Application of dehydrogenated harmine derivatives in improving organ fibrosis

Abstract

The invention discloses the application of dehydroharmine derivatives (HMD) in improving organ fibrosis, belonging to the field of compound application. The dehydroharmine derivatives disclosed in the invention include 1-(2-chloro)phenyl-9-butyl-β-carboline (HMD3) and / or 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide (HMD4). Animal experiments have found that it can significantly improve the problem of compensatory formation of liver and lung fibrosis in echinococcosis due to parasitic squeezing and damage, so as to be used for the treatment of related diseases of tissue and organ fibrosis, and its toxic and side effects are relatively low, which provides a theoretical basis for the development of clinical drugs to improve and treat organ fibrosis.

Description

Technical Field

[0001] The invention relates to the application field of compounds, and in particular to the application of dehydrogenated harmine derivatives in improving organ fibrosis. Background Art

[0002] Fibrosis is a pathological process characterized by tissue scarring. It can be caused by trauma and a variety of diseases, and manifests as excessive tissue growth, scarring or sclerosis. Fibrosis can occur in almost all organs in the human body, thereby damaging tissue structure and function. In mild cases, it becomes organ fibrosis, and in severe cases, it can develop into organ sclerosis, which seriously threatens human health and life. A large number of studies have clarified the key role of the mechanism centered on the overactivation of transforming growth factor-β (TGF-β) signaling in organ fibrosis. At present, drugs developed based on the above research results, such as pirfenidone and imatinib, have been marketed for the treatment of fibrotic diseases such as idiopathic pulmonary fibrosis, but there are still problems of poor efficacy and serious adverse drug reactions.

[0003] Harmine (HM) is a natural alkaloid extracted from the seeds of Peganum harmala, a plant of the Tribulus terrestris family. It has multiple beneficial effects, including anti-hydatid, antibacterial, anti-inflammatory, broad anti-tumor activity, central nervous system effects, and radioprotection. In recent years, research on compound derivatives has become a hot topic. There are literature reports that harmine derivatives can be used to treat cystic echinococcosis. There are also literature reports that chemical modification of harmine can enhance anti-tumor activity. However, there are currently no reports that harmine derivatives can improve organ fibrosis. Therefore, in-depth research on whether harmine derivatives can improve organ fibrosis is beneficial to expanding the pharmaceutical use of harmine derivatives and providing a new direction for the improvement of organ fibrosis. Summary of the invention

[0004] The purpose of the present invention is to provide the use of dehydrogenated harmine derivatives in improving organ fibrosis to solve the problems existing in the above-mentioned prior art. The present invention finds that dehydrogenated harmine derivatives can significantly improve the problem of compensatory liver and lung fibrosis caused by parasite squeezing and damage in echinococcosis, thereby being used to treat diseases related to tissue and organ fibrosis, and providing a theoretical basis for the development of clinical drugs to improve and treat organ fibrosis.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides use of hydroharmine derivatives (HMD) in preparing a drug for improving and / or treating organ fibrosis.

[0007] Further, the dehydrogenated harmine derivative includes one or more of 1-(2-chloro)phenyl-9-butyl-β-carboline (HMD3) and / or 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide (HMD4);

[0008] The chemical structural formula of the 1-methyl-9-(3-pyridyl)methyl-β-carboline is:

[0009]

[0010] The chemical structural formula of the 1-(4-methoxy)phenyl-9-butyl-β-carboline is:

[0011]

[0012] The chemical structural formula of the 1-(2-chloro)phenyl-9-butyl-β-carboline is:

[0013]

[0014] The chemical structural formula of the 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide is:

[0015]

[0016] Furthermore, the organ fibrosis includes organ fibrosis caused by echinococcosis.

[0017] Furthermore, the organ fibrosis caused by echinococcosis includes compensatory organ fibrosis caused by squeezing and damage by parasites.

[0018] Furthermore, the organs include lungs and liver.

