Use of NRF2 or derivatives thereof as diagnostic markers and inhibitors of NRF2 or derivatives thereof as therapeutic targets

Abstract

The invention provides application of NRF2 or a derivative thereof as a diagnostic marker and an inhibitor of the NRF2 or the derivative thereof as a therapeutic target, and belongs to the field of medicine. Experiments find that the p-NRF2 can be used for predicting the curative effect of the photodynamic therapy on oral leukoderma, the lower the expression level of the p-NRF2 is, the better the treatment effect of the photodynamic therapy is, and the higher the expression level of the p-NRF2 is, the poorer the treatment effect of the photodynamic therapy is. And after the p-NRF2 inhibitor is used, the treatment effect of the photodynamic therapy can be obviously improved. Clinical doctors can be helped to better predict and / or improve the sensitivity of patients to photodynamic therapy treatment, better treatment decisions can be made, individualized treatment schemes can be made, and the prognosis and life quality of oral leukoderma patients can be improved.

Description

Technical Field

[0001] This invention belongs to the medical field, specifically relating to the use of NRF2 or its derivatives as diagnostic markers and the use of inhibitors of NRF2 or its derivatives as therapeutic targets. Background Technology

[0002] Oral leukoplakia (OLK) refers to plaque-like, white or grayish-white keratotic lesions occurring solely on the oral mucosa. It is a common, non-infectious, chronic disease that can occur on any part of the oral mucosa, but is most common on the buccal and lingual areas. The incidence of oral leukoplakia in the general population is approximately 0.57%–3.6%, and the rate of malignant transformation is approximately 0.13%–34%. The clinical goal of oral leukoplakia treatment is to minimize or eliminate the lesions and prevent malignant transformation.

[0003] Currently, treatment methods for oral leukoplakia include surgical and non-surgical approaches. Surgical treatments for oral leukoplakia include traditional lesion excision, electrocautery, laser ablation, and cryotherapy. However, in some patients with large lesions or extensive involvement, invasive surgical methods are not suitable. Furthermore, surgical treatment can cause adverse reactions such as postoperative pain, edema, and bleeding, and may also lead to complications such as extensive scarring. These are some reasons why some patients do not undergo surgical treatment.

[0004] Photodynamic therapy (PDT) has been widely used in the past two decades to treat precancerous lesions and cancers of the skin, digestive tract, and genitourinary tract. PDT has been used to treat oral cancer, oral leukoplakia, and other diseases. For oral leukoplakia, PDT has good targeting and reproducibility, mild postoperative adverse reactions, and almost no severe scarring. PDT shows promise as a routine or adjuvant treatment for oral leukoplakia.

[0005] PDT treatment is divided into two stages. First, an appropriate type and dose of photosensitizer is used at the lesion site. Then, light of a specific wavelength is used to irradiate the lesion site. After being activated by light, the photosensitizer will cause a series of photochemical and photobiological reactions. Through oxidative stress-related mechanisms, it will cause the death of abnormally proliferating cells, thereby reducing or eliminating the lesion.

[0006] Currently, the complete remission rate of phototherapy (PDT) for oral leukoplakia is approximately 26%, and the overall response rate is approximately 75%. Therefore, exploring the mechanisms of PDT resistance in oral leukoplakia, discovering molecular markers for the prognosis of PDT treatment, and enhancing the sensitivity of oral leukoplakia patients to PDT treatment are of great significance for improving patient prognosis and enhancing their quality of life.

[0007] However, there are currently no effective molecular markers to predict the sensitivity of oral leukoplakia to photodynamic therapy (PDT), and there are no biological adjuvants for PDT treatment of OLK patients, which poses a great obstacle to improving the treatment prognosis of oral leukoplakia patients.

[0008] NRF2 protein is a transcription factor that plays a key role in oxidative stress response. When cells encounter oxidative stress, NRF2 protein can be phosphorylated to form p-NRF2 protein.

[0009] Brusatol (CAS No. 14907-98-3) is an Nrf2 inhibitor that effectively inhibits the Nrf2 protein. Currently, there are no reports on the Nrf2 inhibitor Brusatol inhibiting p-NRF2.

[0010] There are currently no reports on the use of p-NRF2 in PDT treatment of oral leukoplakia. Summary of the Invention

[0011] The purpose of this invention is to provide the use of NRF2 or its derivatives as diagnostic markers and inhibitors of NRF2 or its derivatives as therapeutic targets.

