Application of isoaurantianone as TNIK (Tumor Necrosis Inhibitor Kinase) inhibitor in preparing medicine for treating fibrosis

Abstract

The invention discloses application of isoaurantianone as a TNIK inhibitor in preparation of a medicine for treating fibrosis, and relates to the technical field of application of natural compounds. The research result shows that the isoorange flavone is a novel TNIK inhibitor, can reduce the expression of TNIK in a dose-dependent manner within a certain concentration range, can improve the fibrosis phenotype induced by TGF beta1 and BLM at the cellular level, and can inhibit the 3T6 cell activation stimulated by the TGF beta1 and the RLE-6TN epithelial-mesenchymal transition induced by the BLM in a dose-dependent manner, so that the isoorange flavone can be used for preparing the TNIK inhibitor. And collagen production of the 3T6 and RLE-6TN cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of the application of natural compounds, and particularly relates to the application of isosinensetin as a TNIK inhibitor in the preparation of a medicament for treating fibrosis. Background Art

[0002] Traf2 and Nck interacting kinase (TNIK) belongs to a member of the germinal center kinase family (GCKs), and is a serine / threonine kinase, which is involved in the processes of cytoskeleton formation, cell migration, cell proliferation and the extension of neuronal dendritic cells. The functional alteration of TNIK is associated with chronic inflammation, dysregulated nutrient sensing, cellular senescence and altered intercellular communication, and is one of the key factors driving the occurrence and development of various diseases such as cancer, metabolic diseases and neurodegenerative diseases.

[0003] In recent years, studies have shown that TNIK is closely related to the fibrosis-driven pathways, including the transduction of signals such as WNT, TGFβ, JNK and NF-κB. As a downstream effector of the WNT signal transduction, TNIK activates the transcription of WNT target genes by phosphorylating the T cell factor 4 (TCF4) and β-catenin transcription complex, promoting the generation of extracellular matrix and inducing epithelial-mesenchymal transition (EMT). In addition, TNIK can also indirectly maintain TGFβ signal transduction by phosphorylating the TGFβ receptor, promoting the proliferation and differentiation of fibroblasts. In liver fibrosis, TNIK induces the activation of stellate cells, thereby driving the pathological fibrosis of the liver; in pulmonary fibrosis and renal fibrosis, TNIK has been shown to promote fibroblast activation and collagen production, while the intervention of the TNIK small molecule inhibitor (INS018_055) can effectively alleviate the fibrotic lesions in the lung and kidney tissues of mice. Therefore, TNIK is a promising drug target, and exploring more strategies that can effectively inhibit TNIK is of great significance for the prevention and treatment of fibrotic diseases.

[0004] Isosinensetin (ISO) is a methoxyflavonoid compound extracted from the traditional Chinese medicine Pericarpium Citri Reticulatae Viride, and has diverse pharmacological activities. Modern pharmacological studies have shown that isosinensetin has anti-inflammatory and antioxidant effects, can effectively reduce the damage of PM2.5 to human bronchial epithelial cells by inhibiting inflammation and ROS release, and can reduce estrogen deficiency-induced osteoporosis by inhibiting the ROS-mediated NF-κB / MAPK signaling pathway. At the same time, isosinensetin also exhibits good anti-cancer activity, and can inhibit the proliferation of cervical cancer Hela cells and gastric cancer AGS cells, and induce apoptosis and cell cycle arrest.

[0005] At present, there is no relevant report on the effect of isosinensetin in improving fibrotic diseases. Summary of the Invention

[0006] To solve the above technical problems, the object of the present invention is to provide a new application of isoorientin, specifically to provide the application of isoorientin as a TNIK inhibitor in the preparation of drugs for treating fibrosis. The research of the present invention shows that isoorientin can significantly reduce the protein expression of TNIK, and can significantly improve the fibrosis level and epithelial-mesenchymal transition (EMT) in the cell model induced by TGFβ 1 and BLM. Isoorientin has the characteristics of simple structure and good safety, and has good application value and development prospect as a TNIK inhibitor.

