Hydroxychloroquine treatment of oral lichen planus synergistic and attenuated research method, device and application

Abstract

The application provides a synergistic and attenuated research method, device and application of hydroxychloroquine in treating oral lichen planus, relates to the biomedical technical field, and the key target point of hydroxychloroquine in treating oral lichen planus can be determined based on network pharmacology and molecular docking technology combined with a GEO database and clinical samples in the application, and potential adverse reaction target points of hydroxychloroquine can also be determined based on potential adverse reaction target points commonly shared in diseases related to toxic reaction of hydroxychloroquine, common target points of candidate combination drugs and hydroxychloroquine are further determined, in the case that the common target points include adverse reaction target points, and the binding ability of the candidate combination drug to the adverse reaction target points is better than that of hydroxychloroquine, it is determined that the candidate combination drug can exert synergistic curative effect and attenuated effect with hydroxychloroquine. The therapeutic key target point of hydroxychloroquine, the potential adverse reaction target point and the common target point between hydroxychloroquine and the candidate combination drug are explored, the curative effect of hydroxychloroquine is determined from the pharmacological point of view, and the combination drug exerting synergistic curative effect and attenuated effect is sought.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biomedical technology, and in particular to research methods, devices and applications for enhancing the efficacy and reducing the toxicity of hydroxychloroquine in the treatment of oral lichen planus. Background Technology

[0002] Oral lichen planus (OLP) is a common chronic inflammatory disease of the oral mucosa, often presenting as bilateral symmetrical lesions with diverse morphology and malignant potential. Patients with erosive oral lichen planus (EOLP), in particular, are characterized by large-area single or multifocal erosions, facing a higher risk of malignancy and recurrence. Furthermore, the chronic pain significantly reduces their quality of life, placing a heavy psychological burden on them. In clinical practice, systemic glucocorticoids are recommended as first-line treatment for severe EOLP. However, their use is often limited by contraindications, serious adverse reactions, and treatment resistance. Domestic and international guidelines for the diagnosis and treatment of oral lichen planus suggest that hydroxychloroquine (HCQ) can be used as an alternative therapy in such cases. HCQ has been used off-label for the treatment of OLP and has shown effectiveness in several studies. However, interactions between HCQ and targets outside the intended therapeutic effect can cause off-target effects and adverse reactions, including ocular toxicity, cardiotoxicity, hematologic toxicity, and hepatotoxicity. Identifying specific molecular targets may help improve treatment efficacy and guide future drug development. However, the molecular targets and related mechanisms by which HCQ induces a therapeutic response in OLP remain unclear. Summary of the Invention

[0003] This invention provides a method, apparatus, and application for enhancing the efficacy and reducing the toxicity of hydroxychloroquine in the treatment of oral lichen planus. Through the exploration of key targets and adverse reaction targets and the verification of combined drugs, this invention studies combined drugs that have a detoxifying effect on hydroxychloroquine in the treatment of oral lichen planus, providing a theoretical basis for the combined treatment of oral lichen planus.

[0004] In this embodiment of the invention, a first aspect provides a method for studying the synergistic and toxicity-reducing effects of hydroxychloroquine in the treatment of oral lichen planus, the method comprising:

[0005] Network pharmacology and molecular docking techniques were used to screen potential therapeutic targets of hydroxychloroquine in the treatment of oral lichen planus;

[0006] The expression levels of the potential therapeutic targets were validated using the GEO database and clinical samples to identify key targets for hydroxychloroquine in the treatment of oral lichen planus.

[0007] Identify potential adverse reaction targets common to diseases associated with hydroxychloroquine toxicity, including drug-induced liver injury, retinopathy, cardiomyopathy, and leukopenia;

[0008] Identify common targets between hydroxychloroquine and candidate combination drugs;

[0009] In cases where the common target includes the adverse reaction target, the binding energy of the candidate combination drug to the key target and the adverse reaction target is determined based on molecular docking technology;

[0010] When the candidate combination drug has a better binding affinity to the adverse reaction target than hydroxychloroquine, and its binding affinity to the key target does not affect the binding affinity of hydroxychloroquine to the key target, it is determined that when hydroxychloroquine and the candidate combination drug are administered in combination, the candidate combination drug preferentially occupies the adverse reaction target, thereby exerting a synergistic therapeutic effect and reducing toxicity.

[0011] Optionally, network pharmacology and molecular docking techniques can be used to screen potential therapeutic targets of hydroxychloroquine in the treatment of oral lichen planus, including:

[0012] Network pharmacology analysis was performed to identify multiple target genes as potential targets;

[0013] Molecular docking was performed between hydroxychloroquine and the aforementioned potential targets to determine the main interaction modes between the potential targets and hydroxychloroquine, and several potential targets with strong binding ability were selected as potential therapeutic targets.