[0019] The present invention also provides a drug for improving and / or treating organ fibrosis, comprising any combination of more than one of the above-mentioned dehydrogenated harmine derivatives.

[0020] Furthermore, the medicine also includes commonly used excipients, auxiliary materials and other pharmaceutically acceptable salts.

[0021] Application of a pharmaceutical preparation containing the dehydrogenated harmine derivative in preparing a drug for improving and / or treating organ fibrosis.

[0022] The present invention discloses the following technical effects:

[0023] The dehydrogenated harmine derivatives disclosed in the present invention can significantly improve the problem of compensatory liver and lung fibrosis caused by parasite squeezing and damage in echinococcosis, and can be used to treat diseases related to tissue and organ fibrosis, providing a theoretical basis for the development of clinical drugs to improve and treat organ fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 HE staining images of lung tissue sections of mice in each group;

[0026] Figure 2 HE staining images of liver tissue sections of mice in each group;

[0027] Figure 3 Masson staining of lung tissue sections of mice in each group;

[0028] Figure 4 Masson staining of lung tissue sections of mice in each group;

[0029] Figure 5 The mRNA expression levels of TGF-β1 (A), Smad3 (B) and NF-кB p65 (C) in lung tissues of mice in each group (compared with the normal control group, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01).

[0030] Figure 6 Figure 2 is the content of hydroxyproline in the lung tissue of mice, where A is the content of hydroxyproline in the lung tissue of mice in the control group, model group and HMD1-HMD4 treatment group, B is the content of hydroxyproline in the lung tissue of mice in the control group, model group and HMD1 low / high dose group and HMD2 low / high dose group, and C is the content of hydroxyproline in the lung tissue of mice in the control group, model group and HMD3 low / high dose group and HMD3 low / high dose group (compared with the normal control group, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01). DETAILED DESCRIPTION

[0031] Various 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.

[0032] 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.

[0033] 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.

[0034] 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 those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The dehydrogenated harmine derivatives used in the embodiments of the present invention and their chemical structural formulas are as follows:

[0037] 1. HM derivative (HMD1): 1-methyl-9-(3-pyridyl)methyl-β-carboline, its chemical structure is:

[0038]

[0039] 2. HM derivative (HMD2), 1-(4-methoxy)phenyl-9-butyl-β-carboline, its chemical structure is:

[0040]

[0041] 3. HM derivative (HMD3): 1-(2-chloro)phenyl-9-butyl-β-carboline, its chemical structure is:

[0042]

[0043] 4. HM derivative (HMD4): Butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide, its chemical structure is:

[0044]

[0045] The present invention uses intraperitoneal injection of Echinococcus to establish a mouse model of hydatid infection. The specific conditions of establishing the animal model, grouping and drug administration are as follows:

[0046] Eight-week-old female Kunming mice (provided by the Experimental Animal Center of Xinjiang Medical University) were selected, and fresh, naturally infected sheep livers of Echinococcus granulosus were collected from the slaughterhouse in Urumqi, Xinjiang. The Echinococcus granulosus in the liver cysts were collected under sterile conditions to ensure that the activity of Echinococcus granulosus was ≥95%. About 1000 (Echinococcus granulosus) / mouse were injected intraperitoneally to establish a mouse model of echinococcosis infection. Mice that were not injected with Echinococcus granulosus served as the blank control group. The mice with successful modeling were divided into a model group and a dehydrogenated harmaline derivative treatment group 1-4 (50 mg / kg) by a completely random method, with 10 mice in each group, of which the dehydrogenated harmaline derivative treatment groups 1-4 were respectively administered with the above-mentioned HM derivatives (HMD1)-(HMD4). The drugs were mixed into the corresponding concentrations and then administered by gavage, once a day, for 28 days.

[0047] Example 1 Observation of lung tissue pathological changes by HE staining and observation of lung tissue collagen deposition by Masson staining

[0048] 1. Collection and Slice Preparation of Mouse Lung and Liver Tissue Specimens

[0049] 28 days after administration, mice in each group were killed by cervical dislocation, and the lungs and livers of the mice were removed. Some of them were fixed with 4% paraformaldehyde, dehydrated with gradient ethanol, made transparent, paraffin-embedded, and sliced ​​(4 μm thick). The remaining lung tissues were frozen at -80°C for later use.

