Covalent pharmaceutical composition for targeting modification of keap1 agonist nrf2 pathway and application thereof

Abstract

The application relates to a drug composition targeting a KEAP1-Nrf2 pathway and application thereof, and the drug composition comprises tanshinone and a mulberry isoflavone. KEAP1-Nrf2 is a core pathway for maintaining oxidation-reduction balance and detoxification defense of a body, and covalent modification of KEAP1 protein can effectively activate the Nrf2 signal pathway to produce effects such as relieving oxidative stress, resisting inflammation and protecting organs. The drug composition provided in the application can activate the Nrf2 pathway by targeting covalent modification of different sites of KEAP1, and significant antioxidant and anti-inflammatory effects are exhibited in vivo and in vitro, and the drug composition is suitable for treating lung injury or pulmonary fibrosis caused by various factors.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a covalent pharmaceutical composition that targets and modifies KEAP1 to synergistically activate the Nrf2 signaling pathway and its application. Background Technology

[0002] Multiple organ injury (MOI) is a fatal complication of severe illnesses such as trauma, shock, and serious infection. Among these, the lungs are the most vulnerable and have a very high mortality rate in MOI, often manifesting as diffuse lung damage in the acute phase. If uncontrolled oxidative stress and the inflammatory cascade are not addressed in time, the disease can progress to irreversible pulmonary fibrosis and even lead to multiple organ failure through systemic circulation.

[0003] Lung injury and pulmonary fibrosis are severe pathological processes that seriously threaten respiratory function. Their causes encompass a variety of pathways, including inhalation of environmental toxins (such as sulfur dioxide and nitrogen oxides), pathogen infection (bacteria, viruses, and fungi), involvement of autoimmune diseases (such as systemic lupus erythematosus), and iatrogenic injury (such as chest radiotherapy and drug toxicity). Pulmonary fibrosis is an abnormal repair response following persistent or severe damage to lung tissue, manifested as alveolar structural destruction, excessive extracellular matrix deposition, and progressive loss of lung respiratory function.

[0004] Oxidative stress and excessive inflammatory response are the core pathological mechanisms driving the development of lung injury and pulmonary fibrosis. Oxidative stress originates from an imbalance in the generation and clearance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the body, inducing intracellular redox homeostasis dysregulation, which in turn leads to lipid peroxidation, protein denaturation, and DNA damage. In the pathological environment of the lungs, oxidative stress directly damages alveolar epithelial cells and capillary endothelial cells, disrupting the integrity of the alveolar-capillary barrier and causing transmural pulmonary edema and hemorrhagic lesions. Simultaneously, damage signals activate immune effector cells such as neutrophils and macrophages, triggering a cascade release of a large number of pro-inflammatory cytokines and chemokines. This persistent inflammatory microenvironment not only exacerbates cell damage but also significantly induces myofibroblast transdifferentiation, accelerating the process of pulmonary fibrosis. Therefore, targeting and inhibiting oxidative stress and inflammatory response has become a key therapeutic strategy for blocking the pathological evolution of lung injury and pulmonary fibrosis.

[0005] Nrf2 is a key transcription factor that regulates the expression of various antioxidant and detoxification genes. Activation of Nrf2 can enhance cellular antioxidant capacity and mitigate oxidative stress damage, thus playing a crucial role in the pathogenesis of various diseases. Under normal circumstances, Nrf2 is degraded after binding to its negative regulator, KEAP1; however, covalent modification of KEAP1 can block Nrf2 degradation and promote Nrf2 translocation into the nucleus, which further activates the expression of downstream antioxidant and detoxification genes. However, existing Nrf2 agonists (such as omasulolon) still have some shortcomings in clinical applications, such as insufficient efficacy and significant side effects. Therefore, finding more effective and safer Nrf2 agonists by inhibiting the Keap1 protein is currently a hot research topic. Summary of the Invention

[0006] To address the aforementioned technical problems, this application provides a covalent pharmaceutical composition that targets and modifies KEAP1 to synergistically activate the Nrf2 pathway and its application. Tanshinone is derived from Tanshinone (… Salvia Miltiorrhiza Bge The compound isolated from the underground rhizome of [a plant species] exhibits good antioxidant and anti-inflammatory effects, but it still possesses certain cytotoxic and neurotoxic properties. The applicant recently discovered that tanshinone can covalently modify the cysteine ​​residue of KEAP1 to activate the Nrf2 signaling pathway, a process that exposes Cys518, which was previously embedded. Therefore, developing a drug composition that targets KEAP1 and synergistically activates the Nrf2 signaling pathway to enhance Nrf2 activation activity has significant scientific and clinical application value.