[0012] This invention provides the use of reagents for detecting the expression level of NRF2 or its derivatives in the preparation of reagents for predicting the efficacy of photodynamic therapy in treating potential oral malignancies.

[0013] Furthermore, the reagent for detecting the expression level of NRF2 or its derivatives is the same reagent for detecting the expression level of NRF2 or its derivatives in pathological tissues; the derivative of NRF2 is p-NRF2.

[0014] Furthermore, the reagents for detecting the expression level of NRF2 or its derivatives include reagents for enzyme-linked immunosorbent assay (ELISA), reagents for immunoblotting, reagents for immunoelectrophoresis, reagents for tissue immunostaining, reagents for immunoprecipitation analysis, reagents for radioimmunoassay, reagents for radioimmunodiffusion, reagents for complement fixation analysis, reagents for fluorescence-activated cell differentiation, reagents for quality analysis, or reagents for protein microarrays.

[0015] Furthermore, the potential malignant oral disease is oral leukoplakia or oral erythroplakia.

[0016] The present invention also provides the use of an agent that inhibits NRF2 or its derivatives in the preparation of a medicament for treating potential oral malignancies using photodynamic therapy.

[0017] Furthermore, the reagent that inhibits NRF2 or its derivatives is a reagent that reduces the expression level of NRF2 or its derivative proteins; the derivative of NRF2 is p-NRF2.

[0018] Furthermore, the reagent for inhibiting NRF2 or its derivatives is ML385, NRF2-IN1, NRF2-IN3, or Brusatol.

[0019] Furthermore, the potential malignant oral disease is oral leukoplakia or oral erythroplakia.

[0020] Furthermore, the photodynamic therapy uses a photosensitizer, preferably 5-aminolevulinic acid.

[0021] The present invention also provides the use of Brusatol in the preparation of reagents that inhibit the expression level of p-NRF2 protein.

[0022] Experimental results show that this invention provides the use of NRF2 or its derivatives as diagnostic markers and inhibitors of NRF2 or its derivatives as therapeutic targets. Through experiments, this invention found that p-NRF2 can be used to predict the efficacy of PDT in treating oral leukoplakia (OLK). The lower the expression level of p-NRF2, the better the PDT treatment effect; the higher the expression level of p-NRF2, the worse the PDT treatment effect. This invention also found that using the NRF2 inhibitor, crocinol, can significantly reduce p-NRF2 expression and improve the efficacy of PDT in treating OLK. This invention can help clinicians better predict and / or improve patients' sensitivity to PDT treatment, facilitating better treatment decisions, developing individualized treatment plans, and improving the prognosis and quality of life of patients with oral leukoplakia.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 p-NRF2 was upregulated in the PDT-resistant group, **** indicates p < 0.0001.

[0026] Figure 2 ROC curves showing the expression of p-NRF2 and the efficacy of PDT treatment.

[0027] Figure 3 Results of p-NRF2 protein expression levels in the DOK-BRUS and DOK-DMSO groups.

[0028] Figure 4 Inhibiting p-NRF2 can enhance the sensitivity of cells to PDT treatment.

[0029] Figure 5 Results of p-NRF2 protein expression levels in OE-NRF2 cells.

[0030] Figure 6 Increasing p-NRF2 can enhance cellular resistance to PDT treatment.

[0031] Figure 7 This describes a method for constructing a PDT-resistant cell line (DOK-RP).

[0032] Figure 8 p-NRF2 is highly expressed in the PDT-resistant cell line (DOK-RP).

[0033] Figure 9 Results of p-NRF2 protein expression levels in DOK-RP cells.

[0034] Figure 10 The combination of Brusatol, an NRF2 inhibitor, and a photosensitizer can improve the sensitivity of cellular PDT therapy. Detailed Implementation

[0035] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0036] Growth medium (DMEM medium containing 10% fetal bovine serum): Reference catalog number gibco 11995065DMEM, high glucose, containing pyruvate;

[0037] Photosensitizer 5-ALA: 5-aminolevulinic acid: CAS number 106-60-5.

[0038] ML385 CAS No. 846557-71-9.

[0039] NRF2-IN1 CAS No. 1610022-76-8.

[0040] NRF2-IN3 CAS No. 6325-13-9.