[0014] Optionally, the expression levels of the potential therapeutic targets are validated using the GEO database and clinical samples to identify key targets for hydroxychloroquine treatment of oral lichen planus, including:

[0015] Validating RNA expression levels of potential therapeutic targets using publicly available GEO data;

[0016] By using clinical samples to verify the protein expression levels of potential therapeutic targets, the key targets of hydroxychloroquine in treating oral lichen planus were identified.

[0017] Optionally, the expression levels of the potential therapeutic targets are validated using the GEO database and clinical samples to identify key targets for hydroxychloroquine treatment of oral lichen planus, including:

[0018] NOK cells stimulated with LPS were used as an in vitro model to simulate oral lichen planus epithelial cells.

[0019] NOK cells were treated with hydroxychloroquine to simulate a clinical treatment response, and the mRNA expression levels of the potential therapeutic targets before and after hydroxychloroquine treatment were determined to identify the key targets of hydroxychloroquine in the treatment of oral lichen planus.

[0020] In this embodiment of the invention, a second aspect provides a research device for enhancing and reducing the toxicity of hydroxychloroquine in the treatment of oral lichen planus, the device comprising:

[0021] The potential therapeutic target identification module is used to screen potential therapeutic targets of hydroxychloroquine in the treatment of oral lichen planus using network pharmacology and molecular docking technology.

[0022] The key target identification module is used to verify the expression level of the potential therapeutic targets using the GEO database and clinical samples, and to identify the key targets of hydroxychloroquine in the treatment of oral lichen planus.

[0023] A potential adverse reaction target identification module is used to identify potential adverse reaction targets common to diseases associated with hydroxychloroquine toxicity, including drug-induced liver injury, retinopathy, cardiomyopathy, and leukopenia.

[0024] The common target identification module is used to identify common targets between hydroxychloroquine and candidate combination drugs;

[0025] The analysis module is used to determine the binding energy of the candidate combination drug to the key target and the adverse reaction target based on molecular docking technology when the common target includes the adverse reaction target.

[0026] The determination module is configured to, when the candidate combination drug has a better binding energy to the adverse reaction target than hydroxychloroquine, and its binding to the key target does not affect the binding of hydroxychloroquine to the key target, determine that when hydroxychloroquine and the candidate combination drug are administered in combination, the candidate combination drug preferentially occupies the adverse reaction target, thereby exerting a synergistic therapeutic effect and toxicity reduction effect.

[0027] Optionally, the potential therapeutic target determination module is used to:

[0028] Network pharmacology analysis was performed to identify multiple target genes as potential targets;

[0029] Molecular docking was performed between hydroxychloroquine and the aforementioned potential targets to determine the main interaction modes between the potential targets and hydroxychloroquine, and several potential targets with strong binding ability were selected as potential therapeutic targets.

[0030] Optionally, the key target determination module is used for:

[0031] Validating RNA expression levels of potential therapeutic targets using publicly available GEO data;

[0032] By using clinical samples to verify the protein expression levels of potential therapeutic targets, the key targets of hydroxychloroquine in treating oral lichen planus were identified.

[0033] Optionally, the key target determination module is used for:

[0034] NOK cells stimulated with LPS were used as an in vitro model to simulate oral lichen planus epithelial cells.

[0035] NOK cells were treated with hydroxychloroquine to simulate a clinical treatment response, and the mRNA expression levels of the potential therapeutic targets before and after hydroxychloroquine treatment were determined, thus identifying the key targets of hydroxychloroquine in the treatment of oral lichen planus.

[0036] In this embodiment of the invention, a third aspect provides the combined use of hydroxychloroquine and glycyrrhizin in the preparation of a drug for treating oral lichen planus. The key targets of hydroxychloroquine in treating oral lichen planus are HSP90α and HSP90β. Hydroxychloroquine acts as both an HSP90α and HSP90β agonist in treating oral lichen planus. During the treatment of oral lichen planus, the adverse reaction target of hydroxychloroquine is HSP90α. Glycyrrhizin detoxifies hydroxychloroquine by occupying the binding site of HSP90α.

[0037] Glycyrrhizin and hydroxychloroquine bind to different sites on the same residues of HSP90β, and glycyrrhizin does not affect the binding of hydroxychloroquine to HSP90β.

[0038] In this embodiment of the invention, key targets for hydroxychloroquine in treating oral lichen planus can be identified based on network pharmacology and molecular docking technology combined with GEO databases and clinical samples. Furthermore, potential adverse reaction targets of hydroxychloroquine can be identified based on potential adverse reaction targets common to diseases related to hydroxychloroquine toxicity. In addition, common targets of candidate combination drugs and hydroxychloroquine can be identified. If the common targets include adverse reaction targets, and the binding ability of the candidate combination drug to the adverse reaction targets is better than that of hydroxychloroquine, it can be determined that the candidate combination drug can exert synergistic therapeutic effects and toxicity reduction with hydroxychloroquine.

