Application of kushenol F in preparation of medicine combined with gefitinib

Abstract

The invention relates to the field of medicine application, and particularly discloses application of kushenol F in preparation of a medicine combined with gefitinib, the medicine is used for treating non-small cell lung cancer, and the combination is that kushenol F and gefitinib have a synergistic effect so as to inhibit tumor growth or reverse gefitinib drug resistance. According to the invention, the sophora alcohol F monomer is combined with the gefitinib, so that the accuracy of the administration dosage is realized, the mechanism is clear, the synergistic effect is obvious, and a brand-new and targeted treatment strategy is provided for solving the gefitinib drug resistance problem of the non-small cell lung cancer.

Description

Technical Field

[0001] The invention belongs to the field of medical applications and relates to use of matrine F in preparing a drug for use in combination with gefitinib. Background Art

[0002] Kushenol F, also known as kushenol F, sophora flavanone, and norkushenone, is a natural flavonoid monomer compound with a dihydroflavonoid skeleton. It exists in the roots of Sophora flavescens and Sophora angustifolia or other plants of the same genus. Its structural feature is the combination of a benzopyran ring and an isopentenyl side chain, which gives it unique pharmacological activity.

[0003] Currently, there are two main monomer structures, including isoprene structure, which exists on the 6th and 8th C atoms of the flavonoid nucleus respectively. Molecular formula: C 25 H 28 O6, with a molecular weight of 424.49 g / mol and a LogP (lipophilicity) of 5.30-5.74, has anti-tumor activity, inhibiting MAPK signaling pathways (such as the ERK / JNK pathway) to induce apoptosis in breast cancer cells (MDA-MB-231) and leukemia cells (HL-60). It also regulates the expression of inflammatory factors, inhibits pathways such as NF-κB, and scavenges free radicals, alleviating oxidative damage.

[0004] Matrine F's current pharmaceutical applications primarily target psoriasis and atopic dermatitis. In an imiquimod-induced psoriasis mouse model, Matrine F significantly improved skin lesions, reduced the expression of pro-inflammatory cytokines such as IL-17A, IL-22, and IL-23, and increased IL-10. Its efficacy was superior to that of the positive control drug calcipotriol and inhibited the proliferation of HaCaT keratinocytes and the release of inflammatory factors.

[0005] In vitro studies have shown that matrine F induces mitochondrial-mediated apoptosis by activating caspase-3 and caspase-9, downregulating the anti-apoptotic proteins Bcl-2 / Bcl-xL, and upregulating the pro-apoptotic protein Bax. Furthermore, it inhibits the activity of MMP-2 / MMP-9, an enzyme involved in tumor cell migration and invasion.

[0006] When gefitinib is used to treat non-small cell lung cancer (NSCLC), drug resistance symptoms are prone to occur. However, when used in combination with adjuvant drugs, it can effectively inhibit tumor growth and reduce adverse reactions. Its therapeutic effect may involve the synergistic or antagonistic effects of multiple pathways. It is necessary to combine the above-mentioned functional studies of matrine F in inhibiting inflammation and regulating metabolism and propose a combination drug that can synergize with gefitinib to reduce drug resistance in non-small cell lung cancer and enhance efficacy, and to reveal the mechanism behind its action. Summary of the Invention

[0007] To address the above issues, the present invention provides a novel use of matrine F in combination with gefitinib in the preparation of a drug for the treatment of non-small cell lung cancer. This combination regimen significantly inhibits non-small cell lung cancer cell proliferation and tumor growth through the synergistic effect of the two drugs, and can effectively reverse acquired and natural resistance to gefitinib. Compared with compound preparations with complex ingredients and large batch variability, the combination of matrine F monomer and gefitinib achieves precise dosing, a clear mechanism, and a significant synergistic effect, providing a new, more targeted treatment strategy for addressing gefitinib resistance in non-small cell lung cancer.

[0008] In order to achieve the above-mentioned purpose, the technical solution of the present invention is as follows: a use of matrine F in the preparation of a drug for combination with gefitinib, wherein the drug is used to treat non-small cell lung cancer, and the combination is a synergistic effect of matrine F and gefitinib to inhibit tumor growth or reverse gefitinib resistance.