[0007] To achieve the above object, the present invention adopts the following technical scheme:

[0008] The molecular formula of the isoorientin is: C 20 H 20 O 7 , and the structural formula is as follows:

[0009] Preferably, the isoorientin is isoorientin itself and a mixture or extract containing isoorientin.

[0010] The isoorientin and its pharmaceutically acceptable salts or other derivatives are natural compounds for inhibiting TNIK expression to treat TNIK-impaired diseases.

[0011] Further, the dosage form of the drug is liquid, powder for injection, tablet, capsule, powder, pill, plaster, granule or dressing.

[0012] Preferably, when applied, the dosage of the drug is 5-40 μM.

[0013] The research of the present invention shows that isoorientin can dose-dependently reduce the protein expression of TNIK within a certain concentration range. At the same time, isoorientin improves the fibrosis phenotype induced by TGFβ 1 and BLM at the cell level. Isoorientin dose-dependently inhibits the activation of 3T6 cells stimulated by TGFβ 1 , the epithelial-mesenchymal transition of RLE-6TN induced by BLM, and the collagen production of 3T6 and RLE-6TN cells.

[0014] The beneficial effects of the present invention: The research results of the present invention show that isoorientin is a new TNIK inhibitor, which can significantly reduce the protein expression level of TNIK and improve the fibrosis phenotype induced by TGFβ 1 and BLM at the cell level. Description of the Drawings

[0015] Figure 1It is the preliminary verification of the inhibition of TNIK by isoorange flavone, showing the changes in the expression levels of TNIK and its downstream protein c-Myc in fibroblasts 3T6 after treatment with different concentrations of isoorange flavone for 24 hours.

[0016] Figure 2 It is in the 1 cell fibrosis model induced by TGFβ, and it is the further verification of the inhibition of TNIK by isoorange flavone. Different concentrations of isoorange flavone are used to act on fibroblasts 3T6 stimulated by TGFβ 1 for 24 hours respectively, and then the changes in the protein level of TNIK in the cells are observed; among them, indicates p < 0.01, ** indicates p < 0.01, and *** indicates p < 0.001.

[0017] Figure 3 It is in the 1 cell fibrosis model induced by TGFβ. It shows the effects of different concentrations of isoorange flavone on the protein levels of fibrosis-related markers; among them, indicates p < 0.001, and ** indicates p < 0.01.

[0018] Figure 4 It is in the 1 cell fibrosis model induced by TGFβ. It shows the effects of different concentrations of isoorange flavone on the mRNA levels of fibrosis-related markers; among them, indicates p < 0.001, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0019] Figure 5 It is in the alveolar epithelial cell fibrosis model induced by BLM, and it shows the effects of different concentrations of isoorange flavone on the protein expression of EMT markers.

[0020] Figure 6 It is in the alveolar epithelial cell fibrosis model induced by BLM, and it shows the effects of different concentrations of isoorange flavone on the expression of collagen. Specific Embodiments

[0021] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the accompanying drawings and embodiments. The embodiments of the present invention include but are not limited to the following conditions, and can be modified within the acceptable range in the art. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0022] The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are, unless otherwise specified, reagents and materials that can be obtained from commercial channels.

[0023] Example 1 The experiment used mouse embryonic fibroblasts 3T6 to verify the effect of isosakuranetin as a TNIK inhibitor. The 3T6 cells were seeded in a 12-well plate and cultured in an incubator at 37 °C, 5% CO 2 and saturated humidity for 24 h. Then, 3T6 cells were treated with 0, 5, 10, 20 μM of isosakuranetin for 24 h respectively. The 3T6 cells were collected to extract cellular proteins, and the expression levels of TNIK protein and its downstream protein c-Myc were detected by Western blot.

[0025] The experimental results are specifically described as follows: After treatment with isosakuranetin, the expression levels of TNIK and c-Myc proteins in 3T6 cells decreased in a dose-dependent manner. The results are shown in Figure 1 .

