Drug target of tribulus terrestris extract for treating pulmonary fibrosis as well as screening method and application of drug target

Abstract

The invention relates to the technical field of pharmacological research, in particular to a drug target of tribulus terrestris extract for treating pulmonary fibrosis as well as a screening method and application of the drug target. According to the invention, the effective treatment effect of TED on PF is determined through pharmacodynamic research. The SCD2 protein is preliminarily screened as the target protein by using a 4D-SmartDIA quantitative proteomics technology, the protein expression level proves that the SCD2 expression and phosphorylation level in the mouse lung tissue after TED treatment are obviously increased, and the interaction between SCD2 and phosphorylation is investigated and determined. Therefore, the SCD2 can be used as a drug target for predicting the treatment of the TED on the PF. And a basis is provided for development of PF treatment drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmacological research, and in particular to a drug target for treating pulmonary fibrosis with a Tribulus terrestris extract, a screening method and an application thereof. Background Art

[0002] Pulmonary fibrosis (PF) is a chronic, progressive lung disease characterized by abnormal activation and differentiation of lung fibroblasts, persistent alveolar damage, and massive deposition and incomplete repair of extracellular matrix. PF has become a public health issue that needs to be addressed urgently, placing a burden on the entire medical economy. Currently, evidence-based guidelines for the treatment of PF recommend the use of nintedanib and pirfenidone. Clinical trials have shown that both drugs can slow the decline of lung function and have relatively good efficacy. However, due to the side effects and superficial therapeutic effects exhibited during treatment, fundamental cure of this disease remains a challenge. Therefore, research on the prevention, early diagnosis and treatment strategies of PF has become increasingly urgent.

[0003] In recent years, traditional Chinese medicine (TCM), particularly monomeric compounds, has made significant progress in improving PF. Due to its significant efficacy and minimal side effects, it has become an alternative treatment option for PF. Tribulus terrestris L., the dried, mature fruit of the genus Tribulus in the family Tribulaceae, was first described in the Shennong's Herbal Classic. It possesses natural antioxidant and anti-inflammatory properties and has been widely used in traditional Chinese medicine since ancient times. Tribulus terrestris saponin D (TED), a steroidal saponin extracted and isolated from Tribulus terrestris, exhibits pharmacological effects such as anti-inflammatory, anticancer, and antitumor activities, alleviating inflammatory damage and inhibiting oxidative stress. Previous studies have shown that TED has significant anti-inflammatory and collagen fiber production inhibitory effects in PF, potentially improving PF by inhibiting the excessive secretion of cytokines such as transforming growth factor-β (TGF-β) by vascular endothelial cells and platelets during inflammatory damage. However, the underlying mechanism of TED in treating PF remains unclear. Therefore, it is urgent to establish a method to predict the drug targets of Tribulus terrestris extract, namely Tribulus terrestris saponin D, for the treatment of pulmonary fibrosis and to apply and verify it. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a drug target for treating pulmonary fibrosis with a Tribulus terrestris extract, a screening method and application thereof.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for screening drug targets for a Tribulus terrestris extract in treating pulmonary fibrosis, comprising the following steps:

[0007] (1) A pulmonary fibrosis mouse model was established using lipopolysaccharide. The mice with successful modeling were divided into a model group and a drug-treated group. The mice without modeling served as a blank control group. The mice in the drug-treated group were given Tribulus terrestris extract after successful modeling.

[0008] (2) 4D-SmartDIA quantitative proteomics analysis was performed on the lung tissues of the model group, drug-treated group, and blank control group mice. The analysis results were compared to obtain the differentially expressed proteins between each two groups of mice;

[0009] (3) Performing heat map analysis on all differentially expressed proteins obtained in step (2), selecting proteins with significant differential expression between the mice in the drug-treated group and the blank control group, and proteins with a callback differential expression compared with the mice in the model group as significantly differentially expressed proteins;

[0010] (4) performing subcellular structural localization on the significantly differentially expressed proteins obtained in step (3), wherein the subcellular structure with the largest number of significantly differentially expressed proteins is the cell structure associated with the treatment of pulmonary fibrosis with the Tribulus terrestris extract;

[0011] (5) Performing Venn diagram analysis on the differentially expressed proteins between the model group mice and the blank control group mice and the differentially expressed proteins between the model group mice and the drug-treated group mice, screening for overlapping proteins, and obtaining overlapping differentially expressed proteins;

[0012] (6) Selecting significantly differentially expressed proteins that can be located in cell structures related to the treatment of pulmonary fibrosis by Tribulus terrestris extract from the overlapping differentially expressed proteins, and obtaining drug targets for the treatment of pulmonary fibrosis by Tribulus terrestris extract.

