Use of roflumilast for the preparation of a medicament for the treatment of pemphigus

Abstract

The application discloses application of roflumilast in preparation of a pemphigus treatment drug, relates to the technical field of biological medicine, and has the technical scheme as follows: the application discloses that endoplasmic reticulum stress participates in the pathogenesis of pemphigus and the application of roflumilast in preparation of a pemphigus treatment drug for down-regulating endoplasmic reticulum stress and inhibiting keratinocyte apoptosis. The drug down-regulates the expression of endoplasmic reticulum stress marker proteins, inhibits the Caspase family and Fas-mediated pro-apoptotic cascade, and thus significantly reduces the rate of inhibition of keratinocyte apoptosis induced by pemphigus. By blocking the apoptosis process, the degradation and distribution disorder of Dsg3, PG and other desmosome structure proteins are reversed, and the remodeling of the desmosome skeleton is promoted, so that the adhesion between epidermal cells is fundamentally restored. It is proved that roflumilast has the potential as a new type of pemphigus treatment drug, and a new strategy for treating pemphigus by regulating the ''endoplasmic reticulum stress-apoptosis axis'' is provided.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of roflumilast in the preparation of pemphigus treatment drugs. Background Technology

[0002] Pemphigus disease (PD) is a severe, autoantibody-mediated, bullous skin disease characterized by desmosome loss of adhesion between keratinocytes (KCs). In severe cases, it can directly threaten the patient's life. The global incidence of this disease varies significantly by region and ethnicity, ranging from approximately 0.098 to 5 cases per 100,000. The typical clinical manifestations include flaccid vesicles, bullae, and extensive erosive lesions on the skin and mucous membranes.

[0003] Pemphigus vulgaris (PV), as the most representative subtype, is characterized by epidermal acantholysis—loss of intercellular adhesion function of epidermal keratinocytes, ultimately leading to the formation of intraepidermal bullae. This pathological process is primarily driven by IgG-type autoantibodies targeting desmogleins (Dsgs), especially Dsg3. Dsg3, a key transmembrane glycoprotein for intercellular adhesion, is specifically expressed in mucous membranes and the deep epidermis. When autoantibodies bind to it, they can directly disrupt desmosome structure and intercellular connections through multiple mechanisms, including steric hindrance, induction of protein endocytosis and degradation, activation of intracellular signaling pathways such as p38MAPK and RhoGTPases, and triggering complement activation. The academic community has gradually broken through the single "steric hindrance" theory and further confirmed that the intracellular signaling cascade reaction initiated after the binding of autoantibodies plays a core role in the process of acantholysis. It can induce cytoskeleton rearrangement, abnormal activation of proteases such as plasminogen activator, and even directly induce keratinocyte apoptosis. Therefore, the pathogenesis of PV is a complex regulatory network in which humoral immunity, cellular immunity and the intrinsic reaction of keratinocytes are intertwined.

[0004] The current first-line clinical treatment for pemphigus still revolves around systemic corticosteroids (such as prednisone), often combined with immunosuppressants such as azathioprine, mycophenolate mofetil, cyclophosphamide, and methotrexate to rapidly control the disease and gradually reduce the dosage of corticosteroids. For severe or refractory cases, rituximab, which targets CD20, has become a key treatment option, significantly reducing the production of pathogenic autoantibodies by depleting B cells. In addition, intravenous immunoglobulin (IVIG) and immunoadsorption therapy are also used in specific clinical scenarios. However, the existing treatment system still has significant clinical limitations: long-term use of high-dose corticosteroids can induce serious adverse reactions such as metabolic disorders (diabetes, hypertension, central obesity), osteoporosis, increased risk of infection, peptic ulcers, and mental abnormalities; conventional immunosuppressants also have potential toxicities such as bone marrow suppression, liver and kidney damage, and increased risk of malignant tumors; although rituximab has outstanding efficacy, the treatment cost is high, and there are safety risks such as hepatitis B virus reactivation and opportunistic infections, and some patients still experience disease relapse after treatment. Therefore, developing new, more efficient, and safer treatment strategies, especially specific drugs that target downstream effects of the disease—namely, keratinocyte dysfunction and structural damage caused by antibody binding—has become a core direction that urgently needs to be broken through in the field of pemphigus research.