[0039] In this embodiment of the invention, by exploring the key therapeutic targets, potential adverse reaction targets, and common targets between hydroxychloroquine and candidate combination drugs, the efficacy of hydroxychloroquine is determined from a pharmacological perspective, and combination drugs that can exert synergistic effects and reduce toxicity are sought. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 This is a flowchart of the steps in the research method for enhancing the efficacy and reducing the toxicity of hydroxychloroquine in treating oral lichen planus provided in the embodiments of the present invention;

[0042] Figure 2This is a schematic diagram of molecular target screening and molecular docking related to hydroxychloroquine treatment of oral lichen planus provided in an embodiment of the present invention;

[0043] Figure 3 This is a schematic diagram of the expression data of potential targets of hydroxychloroquine for treating oral lichen planus in the GEO dataset provided in an embodiment of the present invention;

[0044] Figure 4 This is a schematic diagram illustrating the expression verification of potential targets of hydroxychloroquine in treating oral lichen planus in clinical samples, as provided in an embodiment of the present invention.

[0045] Figure 5 This is a schematic diagram illustrating the in vitro efficacy verification of hydroxychloroquine in treating oral lichen planus provided in an embodiment of the present invention;

[0046] Figure 6 This is a schematic diagram of the potential targets of hydroxychloroquine toxicity and its detoxification effect provided in the embodiments of the present invention. Detailed Implementation

[0047] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0048] Specifically, such as Figure 1 The diagram illustrates a flowchart of the steps involved in a study on the synergistic and toxicity-reducing method of hydroxychloroquine for treating oral lichen planus, as provided in an embodiment of the present invention. The method includes the following steps:

[0049] S101 uses network pharmacology and molecular docking technology to screen potential therapeutic targets of hydroxychloroquine in the treatment of oral lichen planus;

[0050] S102, using the GEO database and clinical samples to verify the expression level of the potential therapeutic targets and identify the key targets of hydroxychloroquine in the treatment of oral lichen planus;

[0051] S103, Identify potential adverse reaction targets common to diseases associated with hydroxychloroquine toxicity, including drug-induced liver injury, retinopathy, cardiomyopathy, and leukopenia;

[0052] S104 identifies a common target between hydroxychloroquine and candidate combination drugs;

[0053] S105, in the case where the common target includes the adverse reaction target, determine the binding energy of the candidate combination drug to the key target and the adverse reaction target based on molecular docking technology;

[0054] S106, where the binding ability of the candidate combination drug to the adverse reaction target is superior to that of hydroxychloroquine, and the binding of the drug to the key target does not affect the binding of hydroxychloroquine to the key target; it is determined that when hydroxychloroquine and the candidate combination drug are administered in combination, the candidate combination drug preferentially occupies the adverse reaction target, thereby exerting synergistic therapeutic effect and toxicity reduction effect.

[0055] In this embodiment of the invention, computational analysis, histological evaluation, and in vitro studies are proposed to determine the key targets for HCQ treatment of oral lichen planus.

[0056] In this embodiment of the invention, potential therapeutic targets of HCQ in OLP treatment are screened through network pharmacology and molecular docking.

[0057] Specifically, in one optional implementation, based on the above embodiments, step S101 includes the following sub-steps:

[0058] S1011, network pharmacology analysis was performed to identify multiple target genes as potential targets;

[0059] S1012, Molecular docking of hydroxychloroquine with the multiple potential targets is performed to determine the main interaction modes between the multiple potential targets and hydroxychloroquine, and multiple potential targets with strong binding ability are selected as potential therapeutic targets.

[0060] Figure 2 This diagram illustrates the screening and molecular docking of molecular targets related to hydroxychloroquine in the treatment of oral lichen planus. (A) shows the chemical structure of hydroxychloroquine; (B) shows the screening of potential OLP target genes; (C) shows the Venn diagram of the intersection of hydroxychloroquine and potential OLP target genes; (D) shows the stepwise screening of the top 20 target genes in the PPI network; and (E) shows the visualization of molecular docking between hydroxychloroquine and potential targets including HSP90β, HSP90α, EGFR, and VCP. Blue bars represent hydroxychloroquine; red bars represent amino acid residues at the binding site; and yellow dashed lines represent hydrogen bonds and distances at the binding site.

[0061] In this embodiment of the invention, network pharmacology analysis is first performed to select 20 target genes as potential targets (e.g., Figure 2 (As shown in (A)-(D)). Then, HCQ was molecularly docked with these 20 candidate targets. The main interaction mode between the candidate targets and HCQ was hydrogen bonding, with HSP90AB1 exhibiting the strongest binding affinity (e.g., ...). Figure 2 (As shown in (E)).