[0013] Preferably, the intervention dose of the Tribulus terrestris extract is 5 to 20 mg / kg / d.

[0014] Preferably, the intervention time is 7 to 42 days.

[0015] Preferably, the Tribulus terrestris extract is Tribulus terrestris saponin D.

[0016] The present invention also provides that the drug target obtained by screening according to the method is SCD2.

[0017] The present invention also provides the use of the SCD2 in preparing a product for detecting the therapeutic effect of a drug on pulmonary fibrosis, wherein the drug is a Tribulus terrestris extract.

[0018] The present invention also provides the use of the SCD2 in preparing a medicine for treating and / or preventing pulmonary fibrosis.

[0019] The present invention also provides use of a reagent for promoting the expression of SCD2 and the phosphorylation level of SCD2 in the preparation of a drug for treating and / or preventing pulmonary fibrosis.

[0020] The present invention also provides a kit for detecting the therapeutic effect of Tribulus terrestris extract on pulmonary fibrosis, comprising any one of the following:

[0021] (1) Reagents for detecting SCD2 expression;

[0022] (2) Reagents for detecting the phosphorylation level of SCD2.

[0023] The present invention provides a drug target for the treatment of pulmonary fibrosis using a Tribulus terrestris extract, a screening method, and an application thereof. The present invention clarifies the effective therapeutic effect of the Tribulus terrestris extract (TED) on pulmonary fibrosis (PF) through pharmacodynamic studies. Using 4D-SmartDIA quantitative proteomics technology, SCD2 protein was preliminarily screened as the target protein. Protein expression levels demonstrated a significant increase in SCD2 expression and phosphorylation levels in mouse lung tissue after TED treatment. The interaction between SCD2 and phosphorylation was investigated and determined. It can be seen that SCD2 can be used as a drug target for predicting TED treatment of PF. This provides a basis for the treatment of PF. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The pharmacodynamic study of TED in the treatment of pulmonary fibrosis found that TED improved lung damage and lung function in lipopolysaccharide (LPS)-induced PF mice. Figure 1 A is the experimental flow chart; Figure 1 B is a graph showing changes in mouse body weight; Figure 1 C is the survival rate of mice; Figure 1 D is the mouse lung coefficient; Figure 1 E shows HE staining and Masson staining of mouse lung tissue.

[0025] Figure 2 TED can reduce lung inflammation and EMT in PF mice. Figure 2 AD are ELISA detection of IL-10, CTGF, VEGF and α-SMA in mouse plasma; Figure 2 EG were quantified by qRT-PCR for the relative mRNA expressions of α-SMA, Vimentin, and Collagen I in mouse lung tissues; Figure 2 HK used Western blot to detect the protein levels of α-SMA, Vimentin and Collagen I in mouse lung tissue.

[0026] Figure 3 4D-SmartDIA quantitative proteomics analysis. Figure 3A is the relative standard deviation (RSD) of the control group (Control), model group (Model), and tribulus terrestris saponin D high-dose group (TED-H) based on the identified proteins; Figure 3 B is the heat map analysis of the Control, Model, and TED-H groups; Figure 3 C is the subcellular localization prediction of proteins identified after TED intervention; Figure 3 Figure 3D and Figure 4D are the volcano plot analysis of identified proteins; Figure F is the Venn diagram analysis of differentially expressed proteins between groups; Figure G is the quantitative expression of SCD2 protein.

[0027] Figure 4 KEGG enrichment analysis after TED intervention.

