Application of parthenolide as CMA activator in preparation of medicine for treating fibrosis

Abstract

The invention discloses application of parthenolide as a CMA activator in preparation of a medicine for treating fibrosis, and relates to the technical field of application of natural compounds. Research results show that parthenolide is a novel CMA activator, can improve expression of LAMP2A in a dose-dependent manner within a certain concentration range, can significantly improve mRNA and protein levels of LAMP2A, can improve TGF-beta induced fibrosis phenotypes at a cellular level, and can inhibit TGF-beta stimulated HK-2 cell epithelial-mesenchymal transition (EMT) in a dose-dependent manner, so that the parthenolide can be used for preparing a medicine for treating leukocyte apoptosis. And 3T6 cell and HK-2 cell fibrogenesis.

Description

Technical Field

[0001] The present invention relates to the technical field of application of natural compounds, and in particular to the application of parthenolide as a CMA activator in the preparation of a drug for treating fibrosis. Background Art

[0002] Chaperone-Mediated Autophagy (CMA) is a finely regulated protein degradation pathway in cells. It plays a vital role in maintaining cell homeostasis, coping with stressful environments, and promoting cell survival. The core of this process lies in its high selectivity. It relies on a specific molecular chaperone, heat shock homologous protein 70 (Hsc70), which can accurately recognize and bind to substrate proteins containing the KFERQ pentapeptide sequence. This specific sequence is like a "tag", guiding these selected proteins to be safely transported to the lysosome, the "intracellular garbage disposal plant", through a series of complex molecular interactions.

[0003] On the inner membrane of the lysosome, there is a key receptor, lysosomal associated membrane protein 2A (LAMP2A), which is the portal in the CMA pathway and is responsible for binding to the Hsc70-substrate protein complex, thereby allowing the substrate protein to enter the lysosome for hydrolysis and degradation. This process not only helps to remove damaged or excess proteins, but also provides cells with necessary amino acids and other small molecules as raw materials for synthesizing new proteins and biomolecules, reflecting the high efficiency of cell waste utilization.

[0004] However, as the organism ages, the function of CMA gradually declines, which is considered to be one of the important factors in the development of various age-related diseases. In neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, reduced CMA activity is often observed in the brain cells of patients, leading to accumulation of abnormal proteins and impaired neuronal function. In addition, complex diseases such as metabolic disorders, immune deficiencies, and cancer are also closely related to CMA dysfunction. Therefore, exploring strategies that can effectively regulate CMA activity is of great significance for the prevention and treatment of these diseases.

[0005] Parthenolide (PTL), as a sesquiterpene lactone compound derived from the natural plant feverfew, has attracted much attention in recent years due to its wide range of pharmacological activities. The anti-inflammatory effect of PTL has been widely studied. It can effectively inhibit the production of multiple inflammatory mediators and reduce inflammatory responses, which is of great significance for the treatment of autoimmune diseases and inflammation-related diseases. At the same time, PTL also exhibits significant anti-tumor activity, can inhibit the proliferation of tumor cells, induce apoptosis, and interfere with tumor angiogenesis, providing a new idea for cancer treatment. In addition, its antiviral activity also makes it show potential in the treatment of infectious diseases.

[0006] In the prior art, parthenolide has an effect on the transdifferentiation of rat lung fibroblasts induced by transforming growth factor-β1 (TGF-β1). However, there is no report on its application to embryonic fibroblasts and other fibrotic diseases. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a new application of parthenolide, specifically providing the application of parthenolide as a CMA activator in the preparation of a drug for treating fibrosis. The present invention points out that parthenolide can significantly increase the transcription and protein expression of LAMP2A, and can significantly improve the fibrosis level and epithelial-mesenchymal transition (EMT) in a TGF-β-induced cell model. Parthenolide has the characteristics of simple structure and good safety, and has good application value and development prospects of CMA activators.

[0008] The present invention provides the use of parthenolide as a CMA activator in the preparation of a medicament for treating fibrosis.