[0041] The Western blotting method of this invention is as follows: Cells are lysed using a suitable amount of protein lysis buffer containing protease inhibitors for protein quantification, followed by SDS-PAGE electrophoresis. After electrophoresis, the gel is gently removed and transferred to a semi-dry transfer apparatus. After transfer, the PVDF membrane is rinsed in Western blotting wash buffer for 30 seconds, then placed in blocking buffer and incubated on a horizontal shaker for 1 hour. The membrane is then cut to the appropriate size according to the experimental purpose and placed in a primary antibody solution of the target protein at an appropriate dilution, and incubated overnight at 4°C. The next day, the PVDF membrane is removed from the refrigerator and washed in Western blotting wash buffer for 10 minutes, repeated three times. It is then placed in a secondary antibody solution of an appropriate dilution and incubated on a horizontal shaker at room temperature for 1 hour. After secondary antibody incubation, the PVDF membrane is removed and placed in a chemiluminescence analyzer for chemiluminescence analysis and result analysis.

[0042] NRF2 is rapidly degraded by ubiquitination within cells, making its expression difficult to detect. When NRF2 is activated, it is phosphorylated to p-NRF2 and accumulates within the cell. At this point, the expression of p-NRF2 can be detected. Therefore, this invention primarily examines the expression of p-NRF2.

[0043] Example 1: Sensitivity of p-NRF2 in PDT treatment of oral leukoplakia

[0044] I. Experimental Methods

[0045] 1. p-NRF2 expression level

[0046] Lesion tissues were collected from patients with oral leukoplakia diagnosed by pathology at the Department of Oral Mucosa, West China Hospital of Stomatology, Sichuan University, who had not undergone photodynamic therapy, as well as pathological tissues that had undergone photodynamic therapy. Paraffin sections were prepared from these tissues.

[0047] Immunohistochemical staining was used to detect p-NRF2 expression in lesion tissues: Paraffin sections were placed in a 65°C oven for 2–4 hours. After dewaxing with xylene, they were followed by graded alcohol dehydration. The sections were washed three times with 1×PBS for 5 min each. Antigen retrieval was performed using sodium citrate retrieval buffer; the sections were then blocked with 3% hydrogen peroxide. Commercially available p-NRF2 antibody was added at the specified dilution ratio, and the sections were incubated overnight at 4°C. The next day, the sections were removed, washed three times with PBS for 5 min each, and then incubated with secondary antibody at 37°C for 60 min. DAB staining was performed, followed by rinsing with running water for 10 min. The sections were counterstained with hematoxylin, rinsed with running water to achieve blue staining, hydrated with graded alcohol, cleared with xylene, and mounted.

[0048] 2. PDT Treatment Response Assessment Criteria Grouping

[0049] Oral leukoplakia was treated with 20% 5-aminolevulinic acid (5-ALA) as a photosensitizer. After a 3-hour wet compress on the lesion surface, phototherapy was applied at a wavelength of 630nm ± 5nm and a power of 100mW / cm². 2 The light dose is 100 J / cm. 2 Patients were treated with irradiation every two weeks for a total of three treatments. Treatment response was recorded four weeks after the last treatment. Complete remission was defined as the disappearance of visible lesions; partial remission was defined as a reduction in lesion volume greater than 20%; and no response was defined as a reduction in lesion volume less than 20% or an increase in lesion volume. Patients assessed as having complete or partial remission were included in the PDT-sensitive group, while those assessed as having no response were included in the PDT-resistant group.

[0050] II. Experimental Results

[0051] 1. p-NRF2 expression level

[0052] The expression levels of p-NRF2 in each group of samples were detected using immunohistochemical staining. The results are as follows: Figure 1 As shown, the expression level of p-NRF2 in the tissues of patients in the PDT-resistant group was significantly upregulated compared to that in the PDT-sensitive group.

[0053] 2. p-NRF2 expression level and the efficacy of PDT treatment

[0054] Based on the expression level of p-NRF2 in the patient's pathological tissue and the efficacy of PDT treatment, receiver operating characteristic (ROC) curves were plotted. The results showed that the expression level of p-NRF2 could be used to predict the sensitivity of OLK patients to PDT treatment, with an area under the curve (AUC) of 0.825. Figure 2 p-NRF2 can be used to predict the efficacy of PDT in treating OLK. The lower the expression level of p-NRF2, the greater the probability of good PDT treatment effect, and the higher the expression level of p-NRF2, the greater the probability of poor PDT treatment effect.

[0055] Example 2: Inhibition of p-NRF2 can enhance the sensitivity of cells to PDT therapy.

[0056] I. Experimental Methods

[0057] 1. Experimental Grouping

[0058] DOK cells were harvested, digested, and counted. Cells were seeded at a density of 3000 cells / well in 96-well plates and cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a cell culture incubator containing 5% CO2. Cells were grouped on day 2 post-seeding.

