Application of N-beta-alanyl dopamine or salt thereof in preparation of products for preventing and treating hepatic fibrosis

Abstract

The invention discloses an application of N-beta-alanyl dopamine or a salt thereof in preparation of a product for preventing and treating hepatic fibrosis, and belongs to the technical field of medicines. The invention provides an application of N-beta-alanyldopamine hydrochloride (N-beta-alanyldopamine hydrochloride) in preparation of a product for preventing and treating hepatic fibrosis, and experiments prove that the N-beta-alanyldopamine hydrochloride is given, so that the content level of hydroxylysylpyridinine (Pyr) is reduced, the liver hardness is reduced, the collagen solubility is improved, the collagen deposition of liver tissues is further reduced, and the liver fibrosis prevention and treatment effect is improved. The application of N-beta-alanyldopamine hydrochloride (N-beta-alanyldopamine hydrochloride (N-beta-alanyldopamine hydrochloride (N-beta-alanyldopamine hydrochloride (N-beta-alanyldopamine hydrochloride) in preparation of the product for preventing and treating hepatic fibrosis is realized. The traditional Chinese medicine composition can be used for relieving the liver fibrosis symptom of a mouse induced by carbon tetrachloride (CCl4), and can be used for relieving the liver fibrosis symptom of the mouse induced by the CCl4.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of N-β-alanyl dopamine or its salt in preparing products for preventing and treating liver fibrosis. Background Art

[0002] Liver fibrosis is a dynamic pathological process in all chronic liver diseases. It can be induced by hepatitis B or C virus, drugs, autoimmune and biliary diseases, alcoholic and non-alcoholic fatty hepatitis, etc. If it is not effectively treated, it can eventually lead to cirrhosis and related complications. The main feature of liver fibrosis is the excessive accumulation of extracellular matrix (ECM) maintained by heterogeneous liver myofibroblasts (MFs). The gradual deposition and remodeling of these ECM proteins in the interstitial space of lesions further leads to increased ECM stiffness and disease progression. Among the complex components of ECM, collagen is the most important structural component. Together with other components (elastic fibers, fibronectin, laminin, proteoglycans, etc.), it provides a scaffold to support the liver tissue structure and is the main determinant of tissue stiffness.

[0003] ECM is a structural scaffold composed of non-cellular, fibrin and non-fibrous proteins that can affect cell function and tissue homeostasis. Collagen is a large family of proteins that make up the ECM and plays an important role in providing the structural and biomechanical integrity of tissues. Increased cross-linking of collagen is also an important factor in preventing the regression of fibrosis. The pattern and degree of collagen lysyl hydroxylation affect the stability of collagen cross-linking, which provides tensile strength and mechanical stability of collagen fibrils. Two collagen cross-linking pathways have been identified, namely lysine aldehyde (Lys ald )-mediated cross-linking pathway and hydroxylysine aldehyde (Hyl ald )-mediated cross-linking pathway, Hyl ald The formed cross-links are difficult to degrade. In fibrotic tissue, the lysine hydroxylation of collagen increases significantly, the terminal peptide lysine residues are modified by procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (Plod2), and the collagen cross-links form a mature cross-link product: hydroxylysylpyridinoline (Pyr). Our studies have confirmed that Plod2 expression increases and Pyr content increases in liver fibrosis, liver stiffness increases, and collagen solubility decreases. Targeted inhibition of Plod2 activity and function can improve liver fibrosis.

[0004] Although several drugs with anti-liver fibrosis efficacy have entered clinical research, most of them have toxic side effects and fail to reach the end point of clinical trials. So far, no drugs targeting liver fibrosis have been marketed. Therefore, it is of great value to develop anti-liver fibrosis drugs with new targets and mechanisms of action.

[0005] N-β-alanyldopamine (hydrochloride) is an organic compound with the chemical formula C 11 H 17 ClN2O3 is the main derivative of dopamine in blood and lymph. However, the prior art does not disclose any report on the use of N-β-alanyldopamine (hydrochloride) or its derivatives to alleviate liver fibrosis. Summary of the invention

[0006] The purpose of the present invention is to provide an application of N-β-alanyl dopamine or its salt in the preparation of a product for preventing and treating liver fibrosis, so as to solve the problems existing in the above-mentioned prior art. The present invention provides an application of N-β-alanyl dopamine hydrochloride (N-β-alanyldopamine (hydrochloride)) in the preparation of a product for preventing and treating liver fibrosis. Experiments have confirmed that the administration of N-β-alanyl dopamine hydrochloride reduces the content of hydroxylysyl pyridinoline, reduces liver hardness and increases collagen solubility, thereby being able to alleviate the symptoms of liver fibrosis in mice induced by carbon tetrachloride (CCl4).

