Application of blood fat reducing medicine in preparation of medicine for treating pemphigus

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a blood fat reducing medicine in preparation of a medicine for treating pemphigus. According to the application disclosed by the invention, the hypolipidemic drug is found to be used for treating pemphigus for the first time, and by analyzing the blood fat of a PV patient, the hypolipidemic metabolism of the patient is found to be abnormal, and the oxidative stress level is related to the classical biomarker level of PV. Lipid-induced oxidative stress was confirmed by in vitro experiments using HaCaT cells treated with serum of PV patients, and it was found that it results in STAT3 activation and mitochondrial dependent apoptosis. ATO intervention can effectively reverse the pathological process: by correcting lipid metabolism disorder, ATO significantly reduces the oxidative stress level, inhibits STAT3 phosphorylation, and reduces the occurrence of mitochondrial dependent apoptosis. The invention discloses an important relationship among lipid disorder, oxidative stress, apoptosis and STAT3 activation in PV, and prompts that statins may become a potential treatment strategy for PV.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of lipid-lowering drugs in the preparation of drugs for treating pemphigus. Background Art

[0002] Pemphigus Vulgaris (PV) is the most common and severe type of pemphigus, a rare autoimmune blistering disease that poses a significant threat to human health and may even be life-threatening. PV usually leads to the loss of keratinocyte adhesion function or acantholysis. Pathomechanism studies have shown that PV-related pathogenic autoantibodies mainly target desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3), and the inactivation of these two cadherin-type adhesion molecules directly leads to the breakdown of intercellular adhesion. Although some progress has been made in targeted therapy against autoantibodies, some patients still respond poorly to existing therapies, suggesting that the pathophysiological process of PV may involve a more complex regulatory network.

[0003] In recent years, the role of metabolic disorders in autoimmune diseases has gradually attracted attention. Clinical observations have found that PV patients often have dyslipidemia, but its correlation with disease activity has not been clarified. There are literature reports that lipid abnormalities can cause oxidative stress (doi:10.1016 / j.redox.2024.103230, doi:10.1016 / j.jnutbio.2025.109866). Oxidative stress is defined as the imbalance between oxidants such as reactive oxygen species (ROS) and the body's antioxidant capacity, leading to tissue damage. In recent years, its role in autoimmune diseases has attracted increasing attention (doi:10.3390 / ijms22020723). There is a hypothesis that oxidative stress may trigger apoptosis of PV epithelial cells by increasing the production of ROS (doi:10.11786 / sypfbxzz.1674-1293.20190112), which is a key dynamic balance process in disease progression. However, the specific mechanisms of oxidative stress and apoptosis in PV still need to be further elucidated.

[0004] Lipid-lowering drugs are mainly used for cardiovascular-related diseases, and the application of lipid-lowering drugs in the treatment of autoimmune blistering diseases has not been reported in the existing technology. Therefore, it is necessary to develop new drugs for treating pemphigus based on the pathway of lipid abnormality and oxidative stress abnormality. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a new use of lipid-lowering drugs in the treatment of pemphigus, and the technical solutions adopted are as follows:

[0006] The first aspect of the present invention provides the use of lipid-lowering drugs in the preparation of drugs for treating pemphigus.

[0007] In some embodiments of the present invention, the pemphigus includes one of pemphigus vulgaris, pemphigus vegetans, pemphigus foliaceus, pemphigus herpetiformis, and paraneoplastic pemphigus.

[0008] In some embodiments of the present invention, the lipid-lowering drugs include statins, cholesterol absorption inhibitor drugs, fibrates, or PCSK9 inhibitors.

[0009] In some embodiments of the present invention, the lipid-lowering drug is a statin.

[0010] In some embodiments of the present invention, the statins include at least one of atorvastatin (Atorvastatin, ATO), rosuvastatin, simvastatin, pravastatin, and fluvastatin.

[0011] In some embodiments of the present invention, the statin includes atorvastatin or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments of the present invention, the pharmaceutically acceptable salts include at least one of metal salts, ammonium salts, salts formed with inorganic acids, salts formed with organic bases, salts formed with organic acids, salts formed with basic amino acids, and salts formed with acidic amino acids.

[0013] In some embodiments of the present invention, the metal salts include alkali metal salts and alkaline earth metal salts.

[0014] In some embodiments of the present invention, the alkali metal salts include at least one of sodium salts and potassium salts.

[0015] In some embodiments of the present invention, the alkaline earth metal salts include at least one of calcium salts, magnesium salts, barium salts, and aluminum salts.