[0062] In this embodiment of the invention, based on pharmacological analysis and molecular docking technology, HSP90β, HSP90α, EGFR, and VCP are screened as potential therapeutic targets. The expression levels of these potential therapeutic targets are further verified using the GEO database and clinical samples to determine the key targets for hydroxychloroquine treatment of oral lichen planus. Specifically, in an optional implementation, based on the above embodiments, S102 includes the following sub-steps:

[0063] S1021, using publicly available GEO data to validate the RNA expression levels of potential therapeutic targets;

[0064] S1022, Using clinical samples to verify the protein expression level of potential therapeutic targets, and to determine the key targets of hydroxychloroquine in treating oral lichen planus;

[0065] S1023 uses LPS-stimulated NOK cells as an in vitro model to simulate oral lichen planus epithelial cells.

[0066] S1024, NOK cells were treated with hydroxychloroquine to simulate a clinical treatment response, and the mRNA expression levels of the potential therapeutic targets before and after hydroxychloroquine treatment were determined, thus identifying the key targets of hydroxychloroquine in the treatment of oral lichen planus.

[0067] In this embodiment of the invention, the expression levels of potential targets were validated using the GEO database and clinical samples. Specifically, the GEO datasets GSE38616, GSE52130, and GSE213346, which are related to OLP, were used.

[0068] Figure 3 This diagram illustrates the changes in expression levels of potential OLP targets for HCQ treatment in the GEO database.

[0069] In this context, (A) represents differentially expressed genes in the three OLP datasets, (B) represents the expression levels of the top 20 candidate targets in the three datasets and a representative heatmap (GSE52130), and (C) represents the significant differences in the expression of HSP90AB1, HSP90AA1, EGFR and VCP among the 20 candidate targets (fold change ≥ 1.5; P < 0.1).

[0070] In this embodiment of the invention, the heatmap shows the differential expression of the top 20 selected targets in the GSE52130 dataset (e.g., Figure 3 (As shown in (B)). The expression differences of HSP90AB1, HSP90AA1, EGFR, and VCP in the three databases were statistically significant (P<0.1, as shown in (B)). Figure 3 (as shown in C).

[0071] In this embodiment of the invention, paraffin-embedded sections of normal mucosal tissue and oral lichen planus tissue were obtained from clinical samples and used for hematoxylin-eosin (H&E), immunohistochemistry (IHC), and four-color multiplex immunohistochemistry (mIHC) staining.

[0072] Figure 4 A schematic diagram illustrating the expression validation of potential targets in clinical samples is shown. (A) represents representative H&E staining of human samples, including normal oral mucosa tissue (n=10) and oral precancerous lesion (OLP) tissue (n=18); (B) represents representative IHC images and statistical analysis of HSP90β, HSP90α, EFGR, and VCP (scale bar, 100 μm); and (C) represents representative mIHC staining images (DAPI, PAN-CK, HSP90β, and HSP90α) of OLP and normal tissue sections. Within the same field of view, HSP90β and HSP90α exhibit some co-localization, but HSP90β shows a more widespread expression distribution (scale bar, 50 μm). All data are expressed as mean ± standard error.

[0073] In this embodiment of the invention, a significant decrease in the expression of HSP90β in the spinous and basal layers of the OLP epithelium was observed (e.g., Figure 4 (As shown in (B)). Similarly, HSP90α expression in OLP tissues was significantly reduced in both the spinous and basal layers of the epithelium. There was no significant difference in EGFR or VCP expression between the two groups (e.g., ...). Figure 4 (As shown in (B)). Subsequently, mIHC showed that HSP90β was localized in the cytoplasm in both the normal and OLP groups, while HSP90α translocated from the nucleus of the normal group to the cytoplasm of the OLP group (as shown in (B)). Figure 4 (As shown in C). The above findings reveal that the expression of HSP90α and HSP90β in OLP epithelium is significantly downregulated, suggesting that they may be key targets for HCQ therapy of OLP.

[0074] In this embodiment, to further verify the therapeutic effect of HCQ on OLP, a normal human oral keratinocyte (NOK) cell line was obtained from the State Key Laboratory of Oral Diseases. NOK cells were treated with lipopolysaccharide (LPS, Sigma-Aldrich, L2880, 10 μg / ml) or HCQ (MCE, HY-B1370) to stimulate a clinical therapeutic response mimicking OLP epithelium or HCQ. Cell proliferation was assessed using Ki67 immunofluorescence (IF) staining. NOK cells were stained with PI / RNase A (9:1) solution, and cell cycle changes were assessed by flow cytometry. Total RNA was extracted and detected by qRT-PCR.

[0075] Figure 5A schematic diagram illustrating the in vitro efficacy validation of hydroxychloroquine (HCQ) is shown. (A) shows Ki67 immunofluorescence staining (scale bar, 100 μm) after treating NOK cells with LPS and different concentrations of HCQ; (B) shows representative images of the cell cycle analyzed by flow cytometry using PI staining and histograms of cell numbers in G1, S, and G2 phases; (C) shows the mRNA expression levels of inflammatory cytokines TNF-α and IL6 after treating NOK cells with LPS and 10 μM HCQ for 24 hours, analyzed by qRT-PCR; and (D) shows the mRNA expression levels of HSP90AB1 and HSP90AA1 after treating NOK cells with LPS and 10 μM HCQ for 24 hours, analyzed by qRT-PCR.