[0009] Preferably, the non-small cell lung cancer is EGFR mutant non-small cell lung cancer.

[0010] Preferably, the non-small cell lung cancer is gefitinib-resistant non-small cell lung cancer.

[0011] Preferably, the matrine F reverses gefitinib resistance by inhibiting the expression or function of stromal interaction molecule 1 (STIM1).

[0012] Preferably, the inhibition of STIM1 by matrine F leads to a decrease in intracellular calcium ion concentration, downregulation of nuclear factor κB (NF-κB) signaling pathway activity and / or downregulation of tumor necrosis factor (TNF) signaling pathway activity.

[0013] Preferably, the matrine F and gefitinib are prepared into a single pharmaceutical composition for simultaneous administration.

[0014] Preferably, the matrine F and gefitinib are prepared into independent dosage units for sequential administration.

[0015] Preferably, the dosage ratio of matrine F to gefitinib is 3:2.

[0016] The principle of the basic scheme is: Matrine F directly targets the STIM1 protein, inhibiting its function, resulting in a decrease in intracellular calcium ion concentration, thereby inhibiting the activity of the CaMKII, IKKβ, and NF-κB signaling axis, destroying the TNF-α autocrine loop, and weakening the conduction of the Ras-ERK signaling pathway, ultimately leading to the downregulation of EGFR expression and the inhibition of its activation, thereby restoring the sensitivity of drug-resistant tumor cells to gefitinib.

[0017] The beneficial effects of the present invention are: 1. The present invention first discovered that the combination of matrine F and gefitinib has a significant synergistic effect, which can significantly reduce the half-inhibitory concentration of the two drugs when used alone, and shows strong anti-tumor activity against both gefitinib-sensitive and resistant strains, especially the IC of gefitinib against the resistant strain PC9-GR. 50 It was reduced by 24 times, effectively reversing the common clinical problem of gefitinib resistance.

[0018] 2. The present invention breaks through the bottleneck of the unknown mechanism of action of compound preparations. Through advanced technologies such as DARTS and Lip-MS, it accurately identifies that the direct molecular target of matrine F is STIM1, and fully elucidates its molecular mechanism of downregulating EGFR expression and activation through the "STIM1-calcium signal-NF-κB / TNF / ERK" pathway axis, providing a solid theoretical basis for combined drug use and achieving a leap from "extensive compound" to "precise targeting".

[0019] 3. Compared with traditional Chinese medicine compound preparations with complex ingredients and large fluctuations in the content of main ingredients between batches, the present invention uses high-purity monomer of matrine F in combination with gefitinib, which increases the dosage from the rough milliliter level to the precise milligram level, greatly ensuring the consistency and reliability of the drug treatment effect, and laying the foundation for the standardized production and clinical transformation of the drug.

[0020] 4. IC of matrine F on normal lung cells, such as BEAS-2B and HPMEC 50 Much higher than the IC for tumor cells 50 , showing good selective cytotoxicity, indicating that this combination strategy has a wider therapeutic window and lower potential risk of adverse reactions.

[0021] 5. The present invention provides two feasible dosage forms. Formulation into a single pharmaceutical composition, such as a compound tablet or capsule, simplifies dosing regimens, improves patient compliance, and ensures optimal absorption and utilization of the two active ingredients. Formulation into separate dosage units, such as separate tablets or separately packaged injections, provides greater clinical flexibility, allowing physicians to adjust the dosage and timing of both drugs based on the patient's specific condition, tolerance, and other factors, enabling personalized, precision treatment.

[0022] 6. Developing the two drugs in combination as independent pharmaceutical units can be based on the existing safety data of gefitinib, focusing on the additional efficacy and safety of the combination, which may accelerate the clinical trial process and regulatory approval speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the chemical structural formula of matrine F. Figure 2 is the chemical structural formula of gefitinib.

[0024] Figure 3 This is a schematic diagram of the mechanism by which matrine F reverses gefitinib resistance.

[0025] Figure 4 This is the inhibitory effect of matrine F on PC9 cell proliferation.