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides the use of N-β-alanyl dopamine or a salt thereof in preparing a product for preventing and treating liver fibrosis. The structural formula of the N-β-alanyl dopamine is:

[0009]

[0010] Optionally, the salt comprises N-β-alanyl dopamine hydrochloride.

[0011] Furthermore, the structural formula of the N-β-alanyl dopamine hydrochloride is:

[0012]

[0013] Furthermore, the N-β-alanyl dopamine hydrochloride reduces the content of hydroxylysylpyridinoline in liver tissue, reduces liver hardness and increases collagen solubility, thereby reducing collagen deposition in liver tissue, thereby preventing and treating liver fibrosis.

[0014] Optionally, the product comprises a medicine.

[0015] Optionally, the dosage form of the drug includes injection.

[0016] Optionally, the drug also includes pharmaceutically acceptable excipients.

[0017] The present invention discloses the following technical effects:

[0018] The present invention uses N-β-alanyldopamine hydrochloride (N-β-alanyldopamine (hydrochloride)) as a representative compound, and evaluates the preventive and therapeutic effect of N-β-alanyldopamine hydrochloride (N-β-alanyldopamine (hydrochloride)) on liver fibrosis through mouse experiments. N-β-alanyldopamine (hydrochloride) was intraperitoneally injected into a liver fibrosis mouse model induced by carbon tetrachloride for two consecutive weeks. The results showed that N-β-alanyldopamine (hydrochloride) significantly reduced the content of hydroxylysylpyridinoline, reduced liver hardness and increased collagen solubility, and improved the pathological state of liver fibrosis.

[0019] The present invention discloses the preventive and therapeutic effects and action mechanism of N-β-alanyldopamine (hydrochloride) on liver fibrosis, and lays a theoretical foundation and technical support for the development of new drugs for liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The figures are the test results of serum levels and liver tissue sections of mice after administration of different doses of N-β-alanyldopamine (hydrochloride); wherein A is the test result of alanine aminotransferase (ALT) level in the serum of mice in each dose group; B is the test result of aspartate aminotransferase (AST) level in the serum of mice in each dose group; C is the observation picture of liver tissue sections of mice in each dose group;

[0022] Figure 2The following are observation pictures of liver tissue sections of mice in the CCl4+Vehicle group and the CCl4+N-β-alanyldopamine (hydrochloride) group, wherein A is a representative picture of picrosirius red staining of mouse liver; B is the statistical result of the fibrosis area of ​​mouse liver sections;

[0023] Figure 3 The results of the detection of hydroxylysylpyridinoline (Pyr) content in the liver tissue of mice in the CCl4+Vehicle group and the CCl4+N-β-alanyldopamine (hydrochloride) group;

[0024] Figure 4 The results of the mouse liver hardness test in the CCl4+Vehicle group and the CCl4+N-β-alanyldopamine (hydrochloride) group; A is a schematic diagram of the mouse liver hardness test process; B is the statistical results of the mouse liver hardness;

[0025] Figure 5 These are the results of mouse liver collagen solubility test in the CCl4+Vehicle group and the CCl4+N-β-alanyldopamine (hydrochloride) group. DETAILED DESCRIPTION

[0026] Several exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] N-β-alanyldopamine: N-β-alanyl dopamine, CAS registration number is 54653-62-2, and its structural formula is as follows:

[0032]

[0033] N-β-alanyldopamine (hydrochloride): N-β-alanyl dopamine hydrochloride, CAS registration number is 58077-93-3, its structural formula is as follows:

[0034]

[0035] Example 1

[0036] The experiment in this example was approved by the Ethics Committee of Capital Medical University. All experimental data were expressed as mean ± standard error. SPSS25 statistical software was used to analyze the experimental results. The independent sample t test was used for comparison between two groups, and the one-way analysis of variance was used for comparison between multiple groups. P < 0.05 was considered to be significantly different.

[0037] The animals used in the experiment were fed with a normal diet and had free access to water and food. Mice were kept in an SPF-level animal room of the Capital Medical University Experimental Animal Center at (20±2)℃ and 12 hours of light / darkness. After one week of pre-adaptation, the mice were randomly divided into four groups and given four doses (0, 2.5 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight) of N-β-alanyldopamine (hydrochloride). The mice in the 0-dose group were given an equal volume of PBS solution. Each group of mice was given the drug by intraperitoneal injection twice a week for 2 weeks. The mice were killed on the 14th day for sampling, and they were fasted for 6 hours before being killed and had regular drinking water.