[0028] Figure 5 Validation of predicted targets. Figure 5 A is the molecular docking result of TED and SCD2; Figure 5 B is the relative expression of SCD2 mRNA in mouse lung tissue quantified by qRT-PCR; Figure 5 C shows the changes in phosphorylation screening and SCD2 expression levels; Figure 5 DE respectively represent the changes in SCD2 protein phosphorylation verified by Co-IP experiments. DETAILED DESCRIPTION

[0029] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] A PF mouse model was established using lipopolysaccharide (LPS). Successfully established mice were divided into a positive control group (Positive), a model group (Model), and a drug group (administered with Tribulus terrestris saponin D (TED)). The drug groups were further divided into a high-dose drug group (TED-H), a medium-dose drug group (TED-Z), and a low-dose drug group (TED-L). Each group was controlled with untreated blank mice as a blank control group (Control). After grouping, control mice were administered 0.3% CMC-Na daily, model mice were administered 0.3% CMC-Na daily, TED-H mice were administered 20 mg / kg TED daily, TED-Z mice were administered 10 mg / kg TED daily, TED-L mice were administered 5 mg / kg TED daily, and positive mice were administered 50 mg / kg pirfenidone daily.

[0032] The drug intervention lasted for 6 weeks. The specific flow chart is as follows Figure 1 As shown in A.

[0033] During the drug intervention process, the body weight of each group of mice was measured every day. Figure 1 As shown in B, the vertical axis is weight. Figure 1 B shows that except for the Control group, the weight of mice in other groups decreased significantly in the first week after modeling. From the second week onwards, the weight of mice in the TED group gradually increased in a dose-dependent manner, gradually approaching that of the Positive group, and significantly increased compared with the Model group.

[0034] During the drug intervention, the death rate of mice in each group was detected every day, and the survival rate was calculated. Survival rate = number of existing mice / total number of mice × 100%. Death rate = 1 - survival rate. The results of survival rate are shown in Figure 1 C. The vertical axis represents the survival rate. Figure 1 C shows that after TED administration, the mortality rate of PF mice was significantly lower than that of the Model group.

[0035] 30 minutes after the last administration, the body weight of the mice was recorded, the lung tissue was dissected and separated, and the lung wet weight was weighed using an electronic analytical balance. The lung coefficient of each group of mice was calculated according to the following formula: lung coefficient = lung wet weight (mg) / mouse body weight (g). Figure 1 D. The vertical axis represents the lung coefficient. Figure 1 D shows that the lung coefficient of mice in the Model group was higher than that in the Control group (P < 0.001). Compared with the Model group, the TED group significantly improved the abnormal increase in the lung coefficient of PF mice, and the TED-H group had the best improvement effect (P < 0.01).

[0036] After the drug intervention, the lung tissues of mice in each group were taken out and stained with HE. Figure 1 E. HE staining showed that the lung tissue structure of mice in the Control group was normal, with clear alveolar structure. However, the lung structure of the Model group was distorted, with significant thickening of the alveolar walls, partial disappearance of alveoli, and visible inflammatory cell infiltration. Compared with the Model group, the thickness of the alveolar septa and inflammatory cell infiltration in the Positive and TED groups were reduced, and the lung tissue lesions of the mice were improved. Masson staining microscopic observation showed that the collagen fibers in the lung tissue of the Model group mice were significantly increased compared with the Control group, with a strip-like distribution. Significant fibrosis was observed between the pulmonary interstitium and the bronchi. After TED intervention, the integrity of the mouse lung tissue structure was improved, and collagen fiber deposition was reduced. The TED-H group had the best improvement, approaching that of the Positive group.

[0037] Example 2

[0038] After the drug intervention, plasma was collected from each group of mice, and the levels of the inflammatory cytokine IL-10, and the fibrosis-related factors CTGF, VEGF, and α-SMA in the plasma were measured by ELISA. The results are shown in Figures 2A-D. The results showed that compared with the Control group, the Model group significantly reduced the level of IL-10 and increased the levels of CTGF, VEGF, and α-SMA. However, after TED intervention, the levels of these four cytokines were significantly reduced. This shows that TED can effectively inhibit lung inflammation and epithelial-mesenchymal transition (EMT) levels in mice, preventing the formation of PF.

[0039] qRT-PCR further verified the expression changes of EMT-related markers α-SMA, vimentin and collagen I. Figure 2 EG showed that LPS induced an increase in the expression of α-SMA, Vimentin and Collagen Ⅰ in the lung tissue of mice, while the TED group showed a downward trend compared with the Model group, and the TED-H group had the best inhibitory effect.

[0040] Western blot was used to detect the effect of TED on the expression of α-SMA, Vimentin, and CollagenⅠ. Interestingly, the results showed the same trend as the qRT-PCR results ( Figure 2 This confirms the conclusion that TED effectively inhibits EMT in the mouse lungs at the protein level. Therefore, the TED-H group, which showed the best pharmacodynamics, was selected as a representative for 4D-SmartDIA quantitative proteomics analysis.