[0026] Figure 5 This is the inhibitory effect of matrine F on the proliferation of PC9-GR cells.

[0027] Figure 6 The inhibitory effect of gefitinib on PC9 cell proliferation.

[0028] Figure 7 This is the inhibitory effect of gefitinib on the proliferation of PC9-GR cells.

[0029] Figure 8 This is the synergistic inhibitory effect of matrine F combined with gefitinib on PC9-GR cell proliferation.

[0030] Figure 9 Photos of tumor sizes of CDX models in each group of mice.

[0031] Figure 10 A bar graph showing tumor growth in the in vivo CDX model.

[0032] Figure 11 This is the principal component analysis diagram of the global gene expression profile of tumor samples.

[0033] Figure 12 The DARTS experiment verifies the direct binding results of matrine F and STIM1.

[0034] Figure 13 This is the volcano plot of differentially expressed genes in the combination drug group and the control group.

[0035] Figure 14 This is the cluster heat map of significant differential expression after combined treatment of matrine F and gefitinib.

[0036] Figure 15 This is the KEGG pathway enrichment analysis diagram. DETAILED DESCRIPTION

[0037] The following is a detailed description of the embodiments with reference to the accompanying drawings.

[0038] Example 1 Basically as attached Figures 1 to 4Shown is a use of matrine F in preparing a drug for use in combination with gefitinib, wherein the drug is used to treat non-small cell lung cancer. The combination is a synergistic effect of matrine F and gefitinib to inhibit tumor growth or reverse gefitinib resistance.

[0039] The non-small cell lung cancer is EGFR mutant non-small cell lung cancer, and the non-small cell lung cancer is gefitinib-resistant non-small cell lung cancer.

[0040] The matrine F reverses gefitinib resistance by inhibiting the expression or function of matrix interaction molecule 1. The inhibition of STIM1 by matrine F leads to a decrease in intracellular calcium ion concentration, downregulation of nuclear factor κB signaling pathway activity, and / or downregulation of tumor necrosis factor signaling pathway activity.

[0041] This study focused on the structure of isoprene at the C-8 position of the flavonoid nucleus. In non-small cell lung cancer cells and animal models, it was found that matrinol F monomer could inhibit the proliferation of non-small cell lung cancer and tumor growth at low doses, and also had an inhibitory effect on EGFR-TKI-resistant non-small cell lung cancer. When combined with gefitinib, it had a better effect on gefitinib-resistant non-small cell lung cancer than gefitinib monotherapy. The two may synergistically exert anti-tumor effects.

[0042] The molecular formula of Kushenol F is C 25 H 28 O5, molecular weight 424.49 g / mol, pale yellow to colorless crystalline powder, soluble in DMSO. Gefitinib, molecular formula C 22 H 24 ClFN4O3, molecular weight 446.9 g / mol, white to off-white crystalline powder, soluble in DMSO.

[0043] The specific experimental process is as follows: 1. Cell experiments Experimental methods 1. Cell lines and culture conditions The following five human non-small cell lung cancer (NSCLC) cell lines were used in this study: A549: EGFR wild-type, carrying KRAS G12S activating mutation.

[0044] H1650: Carries EGFR exon 19 deletion mutation (ex19del) and PTEN gene deletion.

[0045] PC9: Carries EGFR ex19del mutation and is highly sensitive to gefitinib.

[0046] H1975: Carries EGFR L858R and T790M double mutations and is naturally resistant to gefitinib.

[0047] HCC827: Carries EGFR ex19del mutation.

[0048] All cell lines were cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution in a constant temperature and humidity incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0049] 2. Compounds and treatment options Kushenol F is dissolved in dimethyl sulfoxide (DMSO) to prepare a high-concentration stock solution, which is then aliquoted and stored at -20°C. Upon use, dilute the solution to the desired working concentration in complete culture medium. Ensure that the final DMSO concentration in each experimental group is consistent and below 0.1% (v / v) to eliminate solvent effects on cell viability.

[0050] After cells were seeded into culture plates and allowed to adhere overnight, they were treated with fresh complete culture medium containing various concentrations of matrine F (5 μM, 10 μM, and 20 μM) or an equal volume of DMSO (as a solvent control). The treatment time was determined based on subsequent assays (e.g., MTT assay for cell viability, typically 48-72 hours).