[0059] DOK+BRUS group (experimental group): The culture medium in the well plate was replaced with serum-free medium containing 0.5 mM 5-ALA and 30 nM Brusatol (Brusatol was first dissolved in DMSO and then added to the culture medium).

[0060] The DOK+DMSO group (control group) had its cell growth medium in the well plates replaced with serum-free medium containing 0.5 mM 5-ALA, and an appropriate amount of DMSO was added. The volume of DMSO added to the control group was the same as that in the experimental group.

[0061] Both groups of cells were incubated in the dark for 5 hours before undergoing PDT treatment. The specific treatment method for PDT was as follows: each group of cells received treatment at corresponding power densities (0, 3, 6, 9, 12 J / cm²). 2 ).

[0062] 2. Cell viability detection methods

[0063] Cells treated with PDT were cultured in a cell culture incubator containing 5% CO2 for 12 hours. After 12 hours, cell viability was assessed using the CCK8 reagent. The CCK8 reagent was added according to the CCK8 instructions, and the absorbance (OD value) at 450 nm was measured using a microplate reader and statistical analysis was performed.

[0064] Cell viability (%) = (OD value of experimental group - OD value of blank control) / (OD value of control group - OD value of blank control) × 100%. Wherein, the experimental group consists of cells subjected to different PDT irradiation conditions, the control group consists of cells not subjected to PDT irradiation, and the blank control consists of cell-free culture medium. Cell viability curves were plotted under each power condition. Three biological replicates were set up for each group in the above experiments.

[0065] Western blot was used to detect the expression level of p-NRF2 in the DOK-BRUS and DOK-DMSO groups before PDT treatment, verifying that p-NRF2 protein expression was inhibited in DOK-BRUS.

[0066] II. Experimental Results

[0067] 1. p-NRF2 protein expression level

[0068] Experimental results are as follows Figure 3 As shown, Brusatol can significantly inhibit the expression level of p-NRF2 protein.

[0069] 2. Inhibition of p-NRF2 can enhance the sensitivity of cells to PDT therapy.

[0070] Experimental results are as follows Figure 4As shown, under the same PDT treatment environment, the survival rate of DOK cells in the DOK-BRUS group was significantly lower than that in the DOK-DMSO group.

[0071] Experimental results show that the NRF2 inhibitor Brusatol can significantly inhibit the expression level of p-NRF2 protein, which can significantly reduce the survival rate of human oral mucosal precancerous lesion cells (DOK cells), enhance the therapeutic effect of PDT, and enhance the efficacy of PDT in treating OLK.

[0072] Example 3: Increasing p-NRF2 can enhance cellular resistance to PDT therapy.

[0073] I. Experimental Methods

[0074] 1. Constructing a p-NRF2 overexpressing cell line

[0075] NRF2 is rapidly degraded by ubiquitination within cells, making it difficult to detect its bulk expression. When NRF2 is activated, it is phosphorylated to p-NRF2 and accumulates within the cell. At this point, the expression of p-NRF2 can be detected, so this invention primarily focuses on detecting the expression of p-NRF2.

[0076] This invention constructs an NRF2 overexpression vector, and then constructs a cell line infected with lentivirus containing the NRF2 overexpression vector (OE-NRF2) and a cell line infected with lentivirus containing the control vector (CONTROL). The successful construction of the p-NRF2 overexpression cell line is confirmed by detecting p-NRF2 expression. The specific construction method is as follows:

[0077] NRF2 overexpression vector

[0078] pLV[Exp]-EGFP:T2A:Puro-EF1A>hNFE2L2[NM_006164.5]

[0079] The vectors VB900004-1506ejy and the control vector VB010000-9298rtf were constructed by Yunzhou Biotechnology. After the vector construction was completed, Yunzhou Biotechnology was commissioned to perform lentiviral packaging, concentration, purification, and titer determination. DOK cells (human oral mucosal precancerous cells) were infected with the virus and screened. Based on the type of virus and cell type, OE-NRF2 and CONTROL were constructed. Western blot was used to detect the expression level of p-NRF2 in OE-NRF2 and CONTROL cells to verify the overexpression of p-NRF2 protein in OE-NRF2.

[0080] 2. Experimental Grouping

[0081] The experiment was divided into the OE-NRF2 group and the CONTROL group. OE-NRF2 and CONTROL cells were taken, digested, and counted. Cells were seeded in 96-well plates at a density of 3000 cells / well and cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a cell culture incubator containing 5% CO2.