[0076] like Figure 5 As shown, treatment with different concentrations of HCQ significantly increased the proliferation of LPS-treated NOK cells, a finding confirmed by increased Ki67 expression (e.g., ...). Figure 5 (As shown in Figure (A)). Among them, 10 μM HCQ showed the most significant effect in promoting cell proliferation. 24 hours after treatment, 10 μM HCQ alleviated LPS-induced S-phase arrest in NOK cells and significantly reduced the expression of typical inflammatory cytokines, including TNF-α and IL-6 (e.g., TNF-α and IL-6). Figure 5 (As shown in (B)-(C)). Mechanistically, LPS stimulation downregulates the RNA levels of HSP90α / β (representing HSP90AA1 and HSP90AB1, respectively) (e.g. Figure 5 As shown in (D), this is consistent with IHC findings (as shown in...). Figure 5 (As shown in Figure B). HCQ treatment significantly restored the levels of HSP90AA1 and HSP90AB1, especially the increase in HSP90AB1 (as shown in Figure B). Figure 5 (As shown in D). In vitro studies have shown that HCQ stimulated cell proliferation in OLP-mimicking epithelial cells and inhibited the secretion of TNF-α and IL6, accompanied by enhanced HSP90α / β expression. These findings suggest that HCQ may exert its therapeutic effect on OLP through HSP90α and HSP90β.

[0077] Therefore, in this embodiment of the invention, the key targets of hydroxychloroquine in treating oral lichen planus are determined to be HSP90α and HSP90β.

[0078] In this embodiment of the invention, it is proposed that the interaction between HCQ and its targets other than its expected therapeutic effect may cause off-target effects and adverse reactions, including ocular toxicity, cardiotoxicity, hematologic toxicity and hepatotoxicity. Based on this, this embodiment of the invention proposes to identify potential adverse reaction targets common to hydroxychloroquine in drug-induced liver injury, retinopathy, cardiomyopathy and leukopenia.

[0079] In an optional implementation, based on the above embodiments, step S104 includes: screening potential therapeutic targets of candidate combination drugs in the treatment of oral lichen planus through network pharmacology, and thereby determining common targets between hydroxychloroquine and candidate combination drugs.

[0080] In this embodiment of the invention, by exploring the key therapeutic targets, potential adverse reaction targets, and common targets between hydroxychloroquine and candidate combination drugs, the efficacy of hydroxychloroquine is determined from a pharmacological perspective, and combination drugs that can exert synergistic effects and reduce toxicity are sought.

[0081] In this embodiment of the invention, when the common target between hydroxychloroquine and the candidate combination drug includes the adverse reaction target and the binding ability of the candidate combination drug to the adverse reaction target is better than that of hydroxychloroquine, it indicates that the candidate combination drug can preferentially bind to the adverse reaction target, making it impossible for hydroxychloroquine to bind to the adverse reaction target, eliminating its possible adverse reactions, and without affecting the binding of hydroxychloroquine to the key therapeutic target, and without affecting its therapeutic effect.

[0082] In this embodiment of the invention, steps S103 to S108 are described using glycyrrhizic acid (GL) as an example of a candidate combination drug: Specifically, Figure 6 This diagram illustrates the toxic reactions of HCQ and potential targets for its detoxification. (A) shows a schematic diagram of licorice and a Venn diagram of the intersection of GL target genes with the top 20 genes in the HCQ PPI network; (B) shows a schematic diagram of the molecular docking of HCQ and GL with HSP90β and HSP90α, with blue bars representing HCQ or GL, red bars representing amino acid residues at the binding site, and yellow dashed lines representing hydrogen bonds and distances at the binding site. The bottom indicates the molecular docking binding energy; (C) shows the gene set involved in the toxic reactions of HCQ; and (D) shows an overview diagram of HCQ and GL's treatment of OLP via HSP90α / β.

[0083] In this embodiment of the invention, six common targets were found between HCQ and GL, namely HSP90AA1, HSP90AB1, CDK2, EGFR, ESR1, and HSPA8 (e.g., Figure 6 (As shown in (A)). Furthermore, GL exhibits superior binding affinity to HSP90α / β compared to HCQ, as demonstrated by the molecular docking results (e.g., Figure 6(As shown in (B)). However, the binding effect of GL on HSP90α or HSP90β differs from that of HCQ. GL and HCQ bind to different sites on the same residues of HSP90β, meaning that GL may not affect the binding of HCQ to HSP90β. Conversely, GL binds to the same residue sites on HSP90α as HCQ. In step S103, based on network pharmacology and molecular docking techniques, eight common targets (such as...) in several diseases associated with HCQ toxicity (including drug-induced liver injury, retinopathy, cardiomyopathy, and leukopenia) were identified. Figure 6 (As shown in (C)). The inclusion of HSP90α indicates that HSP90α is a potential target for HCQ-induced toxicity. HSP90α was identified as a common potential adverse reaction target in diseases associated with hydroxychloroquine toxicity.