[0051] 3. Induction and culture of PC9 gefitinib-resistant cell line (PC9-GR) The "Pulse-Recovery" method with increasing concentration gradient was used to induce acquired drug resistance in PC9 cells.

[0052] Initial IC 50 Determination: The half inhibitory concentration (IC50) of gefitinib on parental PC9 cells was determined by preliminary experiments. 50 ), determined to be 10 nM.

[0053] Induction process: IC 50 The concentration (10 nM) was used as the starting point for induction and passage. The treatment process for each passage of cells is as follows: (1) Pulse exposure: When the cells grow to about 60-70% confluence, replace them with complete culture medium containing 1 μM gefitinib and continue treatment for 24 hours.

[0054] (2) Recovery culture: Discard the drug-containing culture medium, gently wash the cells twice with pre-warmed PBS, then replace with complete culture medium without drugs and continue culturing for 48 hours.

[0055] (3) Concentration increase and continuous induction: The above-mentioned “pulse-recovery” process is repeated. As the cells gradually adapt, the gefitinib concentration during pulse exposure can be gradually increased (e.g., from 1 μM to 2 μM, 4 μM, etc.) according to the cell survival status, until the cells can stably proliferate under high concentrations of gefitinib (e.g., 2 μM). The drug is withdrawn for 7 days before the experiment to eliminate the residual drug effect.

[0056] 4. Experimental results Combine Figures 4 to 8 As shown in Table 1, the IC values ​​of matrine F on normal lung cell lines BEAS-2B and HPMEC were 50 The values ​​were 106.67 μM and 70.19 μM, respectively, indicating that its toxicity to normal cells was relatively low. In contrast, matrine F showed stronger proliferation inhibition ability against all tested NSCLC cell lines, with its IC 50 The values ​​ranged from 14.07 μM (H1975) to 22.68 μM (H1650). Notably, the antitumor activity of matrine F did not appear to be clearly associated with the type of EGFR mutation (including wild-type, ex19del, and L858R / T790M), suggesting that its mechanism of action may not be dependent on the EGFR pathway.

[0057] Table 1. The half-maximal inhibitory concentration (IC) of matrine F on different cell lines 50 )

[0058] The “pulse-recovery” concentration gradient method was used to successfully induce a PC9 cell line (PC9-GR) with acquired resistance to gefitinib. The parental PC9 cells were highly sensitive to gefitinib, and IC 50 The induced PC9-GR cells showed strong drug resistance, with an IC 50 As high as 11.03 μM, the drug resistance index exceeded 60 times, confirming that the PC9-GR cell model was successfully constructed and can be used for subsequent combination drug studies.

[0059] To investigate the combined effect of matrine F and gefitinib, a co-administration experiment was conducted on the sensitive PC9 cell line and the drug-resistant PC9-GR cell line. The combination of low-concentration gefitinib (10 nM) and matrine F significantly enhanced the killing effect of the latter on PC9 cells. 50 was 16.65 μM, while the combined use of the drugs increased its IC 50 The sensitivity was reduced to 4.1 μM, and the sensitivity was increased by 4.06 times. The combined treatment strategy showed a stronger effect of reversing drug resistance. Figure 4 、 Figure 5 、 Figure 6 and Figure 7 shown.

[0060] The concentration of matrine F was fixed at 5 μM. The inhibitory rate of matrine F alone on PC9-GR cells was low at this concentration. However, when combined with gefitinib, the IC of gefitinib on PC9-GR cells was reduced. 50 The concentration of the drug was significantly reduced from 11.03 μM to 458 nM, reducing drug resistance by 24 times and effectively reversing its drug-resistant phenotype.

[0061] The fixed concentration of gefitinib was 1 μM, and the fixed concentration of gefitinib was combined with matrine F, which also increased the IC of matrine F to PC9-GR. 50 The sensitivity was increased 4-fold from 23.29 μM to 6.5 μM.