[0082] On the second day after cell seeding, microscopic observation showed that the cells adhered well and grew well. At this time, the growth medium for both groups of cells in the plate was changed to serum-free medium containing 0.5 mM 5-ALA. After incubation in the dark for 5 hours, PDT treatment was performed. The specific treatment method for PDT was as follows: each group of cells received treatment at the corresponding power density (0, 3, 6, 9, 12 J / cm²). 2 ).

[0083] 3. Cell viability detection methods

[0084] Cells treated with PDT were cultured in a cell culture incubator containing 5% CO2 for 12 hours. After 12 hours, cell viability was assessed using the CCK8 reagent. The CCK8 reagent was added according to the CCK8 instructions, and the absorbance (OD value) at 450 nm was measured using a microplate reader and statistical analysis was performed.

[0085] Cell viability (%) = (OD value of experimental group - OD value of blank control) / (OD value of control group - OD value of blank control) × 100%. Plot cell viability curves under each power condition. Three biological replicates were set up for each group in the above experiments.

[0086] II. Experimental Results

[0087] 1. The p-NRF2 overexpressing cell line OE-NRF2 was successfully constructed.

[0088] Western blot analysis showed that p-NRF2 protein expression in OE-NRF2 cells and control cells was as follows: Figure 5 As shown in the figure, the expression level of p-NRF2 protein in OE-NRF2 cells is significantly higher than that in control cells.

[0089] 2. Cell viability test results

[0090] Experimental results are as follows Figure 6As shown, under the same PDT treatment environment, the survival rate of DOK cells in the OE-NRF2 group was significantly higher than that in the CONTROL group. This indicates that increasing the expression level of p-NRF2 protein significantly improves the survival rate of precancerous lesion cells (DOK cells) in the human oral mucosa. The experimental results suggest that increasing p-NRF2 can enhance cellular resistance to PDT treatment.

[0091] Experimental results show that p-NRF2 overexpression enhances the resistance of cells to PDT, thereby reducing the therapeutic effect of PDT. The NRF2 inhibitor Brusatol can significantly inhibit the expression level of p-NRF2 protein, and inhibiting the expression level of p-NRF2 protein can significantly reduce the survival rate of human oral mucosal precancerous lesion cells (DOK cells). Inhibiting the expression level of p-NRF2 protein can enhance the therapeutic effect of PDT, and Brusatol can enhance the efficacy of PDT in treating OLK.

[0092] Example 4: Brusatol, an inhibitor of NRF2, as an adjunct to photosensitizers

[0093] I. Experimental Methods

[0094] 1. Constructing PDT-resistant DOK cells: DOK-RP cells

[0095] DOK-RP cell construction method, such as Figure 7 As shown, human oral mucosal precancerous lesion cells (DOK cells) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a cell culture incubator containing 5% CO2 until 90% confluence. The growth medium was then replaced with serum-free medium containing 0.5 mM 5-ALA and incubated in the dark for 5 hours. DOK cells were irradiated and screened according to the power increment method in Table 1. The irradiation power was set so that 90% of the cells died after each round of irradiation. The surviving cells after each round of irradiation were continued to be cultured, expanded, and passaged. A total of 13 rounds of PDT irradiation were performed to obtain PDT-resistant DOK cells, which were named DOK-RP cells.

[0096] Table 1 PDT Irradiation Information

[0097]

[0098] 2. DOK-RP cells successfully constructed

[0099] The expression level of p-NRF2 in DOK-RP cells and DOK cells was detected using Western blot.

[0100] DOK and DOK-RP cells were cultured in DMEM complete medium containing 10% fetal bovine serum to 90% confluence in culture dishes, and then treated under the following conditions:

[0101] Base group: The cell growth medium was replaced with serum-free DMEM medium, and the cells were incubated in the dark for 5 hours. After 5 hours, the cells were collected, lysed, and proteins were extracted to detect the p-NRF2 expression level.

[0102] ALA group: The cell growth medium was replaced with serum-free medium containing 0.5 mM 5-ALA, and the cells were incubated in the dark for 5 hours. After 5 hours, the cells were collected, lysed, and proteins were extracted to detect the p-NRF2 expression level.

[0103] PDT-0, 0.5, 1, 2, 4, 6h groups: The cell growth medium was replaced with serum-free medium containing 0.5mM 5-ALA and incubated in the dark for 5 hours. After 5 hours, PDT treatment was performed at a power of 100mw for 5 minutes. Cells were lysed at 0, 0.5, 1, 2, 4, and 6h after PDT, and proteins were extracted to detect the expression level of p-NRF2.

