Application of chloroquine in reducing potential pulmonary toxicity of toxic component Pterocephin A of Tibetan medicine pterocephalus hookeri
By using chloroquine to intervene in cell experiments in the potential lung toxicity of Pterocephin A, the damage problem of this toxic component to lung cells was solved, significantly improving the safety of the drug, and providing a basis for scientific evaluation for its clinical application.
Patent Information
- Application Number
- CN202411788779.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-02-28
AI Technical Summary
The toxic component of Tibetan medicine Yishoucao Pterocephin A has potential pulmonary toxicity, which limits its wide application in clinical practice.
The potential pulmonary toxicity of Pterocephin A is reduced by using chloroquine to intervene in the effects of Pterocephin A on human normal lung epithelial cells. Specific methods include detecting the effect of the combination of Pterocephin A and chloroquine on cell viability, apoptosis and related protein expression in cell experiments.
Chloroquine significantly reduced the apoptosis and related toxic effects induced by Pterocephin A, improved cell survival rate, and provided guarantee for the safety assessment and clinical application of yshoucao.
Smart Images
Figure CN119530334A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of toxicological evaluation of monomeric compounds, and in particular to the application of chloroquine in reducing the potential pulmonary toxicity of Pterocephin A, a toxic component of the Tibetan medicine Pterocephin. Background Art
[0002] Traditional Tibetan medicine in my country has a long history of more than 2,000 years and is an important part of the great treasure house of Chinese medicine. As a representative ethnic medicine, Tibetan medicine has coexisted with traditional Chinese medicine in the process of development, achieved multicultural development, and gradually matured in the domestic and foreign markets. However, there are still problems, one of which is that the research on toxic side effects and toxicological mechanisms is relatively blank, and basic toxicological research is still in its infancy, which has greatly affected its safe and effective use.
[0003] Pterocephalus hookeri (CBClarke) Hoeck, a plant of the genus Pterocephalus of the family Dipsaciaceae, is a commonly used Tibetan medicine with the effects of detoxification, antipyretic, heat-clearing, anti-dysentery, and wind-clearing and arthralgia-relieving. It is bitter, cold, and slightly toxic. Pterocephalus hookeri has a long history of folk medicine use, mainly for the treatment of rheumatoid arthritis. Many medical books, such as Tibetan Medicine Records, Commonly Used Chinese Herbal Medicines in Tibet, and Jingzhu Materia Medica, have detailed records of its use. Prescription drugs such as Ranjiang Duoji Capsules, Jiebai Pills, and Ershiwuwei Donkey Blood Pills, which use it as the main ingredient, have been used clinically for many years. However, the "toxicity" of Pterocephalus hookeri is a major drawback that restricts its development and widespread application.
[0004] Chloroquine is an organic compound that is widely used as an anti-malarial and anti-inflammatory agent for the treatment of malaria and rheumatoid arthritis. It is also an autophagy inhibitor. In order to increase the efficacy of drugs or reduce the toxic side effects of drugs, it is urgent to carry out relevant research on the combined use of chloroquine and Pterocephalus. Summary of the invention
[0005] In view of this, the purpose of the present invention is to provide the use of chloroquine in reducing the potential lung toxicity of the toxic component Pterocephin A of the Tibetan medicine Pterocephin. The present invention clarifies that the toxic component Pterocephin A of the Tibetan medicine Pterocephin has potential lung toxicity, investigates its potential lung toxicity by means of in vitro experiments, and relates to a use of chloroquine in reducing the potential lung toxicity of the toxic component Pterocephin A of the Tibetan medicine Pterocephin, establishes a basis for the safety assessment of Pterocephin drugs, and provides a guarantee for its rational clinical use.
[0006] In order to achieve the above object, the present invention provides the following technical solutions: 1. The application of chloroquine in reducing the potential lung toxicity of Pterocephin A, a toxic component of the Tibetan medicine Pterocephin. The specific cell verification process includes the following steps: 1) Pterocephin A directly acts on human normal lung epithelial cells to detect relevant indicators; 2) Pterocephin A was used to act on normal human lung epithelial cells under the intervention of chloroquine to detect relevant indicators; 3) The experimental results comprehensively determined the role of chloroquine in reducing the potential lung toxicity of Pterocephin A, a toxic component of the Tibetan medicine Pterocephin.