[0062] Combine Figure 8 As shown in the figure, the combination index (CI) was calculated to quantitatively evaluate the nature of the combined effect. Under the above two combination schemes: Immobilized 5 μM matrine F+458 nM gefitinib (IC 50 points): CI=(458 / 11030)+(5 / 23.29)=0.256 Fixed 1 μM gefitinib + 6.5 μM matrine F (IC 50 points): CI=(1000 / 11030)+(6.5 / 23.29)=0.37 Both CI values ​​were far less than 1, clearly indicating a strong synergistic effect between matrine F and gefitinib in inhibiting PC9-GR cell proliferation. The synergistic effect was even more pronounced when the concentration of matrine F was fixed (CI = 0.256).

[0063] Example 2 The difference from the above embodiment is that, as shown in the attached Figure 5 、 Figure 6 As shown: the matrine F and gefitinib are prepared into a single pharmaceutical composition, or are prepared into independent pharmaceutical units for simultaneous or sequential administration.

[0064] In this embodiment, a simultaneous administration strategy was adopted, and the dosage ratio of matrine F to gefitinib was 3:2.

[0065] Following the above experimental results, animal experiments were conducted: 2. In vivo mouse CDX model experiments 1. Establishment of xenograft tumor (CDX) model PC9-GR cells in the logarithmic growth phase were obtained, trypsinized, counted, and resuspended in pre-chilled serum-free medium. The cell suspension was thoroughly mixed with Matrigel at a 1:1 volume ratio on ice, keeping the temperature low to prevent gel solidification. The final cell density was adjusted to 5 × 10 cells per mouse. 6 Using a sterile 1 mL syringe, inject 200 μL of the cell-matrix gel mixture subcutaneously into the right dorsal flank of each mouse. Observe the mice's condition and tumor formation daily.

[0066] 2. Grouping and Dosing Grouping: When the tumor volume grew to approximately 100 mm³, the tumor-bearing mice were randomly assigned (n=6 / group) to the following four experimental groups: Control group: An equal volume of drug solvent (such as normal saline containing 5% DMSO + 5% Cremophor EL) was administered.

[0067] Gefitinib group: 20 mg / kg / d, oral gavage.

[0068] Sophoflavescenol F group: 30 mg / kg / d, oral gavage.

[0069] Combination group: 20 mg / kg / d gefitinib and 30 mg / kg / d matrine F were given simultaneously, and the administration method was the same as that of the single-drug group.

[0070] Dosage regimen: Each group of mice was administered the drug for 17 consecutive days. The body weight and tumor volume of the mice were recorded daily.

[0071] 3. Tumor volume and body weight monitoring Tumor measurement: The longest diameter (a) and shortest diameter (b) of the tumor were measured every 2-3 days using a vernier caliper. Tumor volume (TV) was calculated using the formula: TV (mm³) = 0.5 × a × b².

[0072] Body weight monitoring: The mice were weighed daily to assess possible systemic toxicity caused by the drug.

[0073] Experimental endpoint: 17 days after dosing, all mice were humanely sacrificed by carbon dioxide inhalation. Tumor tissue was excised, weighed, and photographed. A portion of the tumor tissue was frozen at -80°C or fixed in 4% paraformaldehyde for subsequent analysis.

[0074] 4. Transcriptome sequencing (RNA-Seq) analysis Sample preparation: PC9-GR tumor tissues (n=3 / group) from the four groups of mice (control group, gefitinib group, matrine F group, and combined drug group) in the above experiment were obtained and quickly ground into powder in liquid nitrogen.

[0075] RNA Extraction: Total RNA was extracted from tumor tissues in each group using the TRIzol method. RNA concentration was determined using NanoDrop, and RNA integrity (RIN value) was assessed using an Agilent 2100 Bioanalyzer to ensure that sample quality met library construction requirements.

[0076] Library Construction and Sequencing: Qualified samples were subjected to mRNA enrichment, fragmentation, and cDNA synthesis, and PE150 sequencing libraries were constructed. High-throughput sequencing was performed on the Illumina NovaSeq 6000 platform.

[0077] Bioinformatics Analysis: Raw data were quality-controlled, aligned to the reference genome GRCh38, and gene expression quantified. Differentially expressed genes (DEGs) were screened for a |log2(Fold Change)| > 1 and an adjusted p-value < 0.05. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on DEGs to reveal significant signaling pathway changes between groups.