[0007] To achieve this objective, the following technical solution is adopted in this application: In a first aspect, this application provides a covalent pharmaceutical composition that targets and modifies KEAP1 to co-activate the Nrf2 signaling pathway, the pharmaceutical composition comprising tanshinone and verbascoside isoflavone.

[0008] The pharmaceutical composition provided in this application, comprising tanshinone and verbascoside, can target KEAP1 and co-activate the Nrf2 signaling pathway, exhibiting significant antioxidant and anti-inflammatory effects. It can be used to treat organ damage, and is particularly suitable for treating lung injury or pulmonary fibrosis caused by multiple factors.

[0009] In some embodiments, the molar ratio of tanshinone and verbascoflavonoid is 1:3 to 1:6, for example, it can be 1:3, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, etc.

[0010] In some embodiments, the optimal molar ratio of tanshinone and verbascoflavonoid is 1:4.5-1:5.5.

[0011] In this application, when the preferred molar ratio of tanshinone and verbascoside isoflavone is 1:4.5-1:5.5, especially the optimal molar ratio (1:5), the composition can not only effectively reduce the cytotoxicity of tanshinone, but also achieve the maximum synergistic and detoxification effect of the two in terms of their biological effects such as anti-oxidation, anti-inflammation and anti-fibrosis.

[0012] In some embodiments, based on the total mass of tanshinone and verbascoside being 100%, the mass percentage content of verbascoside is 75%-90%, and the mass percentage content of tanshinone is 10%-25% (preferably 15%-18%). Controlling the active ingredients within the above molar ratio and mass percentage range can significantly reduce the toxic side effects of single drugs while efficiently leveraging the synergistic activation potential of verbascoside and tanshinone on the Nrf2 pathway.

[0013] In some embodiments, the purity of the tanshinone is ≥98%, and the purity of the verbascoside isoflavone is ≥98%.

[0014] Both tanshinone and verbascoflavonoids described in this application can be prepared by conventional chemical synthesis methods in the field, or obtained through commercial extraction and procurement.

[0015] In some embodiments, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients.

[0016] In some embodiments, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, hydrosolvents, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, anti-inflammatory agents, antibacterial agents, or buffers. In this application, the pharmaceutical composition may contain excipients commonly used in the pharmaceutical field, and these excipients are added at amounts typically used in the pharmaceutical field.

[0017] In some embodiments, the dosage form of the pharmaceutical composition includes tablets, capsules, oral liquids, injections, dialysate, inhalers, syrups, or nebulizers. The dosage form of the pharmaceutical composition in this application may also be other forms, and is not particularly limited thereto.

[0018] The pharmaceutical composition described in this application also contains substances that promote drug absorption in order to increase therapeutic efficacy.

[0019] In a second aspect, this application provides a method for preparing a pharmaceutical composition targeting the KEAP1-Nrf2 pathway according to the first aspect, the method comprising: mixing tanshinone and verbascoside to obtain the pharmaceutical composition.

[0020] In some embodiments, the preparation method includes the following steps: (1) Dissolve tanshinone and verbascoside in solvents to obtain solution A and solution B respectively; (2) Mix solution A and solution B, and then dry to obtain a powder, thereby obtaining the pharmaceutical composition.

[0021] In some embodiments, after obtaining the powder in step (2), the powder is further mixed with pharmaceutically acceptable excipients to prepare the desired dosage form.

[0022] Thirdly, this application provides the use of the pharmaceutical composition targeting the KEAP1-Nrf2 pathway according to the first aspect in the preparation of a medicament for treating lung injury or pulmonary fibrosis.