[0050] 2. HE staining to observe pathological changes in lung tissue

[0051] The paraffin sections were baked in a 65°C oven, dewaxed and rehydrated, then stained in hematoxylin solution for 1 min, washed 3 times, differentiated in hydrochloric acid and ethanol for 2 s, blued in PBS (pH 7.2), washed; stained with eosin solution for 1 min, washed, dehydrated, dried naturally after transparency, sealed with neutral resin, and examined under a microscope.

[0052] Results: The lung tissue structure of mice in the blank control group was basically normal, the alveoli were uniform in size, no inflammatory exudate was observed, and no pulmonary fibrosis was observed. However, the degree of pulmonary fibrosis in the model group infected with Echinococcus granulosus was significantly aggravated, as shown by severe damage to the alveolar structure, a large number of red blood cells in some alveolar cavities, obvious thickening of the alveolar septa, the appearance of a large number of inflammatory cells, and more severe inflammatory infiltration. The inflammatory changes in the lung tissue of mice treated with 1-(2-chloro)phenyl-9-butyl-β-carboline (HMD3) or 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide (HMD4) were significantly alleviated compared with the model group in the same period, the alveolar structure was relatively complete and uniform in size, the congestion in the alveolar cavity was improved, as shown by reduced thickening of the alveolar septa, reduced infiltration of peripheral inflammatory cells, and significantly reduced degree of fibrosis. The improvement of lung tissue inflammation in mice treated with 1-methyl-9-(3-pyridyl)methyl-β-carboline (HMD1) and 1-(4-methoxy)phenyl-9-butyl-β-carboline (HMD2) was not significant compared with the model group at the same period. Figure 1 HE staining of lung).

[0053] The model group mice had severe pathological damage to liver tissue, with edema, necrosis, inflammatory cell infiltration, irregular arrangement of hepatic cords, disordered hepatic lobule structure, increased hepatic intercellular matrix, and excessive deposition of blue collagen between hepatocytes. Compared with the model group, the inflammatory changes in liver tissue of mice in the medication group (1-(2-chloro)phenyl-9-butyl-β-carboline (HMD3) or 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide (HMD4)) were significantly alleviated compared with the model group in the same period, and the nuclear structure, cell arrangement, and liver plate structure were improved, which was manifested by reduced thickening of the hepatic intercellular matrix, reduced infiltration of surrounding inflammatory cells, and reduced fibrosis. The inflammatory changes in liver tissue of mice in the 1-methyl-9-(3-pyridyl)methyl-β-carboline (HMD1) and 1-(4-methoxy)phenyl-9-butyl-β-carboline (HMD2) treatment groups were not significantly improved compared with the model group in the same period ( Figure 2 Liver HE staining).

[0054] 3. Masson staining to observe collagen deposition in lung tissue

[0055] According to the Masson trichrome staining kit operating procedures, the paraffin sections were baked in a 65°C oven, dewaxed and rehydrated; hematoxylin staining for 8 minutes, washed with water; differentiated for 10 seconds, rinsed with distilled water; ammonia aqueous solution to blue for 4 minutes, washed with distilled water; stained with Ponceau fuchsin for 8 minutes, washed with weak acid working solution; differentiated with phosphomolybdic acid for 2 minutes, washed with distilled water; stained with aniline blue staining solution for 2 minutes; excess aniline blue staining solution was washed with weak acid working solution; conventional dehydration to make it transparent, naturally dried, sealed with neutral resin, and examined under a microscope.