[0038] ALT and AST detection kits (Nanjing Jiancheng, Nanjing, China) were used to detect the ALT and AST levels in the serum of the above mice. Before use, R1 solution and R2 solution were prepared in a ratio of 200 μL:50 μL and preheated at 37°C. 10 μL of the serum to be tested and the blank control (normal saline) were placed in a 96-well ELISA plate, and 250 μL of the R1 and R2 mixed solution were added. After mixing, the OD value was detected at a wavelength of 340 nm, and the OD value and detection time point of continuous detection within 6 minutes were recorded. Enzyme activity = (OD change per minute - blank OD change) × 1746. The final results are expressed as mean ± standard error. The experiment was repeated 3 times.

[0039] Test results such as Figure 1 A and Figure 1 As shown in B, it can be seen that compared with normal mice (0 dose group), administration of 2.5 mg / kg body weight, 5 mg / kg body weight, and 10 mg / kg body weight of N-β-alanyldopamine (hydrochloride) had no significant effect on ALT and AST levels in the liver, indicating that N-β-alanyldopamine (hydrochloride) has no negative impact on liver function and has high biosafety.

[0040] The liver tissues of mice in each group were further taken for pathological sections, and the liver damage was evaluated by hematoxylin-eosin (H&E) staining. The steps of H&E staining are as follows:

[0041] (1) Conventional dewaxing of paraffin sections: xylene I for 10 min, xylene II for 10 min, 100% ethanol for 3-5 min, 100% ethanol for 3-5 min, 95% ethanol for 3-5 min, 90% ethanol for 3-5 min, 80% ethanol for 3-5 min, 70% ethanol for 3-5 min, 50% ethanol for 3-5 min, distilled water for 2 min;

[0042] (2) H&E staining:

[0043] 1) Place the dewaxed sections in hematoxylin staining solution for 5 minutes;

[0044] 2) Put the slices into 0.5% hydrochloric acid alcohol for color separation for 5 seconds;

[0045] 3) Rinse the slices in tap water for 15 minutes and then in distilled water for a while;

[0046] 4) Stain the sections in eosin solution for 10 minutes;

[0047] (3) Rinse with running water;

[0048] (4) Gradient alcohol dehydration: 50% ethanol for 3-5 minutes, 70% ethanol for 3-5 minutes, 80% ethanol for 3-5 minutes, 90% ethanol for 3-5 minutes, 95% ethanol for 3-5 minutes, 100% ethanol for 3-5 minutes, 100% ethanol for 3-5 minutes;

[0049] (5) Place the sections in xylene I for 10 minutes and xylene II for 10 minutes;

[0050] (6) Seal the slides with neutral gum to avoid air bubbles.

[0051] The results of pathological sections observation of mice in each group are as follows Figure 1 As shown in C, it can be seen that the hepatocytes of each group of mice have regular morphology, uniform size, neat arrangement, normal hepatocyte nuclear morphology, uniform chromatin distribution, no nuclear division or abnormal nucleoli, no obvious signs of inflammatory cell infiltration, fibrous tissue hyperplasia or fibrosis, etc. This shows that the administration of 2.5mg / kg body weight, 5mg / kg body weight, and 10mg / kg body weight of N-β-alanyldopamine (hydrochloride) has no toxic side effects on the liver of the normal body and is highly safe.

[0052] Example 2

[0053] The experiment in this example was approved by the Ethics Committee of Capital Medical University. All experimental data were expressed as mean ± standard error. SPSS25 statistical software was used to analyze the experimental results. The independent sample t test was used for comparison between two groups, and the one-way analysis of variance was used for comparison between multiple groups. P < 0.05 was considered to be significantly different.

[0054] The animals used in the experiment were fed with a normal diet and had free access to water and food. Mice were kept in an SPF-grade animal room at the Capital Medical University Experimental Animal Center at (20±2)℃ and 12 hours of light / darkness. After the mice were adapted for one week in advance, they were randomly divided into a model group and a drug-treated group.

[0055] The model group used CCl4 to induce liver fibrosis in mice, and intraperitoneal injection of CCl4 [CCl4 / olive oil (OO) = 1:9 (v / v)] was performed at a dose of 1 mL / kg body weight twice a week. An equal volume of PBS solution was given 2 hours before intraperitoneal injection of CCl4, which was recorded as the CCl4+Vehicle group. The drug administration group also used CCl4 to induce liver fibrosis in mice, and the administration of CCl4 was the same as that of the model group; at the same time, N-β-alanyldopamine (hydrochloride) was intraperitoneally injected twice a week, 2 hours before intraperitoneal injection of CCl4, and the dosage was 10 mg / kg body weight, which was recorded as the CCl4+N-β-alanyldopamine (hydrochloride) group. On the 14th day of modeling, the mice were killed for sampling, and they were fasted for 6 hours before being killed and had regular drinking water.