[0023] The pharmaceutical composition provided in this application can be used to prepare drugs for treating multiple organ injuries, such as drugs for drug-induced lung injury or pulmonary fibrosis.

[0024] Fourthly, this application provides the use of the pharmaceutical composition targeting the KEAP1-Nrf2 pathway according to the first aspect in the preparation of an Nrf2 agonist.

[0025] Compared with the prior art, this application has at least the following beneficial effects: The pharmaceutical composition comprising verbascoside and tanshinone provided in this application is an Nrf2 agonist with significant antioxidant effects, particularly suitable for treating lung injury and liver fibrosis caused by various reasons. Moreover, the composition has low toxicity and significant therapeutic effects. Attached Figure Description

[0026] Figure 1 This is a molecular dynamics analysis diagram of the KEAP1-Cys518 heteroform pocket exposure induced by tanshinone (MTR) covalent modification; A is a histogram comparing the pKa values ​​of each cysteine ​​residue in the KEAP1 protein in the unmodified state (APO) and the tanshinone-modified state (MTR-modified) (where the pKa of Cys518 is significantly reduced); B is a probability density distribution diagram of the χ1 dihedral angle (N-Cα-Cβ-Sγ) of the Cys518 residue side chain in the unmodified state (APO) and the tanshinone-modified state (MTR-modified) (showing that the side chain conformation is drastically flipped and exposed after modification).

[0027] Figure 2A is a high-throughput virtual screening and Hits compound identification diagram based on the KEAP1-Cys518 sensitized pocket; A is a scatter plot of the predicted binding energy distribution of small molecules from the TargetMol compound library docking with the KEAP1-Cys518 pocket in the APO state and MTR-modified state, respectively; B is a scatter plot of 103 initial screening seed compounds (Hits) obtained by screening based on dual energy thresholds (binding energy < -6.0 kcal / mol and ΔEnergy < -2.0 kcal / mol).

[0028] Figure 3 This is a covalent molecular docking screening targeting the heterologous multivalent site of Cys518 and structural diagrams of 9 core candidate compounds; A is a bar chart showing the predicted covalent binding energy between candidate compounds with nucleophilic reactive groups (α,β-unsaturated ketones or orthoquinone structures) and Cys518 residues (the predicted binding energy is -3.0 kcal / mol as the threshold for secondary screening); B is the chemical structural formula of 9 core candidate compounds with strong covalent binding ability obtained through secondary covalent screening.

[0029] Figure 4 This is a diagram showing the agonistic effect of candidate compounds combined with tanshinone (MTR) on Nrf2 transcriptional activity at different molar ratios.

[0030] Figure 5 This is a flowchart of a high-throughput screening process for drug compositions.

[0031] Figure 6 This is a graph showing the effect of the optimal combination of verbenafil (CCS) and tanshinone (MTR) on the ROS levels of bleomycin (BLM)-induced MLE-12 cells. The left graph shows the histogram distribution of intracellular ROS fluorescence intensity as detected by flow cytometry when the following groups were treated: solvent control group, model group, verbenafil alone (5 μM CCS), tanshinone alone (1 μM MTR), low concentration group of verbenafil and tanshinone 5:1 (CMCL, 1.25 μM CCS + 0.25 μM MTR), and high concentration group of verbenafil and tanshinone 5:1 (CMCH, 5 μM CCS + 1 μM MTR). The right graph shows the quantitative statistical graph of the mean fluorescence intensity (MeanFL-1) of each group.

[0032] Figure 7The left image shows the inhibitory effect of the combination of verrucoside isoflavone (CCS) and tanshinone (MTR) on the expression of TGF-β1-induced fibrosis markers in MRC-5 cells; the right image shows the Western blot results of the expression levels of Fibronectin and Collagen-I α-SMA proteins in each treatment group (Veh, Model, CCS, MTR, CMCL, CMCH) (with α-tubulin as an internal control); the right image shows the quantitative statistical graph of the relative expression levels of each protein (Normalize to α-tubulin). Detailed Implementation

[0033] The technical solution of this application will be further described below with reference to the accompanying drawings and specific embodiments. However, the examples below are merely simplified examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application shall be determined by the claims.

