with APAP-induced liver injury

[0126] like Fig. 9 As shown: *p<0.05, **p<0.01 compared with the control group; n=3. 24h after APAP injection, the mice in the control group showed congestion around the central vein and hepatocyte necrosis, and a large number of inflammatory cells appeared in the necrotic area; the positive control group (NAC) group was similar to the control group, with congestion and cell necrosis around the central vein; after 1-lactulose treatment, the liver of the mice in the 1-lactulose group showed only slight congestion and a small amount of cell necrosis. It shows that 1-lactulose can reduce the congestion and cell necrosis caused by APAP drug-induced liver injury, and it still has a good therapeutic efficiency when administered 12h after modeling, which is better than NAC.

[0127] like Fig.10 As shown: *p<0.05, **p<0.01 compared with the control group; n=3. On the 7th day after administration, there were still a large number of hepatocyte necrosis areas in the control group. However, the inflammation and necrosis of the liver tissue in the 1-lactulose group were improved, and the effect was better than that of NAC.

[0128] 3.2.5 Effects of 1-lactulose on hepatocyte regeneration-related indices in mice with APAP-induced liver injury

[0129] According to the HE results, it is speculated that 1-lactulose promotes liver cell regeneration, thereby reducing cell necrosis caused by APAP drug-induced liver injury. Therefore, the present invention detects the expression of Ki67 in liver tissue by immunohistochemistry.

[0130] like Fig.11As shown: 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 Ki67 in the control group was reduced. After 1-lactulose administration, the expression of Ki67 in the liver tissue of mice increased significantly (increased by 215.6% compared with the control group). The results show that 1-lactulose can promote the proliferation of hepatocytes in mice with liver damage.

[0131] in conclusion:

[0132] 1. Use CCl 4 Several common acute liver injury models were established with ethanol and APAP to explore the therapeutic effect of 1-lactulose on acute liver injury. 1-lactulose can significantly reduce the levels of AST and ALT in the serum of model mice, indicating that 1-lactulose can significantly improve the conditions of several acute liver injuries.

[0133] 2. HE staining of the livers of three acute liver injury model mice revealed that 1-lactulose can reduce the inflammatory response of the mouse liver, thereby improving the symptoms of acute liver injury.

[0134] 3. By measuring the relevant indicators of promoting hepatocyte regeneration in the liver of mice with drug-induced acute liver injury model, 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.

[0135] 4. Based on all the above results, the present invention believes that 1-lactulose can reduce inflammatory response and promote liver cell proliferation, thereby playing a role in resisting different types of acute liver injury. Among them, direct promotion of liver cell proliferation is the main reason, which is different from other clinical anti-inflammatory drugs that reduce inflammatory response and promote liver cell repair to treat acute liver injury.

[0136] Example 41 - Lactulose Anti-CC1 4 Study on the effect and mechanism of induced liver fibrosis in mice

[0137] 4.1 Experimental methods

[0138] 4.1.1 Animal Experiment Design

[0139] This experiment used SPF grade male Kunming mice, 8 weeks old, weighing about 20-25g, and the animal room was kept in a constant temperature (22±2℃), constant humidity (50±10%), and a day and night cycle of light (12 / 12h light / darkness). The mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. After 7 days of adaptive feeding, the mice were randomly divided into 4 groups, normal group n=3, control group, 1-lactulose group and positive control group n=7. The animals were divided into normal group, control group, positive control group (recorded as silymarin), 1-lactulose group, and then subsequent experiments were carried out.

[0140] The mice in the normal group were intraperitoneally injected with the same dose of corn oil as in the other groups, and the mice in the other groups were intraperitoneally injected with 25% CCl 4 Corn oil solution (1 ml / kg) was administered 3 times a week to ensure consistent dosing intervals for 6 weeks. Starting from the 5th week, 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 a day for 14 consecutive days. The last injection of CCl 4 Two days later, the mice were fasted but not watered for 16 h, blood was collected from their orbits after being anesthetized, and the livers were dissected and weighed after being killed.

[0141] 4.1.2 Determination of mouse liver weight ratio

[0142] Before killing the mice, weigh the weight of mice in each group. After anesthesia, kill all mice and remove the liver to weigh the weight. After collecting the data, calculate the liver weight ratio of mice in each group.

[0143] Liver weight ratio = mouse liver weight (g) / mouse body weight (g) × 100%

[0144] 4.1.3 Determination of liver function-related enzyme activities in mouse serum

[0145] The mouse blood obtained by orbital blood sampling was placed in a 1.5 ml EP tube at room temperature for 2 hours, and then centrifuged at 4°C and 2000 rpm for 20 minutes, and the supernatant was placed in a 1.5 ml EP tube. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were determined using the corresponding kit according to the instructions.

[0146] 4.1.4 Immunohistochemical staining of mouse liver

[0147] Immunohistochemical staining of liver tissues. Liver specimens fixed in formalin were embedded in paraffin blocks. For immunohistochemical analysis, liver sections were separated, rehydrated, and then incubated with rabbit α-SMA and CollagenⅠ primary antibodies and goat anti-rabbit IgG secondary antibodies. The positive staining area was measured in the high-power field of each slide and quantified using ImageJ.

[0148] 4.1.5 HE staining, Masson staining and Sirius red staining of mouse liver

[0149] Hematoxylin and eosin (H&E) staining, Masson staining, and Sirius red staining were used to stain liver tissue sections. The liver tissue fixed in formalin solution was removed and the sections were prepared through dehydration, transparency, paraffin embedding, sectioning, dewaxing, staining, washing, dehydration, fixation, and sealing. The physiological and pathological states of the sections were observed using an optical microscope.