[0078] 5. Drug Affinity Target Stability Technology (DARTS) and LiP-MS Target Fishing Cell lysate preparation Culture PC9-GR cells, harvest cells during the logarithmic growth phase, wash with pre-chilled PBS, and lyse on ice in a lysis buffer (e.g., NP-40 buffer containing protease inhibitors). Centrifuge and collect the supernatant to quantify the total protein concentration.

[0079] 6. DARTS Experiment Drug incubation: Total proteins were gently incubated with different concentrations of matrine F (0 μM, 20 μM, 200 μM) at 4°C for 1 hour to allow the drug to bind to potential targets.

[0080] Enzymatic hydrolysis: Divide the above reaction system into two tubes. Add Pronase (final concentration 0.5 μg / μL) to one tube and add an equal volume of solvent to the other tube as a control. Perform limited enzymatic hydrolysis at room temperature (the time needs to be optimized in advance, such as 30 minutes).

[0081] Termination and denaturation: Add SDS loading buffer and boil to terminate the enzymatic reaction.

[0082] Western Blotting (WB) analysis: SDS-PAGE and WB techniques were used to detect the degree of degradation of specific candidate target proteins. Compared with the control group, the protein bands protected by matrine F were brighter in the enzyme-added group.

[0083] 7. Limited proteolysis mass spectrometry (LiP-MS) Drug incubation and enzymatic hydrolysis: After incubating the protein extract with 20 μM matrine F or solvent control, Pronase was added for brief enzymatic hydrolysis.

[0084] Protease inactivation and peptide preparation: After terminating the reaction, conventional trypsin was used for thorough enzymatic digestion to generate peptides.

[0085] Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: The peptides were analyzed by LC-MS / MS.

[0086] Data analysis: By comparing the differences in peptide abundance between the matrine F-treated group and the control group, we identified peptides whose proteolysis patterns changed after being bound by matrine F, thereby directly identifying the direct target proteins of matrine F in cells.

[0087] 8. Experimental Results (1) Combination of matrine F and gefitinib significantly inhibited the growth of PC9-GR resistant tumors in vivo To evaluate the in vivo efficacy of matrine F alone and in combination with gefitinib against gefitinib-resistant tumors, we established a subcutaneous xenograft (CDX) model of PC9-GR cells in mice. Figure 9 and Figure 10 As shown, compared with the control group, gefitinib monotherapy (20 mg / kg / day) only slightly inhibited tumor growth, consistent with the drug-resistant nature of PC9-GR cells. Matrine F monotherapy (30 mg / kg / day) exhibited a moderate antitumor effect, indicating that it possesses some tumor suppressive activity on its own. However, the combination therapy group had the most significant tumor growth inhibition effect, with tumor volume growth nearly stagnating throughout the entire dosing cycle (17 days), and the final tumor weight was significantly lower than that of the other groups. There was no significant weight loss in mice in any group during treatment, indicating that the treatment regimen was well tolerated. This in vivo experiment confirmed that matrine F effectively enhanced the efficacy of gefitinib against drug-resistant tumors, demonstrating a clear synergistic effect between the two.

[0088] (2) Transcriptome sequencing reveals that combined drug therapy synergistically regulates multiple pro-survival signaling pathways To explore the mechanism of synergy at the molecular level, we performed transcriptome sequencing (RNA-Seq) on four groups of in vivo tumor tissues. Figure 11The results showed that the gene expression profile of the combination drug group was significantly separated from that of the control group and the two single-drug groups, indicating that the combination treatment triggered a unique global transcriptional reprogramming.

[0089] Combine Figure 13 、 Figure 14 and Figure 15 As shown, gene set enrichment analysis (GSEA) further revealed that, compared with monotherapy, combination therapy synergistically downregulated multiple signaling pathways closely associated with tumor proliferation, survival, and drug resistance. Among them, the TNF, NF-κB, and MAPK signaling pathways were most significantly downregulated. This suggests that the synergistic effect of combination therapy is not achieved through a single pathway, but rather by simultaneously targeting multiple redundant pro-survival signaling networks, thereby more thoroughly dismantling the tumor's drug resistance defense system.