[0034] Example 1 To identify the newly exposed cysteine ​​reaction sites of Keap1 protein after modification with tanshinone (MTR, drug A), based on the results of tanshinone binding to Keap1, this embodiment constructed an allosteric recognition model using molecular dynamics simulations and computational chemistry methods, combining the analysis of cysteine ​​residue reactivity (PropKa) and cysteine ​​side chain dihedral angle (χ1 dihedral angle, N-Cα-Cβ-Sγ) of the KEAP1 protein. This successfully located the heterologous allosteric binding sites exposed by tanshinone modification of KEAP1, and used these sites as targets for high-throughput virtual screening and covalent molecular docking. The specific steps are as follows: 1. Structural pretreatment and energy minimization: The crystal structure of human KEAP1 protein (PDBID: 1U6D) was obtained from the PDB database. AmberTools software was used to perform hydrogenation, charge addition, solvation pretreatment and energy minimization optimization on the unmodified (APO) and tanshinone covalently modified (MTR-modified) KEAP1 proteins.

[0035] 2. Calculation of cysteine ​​thiol pKa: The pKa values ​​of the side chain thiol groups (-SH) of all cysteine ​​(Cys) residues in the Keap1 protein in the APO and MTR-modified states were calculated using PropKa 3.0 software, and the pKa change before and after modification was calculated (ΔpKa = pKa). MTR-modified -pKa APO ), the result is as follows Figure 1 As shown in Figure A.

[0036] 3. Conformation inversion and dihedral angle analysis: The χ1 rotation angle of the cysteine ​​side chain was calculated to evaluate the conformation inversion of the cysteine ​​side chain and changes in solvent accessibility before and after tanshinone modification. The results are as follows: Figure 1 As shown in Figure B.

[0037] 4. High-throughput virtual screening: Based on the heterologous multivalent site Cys518 with significantly reduced pKa and allosteric inversion of side chains, a library of 4617 natural products and small molecule compounds with known biological activities (TargetMol) was introduced. After structural optimization, hydrogenation, and charge partitioning of the small molecules in the library, the small molecules were docked into the Cys518 pockets of APO-KEAP1 or MTR-modified KEAP1 proteins using AutoDock Vina. Hits with significantly enhanced binding affinity in the MTR-modified state were screened by predicting binding energies. The results are shown below. Figure 2 As shown.

[0038] 5. Secondary screening using covalent molecular docking: Based on the 103 lead compounds obtained from the initial screening, AutoDock-Covalent software was used, with the Cys518 residue of the MTR-modified KEAP1 protein as the covalent reaction site, and the covalent reaction type was set to electrophilic addition reactions such as Michael addition. Covalent molecular docking was primarily performed on compounds containing α,β-unsaturated ketones and O-quinone groups. The predicted binding energy (kcal / mol) after covalent binding was calculated, and -3.0 kcal / mol was used as the screening threshold. The core candidate compounds for co-operation with tanshinone were finally obtained, and the results are shown below. Figure 3 As shown.

[0039] The results of heterologous multivalent site calculation and analysis are as follows: Figure 1 As shown in Figures A and B, among the multiple cysteine ​​residues in the KEAP1 protein, the pKa of Cys518 in the unmodified APO-KEAP1 is 9.2; however, after covalent modification of the KEAP1 protein with tanshinone (MTR), the pKa of Cys518 decreases to 6.1, and the ΔpKa reaches -3.1. Conformational analysis shows that the side chain conformation of Cys518 in the native state is mainly concentrated around -59°. After covalent modification of the KEAP1 protein with tanshinone, the χ1 rotation angle of the Cys518 side chain becomes -168°. The results indicate that tanshinone modification causes a local conformational change in the KEAP1 protein, prompting a dramatic flipping of the Cys518 side chain to expose it, significantly enhancing its thiol nucleophilic reactivity.