[0056] Results: The lung tissue structure of mice in the blank control group was normal, the alveoli were uniform in size, and there was no significant change. A small amount of blue collagen deposition was observed in the alveolar septa. The blue collagen staining in the lung tissue of mice in the model group increased compared with that in the normal control group, and a large number of proliferating blue collagen fibers could be seen in the alveolar septa and alveolar walls. Compared with the model group, the fibrosis area of ​​mice treated with 1-(2-chloro)phenyl-9-butyl-β-carboline (HMD3) or 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide (HMD4) was reduced, and the alveolar structure remained relatively intact, which was significantly better than the model group. The degree of collagen deposition and proliferation in the alveolar septa was reduced. Compared with the model group, there was no significant difference in the changes of lung fibrosis area, alveolar structure, and the degree of collagen deposition and proliferation in the alveolar septa of mice treated with 1-methyl-9-(3-pyridyl)methyl-β-carboline (HMD1) and 1-(4-methoxy)phenyl-9-butyl-β-carboline (HMD2) in the dehydrogenated harmaline derivatives group ( Figure 3 Masson staining of lung).

[0057] In the model group, the nuclei and proliferating collagen fibers of the liver cells of the mice were stained blue, while the cytoplasm, muscles, and red blood cells were stained red. Compared with the model group, the blue matrix collagen deposition was reduced and the degree of fibrosis was alleviated in the (1-(2-chloro)phenyl-9-butyl-β-carboline (HMD3) or 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide (HMD4)) treatment group. There was no significant improvement in the mice treated with the dehydrogenated harmaline derivatives group, 1-methyl-9-(3-pyridyl)methyl-β-carboline (HMD1), and 1-(4-methoxy)phenyl-9-butyl-β-carboline (HMD2) treatment group compared with the model group ( Figure 4 Masson staining of liver).

[0058] Example 2 Real-time fluorescence quantitative PCR detection of lung tissue TGF-β1 mRNA, Smad3 mRNA and NF-кB p65 mRNA expression levels

[0059] In this embodiment, the real-time fluorescence quantitative PCR experiment for detecting the expression levels of TGF-β1 mRNA, Smad3 mRNA and NF-кBp65 mRNA in lung tissue mainly includes the following steps:

[0060] The total RNA of mouse lung tissue was extracted by Trizol method, the total RNA concentration was determined by nucleic acid quantification instrument, and the PrimeScriptKitTM RTReagent Kit was used to reverse transcribe and synthesize cDNA. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequences are shown in Table 1. The TakaRa (RR030A) PCR kit was used for the experiment, and 3 parallel wells were made for each sample to be tested. Reaction system: Premix Ex-TaqTMⅡ5μL, cDNA1μL, Forward Primer 0.4μL, Reverse Primer 0.4μL, ddH2O 3μL, ROX fluorescent label 0.2μL, total volume 10μL. Reaction conditions: 95℃30s, 95℃5s, 60℃34s, 40 cycles in total. -ΔΔCt The amplification results were analyzed by the method. The results are shown in Table 2 and Figure 5 .

[0061] Table 1

[0062]

[0063] Table 2

[0064]

[0065] Results: Real-time fluorescence quantitative PCR was used to detect the expression levels of TGF-β1mRNA, Smad3mRNA and NF-кB p65mRNA in the lung tissue of mice. The model group was significantly increased compared with the blank control group (F values ​​were 37.641, 39.914 and 362.290, all P<0.01); the dehydrogenated harmaline derivative treatment group was decreased compared with the model group, and the (1-(2-chloro)phenyl-9-butyl-β-carboline (HMD3) or 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide (HMD4)) treatment group had a significantly increased expression of TGF-β1mRNA, Smad3mRNA and NF-кB p65mRNA in the lung tissue of mice. The expression level of p65mRNA was significantly decreased compared with that in the model group (P<0.01). There was no significant difference in the expression levels of TGF-β1mRNA, Smad3mRNA and NF-кB p65mRNA in the lung tissue of mice in the dehydrogenated echinopsine derivative treatment group and the 1-methyl-9-(3-pyridyl)methyl-β-carboline (HMD1) and 1-(4-methoxy)phenyl-9-butyl-β-carboline (HMD2) treatment groups compared with those in the model group.