[0150] 4.2 Experimental Results

[0151] 4.2.11-Lactulose on CCl 4 Effect of liver-to-weight ratio on hepatic fibrosis in mice

[0152] As shown in Table 4, the experimental results show that compared with the normal group, the liver weight ratio of the control group increased significantly (p<0.05), indicating that the liver of the mice in the control group was damaged. Compared with the control group, the liver weight ratio of the mice in the 1-lactulose group decreased significantly (p<0.05), and was lower than that of the mice in the positive control group. This shows that 1-lactulose can alleviate CCl 4 It induces an increase in liver weight in mice with hepatic fibrosis, and the effect is better than silymarin.

[0153] Table 41 - Lactulose to CCl 4 Effect of liver-to-weight ratio on hepatic fibrosis in mice

[0154]

[0155] 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, 1-lactulose group and positive control group n=7.

[0156] 4.2.21-Lactulose on CCl 4 Effect of induced liver fibrosis on the survival of mice

[0157] like Fig.12 As shown, 1-lactulose treatment can improve CCl 4 It can reduce the mortality of mice with liver fibrosis and the number of deaths of mice.

[0158] 4.2.31-Lactulose on CCl 4 Effect of ALT and AST activities in serum of mice with hepatic fibrosis

[0159] The experimental results are as follows Fig.13As shown: 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 increased significantly (p<0.05), indicating that the liver of the control group mice was damaged. Compared with the control group, after 1-lactulose treatment, ALT and AST indicators decreased, and there was a significant difference (the decrease rate was 56.93% and 36.61%).

[0160] 4.2.41-Lactulose on CCl 4 Effect of induced liver fibrosis on liver pathological changes in mice

[0161] like Fig.14 Shown: Control group mice were continuously injected with CCl for 6 weeks 4 After treatment, the mice showed obvious liver cell necrosis, and a large number of inflammatory cells appeared in the necrotic area. After 1-lactulose treatment, the liver cell necrosis of mice in the 1-lactulose group was significantly improved. This shows that 1-lactulose can reduce CCl 4 Induced liver necrosis in mice.

[0162] 4.2.51-Lactulose on CCl 4 Effect of induced liver fibrosis on the degree of liver fibrosis in mice

[0163] like Fig.15 As shown: Compared with the control group, *p<0.05, **p<0.01; n=3. The collagen fibers of the mice in the control group were significantly proliferated, and the fibrotic mice were at the S2 stage, and the liver fibrosis model was successfully established. After 1-lactulose treatment, the collagen fibers in the liver tissue of mice decreased. 1-lactulose can reduce the degree of liver fibrosis in mice, and the decrease rate is 58.73%.

[0164] like Fig.16 As shown: 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 type I collagen and type III collagen in the control group increased significantly, and the mice in the control group developed liver fibrosis. After 1-lactulose treatment, the collagen synthesis in the liver of the mice in the 1-lactulose group was relatively reduced (the decrease rate was 43.75%), indicating that 1-lactulose can reduce the degree of liver fibrosis in mice.

[0165] 4.2.6 Effect of 1-lactulose on fibrosis markers α-SMA and Collagen Ⅰ in CCl4-induced fibrosis mice

[0166] like Fig.17As shown: 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 the control group and the drug-treated group was significantly increased. This indicates that the degree of liver fibrosis increased. Compared with the control group, the expression of α-SMA in the 1-lactulose group and the silymarin group decreased, and the decrease rate of α-SMA expression in the 1-lactulose group was 44.75%.

[0167] like Fig.18 As shown: 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 CollagenⅠ in the control group and the drug-treated group was significantly increased. This indicates that the degree of liver fibrosis increased. Compared with the control group, the expression of CollagenⅠ in the 1-lactulose group and the silymarin group decreased, and the decrease rate of CollagenⅠ in the 1-lactulose group was 41.07%.

[0168] in conclusion:

[0169] 1. The present invention successfully constructed CCl by detecting the activity of ALT and AST in mouse serum and performing HE staining, Masson staining and Sirius red staining on liver tissue. 4 Mouse model of hepatic fibrosis.

[0170] 2. The results of Masson staining and picrosirius red staining showed that the fibrosis degree of the mice in the control group was at S2 grade moderate fibrosis. After treatment with 1-lactulose, the fibrosis degree of the mice was alleviated.

[0171] 3. Further immunohistochemistry of mouse livers revealed that 1-lactulose can reduce the expression of α-SMA and Collagen Ⅰ. This indicates that after 1-lactulose administration, the degree of fibrosis in mice was lighter than that in the control group, and 1-lactulose can inhibit the progression of liver fibrosis in mice.

[0172] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. 1- Use of lactulose in the preparation of a drug for preventing and treating liver damage or liver fibrosis, characterized in that: The structure of the 1-lactulose is at least one of the following structures:

2. The use according to claim 1, characterized in that: The liver injury includes chemical liver injury.

3. The use according to claim 2, characterized in that: The chemical liver injury includes carbon tetrachloride-induced liver injury.

4. The use according to claim 1, characterized in that: The liver damage includes alcoholic liver damage.

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

6. The use according to claim 5, characterized in that: The drug-induced liver injury is liver injury induced by acetaminophen.

7. The use according to claim 1, characterized in that: The medicine is an oral medicine or an injectable medicine.

8. The use according to claim 1, characterized in that: The preparation method of 1-lactulose comprises the following steps: o-Nitrophenol-β-D-galactoside, fructose and PsaGal protease are added to a buffer solution for catalytic reaction, and then the precipitate is removed by centrifugation; the supernatant is extracted, and the extracted solution is freeze-dried to obtain; the amino acid sequence of the PsaGal protease is SEQ ID NO:

2.

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

1.

10. The use according to claim 8, characterized in that: 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.

Images