[0007] In some embodiments of the present invention, the cells directly administered with Pterocephin A are subjected to cell viability detection, reactive oxygen detection, mitochondrial membrane potential detection, cell apoptosis and related protein detection to comprehensively determine the potential pulmonary toxicity of Pterocephin A.
[0008] In some embodiments of the present invention, cell viability, apoptosis and related protein detection are performed on cells after administration of Pterocephin A under the intervention of chloroquine to comprehensively determine the effect of chloroquine in reducing the potential pulmonary toxicity of Pterocephin A.
[0009] In some embodiments of the present invention, the IC 50 The values were calculated and the subsequent Pterocephin A administration concentrations were determined to be 1, 3, and 5 μM.
[0010] In some embodiments of the present invention, the dosage of chloroquine administered is 10 μM.
[0011] In some embodiments of the present invention, the expression levels of apoptosis marker proteins Bax, Bcl-2, and Cleaved-caspase 3 are detected in the detection of cell apoptosis and related proteins.
[0012] The human normal lung epithelial cells are Beas-2B cell line.
[0013] The beneficial effect of the present invention is that the present invention provides a use of chloroquine in reducing the potential lung toxicity of Pterocephin A, a toxic component of the Tibetan medicine Pterocephin. The present invention determines that Pterocephin A has potential lung toxicity by examining a variety of toxicity-related indicators, and comprehensively evaluates its use in reducing lung toxicity in combination with the experimental results under chloroquine intervention, thereby providing an effective method for the intervention of toxic components of Pterocephin. The method is convenient and fast, simple to operate, effective, and reproducible. The present invention finds and confirms that when the concentration of Pterocephin A reaches a certain threshold, it will show lung toxicity, and after chloroquine intervention, its toxicity is significantly alleviated. The invention can provide a comprehensive and effective evaluation method for the toxicity study of Chinese medicine monomers, and provide a reasonable scientific evaluation for the clinical application of Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration: Figure 1 The effects of different doses of Pterocephin A intervention on the viability of Beas-2B cells are shown.
[0015] Figure 2 The figure shows the effect of different doses of Pterocephin A intervention on the expression of reactive oxygen species in Beas-2B cells (microscope magnification 200 times).
[0016] Figure 3 The figure shows the effect of different doses of Pterocephin A intervention on mitochondrial membrane potential of Beas-2B cells (microscope magnification 200 times).
[0017] Figure 4 The effects of different doses of toxic monomer intervention on the apoptosis of Beas-2B cells are shown.
[0018] Figure 5 The results show the effects of different doses of toxic monomer intervention on the expression of apoptosis marker proteins in Beas-2B cells (ns<0.12, *p<0.033, **p<0.002, ***p<0.001).
[0019] Figure 6 The results show the effect of toxic monomers on Beas-2B cell viability under chloroquine intervention (ns<0.12, *p<0.033, **p<0.002, *** p<0.001, PA represents Pterocephin A group, HCQ represents chloroquine group, PA+HCQ represents Pterocephin A and chloroquine combination group).
[0020] Figure 7The figure shows the effect of toxic monomers on apoptosis of Beas-2B cells under the intervention of chloroquine (PA represents the Pterocephin A group, HCQ represents the chloroquine group, and PA+HCQ represents the combined use of Pterocephin A and chloroquine group).
[0021] Figure 8 The data show the effect of toxic monomers on the expression of marker proteins of apoptosis in Beas-2B cells under the intervention of chloroquine (ns<0.12, *p<0.033, **p<0.002, *** p<0.001, PA represents the Pterocephin A group, HCQ represents the chloroquine group, and PA+HCQ represents the combined use of Pterocephin A and chloroquine group). DETAILED DESCRIPTION
[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
[0023] Example 1: Pterocephin A direct action cell experiment 1. Dosage and time of administration Human normal lung bronchial epithelial cells Beas-2B were selected as the cell line. The culture medium was DMEM-F12 / (1:1). The cells were starved for 2 h before administration. The drug concentrations were 1, 3, and 5 μM, and the drug administration time was 24 h.