[0024] Example 2 The 3T6 cells were seeded in a 12-well plate and cultured in an incubator at 37 °C, 5% CO 2 and saturated humidity for 24 h. Then, the cells were stimulated with 10 ng / mL of TGFβ 1 for 24 h. The original culture medium was discarded, and the cells were treated with complete medium containing 5, 10, 20 μM of isosakuranetin for 24 h respectively. The expression level of TNIK protein was detected by Western blot.

[0025] The experimental results are specifically described as follows: The expression level of TNIK protein in 3T6 cells stimulated with TGFβ 1 increased significantly. After treatment with isosakuranetin, the expression level of TNIK protein decreased significantly in a dose-dependent manner. The results are shown in Figure 2 .

[0026] Example 3 On fibroblasts 3T6, a fibroblast activation model was constructed by treating with 10 ng / mL of TGFβ 1 for 24 h. After discarding the original culture medium, the cells were treated with complete medium containing 5, 10, 20, 40 μM of isosakuranetin for 24 h respectively. The protein and mRNA levels of fibroblast activation markers α-SMA and type I collagen (COL1A1) were detected by Western blot and RT-qPCR. The primer information is shown in Table 1.

[0027] Table 1

[0028] The experimental results are specifically described as follows: TGFβ 1The protein and mRNA expression levels of α-SMA and COL1A1 in stimulated 3T6 cells were significantly increased. After treatment with isosakuranetin, the expression levels of α-SMA and COL1A1 were significantly decreased in a dose-dependent manner. The results are shown in Figure 3 and Figure 4 .

[0029] Example 4 Rat alveolar epithelial cells RLE-6TN were seeded in 6-well plates and cultured in an incubator at 37 °C, 5% CO 2 and saturated humidity for 24 h. Then, a fibrosis model of alveolar epithelial cells was established by treating with 50 μg / mL bleomycin (BLM) for 24 h. After discarding the original medium, complete media containing 5, 10, and 20 μM isosakuranetin were added and incubated for 24 h. Cell proteins were collected to detect the protein expression levels of EMT markers α-SMA, Vimentin, and N-cadherin.

[0030] The experimental results are specifically described as follows: The protein expressions of α-SMA, Vim, and N-Cad in BLM-stimulated RLE-6TN cells were significantly increased. After treatment with isosakuranetin, the protein levels of α-SMA, Vimentin, and N-cadherin were decreased in a dose-dependent manner. The results are shown in Figure 5 .

[0031] Example 5 On RLE-6TN cells, a fibrosis model of alveolar epithelial cells was established by treating with 50 μg / mL BLM for 24 h. After discarding the original medium, complete media containing 5, 10, and 20 μM isosakuranetin were added and incubated for 24 h. Cell proteins were collected to detect the protein expression level of COL1A1.

[0032] The experimental results are specifically described as follows: The protein expression of COL1A1 in BLM-stimulated RLE-6TN cells was significantly increased. After treatment with isosakuranetin, the protein level of COL1A1 was decreased in a dose-dependent manner. The results are shown in Figure 6 .

[0033] In summary, the present invention provides a novel natural inhibitor of TNIK - isosakuranetin, which can dose-dependently reduce the expression of TNIK within a certain concentration range. Isosakuranetin shows good application value and development prospects in inhibiting TNIK and is a potential therapeutic drug for fibrotic diseases.

Claims

1. An application of isocinerin as a TNIK inhibitor in the preparation of a drug for treating fibrosis.

2. The use according to claim 1, characterized in that: The isoauricularia flavonoids are isoauricularia flavonoids themselves and a mixture or extract containing isoauricularia flavonoids.

3. The use according to claim 1, characterized in that: Isogranin improved the fibrotic phenotype induced by TGFβ1 and BLM at the cellular level, and dose-dependently inhibited TGFβ1-stimulated 3T6 cell activation, BLM-induced RLE-6TN epithelial-mesenchymal transition, and collagen production in 3T6 and RLE-6TN cells.

4. The use according to claim 1, characterized in that: The use according to claim 1 is characterized in that the dosage form of the drug is liquid, powder injection, tablet, capsule, powder, pill, paste, granule or dressing.

5. The use according to claim 1, characterized in that: When used, the concentration of the drug is 5 to 40 μM.

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