[0016] In some embodiments of the present invention, the salts formed with organic bases include salts formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0017] In some embodiments of the present invention, the salts formed with inorganic acids include salts formed with the following inorganic acids: at least one of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0018] In some embodiments of the present invention, the salts formed with organic acids include salts formed with at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid.

[0019] In some embodiments of the present invention, the salts formed with basic amino acids include salts formed with at least one of the following basic amino acids: arginine, lysine, ornithine.

[0020] In some embodiments of the present invention, the salts formed with acidic amino acids include salts formed with at least one of the following acidic amino acids: aspartic acid, glutamic acid.

[0021] In some embodiments of the present invention, the pharmaceutical product includes pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

[0022] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, carriers.

[0023] Furthermore, for the convenience of drug administration, the active ingredient statin drug (such as atorvastatin) can be processed into a specific dosage form with any one or several pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch paste, and syrup, etc.), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and cross-linked povidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and colloidal silicon dioxide, etc.), coloring agents (such as titanium dioxide, sunset yellow, methylene blue, and medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol, and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate, etc.), bacteriostatic agents (such as phenol, benzyl alcohol, and thimerosal, etc.), or can also be isotonicity regulators (such as sodium chloride and glucose, etc.).

[0024] The above-mentioned pharmaceutically acceptable excipients are generally recognized for this purpose and serve as the inactive ingredients of the pharmaceutical agent. Compilations of pharmaceutically acceptable excipients can be found in tools such as "Handbook of Pharmaceutical Excipients" (2nd edition, edited by A. Wade and P. J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994); "List of Medicinal Excipients in the Pharmacopoeia of the People's Republic of China", etc.

[0025] In some embodiments of the present invention, the dosage form of the drug includes a dosage form for gastrointestinal administration or a dosage form for non-gastrointestinal administration.

[0026] In some embodiments of the present invention, the dosage form for gastrointestinal administration includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0027] In some embodiments of the present invention, the dosage form for non-gastrointestinal administration includes at least one of injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0028] Furthermore, the injectable pharmaceutical dosage forms include, but are not limited to, injection solutions, injectable solutions, intravenous infusion injection solutions, injectable suspensions, sterile powders for injection, intravenous injection preparations, aqueous injections, injectable emulsions, powder injections, injections, sterile powder injections, lyophilized powder injections, etc.

[0029] In a second aspect of the present invention, there is provided a method for reducing the level of cellular oxidative stress for non-therapeutic purposes in vitro:

[0030] Treat HaCaT cells with atorvastatin.

[0031] In some embodiments of the present invention, the concentration of atorvastatin is 1 - 50 μM.

[0032] In some embodiments of the present invention, the treatment time is 12 - 72 hours.

[0033] In a third aspect of the present invention, there is provided the use of a biomarker in the detection of pemphigus:

[0034] The biomarker includes serum cholesterol, triglycerides, and / or apolipoprotein B.

[0035] The beneficial effects of the present invention are:

[0036] The present invention discovers for the first time that lipid-lowering drugs can be used to treat pemphigus. By analyzing the blood lipids of PV patients, it is found that they have significant lipid metabolism disorders, manifested as elevated levels of cholesterol, triglycerides, and apolipoprotein B, while the levels of high-density lipoprotein and serum calcium are decreased. Moreover, oxidative stress is correlated with the classical biomarker of PV - anti-DSG1 / DSG3 antibody. Further through in vitro experiments, HaCaT cells treated with PV patient serum were used to confirm lipid-induced oxidative stress, and it was found that it led to the activation of STAT3 and mitochondrial-dependent apoptosis. The characteristics of this apoptosis are the changes in mitochondrial membrane potential and the activation of apoptotic proteins, indicating that oxidative stress plays a key role in the pathogenesis of PV. And atorvastatin (ATO) intervention can effectively reverse the above pathological process: by correcting lipid metabolism disorders, ATO significantly reduces the level of oxidative stress, inhibits STAT3 phosphorylation, and reduces the occurrence of mitochondrial-dependent apoptosis. The present invention reveals an important connection among lipid disorders, oxidative stress, apoptosis, and STAT3 activation in PV, and suggests that statins may become a potential treatment strategy for PV. Description of the Drawings

[0037] The present invention will be further described below in conjunction with the drawings and embodiments, where:

[0038] Figure 1Results are odds ratios (OR) and 95% confidence intervals (CI) of influencing factors of PV in the multi-factor Logistic regression model.