[0040] The results of docking the TargetMol compound library with the KEAP1-Cys 518 activity pocket in its native state or after modification with tanshinone are as follows: Figure 2 As shown in Figures A and B, the predicted binding energies of most compounds in the MTR-modified state are significantly lower than those in the APO state, indicating a tighter covalent bond. Furthermore, using a binding energy of -6 kcal / mol in the MTR-modified state and a binding energy change ΔEnergy < -2.0 kcal / mol after modification as a dual threshold (…),… Figure 2 (Figure B) , 103 seed compounds (Hits) that selectively bind to MTR-modified KEAP1-Cys518 were successfully identified.

[0041] The results of the covalent docking secondary screening of 103 lead compounds are as follows: Figure 3 Figures A and B are shown in the table. The results show that 17 candidate compounds (compound numbers TN1933, T3923, TN2330, TN1774, TN1449, TN1427, T2787, TN2269, and T2898) can undergo covalent addition reactions with the thiol group of the Cys518 side chain through the α,β-unsaturated ketone structure or ortho-quinone structure in their structure. A secondary screening was conducted using a covalent binding energy of -3.0 kcal / mol as the threshold, resulting in 9 candidate compounds (drug B) with strong covalent binding ability.

[0042] In summary, based on computer-aided rational design, nine candidate compounds that have potential synergistic effects with tanshinone were successfully screened.

[0043] Example 2 To verify the agonistic effect of the nine candidate compounds obtained by high-throughput screening on the KEAP1-Nrf2 pathway when combined with tanshinone, this embodiment used the Nrf2 luciferase reporter gene assay. Under the condition that the total dosage concentration was fixed at 1 μM, the activation fold of Nrf2 transcriptional activity of the candidate compounds and tanshinone at different molar ratios (including 1:0 for single drug and combination ratios of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1) was systematically detected.

[0044] The experimental steps are as follows: HEK293 cells stably transfected with the Nrf2 luciferase reporter gene plasmid (Jiman Biotechnology (Shanghai) Co., Ltd.) were seeded in 96-well microtiter plates (10,000 cells / well) and cultured in DMEM medium containing 10% fetal bovine serum until the coverage reached approximately 90%. The drug was dissolved in serum-free DMEM medium at a final concentration of 1 μM.

[0045] The specific final concentrations of tanshinone compositions at different molar ratios are as follows: 1:0, 1 μM candidate compound + 0 μM MTR; 1:1, 0.5 μM candidate compound + 0.5 μM MTR; 2:1, 0.66 μM candidate compound + 0.33 μM MTR; 3:1, 0.75 μM candidate compound + 0.25 μM MTR; 4:1, 0.8 μM candidate compound + 0.2 μM MTR; 5:1, 0.833 μM candidate compound + 0.167 μM MTR; 6:1, 0.857 μM candidate compound + 0.143 μM MTR; 7:1, 0.875 μM candidate compound + 0.125 μM MTR; 8:1, 0.88 μM candidate compound + 0.11 μM MTR.

[0046] Cells were treated with different drug combinations (molar ratios) for 24 hours, and luciferase expression was detected according to the requirements of the luciferase reporter gene assay kit. The results are shown below. Figure 4 The induction fold represents the fold increase in luciferase expression level relative to the solvent control group.