[0041] Example 3

[0042] Screening potential targets of TED on PF

[0043] 4D-SmartDIA quantitative proteomics analysis was performed on lung tissues from control, model, and TED-H groups (n=4). A total of 48,050 peptides, 7,394 proteins, and 7,342 quantifiable proteins were identified in the lung tissues. RSD analysis showed that the overall RSD values ​​of replicate samples in each group were small, indicating good quantitative reproducibility ( Figure 3A). Comparison of the proteins obtained above revealed that compared with the Control group of mice, the Model group had 56 differentially expressed proteins, including 40 upregulated proteins and 16 downregulated proteins; compared with the Model group of mice, 62 differentially expressed proteins were detected in the lung tissues of the TED-H group of mice, including 28 upregulated proteins and 34 downregulated proteins. A heat map analysis was performed on all the differentially expressed proteins obtained above, and the differentially expressed proteins that showed significant differences compared with the Control group of mice after TED intervention and had a callback trend compared with the Model group were selected as the significantly differentially expressed proteins. Finally, 30 significantly differentially expressed proteins were screened. Since two significantly differentially expressed proteins did not have names, they were not displayed in the heat map. The results are as follows Figure 3 As shown in B.

[0044] In order to thoroughly understand the functional characteristics of different proteins, the 30 significantly differentially expressed proteins obtained above were localized in the subcellular structure. The subcellular localization results ( Figure 3 C) shows that after TED intervention, the number of proteins with significant differential expression in cytoplasmic structures accounts for the largest proportion, which indicates that the mechanism of TED in improving PF is related to a certain cytoplasmic protein.

[0045] Figure 3 DE represents the up-regulation and down-regulation of differentially expressed proteins between the Control group and the Model group, as well as the up-regulation and down-regulation of differentially expressed proteins between the TED-H group and the Model group (grey dots indicate no change, blue indicates down-regulation, and red indicates up-regulation). The differentially expressed proteins between the Control group and the Model group and the differentially expressed proteins between the TED-H group and the Model group were analyzed by Venn diagram, and overlapping proteins were screened. Six overlapping differentially expressed proteins were obtained (P < 0.05), namely SCD2, GALNT7, AZI2, Ube2G1, FGGY, and Igκv12-41 ( Figure 3 F). Among these overlapping differentially expressed proteins, SCD2 is located in the endoplasmic reticulum of the cytoplasm. Therefore, SCD2 was selected as a drug target for TED treatment of PF.

[0046] Proteomic data showed that SCD2 abundance was significantly downregulated in the Model group (P<0.05) ( Figure 3 G), which represents a mechanism for the development of PF phenotype. At the same time, after TED intervention, SCD2 abundance was upregulated (P < 0.05), indicating that SCD2 is a key regulatory protein for TED to improve PF. This study conducted KEGG pathway classification analysis on the screened differentially expressed proteins and found that lipid metabolism plays an important role in TED treatment of PF ( Figure 4 ).

[0047] Example 4

[0048] Verification experiment

[0049] The relationship between TED and SCD2 was predicted by molecular docking experiments. The molecular docking of TED and SCD2 active site was verified. Figure 5 As shown in A. It was observed that TED formed a strong hydrogen bond with the active site of SCD2, indicating that there was good binding activity between the receptor and the ligand, strong docking ability, and high molecular stability after docking. The results of proteomics predicted that SCD2 was differentially expressed, so the expression level of SCD2 was detected and pan-phosphorylation screening was performed. The results of RT-qPCR showed that the mRNA expression of SCD2 in the Model group was significantly decreased compared with the Control group (P < 0.001), while compared with the Model group, the TED group significantly increased the mRNA expression level of SCD2 in a dose-dependent manner, and the TED-H group had the best callback effect (P < 0.01) ( Figure 5 B) (The vertical axis represents the mRNA expression level of SCD2). The results of the Western Blot method showed the same trend as that predicted by proteomics. Using tyrosine phosphorylation antibody (Anti-P Try) to screen for pan-phosphorylation through Western Blot experiments, it was found that the phosphorylation level in the Model group was lower than that in the Control group. After TED administration, the phosphorylation level in each treatment group recovered ( Figure 5 C), indicating that changes in phosphorylation levels are the key mechanism of action of TED in improving PF. Co-IP experiments were then performed to further determine the interaction between SCD2 and phosphorylation. Immunoprecipitation was performed using a phosphorylated pan-antibody, and the signal of the SCD2 band was detected by Western blot, while no signal was detected in the IgG control group. Compared with the Control group, the expression level in the Model group was reduced, and a callback trend appeared after TED administration ( Figure 5 DE), the results indicate that the phosphorylation level of SCD2 plays an important role in the TED treatment of PF.