[0024] 2. Cell IC 50 Detection Using the MTT method, cells were given 10 compounds of different concentrations, and the IC values of the toxic compounds on cells were finally obtained by nonlinear curve fitting. 50 The value is 3.35 ± 0.1 μM, as Figure 1 Based on this, the subsequent dosing concentrations were set at 1, 3, and 5 μM.
[0025] 3. Cellular Reactive Oxygen Species (ROS) Detection The cells were tested by ROS kit. Figure 2 As shown in the figure, under the intervention of different concentrations of compounds, the release of reactive oxygen species showed an upward trend and was dose-dependent. ROS can be used as a pre-marker of cell autophagy and apoptosis, indicating that the compound may induce cell autophagy or apoptosis.
[0026] 4. Cell mitochondrial membrane potential (JC-1) detection Cells were detected using JC-1 nucleic acid dye. Figure 3As shown, under the intervention of different concentrations of compounds, the red fluorescence gradually weakened and the green fluorescence gradually increased, indicating that the mitochondrial membrane potential in the cells decreased and was dose-dependent, indicating that the compounds caused damage to the cells, which may be autophagy or apoptosis.
[0027] 5. Cell apoptosis detection The cell cycle and apoptosis detection kit was used for detection. Figure 4 As shown, under the intervention of different concentrations of compounds, the apoptosis level increased in a dose-dependent manner, indicating that the compounds are toxic and can induce cell apoptosis.
[0028] 6. Detection of apoptosis protein levels The protein expression level was detected by Western blotting. Figure 5 As shown, the compound can upregulate the expression of pro-apoptotic protein Bax and Cleaved caspase-3 protein in cells, and downregulate the expression level of anti-apoptotic protein Bcl-2 in a dose-dependent manner, indicating that the compound induces cell apoptosis.
[0029] Example 2: Cell experiment under chloroquine intervention 1. Cell viability detection After Pterocephin A at a concentration of 5 μM acted on cells under the intervention of 10 μM chloroquine, cell viability was detected. Figure 6 As shown, chloroquine intervention significantly improved cell survival rate.
[0030] 2. Cell apoptosis detection The cell cycle and apoptosis detection kit was used for detection. Figure 7 As shown, under the intervention of chloroquine, the level of cell apoptosis was significantly decreased compared with that of Pterocephin A alone, indicating that chloroquine reversed the cell apoptosis induced by Pterocephin A.
[0031] 3. Detection of apoptosis protein levels The protein expression level was detected by Western blotting. Figure 8 As shown, under chloroquine intervention, the expression of Bax protein was reduced compared with single administration, while the expression level of Bcl-2 was increased, indicating that chloroquine reversed Pterocephin A-induced cell apoptosis.
[0032] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.
Claims
1. The use of chloroquine in reducing the potential lung toxicity of the toxic component Pterocephin A of the Tibetan medicine Pterocephin, characterized in that: The specific cell validation process includes the following steps: 1) Pterocephin A directly acts on human normal lung epithelial cells to detect relevant indicators; 2) Pterocephin A was used to act on normal human lung epithelial cells under the intervention of chloroquine to detect relevant indicators; 3) The experimental results comprehensively determined the role of chloroquine in reducing the potential lung toxicity of Pterocephin A, a toxic component of the Tibetan medicine Pterocephin.
2. The use according to claim 1, characterized in that: After direct administration of Pterocephin A, cells were subjected to cell viability detection, reactive oxygen species detection, mitochondrial membrane potential detection, cell apoptosis and related protein detection to comprehensively determine the potential pulmonary toxic effects of Pterocephin A.
3. The use according to claim 1, characterized in that: Cell viability, apoptosis and related protein detection were performed on cells after administration of Pterocephin A under the intervention of chloroquine to comprehensively determine the role of chloroquine in reducing the potential pulmonary toxicity of Pterocephin A.
4. The use according to claim 2, characterized in that: The cell viability assay was used to detect IC 50 The values were calculated and the subsequent Pterocephin A administration concentrations were determined to be 1, 3, and 5 μM.
5. The use according to claim 3, wherein the dosage of chloroquine is 10 μM.
6. The use according to claim 2 or 3, characterized in that: In the detection of cell apoptosis and related proteins, the expression levels of apoptosis marker proteins Bax, Bcl-2, and Cleaved-caspase 3 were detected.
7. The use according to claim 6, characterized in that: The human normal lung epithelial cells are Beas-2B cell line.