[0056] The liver tissues of mice in each group were taken for pathological sections, and the fibrosis of liver tissue was evaluated by picrosirius red staining. The steps of picrosirius red staining are as follows:

[0057] (1) Conventional dewaxing of paraffin sections: xylene I for 10 min, xylene II for 10 min, 100% ethanol for 3-5 min, 100% ethanol for 3-5 min, 95% ethanol for 3-5 min, 90% ethanol for 3-5 min, 80% ethanol for 3-5 min, 70% ethanol for 3-5 min, 50% ethanol for 3-5 min, distilled water for 2 min;

[0058] (2) Slices were placed in 0.1% picrosirius red staining solution and stained at 37°C for 30 minutes;

[0059] (3) Rinse with running water;

[0060] (4) Gradient alcohol dehydration: 50% ethanol for 3-5 minutes, 70% ethanol for 3-5 minutes, 80% ethanol for 3-5 minutes, 90% ethanol for 3-5 minutes, 95% ethanol for 3-5 minutes, 100% ethanol for 3-5 minutes, 100% ethanol for 3-5 minutes;

[0061] (5) Place the sections in xylene I for 10 minutes and xylene II for 10 minutes;

[0062] (6) Seal the slides with neutral gum to avoid air bubbles.

[0063] (7) Image analysis of liver tissue fibrosis:

[0064] Observe and take photos under an optical microscope. Ten different fields of view were randomly selected for each liver tissue slice, and the fibrosis area was analyzed using ImageJ image analysis software. The specific operation was as follows: first, the total area S0 of each field of view was calculated, then the total fiber area S1 of the field of view was circled (red blood vessels were not included in S1), and then the blank area S2 of the field of view (mainly the blank area of ​​the vascular section) was circled, and the quantitative value of fibrosis in the field of view was calculated according to the formula of fibrosis area, and finally the average value of fibrosis values ​​of all fields of view of each slice was calculated as the quantitative value of each slice. The formula of fibrosis area is as follows:

[0065]

[0066] The liver tissue sections of the two groups of mice were observed and statistically analyzed as follows: Figure 2 As shown, it can be seen that the collagen fiber deposition in the liver of mice in the CCl4+Vehicle group was obvious, and the collagen fiber deposition in the liver of mice in the CCl4+N-β-alanyldopamine(hydrochloride) group was less, indicating that after the administration of the compound N-β-alanyldopamine(hydrochloride), the collagen fiber deposition in mice was correspondingly reduced, indicating that N-β-alanyldopamine(hydrochloride) has a significant therapeutic effect on liver fibrosis.

[0067] Example 3

[0068] The ELISA kit was used to detect the level of hydroxylysylpyridinoline (Pyr) in the liver tissues of the two groups of mice in Example 2, and the detection steps were as follows:

[0069] (1) The content of Pyr in liver tissue was detected using a commercial ELISA kit (Huamei Biotechnology, Wuhan, China) according to the manufacturer's instructions. First, the liver sample was ultrasonically treated according to the instructions (ice water bath, 30 minutes);

[0070] (2) Allow all reagents to equilibrate at room temperature for at least 30 minutes, prepare the reagents according to the instructions, and set aside;

[0071] (3) Take out the ELISA plate, set up a blank control well without adding any liquid; set up two wells for each standard point in sequence, add 50 μL of the corresponding standard to each well; directly add 50 μL of the sample to be tested to each of the remaining detection wells;

[0072] (4) Add 50 μL of biotin marker to each well (except the blank control well), mix thoroughly, attach a self-adhesive sealant, and place at 37°C for 1 hour;

[0073] (5) Wash the plate manually and discard the liquid in the well. Inject 200 μL / well of the working solution into the well, let it stand for 10 seconds to spin dry, repeat three times and pat dry; wash the plate with a plate washer, select the three-wash program, and pat dry after washing;

[0074] (6) Add 50 μL of horseradish peroxidase-labeled avidin to each well (except the blank control well), mix thoroughly, attach a self-adhesive sealant, and place at 37°C for 30 minutes;

[0075] (7) Wash the plate manually and discard the liquid in the well. Inject 200 μL of the working solution into the well, let it stand for 10 seconds to spin dry, repeat three times and pat dry; wash the plate with a plate washer, select the three-wash program, and pat dry after washing;

[0076] (8) Add 50 μL of color developer A and 50 μL of color developer B to each well, shake and mix, and develop at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well.