[0039] Figure 2 (A) shows HE staining of normal tissue, perilesional tissue, and lesional tissue of a PV patient (sample from patient ID 9), scale bar: 100 μm; (B) shows quantitative analysis of SOD, CAT, thiol, and MDA levels in biopsy samples extracted from normal tissue, perilesional tissue, and lesional tissue of PV patients (n = 8). Error bars represent standard error (s.e.m). Student's t-test, ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, compared with normal tissue; (C) shows quantitative analysis of SOD, CAT, thiol, and MDA levels in sera of the healthy control group (n = 7) and the PV patient group (n = 8). Results represent at least three independent experiments. Error bars represent standard error (s.e.m). Student's t-test, *P < 0.05, ***P < 0.001, compared with the control group.

[0040] Figure 3 (A) is a heat map showing Pearson correlations between variables. Red indicates positive values, and yellow indicates negative values; the range is from -0.5 to 1, -0.5 indicates a completely negative linear relationship between variables, 1 indicates a completely positive linear relationship, and 0 indicates no correlation between variables. DSG1 Abs: desmoglein 1 antibody; DSG3 Abs: desmoglein 3 antibody; (B) shows the measurement results of SOD, CAT, thiol, and MDA release in the culture medium of HaCaT cells treated with healthy control sera and 20% PV sera. Results represent at least three independent experiments. Error bars represent standard error (s.e.m). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

[0041] Figure 4(A) Immunoblot (IB) analysis of PV patient tissues; (B) HaCaT cells were treated with control serum, 20% PV serum for 36 hours, and after 20% PV serum treatment for 36 hours, ATO (10 μM) was added and the cells were cultured for an additional 48 hours, followed by TMRE staining. MMP was observed by fluorescence microscopy, scale bar: 100 μm; (C) HaCaT cells were treated with control serum, 20% PV serum for 36 hours, and after 20% PV serum treatment for 36 hours, ATO (10 μM) was added and the cells were cultured for an additional 48 hours, followed by Annexin V / PI staining and flow cytometry analysis; (D) HaCaT cells were treated with control serum, 20% PV serum for 36 hours, and after 20% PV serum treatment for 36 hours, ATO (10 μM) was added and the cells were cultured for an additional 48 hours, followed by preparation of cell lysates for immunoblot (IB) analysis; (E) HaCaT cells were treated with control serum, 20% PV serum for 36 hours, and after 20% PV serum treatment for 36 hours, ATO (10 μM) was added and the cells were cultured for an additional 48 hours, followed by RNA extraction for real-time fluorescence quantitative PCR (real-time PCR) analysis. Gene expression levels were expressed as mean ± standard deviation (n = 3). ***P < 0.001, compared with the control serum group; #P < 0.05, P < 0.001, compared with the PV group. Detailed implementation mode

[0042] The concept of the present invention and the technical effects produced will be clearly and completely described below in combination with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0043] The relevant reagent information used in this discovery is as follows:

[0044] Anti-caspase-3 (Cat#19677-1-AP), anti-BAX (Cat#50599-2-Ig), anti-Bcl2 (Cat#60178-1-Ig), anti-PARP1 (Cat#13371-1-AP), anti-STAT3 (Cat#10253-2-AP) and anti-GAPDH (Cat#10494-1-AP) were purchased from Proteintech Group. Anti-phospho-STAT3 (Tyr705) (Cat#9145S) was purchased from Cell Signaling Technology. EDTA solution (Cat#R21345) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Protease inhibitor (Cat#P1005), enhanced chemiluminescence (ECL) kit (Cat#P0018M), Blotting Grade (Cat#P0216), cDNA synthesis kit (Cat.#D7170S) and SYBR Green qPCR Mix (Cat.#D7601S) were purchased from Beyotime Institute of Biotechnology. PVDF transfer membrane (Cat#INCP00010) was purchased from MERCK. Tris (Cat#T1010) was purchased from Solarbio Life Sciences. Sodium chloride (Cat#A501218) and SDS (Cat#A600485) were purchased from Sangon Biotech. Atorvastatin (Cat#S5715) was purchased from Selleck Biologicals. Desmoglein 1 antibody (Cat#YA2873) was purchased from MedChemexpress, and Desmoglein 3 (5G11) Mouse mAb (Cat#20483) was purchased from Cell Signaling Technology.

[0045] Statistical analysis in this invention was performed using GraphPad Prism (GraphPad Software, Version 8.0). The statistical differences between the control group and the experimental group were analyzed by Student's t-test, and P < 0.05 was considered statistically significant. All experiments were independently repeated at least 3 times to ensure reliability.