[0009] The molecular formula of the parthenolide is: 15 H 20 O3, the structural formula is as follows:

[0010]

[0011] Preferably, the parthenolide is parthenolide itself, parthenolide and its pharmaceutically acceptable salts or derivatives of parthenolide.

[0012] The parthenolide and its pharmaceutically acceptable salt or other derivatives are natural compounds that promote LAMP2A expression and thus treat CMA-damaged diseases.

[0013] Furthermore, the dosage form of the drug is liquid, powder injection, tablet, capsule, powder, pill, paste, granule or dressing.

[0014] Preferably, when used, the dosage of the drug is 1.25-10 μM.

[0015] The present invention provides a CMA activator, which is parthenolide itself, parthenolide and a pharmaceutically acceptable salt thereof, or a derivative of parthenolide.

[0016] The present invention provides a drug for treating fibrosis. The active ingredient of the drug for treating fibrosis includes parthenolide itself, parthenolide and its pharmaceutically acceptable salts or derivatives of parthenolide.

[0017] Furthermore, the dosage form of the drug is liquid, powder injection, tablet, capsule, powder, pill, paste, granule or dressing.

[0018] Preferably, when used, the dosage of the drug is 1.25-10 μM.

[0019] Parthenolide can dose-dependently increase the expression of LAMP2A within a certain concentration range. At the same time, parthenolide dose-dependently inhibits TGF-β-stimulated HK-2 cell EMT, as well as 3T6 cell and HK-2 fiber generation.

[0020] Beneficial effects of the present invention:

[0021] The research results of the present invention show that parthenolide is a new CMA activator, which can significantly increase the mRNA and protein levels of LAMP2A and improve the TGF-β-induced fibrotic phenotype at the cellular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a preliminary verification of the activation of CMA by parthenolide of the present invention, and the effects of parthenolide on the mRNA and protein levels of LAMP2A in fibroblast 3T6 were investigated for 0, 12, 24, and 36 h, respectively; wherein, *** indicates that p < 0.001 compared with the 0 h group.

[0023] Figure 2 It is a further verification of the activation of CMA by parthenolide of the present invention, and the effects of 1.25, 2.5, 5, and 10 μM parthenolide on the mRNA and protein levels of LAMP2A in fibroblast 3T6 for 24 hours were respectively investigated; wherein, ** indicates that compared with the Crtl group, p < 0.01, *** indicates that compared with the Crtl group, p < 0.001.

[0024] Figure 3 The present invention shows the effect of parthenolide on the expression of fibrosis-related proteins in the TGF-β-induced cell fibrosis model; wherein ** indicates p < 0.01 compared with the TGF-β group, and ## indicates p < 0.01 compared with the Con group.

[0025] Figure 4 The present invention discloses the effect of parthenolide on the expression of EMT proteins in a TGF-β-induced renal epithelial cell fibrosis model.

[0026] Figure 5 The present invention discloses the effect of parthenolide on the expression of fibrosis-related proteins in a TGF-β-induced renal epithelial cell fibrosis model. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below 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 making any creative work shall fall within the scope of protection of the present invention.

[0028] Example 1: Parthenolide increases the mRNA and protein levels of LAMP2A in fibroblast 3T6 cells in a time-dependent manner

[0029] The experiment used mouse embryonic fibroblasts 3T6 to verify the role of parthenolide as a CMA activator.

[0030] In 3T6 cells, 10 μM parthenolide was used to act on 3T6 cells for 0, 12, 24, and 36 h, and the 3T6 cells were collected to extract total RNA, and the transcription level of LAMP2A was detected by RT-qPCR, or the cell protein was extracted and the protein expression level was examined by Western blot. The primer information is shown in Table 1.

[0031] Table 1

[0032]

[0033] Results Figure 1 ,The results showed that after treatment with parthenolide, the mRNA and protein levels of LMAP2A in 3T6 cells decreased in a dose-dependent manner.