[0084] Observations suggest that HSP90α can serve as an effective target for glycyrrhizin to antagonize HCQ toxicity. Based on these findings, it is hypothesized that when HCQ and glycyrrhizin are administered co-administered, glycyrrhizin preferentially occupies the binding site of HSP90α residues, without affecting the interaction between HCQ and HSP90β, thereby exerting a synergistic therapeutic and detoxifying effect (e.g., Figure 6 (as shown in D)

[0085] In this invention, HSP90α and HSP90β were innovatively identified as potential targets for HCQ therapy in treating OLP. HSP90α and HSP90β belong to the HSP90 protein family, are abundant in normal cells, and are common housekeeping proteins. Nuclear HSP90α is crucial for maintaining genome stability, while cytoplasmic HSP90 has been observed to redistribute to the cell membrane in various cancer cells. In this invention, compared with the control group, decreased expression of HSP90α and HSP90β and increased cytoplasmic translocation of HSP90α were observed in the epithelial cells of OLP patients. This invention demonstrates that HSP90α and HSP90β are closely related to the pathogenesis of OLP and have the potential to serve as therapeutic targets.

[0086] HSP90α and HSP90β share similar structures and functions, guiding the normal folding, intracellular configuration, and proteolytic turnover of many key regulators of cell growth and survival. For example, HSP90α and HSP90β can influence cell proliferation by controlling the TGFβ signaling cascade and regulate inflammation through multiple signaling pathways, such as NF-κB, TLR4, and JAK / STAT. In particular, IL6 and TNF-α levels produced by keratinocytes in OLP patients are 10-20 times higher than in normal mucosa. HSP90 has also been found to regulate IL6 / TNF-α in mouse models of plaque psoriasis and irritant dermatitis. In confirming the therapeutic effect of HCQ on OLP, we observed that HCQ exhibited pro-proliferative and anti-inflammatory effects in vitro. The strong binding of HCQ to HSP90AB1 and HSP90AA1, along with the upregulation of RNA expression after HCQ exposure, together indicate that HCQ has a restorative effect on HSP90α / β. These findings reveal that HCQ may exert a therapeutic effect on OLP by restoring HSP90α / β levels, inhibiting the production of IL6 and TNF-α, and promoting the recovery of epithelial cells.

[0087] While previous studies have not explored the detailed differences between HSP90α and HSP90β, some evidence suggests that HSP90 subtypes exhibit tissue-specific expression patterns and distinct functions under pathological conditions. In this invention, HSP90α was identified as a potential target for adverse reactions associated with HCQ, consistent with findings from other studies linking HSP90α to hepatotoxicity, ocular toxicity, and cardiotoxicity. Furthermore, reducing serum HSP90α levels in mouse models can improve steatohepatitis; HSP90α plays a crucial role in rod cell survival, and long-term inhibition leads to harmful degradation of specific photoreceptor proteins such as PDE6 and GRK1; HSP90α is also considered an optimal core pivotal protein drug target for HCQ-related QT prolongation, and the potassium channel hERG, crucial for cardiac repolarization, interacts only with HSP90α, not HSP90β. These findings indirectly support our conclusion that HSP90α is a potential target for toxic reactions associated with OLP treated with HCQ.

[0088] Due to its recognized anti-inflammatory and hepatoprotective effects, GL is often used in combination with HCQ for the treatment of OLP. Its anti-inflammatory effect is similar to that of hydrocortisone, and its hepatoprotective mechanism involves anti-steatodendrostasis, anti-oxidative stress, and immunomodulatory effects. Therefore, the combined use of HCQ and GL is also considered a reasonable approach for treating COVID-19 or inflammatory skin diseases. This invention reveals that both HCQ and GL target HSP90α and HSP90β. GL may exhibit a greater tendency to bind to HSP90α residues than HCQ. This molecular discovery may contribute to the synergistic and detoxifying effects of combining GL and HCQ. This finding is significant for optimizing the application of HCQ in the treatment of viral infections, dermatological diseases, and OLP.

[0089] In this invention, studies have demonstrated the efficacy of HCQ in treating OLP. Mechanistically, HCQ may exert its therapeutic effect as an HSP90α / β agonist. Furthermore, GL can mitigate the toxicity of HCQ by occupying the HSP90α binding site. These findings provide an evidence-based and theoretical foundation for the optimal clinical application of HCQ in the treatment of OLP.

[0090] In this embodiment of the invention, a research device for enhancing and reducing the toxicity of hydroxychloroquine in the treatment of oral lichen planus is also provided, the device comprising:

[0091] The potential therapeutic target identification module is used to screen potential therapeutic targets of hydroxychloroquine in the treatment of oral lichen planus using network pharmacology and molecular docking technology.