[0050] In summary, the present invention uses a proteomics method to provide a method for predicting the target of Tribulus terrestris extract in treating pulmonary fibrosis, and verifies and applies the predicted target of Tribulus terrestris extract in treating pulmonary fibrosis.

[0051] As can be seen from the above examples, the present invention provides a drug target for the treatment of pulmonary fibrosis using a Tribulus terrestris extract, as well as a screening method and application thereof. The present invention clarifies the effective therapeutic effect of the Tribulus terrestris extract TED on PF through pharmacodynamic studies. Using 4D-SmartDIA quantitative proteomics technology, SCD2 protein was preliminarily screened as the target protein. Protein expression levels demonstrated a significant increase in SCD2 expression and phosphorylation levels in mouse lung tissue after TED treatment, and the interaction between SCD2 and phosphorylation was investigated and confirmed. Therefore, SCD2 can be used as a drug target for predicting TED in the treatment of PF.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for screening drug targets for the treatment of pulmonary fibrosis with a Tribulus terrestris extract, characterized in that: The steps include: (1) A pulmonary fibrosis mouse model was established using lipopolysaccharide. The mice with successful modeling were divided into a model group and a drug-treated group. The mice without modeling served as a blank control group. The mice in the drug-treated group were given Tribulus terrestris extract after successful modeling. (2) 4D-SmartDIA quantitative proteomics analysis was performed on the lung tissues of the model group, drug-treated group, and blank control group mice. The analysis results were compared to obtain the differentially expressed proteins between each two groups of mice; (3) Performing heat map analysis on all differentially expressed proteins obtained in step (2), selecting proteins with significant differential expression between the mice in the drug-treated group and the blank control group, and proteins with a callback differential expression compared with the mice in the model group as significantly differentially expressed proteins; (4) performing subcellular structural localization on the significantly differentially expressed proteins obtained in step (3), wherein the subcellular structure with the largest number of significantly differentially expressed proteins is the cell structure associated with the treatment of pulmonary fibrosis with the Tribulus terrestris extract; (5) Performing Venn diagram analysis on the differentially expressed proteins between the model group mice and the blank control group mice and the differentially expressed proteins between the model group mice and the drug-treated group mice, screening for overlapping proteins, and obtaining overlapping differentially expressed proteins; (6) Selecting significantly differentially expressed proteins that can be located in cell structures related to the treatment of pulmonary fibrosis by Tribulus terrestris extract from the overlapping differentially expressed proteins, and obtaining drug targets for the treatment of pulmonary fibrosis by Tribulus terrestris extract.

2. The method according to claim 1, characterized in that The intervention dose of Tribulus terrestris extract was 5-20 mg / kg / d.

3. The method according to claim 2, characterized in that The intervention duration is 7 to 42 days.

4. The method according to any one of claims 1 to 3, characterized in that: The tribulus terrestris extract is tribulus terrestris saponin D.

5. The drug target screened by the method according to any one of claims 1 to 4 is SCD2.

6. Use of the SCD2 according to claim 5 in the preparation of a product for detecting the therapeutic effect of a drug on pulmonary fibrosis, wherein the drug is a Tribulus terrestris extract.

7. Use of the SCD2 according to claim 5 in the preparation of a medicament for treating and / or preventing pulmonary fibrosis.

8. Use of the agent for promoting the expression of SCD2 and the phosphorylation level of SCD2 according to claim 5 in the preparation of a drug for treating and / or preventing pulmonary fibrosis.

9. A kit for detecting the therapeutic effect of Tribulus terrestris extract on pulmonary fibrosis, characterized in that: Includes any of the following: (1) Reagents for detecting SCD2 expression; (2) Reagents for detecting the phosphorylation level of SCD2.

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