[0066] Example 3 Effect of HMD on Hydroxyproline Content in Mouse Lung Tissue

[0067] In order to study the effect of different doses of HMD on the hydroxyproline content in mouse lung tissue, the dehydrogenated echinopsine derivative treatment groups 1-4 were used as the (HMD1)-(HMD4) high-dose groups, and the mice successfully modeled in the present invention were administered the above-mentioned HM derivatives (HMD1)-(HMD4) at a dose of 25 mg / kg, recorded as the (HMD1)-(HMD4) low-dose groups, and the mouse lung tissues were collected according to the method of Example 1.

[0068] In this embodiment, the experiment of measuring the hydroxyproline content in mouse lung tissue mainly includes the following steps:

[0069] Mouse lung tissue stored in a -80°C refrigerator was taken, thawed at room temperature, about 80 mg of lung tissue specimen was weighed and placed in a test tube, cut into pieces, 1 mL of hydrolysis solution was added, and mixed. The hydroxyproline (HYP) detection kit of Nanjing Jiancheng Bioengineering Institute was used to detect the HYP content in the lung tissue. The specific operation was carried out according to the instructions. Finally, the supernatant was taken at 550 nm, 1 cm light path, and double distilled water was used to zero to measure the absorbance of each tube.

[0070] Pulmonary fibrosis is characterized by abnormal deposition of extracellular matrix, which eventually leads to a significant increase in collagen fibers. HYP is a unique amino acid in the body, mainly present in collagen. Therefore, hydroxyproline is a conventional indicator for evaluating collagen fibers in pulmonary fibrosis.

[0071] Results: Compared with the blank control group, the hydroxyproline content in the lung tissue of the model mice was significantly increased. Compared with the model group, the dehydrogenated harmaline derivatives group (1-methyl-9-(3-pyridyl)methyl-β-carboline (HMD1)), (1-(4-methoxy)phenyl-9-butyl-β-carboline (HMD2)), (1-(2-chloro)phenyl-9-butyl-β-carboline (HMD3) or 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide (HMD4)) could reduce the hydroxyproline content in the lung tissue of the mice (see Figure 6 A); the low and high dose groups of the dehydrogenated harmaline derivative group (1-(2-chloro)phenyl-9-butyl-β-carboline (HMD3) or 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide (HMD4)) could significantly reduce the content of hydroxyproline in the lung tissue of mice (P < 0.01) (see Figure 6 C); the content of hydroxyproline in the lung tissue of mice in the low-dose group of dehydrogenated echinopsine derivatives (1-methyl-9-(3-pyridyl)methyl-β-carboline (HMD1)) and (1-(4-methoxy)phenyl-9-butyl-β-carboline (HMD2)) did not change significantly, and the content of hydroxyproline in the lung tissue of mice in the high-dose group of dehydrogenated echinopsine derivatives (1-methyl-9-(3-pyridyl)methyl-β-carboline (HMD1)) and (1-(4-methoxy)phenyl-9-butyl-β-carboline (HMD2)) was reduced, but the difference was not significant (see Figure 6 B).

[0072] 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 a dehydrogenated harmine derivative in the preparation of a drug for improving and / or treating organ fibrosis, characterized in that: The dehydrogenated harmaline derivative is 1-(2-chloro)phenyl-9-butyl-β-carboline and / or 9-butyl-1-methyl-N-(2-hydroxy)ethyl-β-carboline-3-carboxamide; The organ fibrosis is the organ fibrosis caused by echinococcosis.

2. The use according to claim 1, characterized in that: The organ fibrosis caused by echinococcosis is a compensatory organ fibrosis formed after being squeezed and damaged by parasites.

3. The use according to claim 1, characterized in that: The organs include the lungs and liver.

4. Use of a pharmaceutical preparation containing the dehydrogenated harmine derivative according to claim 1 in the preparation of a drug for improving and / or treating organ fibrosis, wherein the organ fibrosis is organ fibrosis caused by echinococcosis.

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