[0047] The agonistic effects of different molar ratios of nine candidate compounds with tanshinone on Nrf2 activity are as follows: Figure 4 As shown: 1) Capsulin (TN1933): The activation factor of the single drug (1:0) is 1.4 times. Its synergistic activation effect increases with the increase of the molar ratio of capsulin / tanshinone, and reaches the highest activation factor (2.3 times) at 8:1. 2) Versicolor isoflavone (T3923): The activation factor of the single drug (1:0) is 2.8 times. When combined, it exhibits a peak synergistic agonistic effect at a molar ratio of 5:1, with the highest activation factor reaching 3.2 times. 3) Phenethyl ferulate (TN2330): The activation factor of the single drug (1:0) is 2.2 times. When combined, it exhibits a peak synergistic agonistic effect at a molar ratio of 6:1, with the highest activation factor reaching 2.5 times. 4) Psoralen chalcone (TN1774): The activation factor of the single drug (1:0) is 1.1 times. Its synergistic activation effect increases with the increase of the molar ratio of psoralen chalcone / tanshinone, and reaches the highest activation factor (1.5 times) at 8:1. 5) Lejugten (TN1449): The activation factor of the single drug (1:0) is 1.7 times. Its synergistic activation effect increases with the increase of the molar ratio of lejugten / tanshinone, and reaches the highest activation factor (2.8 times) at 8:1. 6) Baicalein methyl ester (TN1427): The activation factor of the single drug (1:0) is 1.8 times. Its synergistic activation effect increases with the increase of the molar ratio of baicalein methyl ester / tanshinone, and reaches the highest activation factor (1.6 times) at 8:1. 7) Picrorhizon 1 (T2787): The activation factor of the single drug (1:0) is 1.5 times. When combined with MTR, it exhibits a peak synergistic agonistic effect at a molar ratio of 6:1, with the highest activation factor reaching 2.0 times. 8) Tetramethylcurcumin (TN2269): The activation factor of the single drug (1:0) is 2.4 times. The Nrf2 agonist activity of the drug combination generally shows an upward trend, reaching the highest activation factor (2.2 times) at 8:1. 9) Andrographolide (T2898): The activation multiple of the single drug (1:0) is 1.9 times, and the synergistic agonistic activity increases significantly with the increase of its ratio, reaching the highest activation multiple (3.1 times) at 8:1.

[0048] The results indicate that tetramethylcurcumin and tanshinone did not exhibit a synergistic advantage; in fact, their Nrf2 activating effect was worse than that of tetramethylcurcumin alone. Combinations of leucovorin, psoralen chalcone, salicylic acid, baicalin methyl ester, and andrographolide with tanshinone showed certain Nrf2 activating activity, which increased with the molar ratio of the candidate compounds. Combinations of picroside I, phenethyl ferulic acid, or verbascoside with tanshinone not only demonstrated excellent synergistic effects but also exhibited a clear optimal ratio window. Among these, verbascoside and tanshinone showed the most significant activating effect on the KEAP1-Nrf2 pathway at the optimal molar ratio of 5:1.

[0049] Therefore, combined with luciferase reporter gene screening experiments, a drug composition with highly efficient synergistic activation of the KEAP1-Nrf2 pathway using isoflavone / tanshinone (optimal molar ratio 5:1) was successfully identified. The screening process is as follows: Figure 5 As shown.

[0050] Test Example 1 Repeated damage and oxidative stress to alveolar epithelial cells are core early events initiating pulmonary fibrosis. This study established a cellular oxidative damage model using mouse alveolar epithelial cells (MLE-12) induced by bleomycin sulfate (BLM). Flow cytometry was used to detect changes in intracellular ROS levels, evaluating the effects of single administration of verrucoside isoflavone (CCS) and tanshinone (MTR), as well as different concentrations of their combinations (CMCL, CMCH), on intracellular ROS levels, thus verifying the synergistic antioxidant effects of the combinations.

[0051] The experimental steps are as follows: MLE-12 cells were seeded in 24-well plates and placed in DMEM / F12 medium containing 4% fetal bovine serum (FBS), 1x insulin-transferrin-sodium selenite (ITS, containing 0.01 mg / mL human recombinant insulin, 5.5 μg / mL human transferrin and 5 ng / mL sodium selenite), and 1x penicillin-streptomycin (concentrations of 100 U / mL and 100 μg / mL, respectively). The cells were cultured at 37°C in a 5% CO2 incubator until the cell confluence reached approximately 70%. The experiment was divided into 6 groups: control group; model group (serum-free DMEM / F12 medium containing 50 μg / mL bleomycin sulfate (BLM); and drug treatment group (serum-free DMEM / F12 medium containing 50 μg / mL BLM and verrucoside isoflavone (CCS) (5 μM) / tanshinone (MTR) (1 μM MTR) / low-concentration drug combination (CMCL) (1.25 μM CCS + 0.25 μM MTR) / high-concentration drug combination (CMCH) (5 μM CCS + 1 μM MTR). The culture medium was discarded, and the corresponding serum-free DMEM / F12 medium was added to each group, and the culture was continued for 24 h.