[0046] Example 1 Blood Analysis of PV Patients

[0047] 1. Sample Source

[0048] In this example, blood biochemical data of healthy individuals (n = 108) and PV patients (n = 108) were collected. All patients included in the study had no history of hypertension, diabetes, or hyperlipidemia and were all from the Sun Yat-sen Memorial Hospital, Sun Yat-sen University. Among them, some samples were selected for further histological and hematological analysis, and the detailed clinical information of these participants is shown in Table 1. This study was conducted in accordance with the standards of the Declaration of Helsinki and was approved by the Ethics Committee of the Sun Yat-sen Memorial Hospital, Sun Yat-sen University (approval number: SYSKY-2024-240-01). All subjects signed a written informed consent form.

[0049] 2. Hematological tests

[0050] Hematological tests were performed on the above samples.

[0051] One hundred and eight healthy individuals served as the control group, including 53 males and 55 females, with an average age of (52.47 ± 11.62) years. The disease group included 108 patients, including 49 males and 59 females, with an average age of (50.15 ± 14.84) years. All these patients were diagnosed with pemphigus vulgaris (PV). There were no significant statistical differences in gender and age between the healthy group and the disease group (P > 0.05). In the disease group, the levels of serum cholesterol (CHOL), triglyceride (TG), and apolipoprotein B (ApoB) in patients were significantly increased, while the levels of high-density lipoprotein cholesterol (HDL-C) and serum calcium (Serum Ca 2+ ) were significantly decreased, and these differences were statistically significant (P < 0.05), as shown in Table 1.

[0052] Table 1

[0053]

[0054]

[0055] Given the significant differences between the two groups in terms of CHOL, TG, ApoB, HDL-C, and Serum Ca 2+ , in this example, a multivariate Logistic regression analysis was performed with PV as the dependent variable. The analysis results showed that elevated levels of CHOL (OR = 3.35), TG (OR = 1.46), and ApoB (OR = 1.59) were risk factors for PV, while higher levels of Serum Ca 2+ (OR = 0.29) and HDL-C (OR = 0.49) were protective factors ( Figure 1 ). These statistically significant differences (P < 0.05) indicated that PV patients had abnormal lipid metabolism.

[0056] Oxidative Stress in PV Patients of Example 2

[0057] 1. Experimental Samples

[0058] To further study the lipid levels and oxidative stress in PV patients, in this example, samples of 15 healthy individuals and PV patients were further selected for experimental analysis (Table 2).

[0059] Table 2

[0060]

[0061]

[0062] 2. Experimental Methods

[0063] Histological analysis: Skin tissues were fixed in 10% neutral formaldehyde solution for 24 hours, followed by embedding, sectioning, and hematoxylin-eosin (HE) staining. All samples were observed using an optical microscope (Olympus Optical Co., Tokyo, Japan).

[0064] Enzyme-linked immunosorbent assay (ELISA): Immediately after sample collection, ELISA kits from Beyotime were used to measure the levels of thiol (Cat.#S0138S), malondialdehyde (MDA, Cat.#S0131S), superoxide dismutase (SOD, Cat.#S0101S), and catalase (CAT, Cat.#S0051) according to the instructions.

[0065] 3. Experimental Results

[0066] The results of skin histological analysis were as Figure 2 shown. Compared with normal skin, the tissue around the lesion showed inflammatory cell infiltration in the superficial dermal blood vessels, while blisters and acantholysis occurred at the lesion site ( Figure 2 A in). Detection of oxidative stress biomarkers in tissues found that the thiol level in the lesion area was lower than that in normal tissues (p<0.05), while no significant change was shown in the tissue around the lesion compared with normal tissues (p>0.05). This indicates that the decrease in thiol level in the lesion tissue may be due to its oxidation and consumption. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) in the lesion tissue and the tissue around the lesion were higher than those in normal tissues ( Figure 2 B in), and the levels in the lesion area were significantly higher than those in the tissue around the lesion (p<0.05). Consistent with these findings, the ELISA test results of serum showed that the levels of MDA, SOD, and CAT in PV patients were increased, while the thiol level was decreased compared with healthy individuals ( Figure 2In C). These results together indicate the presence of oxidative stress in PV patients.

[0067] Verification of the relationship between lipids and pemphigus in the cell model of Example 3

[0068] In view of the increased oxidative stress observed in the lesional tissues and sera of PV patients, this example evaluated the correlation between oxidative stress markers and the autoantibodies of the PV classical biomarkers DSG1 and DSG3.