[0077] (9) Use an ELISA reader to measure the optical density (OD value) of each well in sequence at a wavelength of 450 nm.

[0078] The results of the detection of hydroxylysylpyridinoline (Pyr) levels in the liver tissues of the two groups of mice are as follows Figure 3 As shown, it can be seen that compared with the mice in the CCl4+Vehicle group, the Pyr level per mole of collagen in the liver of the mice in the CCl4+N-β-alanyldopamine (hydrochloride) group was decreased, indicating that the administration of the compound N-β-alanyldopamine (hydrochloride) can reduce the Pyr level in the liver and improve liver fibrosis.

[0079] Example 4

[0080] Atomic force microscopy was used to detect changes in hardness of liver tissues of the two groups of mice in Example 2, and the detection steps were as follows:

[0081] The frozen liver sections (5 μm thickness) of the two groups of mice were immersed in PBS solution containing protease inhibitors and placed on the operating stage of the atomic force microscope. The MLCT probe (6 levers, 0.01-0.5 N / m) was lowered onto the tissue sample. The probe pair at the tip of the cantilever was 50×50 μm. 2 The samples within the area were scanned in two dimensions. For each indentation, the indentation speed was 20.3 μm / sec and the indentation depth was 50-80 nm. Three randomly selected areas were probed for each sample and the hardness data were analyzed using NanoScopeAnalysis 1.9 software.

[0082] The results of hardness test in liver tissue of two groups of mice are as follows Figure 4As shown, it can be seen that compared with the mice in the CCl4+Vehicle group, the hardness of the liver of the mice in the CCl4+N-β-alanyldopamine (hydrochloride) group decreased, indicating that the administration of the compound N-β-alanyldopamine (hydrochloride) can reduce liver hardness and improve liver fibrosis.

[0083] Example 5

[0084] The changes in collagen solubility in the liver tissues of the two groups of mice in Example 2 were detected, and the detection steps were as follows:

[0085] (1) The liver tissue samples of the two groups of mice were placed in a neutral salt solution (Tris-buffered saline solution containing protease inhibitors) and incubated overnight on a rotary shaker at 4°C. The samples were centrifuged at 4°C at 14,000 rpm for 30 min. The supernatant was collected for collagen determination and defined as salt-soluble collagen.

[0086] (2) The precipitate was extracted with 0.5 mol / L acetic acid solution, incubated on a rotary shaker at 4°C overnight, centrifuged at 4°C, speed: 14000 rpm, time: 30 min, and the supernatant was collected for collagen determination and defined as acid-soluble collagen;

[0087] (3) The precipitate was placed in a 0.5 mol / L acetic acid solution containing 2 mg / mL pepsin, incubated on a rotary shaker at 4°C overnight, centrifuged at 4°C, speed: 14000 rpm, time: 30 minutes, and the supernatant was collected for collagen determination and defined as pepsin-soluble collagen. The precipitate was defined as insoluble collagen.

[0088] (4) The collagen content of each part was determined by the hydroxyproline content determination method, and the soluble collagen contents of the three parts were added together to obtain the total soluble collagen content, which was then statistically calculated.

[0089] The results of collagen solubility in liver tissues of the two groups of mice are as follows Figure 5 As shown, compared with the mice in the CCl4+Vehicle group, the insoluble collagen content in the liver of the mice in the CCl4+N-β-alanyldopamine (hydrochloride) group was reduced, and the soluble collagen content was increased, indicating that the administration of the compound N-β-alanyldopamine (hydrochloride) can increase the solubility of collagen and improve liver fibrosis.

[0090] Therefore, the results of this example show that N-β-alanyldopamine (hydrochloride) can play a role in treating liver fibrosis by reducing Pyr content, reducing liver hardness and increasing collagen solubility.

[0091] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of N-β-alanyl dopamine or its salt in the preparation of a product for preventing and treating liver fibrosis, characterized in that: The structural formula of the N-β-alanyl dopamine is:

2. The use according to claim 1, characterized in that: Such salts include N-β-alanyl dopamine hydrochloride.

3. The use according to claim 2, characterized in that: The structural formula of the N-β-alanyl dopamine hydrochloride is:

4. The use according to claim 1, characterized in that: The N-β-alanyl dopamine hydrochloride reduces the content of hydroxylysyl pyridinoline in liver tissue, reduces liver hardness and increases collagen solubility, thereby reducing collagen deposition in liver tissue, thereby preventing and treating liver fibrosis.

5. The use according to claim 1, characterized in that: The products include pharmaceuticals.

6. The use according to claim 5, characterized in that: The dosage form of the drug includes injection.

7. The use according to claim 5, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

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