[0092] The key target identification module is used to verify the expression level of the potential therapeutic targets using the GEO database and clinical samples, and to identify the key targets of hydroxychloroquine in the treatment of oral lichen planus.

[0093] A potential adverse reaction target identification module is used to identify potential adverse reaction targets common to diseases associated with hydroxychloroquine toxicity, including drug-induced liver injury, retinopathy, cardiomyopathy, and leukopenia.

[0094] The common target identification module is used to identify common targets between hydroxychloroquine and candidate combination drugs;

[0095] The analysis module is used to determine the binding energy of the candidate combination drug to the key target and the adverse reaction target based on molecular docking technology when the common target includes the adverse reaction target.

[0096] The determination module is configured to, when the candidate combination drug has a better binding affinity to the adverse reaction target than hydroxychloroquine, and its binding affinity to the key target does not affect the binding affinity of hydroxychloroquine to the key target, determine that when hydroxychloroquine and the candidate combination drug are administered in combination, the candidate combination drug preferentially occupies the adverse reaction target, thereby exerting a synergistic therapeutic effect and reducing toxicity.

[0097] Optionally, the potential therapeutic target determination module is used to:

[0098] Network pharmacology analysis was performed to identify multiple target genes as potential targets;

[0099] Molecular docking was performed between hydroxychloroquine and the aforementioned potential targets to determine the main interaction modes between the potential targets and hydroxychloroquine, and several potential targets with strong binding ability were selected as potential therapeutic targets.

[0100] Optionally, the key target determination module is used for:

[0101] Validating RNA expression levels of potential therapeutic targets using publicly available GEO data;

[0102] By using clinical samples to verify the protein expression levels of potential therapeutic targets, the key targets of hydroxychloroquine in treating oral lichen planus were identified.

[0103] Optionally, the key target determination module is used for:

[0104] NOK cells stimulated with LPS were used as an in vitro model to simulate oral lichen planus epithelial cells.

[0105] NOK cells were treated with hydroxychloroquine to simulate a clinical treatment response, and the mRNA expression levels of the potential therapeutic targets before and after hydroxychloroquine treatment were determined, thus identifying the key targets of hydroxychloroquine in the treatment of oral lichen planus.

[0106] Glycyrrhizin and hydroxychloroquine bind to different sites on the same residues of HSP90β, and glycyrrhizin does not affect the binding of hydroxychloroquine to HSP90β.

[0107] In this embodiment of the invention, based on the above research, a combined application of hydroxychloroquine and glycyrrhizin in the preparation of a drug for treating oral lichen planus is also proposed. The key targets of hydroxychloroquine in treating oral lichen planus are HSP90α and HSP90β. Hydroxychloroquine acts as both an HSP90α agonist and an HSP90β agonist in treating oral lichen planus. During the treatment of oral lichen planus, the adverse reaction target of hydroxychloroquine is HSP90α. Glycyrrhizin detoxifies hydroxychloroquine by occupying the binding site of HSP90α.

[0108] Glycyrrhizin and hydroxychloroquine bind to different sites on the same residues of HSP90β, and the glycyrrhizin does not affect the binding of hydroxychloroquine to HSP90β.

[0109] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0110] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0111] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0112] The above provides a detailed description of the method, apparatus, and application of hydroxychloroquine in the treatment of oral lichen planus, which enhances efficacy and reduces toxicity. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this invention. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. A research method for enhancing the efficacy and reducing the toxicity of hydroxychloroquine in the treatment of oral lichen planus, characterized in that... The method includes: Network pharmacology and molecular docking techniques were used to screen potential therapeutic targets of hydroxychloroquine in the treatment of oral lichen planus; The expression levels of the potential therapeutic targets were validated using the GEO database and clinical samples to identify key targets for hydroxychloroquine in the treatment of oral lichen planus. Identify potential adverse reaction targets common to diseases associated with hydroxychloroquine toxicity, including drug-induced liver injury, retinopathy, cardiomyopathy, and leukopenia; Identify common targets between hydroxychloroquine and candidate combination drugs; In cases where the common target includes the adverse reaction target, the binding energy of the candidate combination drug to the key target and the adverse reaction target is determined based on molecular docking technology; When the candidate combination drug has a better binding affinity to the adverse reaction target than hydroxychloroquine, and its binding affinity to the key target does not affect the binding affinity of hydroxychloroquine to the key target, it is determined that when hydroxychloroquine and the candidate combination drug are administered in combination, the candidate combination drug preferentially occupies the adverse reaction target, thereby exerting a synergistic therapeutic effect and reducing toxicity.

2. The method for enhancing the efficacy and reducing the toxicity of hydroxychloroquine in treating oral lichen planus according to claim 1, characterized in that, Network pharmacology and molecular docking techniques were used to screen potential therapeutic targets of hydroxychloroquine in the treatment of oral lichen planus, including: Network pharmacology analysis was performed to identify multiple target genes as potential targets; Molecular docking was performed between hydroxychloroquine and the aforementioned potential targets to determine the main interaction modes between the potential targets and hydroxychloroquine, and several potential targets with strong binding ability were selected as potential therapeutic targets.