[0069] 1. Experimental method

[0070] Construction of the PV cell model:

[0071] The human keratinocyte cell line HaCaT was purchased from the American Type Culture Collection (CLS Cat#300493 / p800_HaCaT, RRID:CVCL_0038) and used to establish an in vitro model of PV. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, TransGen Biotech Co., Ltd, Beijing, China), 100 U / mL penicillin, and streptomycin. The cells were cultured in an incubator at 37 °C and 5% CO2. During the logarithmic growth phase, the medium was replaced with a mixture containing 20% serum from PV patients and 80% DMEM (supplemented with 10% FBS), and the cells were cultured for 36 hours. The successful establishment of the PV cell model was verified by detecting the autoantibodies of the PV classical biomarkers DSG1 and DSG3.

[0072] Serological detection: The serological detection results of the patients in Table 2 were used for correlation analysis, and the results were presented in the form of a heat map.

[0073] 2. Experimental results

[0074] This example evaluated the correlation between oxidative stress markers and the autoantibodies of the PV classical biomarkers DSG1 and DSG3. The heat map results showed a strong positive correlation between the lipid oxidation marker MDA and the anti-DSG1 / DSG3 antibodies (DSG1 / DSG3 Abs), and significant correlations were also found between thiols and DSG1 antibodies, and SOD and DSG3 antibodies ( Figure 3 In A), indicating a significant association between oxidative stress markers and DSG1 / DSG3 antibodies.

[0075] Furthermore, to verify lipid-induced oxidative stress, HaCaT cells were treated with serum from PV patients in this example. The results showed that the levels of MDA, SOD, and CAT increased, while the level of thiols decreased in the treated cells, which was consistent with the findings in PV tissues and sera, indicating that lipids induced oxidative stress ( Figure 3 In B).

[0076] Example 4 ATO attenuates oxidative stress-triggered mitochondrial-dependent apoptosis and STAT3 activation in HaCaT cells

[0077] 1. Experimental methods

[0078] Measurement of mitochondrial membrane potential (MMP): Before MMP analysis, target cells were exposed to 20% PV serum for 36 h. The mitochondrial membrane potential detection kit (TMRE, Cat.# C2001S, Beyotime) was used to operate according to the instructions. MMP was analyzed by fluorescence microscopy (Zeiss AxioVert A1).

[0079] Flow cytometry analysis: HaCaT cells were incubated in 20% patient-derived PV serum for 36 h. Control serum was used as a vehicle control in the experiment. For flow cytometry detection, cells were collected and washed twice with PBS, and stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) according to the instructions (Cat.# C1062M, Beyotime). The stained cells were analyzed using a flow cytometer (Beckman).

[0080] Immunoblotting: Cell and tissue samples were lysed immediately after collection in lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% SDS, and protease inhibitors. After centrifugation at high speed for 10 min at 4 °C, equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk at room temperature for 1 h. Subsequently, the membranes were incubated with primary antibodies (usually diluted 1:1,000, dilution factor according to the instructions) overnight on a shaker at 4 °C. After washing with TBST, the membranes were incubated with HRP-labeled secondary antibodies (diluted 1:5,000) for 60 min on a shaker at room temperature. Protein bands were developed by enhanced chemiluminescence, and images were captured using a fully automatic gel imaging system (BLT, Model GelView 1500Pro).

[0081] Real-time fluorescence quantitative PCR (real-time PCR) analysis: After cell collection, total RNA was extracted using TRIzol reagent. Subsequently, RNA was reverse transcribed into cDNA using a cDNA synthesis kit (Cat.#D7170S, Beyotime), and the reaction conditions were carried out according to the instructions. Then, a reaction system was prepared using SYBR Green qPCR Mix (Cat.#D7601S, Beyotime), including cDNA template, forward primer, reverse primer, SYBR Green Mix, and RNase-free water, and a real-time fluorescence quantitative PCR reaction was performed. The program was carried out according to the instructions, including pre-denaturation, cycling stage, and melting curve analysis. Finally, data analysis was performed on the relative expression levels of the target genes.

[0082] The relevant primer sequences are as follows:

[0083] DSG1:

[0084] Forward primer: 5'-TGAGCCTGTTCCCGAATGTT-3' (SEQ ID NO: 1);

[0085] Reverse primer: 5'-GACAGTGGTTATATCCCTGGGT-3' (SEQ ID NO: 2).