[0052] After BLM stimulation and drug treatment, the culture medium was discarded, and the cells were washed twice with PBS. 500 μL of serum-free DMEM / F12 medium (containing 10 μM DCFH-DA probe) was added to each well, and the cells were incubated at 37°C in the dark for 20 min. The probe loading solution was discarded, and the cells were washed twice with PBS to remove any probes that had not entered the cells. An appropriate amount of trypsin was added to digest the cells, and the cells were collected in a flow tube. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL of PBS. The fluorescence intensity of DCF was detected using flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. At least 10,000 cells were collected for each sample. The mean fluorescence intensity (FL-A:B525A) was analyzed using FlowJo software, and the results are shown below. Figure 6 ( *P < 0.05, **P < 0.01, ***P < 0.0001 vs. Modelgroup # P < 0.05, ## P < 0.01, ### P < 0.0001 vs Control).

[0053] Flow cytometry histogram ( Figure 6 (Middle left figure) and statistical chart ( Figure 6(Right and middle figures) shows that the proportion of ROS-positive cells to the right of the baseline in the Control group was only 6.37% (P). After BLM-induced stimulation, the P value in the Model group shifted significantly to the right (41.9%), indicating that BLM induced severe oxidative stress and cell damage in MLE-12 cells. In the BLM-induced damage model, the proportions of ROS-positive cells in the CCS (5 μM) monotherapy group and the MTR (1 μM) monotherapy group were 31.6% and 30.9%, respectively, indicating that both monotherapy drugs had certain antioxidant effects. The P values ​​in the CMCL and CMCH groups were 17.6% and 11.2%, respectively, indicating that the antioxidant effect of low-concentration drug combinations was significantly better than that of CCS or MTR alone, and the ROS level after treatment with high-concentration drug combinations was close to that of the normal control group.

[0054] Therefore, the optimal molar ratio of verbascoside-tanshinone (5:1) has a more significant effect on downregulating ROS levels than verbascoside or tanshinone alone, indicating that the combination of verbascoside-tanshinone can effectively reverse lung epithelial damage and has a significant antioxidant effect.

[0055] Test Example 2 Fibronectin, Collagen-I, and α-SMA are classic biomarkers in fibrosis and epithelial-mesenchymal transition (EMT). To further observe the therapeutic effect of a mixture of tanshinone and verbascoside on pulmonary fibrosis, a cellular pulmonary fibrosis model was constructed by inducing human embryonic lung fibroblasts (MRC-5) cells with TGF-β1, and the changes in the expression of Fibronectin, Collagen-I, and α-SMA in cells after different drug treatments were analyzed.

[0056] (1) Cell culture and grouping: Human embryonic lung fibroblasts (MRC-5) cells were cultured at 3.5 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. The experiment consisted of three groups: a control group with normal culture without TGF-β1 or the drug; a model group with 10 ng / mL TGF-β1 to induce a lung injury model; and drug treatment groups with different concentrations of the drug and 10 ng / mL TGF-β1.

[0057] (2) TGF-β1 induction and drug treatment: After the cells adhered to the culture vessel, they were treated according to the groups. The control group was given serum-free DMEM / F12 medium without drugs and TGF-β1; the model group was given serum-free DMEM / F12 medium containing 10 ng / mL TGF-β1; the drug treatment group was given serum-free DMEM / F12 medium containing verbascoside (CCS) (5 μM) / tanshinone (MTR) (1 μM) / low concentration verbascoside-tanshinone combination (CMCL) (1.25 μM CCS + 0.25 μM MTR) / high concentration verbascoside-tanshinone (CMCH) (5 μM CCS + 1 μM MTR) and 10 ng / mL TGF-β1, and cultured for another 24 hours.