3. The method for enhancing the efficacy and reducing the toxicity of hydroxychloroquine in treating oral lichen planus according to claim 1, characterized in that, The expression levels of the potential therapeutic targets were validated using the GEO database, clinical samples, and in vitro cell experiments to identify key targets for hydroxychloroquine treatment of oral lichen planus, including: Validating RNA expression levels of potential therapeutic targets using publicly available GEO data; By using clinical samples to verify the protein expression levels of potential therapeutic targets, the key targets of hydroxychloroquine in treating oral lichen planus were identified.

4. The method for synergistic and toxicity-reducing research of hydroxychloroquine in the treatment of oral lichen planus according to claim 1, characterized in that, The expression levels of the potential therapeutic targets were validated using the GEO database, clinical samples, and in vitro cell experiments to identify key targets for hydroxychloroquine treatment of oral lichen planus, including: NOK cells stimulated with LPS were used as an in vitro model to simulate oral lichen planus epithelial cells. NOK cells were treated with hydroxychloroquine to simulate a clinical treatment response, and the mRNA expression levels of the potential therapeutic targets before and after hydroxychloroquine treatment were determined to identify the key targets of hydroxychloroquine in the treatment of oral lichen planus.

5. A research device for enhancing the efficacy and reducing the toxicity of hydroxychloroquine in the treatment of oral lichen planus, characterized in that, The device includes: The potential therapeutic target identification module is used to screen potential therapeutic targets of hydroxychloroquine in the treatment of oral lichen planus using network pharmacology and molecular docking technology. The key target identification module is used to verify the expression level of the potential therapeutic targets using the GEO database and clinical samples, and to identify the key targets of hydroxychloroquine in the treatment of oral lichen planus. A potential adverse reaction target identification module is used to identify potential adverse reaction targets common to diseases associated with hydroxychloroquine toxicity, including drug-induced liver injury, retinopathy, cardiomyopathy, and leukopenia. The common target identification module is used to identify common targets between hydroxychloroquine and candidate combination drugs; The analysis module is used to determine the binding energy of the candidate combination drug to the key target and the adverse reaction target based on molecular docking technology when the common target includes the adverse reaction target. The determination module is configured to, when the candidate combination drug has a better binding affinity to the adverse reaction target than hydroxychloroquine, and its binding affinity to the key target does not affect the binding affinity of hydroxychloroquine to the key target, determine that when hydroxychloroquine and the candidate combination drug are administered in combination, the candidate combination drug preferentially occupies the adverse reaction target, thereby exerting a synergistic therapeutic effect and reducing toxicity.

6. The synergistic and toxicity-reducing research device for treating oral lichen planus with hydroxychloroquine according to claim 5, characterized in that, The potential therapeutic target identification module is used for: Network pharmacology analysis was performed to identify multiple target genes as potential targets; Molecular docking was performed between hydroxychloroquine and the aforementioned potential targets to determine the main interaction modes between the potential targets and hydroxychloroquine, and several potential targets with strong binding ability were selected as potential therapeutic targets.

7. The synergistic and toxicity-reducing research device for treating oral lichen planus with hydroxychloroquine according to claim 5, characterized in that, The key target determination module is used for: Validating RNA expression levels of potential therapeutic targets using publicly available GEO data; By using clinical samples to verify the protein expression levels of potential therapeutic targets, the key targets of hydroxychloroquine in treating oral lichen planus were identified.

8. The research device for enhancing and reducing the toxicity of hydroxychloroquine in the treatment of oral lichen planus according to claim 5, characterized in that, The key target determination module is used for: NOK cells stimulated with LPS were used as an in vitro model to simulate oral lichen planus epithelial cells. NOK cells were treated with hydroxychloroquine to simulate a clinical treatment response, and the mRNA expression levels of the potential therapeutic targets before and after hydroxychloroquine treatment were determined, thus identifying the key targets of hydroxychloroquine in the treatment of oral lichen planus.

9. The combined use of hydroxychloroquine and glycyrrhizic acid in the preparation of a drug for treating oral lichen planus, characterized in that, The key targets of the hydroxychloroquine treatment for oral lichen planus are HSP90α and HSP90β. The hydroxychloroquine is used as an HSP90α agonist and an HSP90β agonist to treat oral lichen planus. The adverse reaction target of the hydroxychloroquine in the treatment of oral lichen planus is HSP90α. The glycyrrhizin detoxifies hydroxychloroquine by occupying the binding site of HSP90α.

10. The combined use of hydroxychloroquine and glycyrrhizic acid according to claim 9 in the preparation of a medicament for treating oral lichen planus, characterized in that, Glycyrrhizin and hydroxychloroquine bind to different sites on the same residues of HSP90β, and glycyrrhizin does not affect the binding of hydroxychloroquine to HSP90β.

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