[0086] DSG3:

[0087] Forward primer: 5'-TCAGCCGCCTTTTGGAATCT-3' (SEQ ID NO: 3);

[0088] Reverse primer: 5'-GCCCGACATGTGATCAGGAA-3' (SEQ ID NO: 4).

[0089] 2. Experimental results

[0090] Considering that oxidative stress can trigger apoptosis, this example further explored how oxidative stress affects the apoptotic process. Mitochondrial membrane proteins are key indicators of mitochondrial integrity and are mainly regulated by the Bcl2 protein family. Among them, Bcl2 and Bax play key roles in the initiation of apoptosis. In this context, apoptotic proteins were analyzed, and it was found that compared with the surrounding diseased and normal tissues, the level of Bax protein increased, while the level of Bcl2 decreased in the diseased tissues of PV patients, and caspase3 and PARP were cleaved and activated, indicating that the caspase-3-dependent apoptotic pathway was activated (as shown in A below). At the same time, the STAT3 pathway was activated in the tissues of PV patients, suggesting that oxidative stress may synergistically promote the apoptotic process by activating the STAT3 pathway. Figure 4 as shown in A below.

[0091] To investigate the role of abnormal lipid metabolism therein, the changes in mitochondrial membrane potential (MMP) of HaCaT cells treated with PV serum were analyzed by Rhodamine B (RhB) staining. Apoptotic cells failed to be stained with RhB. Meanwhile, flow cytometry further confirmed the increased apoptosis of cells treated with PV serum (as shown in B and C in Figure 4 ), indicating that lipid-induced oxidative stress led to the loss of mitochondrial membrane potential and mitochondrial dysfunction, further promoting apoptosis. Notably, after atorvastatin (ATO) intervention, the changes in mitochondrial membrane potential and apoptosis triggered by oxidative stress could be reduced. The results suggested that ATO might relieve the damage of oxidative stress to mitochondria and reduce apoptosis by improving lipid metabolism disorders (such as reducing the level of oxidized low-density lipoprotein).

[0092] Furthermore, HaCaT cells treated with PV serum and ATO were collected. Immunoblot analysis showed that after adding ATO, in addition to the reversal of apoptotic protein changes, such as the decrease in Bax protein, the increase in Bcl2 level, and the weakening of caspase3 and PARP cleavage, the activation of the STAT3 pathway was also reduced (as shown in D in Figure 4 ). Meanwhile, the treatment with PV serum led to the down-regulation of the expression of DSG1 and DSG3 proteins in HaCaT cells. After ATO intervention, the expression levels of both were increased relative to the PV group. The results of real-time fluorescence quantitative PCR (RT-PCR) were consistent with those of Western blot (as shown in E in Figure 4 ), confirming that ATO restored the expression of adhesion molecules by regulating transcription or post-transcriptional processes. These data together indicated that oxidative stress exacerbated the PV pathological process by regulating the mitochondrial apoptosis pathway and STAT3 signaling, while ATO could achieve the effect of treating pemphigus by correcting lipid metabolism imbalance, inhibiting oxidative stress and its downstream pro-apoptotic effects.

[0093] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments. Various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a hypolipidemic drug in the preparation of a medicament for treating pemphigus.

2. The use according to claim 1, wherein: The pemphigus includes one of pemphigus vulgaris, pemphigus vegetans, pemphigus foliaceus, pemphigus herpetiformis, and paraneoplastic pemphigus.

3. The use according to claim 1, wherein: The hypolipidemic drug includes a statin, a cholesterol absorption inhibitor, a fibrate, or a PCSK9 inhibitor.

4. The use according to claim 3, wherein: The statin includes atorvastatin or a pharmaceutically acceptable salt thereof.

5. The use according to claim 4, wherein: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

6. The use according to claim 1, wherein: The medicament includes pharmaceutically acceptable excipients.

7. The use according to claim 6, wherein: The pharmaceutically acceptable excipients include at least one of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an antiadhesive, a chelating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculant and an anti-flocculant, a filter aid, a release retarder, and a carrier.

8. The use according to claim 1, wherein: The dosage form of the medicament includes a gastrointestinal administration dosage form or a non-gastrointestinal administration dosage form.

9. An in vitro method for reducing the level of cellular oxidative stress for non-therapeutic purposes: Treating HaCaT cells with atorvastatin.

10. Use of a biomarker in the detection of pemphigus: The biomarker includes serum cholesterol, triglyceride, and / or apolipoprotein B.

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