[0090] (3) Target fishing identified STIM1 as the direct target of matrine F To identify the direct molecular target of matrine F, we used the drug affinity target stability technology (DARTS). Figure 11 and Figure 12 The results showed that with the increase of matrine F concentration (0, 20, 200 μM), the resistance of STIM1 protein to Pronase degradation increased, indicating that matrine F can directly bind to STIM1 protein and protect it from enzymatic degradation.

[0091] This finding was further validated by limited proteolysis-mass spectrometry (LiP-MS). In samples treated with matrine F (20 μM), multiple peptides of the STIM1 protein displayed a unique proteolytic protection pattern, confirming that STIM1 is a direct interacting protein of matrine F in cells.

[0092] (4) Mechanism of matrine F inhibiting calcium signaling and reversing drug resistance by targeting STIM1 Based on the above findings, we proposed and verified the molecular mechanism by which matrine F reverses EGFR-TKI resistance by targeting STIM1. Figure 3 As shown: Matrine F targets STIM1 and inhibits calcium signaling: Matrine F binds to and inhibits the function of STIM1, resulting in a weakening of store-operated calcium influx (SOCE) and a sustained decrease in intracellular calcium ion concentration ([Ca²⁺]i).

[0093] Downregulated calcium signaling inhibits the NF-κB pathway, reducing intracellular calcium concentrations and decreasing the activity of the calcium-dependent kinase CaMKII. Inactivated CaMKII is unable to effectively phosphorylate and activate IKKβ, thereby hindering the degradation of IκBα. Stabilization of IκBα traps the NF-κB p65 subunit in the cytoplasm, preventing it from entering the nucleus and initiating transcription of downstream genes, including pro-survival and inflammatory factors. Transcriptome data demonstrating downregulation of the NF-κB pathway align well with this finding.

[0094] Downregulation of calcium signaling disrupts the TNF autocrine loop and affects EGFR. The low calcium environment caused by STIM1 inhibition disrupts the TNF-α autocrine positive feedback loop and reduces the production of TNF-α ligands. This leads to reduced activation of the TNFR1 receptor and decreased expression of its downstream adaptor proteins TRAF2 / 5 and RIPK1. Ultimately, this reduces the production and release of EGFR ligands, including EGF, thereby weakening the abnormal activation of EGFR at the source.

[0095] Downregulation of calcium signaling inhibits the MAPK / ERK pathway. Calcium ions are a key regulator of Ras GTPase activity. Reduced intracellular calcium concentrations directly inhibit Ras-GTP loading, thereby blocking the downstream Raf-MEK-ERK signaling cascade. ERK inactivation not only reduces feedback phosphorylation of EGFR but also affects its protein stability.

Claims

1. A use of matrine F in the preparation of a drug for use in combination with gefitinib, wherein the drug is used to treat non-small cell lung cancer, characterized in that: The combination is a synergistic effect of matrine F and gefitinib to inhibit tumor growth or reverse gefitinib resistance.

2. The use according to claim 1, characterized in that The non-small cell lung cancer is EGFR mutant non-small cell lung cancer.

3. The use according to claim 2, characterized in that The non-small cell lung cancer is gefitinib-resistant non-small cell lung cancer.

4. The use according to claim 1, characterized in that The matrine F reverses gefitinib resistance by inhibiting the expression or function of matrix interaction molecule 1.

5. The use according to claim 3, characterized in that The inhibition of matrine F on matrix interaction molecule 1 leads to a decrease in intracellular calcium ion concentration, downregulation of nuclear factor κB signaling pathway activity and / or downregulation of tumor necrosis factor signaling pathway activity.

6. The use according to claim 1, characterized in that The matrine F and gefitinib are prepared into a single pharmaceutical composition for simultaneous administration.

7. The use according to claim 1, characterized in that The matrine F and gefitinib are respectively prepared into independent dosage units for sequential administration.

8. The use according to claim 1, characterized in that The dosage ratio of matrine F to gefitinib is 3:2.

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