[0104] 3. Experimental Grouping

[0105] DOK-RP+BRUS group: Brusatol dissolved in DMSO was used to treat DOK-RP cells for 5 hours to inhibit the expression of NRF2 protein in DOK-RP cells.

[0106] DOK-RP+DMSO group: DOK-RP cells were treated with the same volume of DMSO as the DOK-RP+BRUS group for 5 hours.

[0107] 4. Cell viability detection methods

[0108] The cell viability of each group was obtained according to the method in Example 2.

[0109] II. Experimental Results

[0110] 1. Successfully constructed DOK-RP cells

[0111] Experimental results are as follows Figure 8 and Figure 9 As shown, PDT-resistant cells (DOK-RP) were successfully constructed. Detection revealed that p-NRF2 was highly expressed in DOK-RP cells compared to DOK cells.

[0112] 2. Cell viability test results

[0113] Experimental results are as follows Figure 10 As shown, under the same conditions, the survival rate of DOK-RP cells in the DOK-RP+BRUS group, which used the NRF2 inhibitor Brusatol, was significantly lower than that in the DOK-RP+DMSO group.

[0114] Experimental results show that despite high p-NRF2 expression in DOK-RP cells, the use of NRF2 inhibitors can still significantly enhance the sensitivity of PDT-resistant cells to PDT treatment, thereby greatly improving the therapeutic effect of PDT. NRF2 inhibitors can serve as adjunctive agents to photosensitizers, increasing the sensitivity of PDT-resistant cells to PDT treatment and significantly enhancing the efficacy of PDT in treating OLK.

[0115] This invention provides the use of NRF2 or its derivatives as diagnostic biomarkers and inhibitors of NRF2 or its derivatives as therapeutic targets. Experiments have shown that p-NRF2 can be used to predict the efficacy of phototherapy (PDT) in treating oral leukoplakia (OLK). Lower p-NRF2 expression levels correlate with better PDT efficacy, while higher p-NRF2 expression levels correlate with poorer PDT efficacy. This invention also found that using the NRF2 inhibitor crocin can significantly reduce p-NRF2 expression and improve the efficacy of PDT in treating OLK. This invention can help clinicians better predict and / or improve patient sensitivity to PDT, facilitating better treatment decisions, developing individualized treatment plans, and improving the prognosis and quality of life for patients with oral leukoplakia.

Claims

1. The use of reagents for detecting the expression level of NRF2 or its derivatives in the preparation of reagents for predicting the efficacy of photodynamic therapy in treating potential oral malignancies.

2. The use as described in claim 1, characterized in that, The reagent used to detect the expression level of NRF2 or its derivatives is used to detect the expression level of NRF2 or its derivatives in pathological tissues; the derivative of NRF2 is p-NRF2.

3. The use as described in claim 1 or 2, characterized in that, The reagents for detecting the expression level of NRF2 or its derivatives include reagents for enzyme-linked immunosorbent assay (ELISA), reagents for immunoblotting, reagents for immunoelectrophoresis, reagents for tissue immunostaining, reagents for immunoprecipitation analysis, reagents for radioimmunoassay, reagents for radioimmunodiffusion, reagents for complement fixation analysis, reagents for fluorescence-activated cell differentiation, reagents for quality analysis, or reagents for protein microarrays.

4. The use as described in any one of claims 1-3, characterized in that, The potential malignant oral diseases mentioned are oral leukoplakia or oral erythroplakia.

5. Use of agents that inhibit NRF2 or its derivatives in the preparation of medicaments for the treatment of potential oral malignancies using photodynamic therapy.

6. The use according to claim 5, characterized in that, The reagent that inhibits NRF2 or its derivatives is a reagent that reduces the expression level of NRF2 or its derivative proteins; the derivative of NRF2 is p-NRF2.

7. The use according to claim 6, characterized in that, The reagent used to inhibit NRF2 or its derivatives is ML385, NRF2-IN1, NRF2-IN3 or Brusatol.

8. The use according to any one of claims 5-7, characterized in that, The potential malignant oral diseases mentioned are oral leukoplakia or oral erythroplakia.

9. The use according to claim 8, characterized in that, The photodynamic therapy uses a photosensitizer, preferably 5-aminolevulinic acid.

10. Use of Brusatol in the preparation of reagents that inhibit the expression level of p-NRF2 protein.

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