[0058] (3) Detection of cell phenotypic markers (Western blot): Cells were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined using a BCA kit. Equal amounts of protein were subjected to SDS-PAGE electrophoresis, transferred to a membrane, and blocked with 5% skim milk powder for 1 hour. Primary antibodies against Fibronectin, Collagen-I, α-SMA, and α-tubulin (internal control) were added, and the mixture was incubated overnight at 4°C. The membrane was washed three times with TBST, and HRP-labeled secondary antibody was added. The membrane was incubated at room temperature for 1 hour, washed three times with TBST, and developed with ECL reagent. Chemiluminescence imaging was used to capture images. The degree of increase in Fibronectin, Collagen-I, and α-SMA expression was quantitatively analyzed using grayscale analysis. The results are shown in [Figure / Table / Insert Figure ... Figure 7 ( *P <0.05, **P < 0.01, ***P < 0.0001 vs. Model group; # P < 0.05, ## P < 0.01, ### P <0.0001 vs Control).

[0059] Figure 7The experimental results showed that, compared with the control group, the expression of fibrosis markers Fibronectin, Collagen-I, and α-SMA was significantly increased in the model group, indicating that the fibroblast activation and fibrosis injury model was successfully constructed. The expression of Fibronectin, Collagen-I, and α-SMA in the CCS group was not significantly different from that in the model group, and it did not provide protection against pulmonary fibrosis. The expression of Fibronectin, Collagen-I, and α-SMA in the MTR group was somewhat reduced compared with the model group, indicating a certain protective effect against pulmonary fibrosis. The optimal drug combination ratios in the CMCL and CMCH groups effectively inhibited the expression of Fibronectin, Collagen-I, and α-SMA. Compared with the single-drug groups, the anti-pulmonary fibrosis effect of the CMCH group was more significant.

[0060] As described above, the tanshinone-verrucous isoflavone composition obtained by the high-throughput screening method for drug compositions targeting the Keap1-Nrf2 pathway to treat multi-organ injury provided in this application exhibits significant synergistic therapeutic effects in activating Nrf2, anti-oxidation, and anti-pulmonary fibrosis, which are superior to the use of either drug alone.

[0061] The pharmaceutical compositions involved in this application can be prepared into various pharmaceutical dosage forms according to clinical treatment needs (especially for oxidative damage and pulmonary fibrosis), including but not limited to inhaled preparations, nebulized inhalation solutions, inhaled powders, injections, tablets, capsules, oral liquids, syrups, and dialysate.

[0062] Application Example 1 This application example provides a tablet with the following component formulation: Application Example 2 This application example provides a capsule formulation with the following components: Application Example 3 This application example provides an oral liquid with the following component formulation (taking 100 mL as an example): Application Example 4 This application example provides an injectable preparation with the following component formulation (taking 10 mL as an example): Application Example 5 This application example provides a dialysate with the following composition (taking 1 L as an example): Application Example 6 This application example provides an inhaler with the following formulation (taking 10 mL as an example): Application Example 7 This application example provides a syrup with the following component formulation (taking 100 mL as an example): The applicant declares that the above description is only a specific implementation of this application, but the protection scope of this application is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application fall within the protection and disclosure scope of this application.

Claims

1. A covalent pharmaceutical composition that targets and covalently modifies KEAP1 to synergistically activate the Nrf2 pathway, characterized in that, The pharmaceutical composition includes tanshinone and verbascoflavonoids.

2. The covalent pharmaceutical composition according to claim 1, characterized in that, The molar ratio of tanshinone and verbascoflavonoid is 1:3-1:

6.

3. The covalent pharmaceutical composition according to claim 2, characterized in that, The optimal molar ratio of tanshinone and verbascoflavonoid is 1:4.5-1:5.

5.

4. The pharmaceutical composition according to any one of claims 1-3, characterized in that, The pharmaceutical composition also includes one or more pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, anti-inflammatory agents, antibacterial agents, or buffers.

6. The covalent pharmaceutical composition according to any one of claims 1-5, characterized in that, The dosage forms of the pharmaceutical composition include tablets, capsules, oral liquids, injections, dialysis solutions, inhalers, syrups, or nebulizers.

7. The use of the covalent pharmaceutical composition targeting KEAP1 according to any one of claims 1-6 in the preparation of a medicament for treating lung injury or pulmonary fibrosis.

8. The use of the covalent pharmaceutical composition targeting KEAP1 according to any one of claims 1-6 in the preparation of Nrf2 agonists.

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