[0034] Example 2: Parthenolide dose-dependently increases the mRNA and protein levels of LAMP2A in 3T6 fibroblasts

[0035] 3T6 cells were treated with 1.25, 2.5, 5, and 10 μM of parthenolide for 24 h, and the effects of parthenolide on the mRNA and protein levels of LMAP2A were detected by Western blot and RT-qPCR. The results are shown in Figure 2 .

[0036] The results showed that after treatment with parthenolide, the mRNA and protein levels of LMAP2A in 3T6 cells increased in a dose-dependent manner.

[0037] Example 3: Parthenolide dose-dependently inhibits TGF-β-stimulated 3T6 cell fibroblast production

[0038] On 3T6 cells, 10 ng / mL TGF-β was used to act for 24 h to establish a fibroblast fibrosis model. The original culture medium was discarded and 2.5, 5, and 10 μM parthenolide was used for 24 h. Cell proteins were collected to detect changes in the levels of fibrosis markers COL1A1 and α-SMA. The results are shown in Figure 3 .

[0039] The results showed that the protein expressions of collagen I and α-SMA in 3T6 cells stimulated by TGF-β were significantly increased, and after treatment with parthenolide, the levels of COL1A1 and α-SMA were significantly decreased in a dose-dependent manner.

[0040] Example 4: Parthenolide dose-dependently inhibits epithelial-mesenchymal transition of renal epithelial cells HK-2 stimulated by TGF-β

[0041] On HK-2 cells, 20 ng / mL TGF-β was used to act for 24 h to establish a renal epithelial cell fibrosis model. The original culture medium was discarded and the cells were treated with 2.5, 5, and 10 μM parthenolide for 24 h. Cell proteins were collected to detect changes in the levels of EMT markers Vim and N-Cad. The results are shown in Figure 4 .

[0042] The results showed that the protein expressions of Vim and N-Cad in HK-2 cells stimulated by TGF-β were significantly increased, and after treatment with parthenolide, the levels of Vim and N-Cad were significantly decreased in a dose-dependent manner.

[0043] Example 5: Parthenolide dose-dependently inhibits TGF-β-stimulated HK-2 cell fibroblast formation

[0044] On HK-2 cells, 20 ng / mL TGF-β was used to act for 24 h to establish a renal epithelial cell fibrosis model. The original culture medium was discarded and 2.5, 5, and 10 μM parthenolide was used for 24 h. Cell proteins were collected to detect changes in the levels of fibrosis markers COL1A1 and α-SMA. The results are shown in Figure 5 .

[0045] The results showed that the protein expressions of collagen I and α-SMA in HK-2 cells stimulated by TGF-β were significantly increased, and after treatment with parthenolide, the levels of COL1A1 and α-SMA were significantly decreased in a dose-dependent manner.

[0046] In summary, the present invention provides a novel natural CMA activator, parthenolide, which can increase the expression of LAMP2A in a dose-dependent manner within a certain concentration range. Parthenolide shows good application value and development prospects in stimulating LAMP2A and is a potential therapeutic drug for fibrotic diseases.

Claims

1. Application of parthenolide as a CMA activator in the preparation of drugs for the treatment of fibrosis.

2. The use according to claim 1, characterized in that: The parthenolide is parthenolide itself, parthenolide and its pharmaceutically acceptable salt or a derivative of parthenolide.

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

4. The use according to claim 1, characterized in that: When used, the dosage of the drug is 1.25-10 μM.

5. A CMA activator, characterized in that: The CMA activator is parthenolide itself, parthenolide and its pharmaceutically acceptable salts or parthenolide derivatives.

6. A drug for treating fibrosis, characterized in that: The active ingredients of the drug for treating fibrosis include parthenolide itself, parthenolide and its pharmaceutically acceptable salts or derivatives of parthenolide.

7. The drug according to claim 6, characterized in that The dosage form of the medicine is liquid, powder injection, tablet, capsule, powder, pill, paste, granule or dressing.

8. The drug according to claim 6, characterized in that When used, the dosage of the drug is 1.25-10 μM.

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