[0005] Roflumilast is a highly selective phosphodiesterase 4 (PDE4) inhibitor. PDE4, a core enzyme in the intracellular hydrolysis of cyclic adenosine monophosphate (cAMP), is widely expressed in various inflammatory cells and epithelial cells. Roflumilast increases intracellular cAMP levels by inhibiting PDE4 activity, activating the protein kinase A (PKA) pathway, and thus exerts a powerful anti-inflammatory and immunomodulatory effect: on the one hand, it inhibits the production of pro-inflammatory mediators such as TNF-α, IL-17, IL-23, and IFN-γ; on the other hand, it promotes the release of anti-inflammatory mediators such as IL-10, while simultaneously blocking the activation and migration of immune cells. Roflumilast is currently approved for the treatment of inflammatory skin diseases such as psoriasis. In recent years, the application value of PDE4 inhibitors in the field of dermatology has continued to receive attention. PDE4 is highly expressed in skin-related cells such as keratinocytes, dendritic cells, and T cells, and is widely involved in the pathogenesis of various inflammatory skin diseases. Clinical studies have confirmed that PDE4 inhibitors (such as criborone ointment) have good efficacy and safety in the treatment of atopic dermatitis. As a potent PDE4 inhibitor, roflumilast has potential therapeutic advantages for skin diseases with systemic immune dysregulation. Existing clinical cases and small studies have confirmed that it has shown certain therapeutic effects on refractory inflammatory skin diseases such as psoriasis, pyoderma gangrenosa, and Behçet's disease. Its mechanism of action covers multiple dimensions, including inhibiting the differentiation and function of pathogenic T cell subsets such as Th1 and Th17, reducing neutrophil activation and infiltration, and directly regulating keratinocyte inflammatory response and barrier function.

[0006] Existing basic research has confirmed that the cAMP signaling pathway is closely related to the regulation of cell adhesion and apoptosis: in keratinocytes, the cAMP / PKA pathway can directly regulate the activity of cytoskeletal proteins, affecting the formation and structural stability of intercellular connections; at the same time, elevated intracellular cAMP levels have been shown to play a clear anti-apoptotic role in various cell types. Based on the core pathogenesis of pemphigus—Dsg3-mediated disruption of cell adhesion and abnormal activation of apoptosis pathways—existing research suggests that roflumilast can intervene in the pathological process of pemphigus at multiple levels through the core mechanism of increasing intracellular cAMP levels: First, by regulating intracellular signaling pathways in keratinocytes, stabilizing Dsg3 protein expression, reducing its endocytosis and degradation, thereby protecting the integrity of desmosomes; second, by inhibiting the increase in the Bax / Bcl-2 ratio and blocking the activation of the Caspase cascade, inhibiting pro-apoptotic signaling pathways and reducing pemphigus IgG-induced keratinocyte apoptosis; third, through broad-spectrum immunomodulation, inhibiting the function of pathogenic autoantibody-generating B cells and helper T cells, reducing the intensity of autoimmune attack at its source; and fourth, by inhibiting the release of pro-inflammatory cytokines such as TNF-α and IL-6, reducing local tissue inflammatory damage.

[0007] However, significant research gaps remain in this field: No systematic experimental studies have yet clearly defined the therapeutic effect of roflumilast on pemphigus, and its specific targets and molecular regulatory mechanisms remain unclear. In particular, whether roflumilast can directly protect keratinocytes from pemphigus IgG attack in classic in vitro and in vivo animal models of pemphigus, whether it can reverse existing cell adhesion disruption and cell damage, and its therapeutic efficacy and safety at the whole-animal level are all critical scientific questions that urgently need to be addressed. Simultaneously, during the pathogenesis of pemphigus, autoantibody attack can induce a large accumulation of unfolded proteins in keratinocytes, triggering excessive activation of endoplasmic reticulum stress. Sustained endoplasmic reticulum stress further amplifies apoptosis signals, accelerating acantholysis and epidermal vesicle formation. Existing clinical and basic research lacks any patents or public reports related to pemphigus treatments that target the endoplasmic reticulum stress pathway and inhibit keratinocyte apoptosis by downregulating endoplasmic reticulum stress levels. The therapeutic potential in this direction has not yet been effectively developed. Summary of the Invention

[0008] The purpose of this invention is to provide the application of roflumilast in the preparation of pemphigus treatment drugs, to demonstrate that ERS participates in mediating the core link of KC damage, and to demonstrate the potential of roflumilast as a novel treatment drug for PV, providing a new strategy for clinical treatment of PV by regulating the "endoplasmic reticulum stress-apoptosis axis".

[0009] The above-mentioned technical objective of the present invention is achieved through the following technical solution: the application of roflumilast in the preparation of a pemphigus treatment drug, wherein the drug significantly reduces the pemphigus-induced apoptosis rate of keratinocytes by downregulating the expression of endoplasmic reticulum stress marker proteins and inhibiting the pro-apoptotic cascade reaction mediated by the Caspase family and Fas.

[0010] The present invention is further configured such that the ERS marker proteins are CHOP, ATF4, and IRE1.

[0011] The present invention is further configured such that the Caspase family is Caspase-3, Cleaved-Caspase-3, and Caspase-7.

[0012] The drug significantly upregulated the protein expression of Caspase-3, Cleaved-Caspase-3, Caspase-7, Bax, and Fas, and downregulated the expression of Bcl-2.

[0013] The present invention is further configured such that the drug can significantly enhance and restore the expression levels of desmosome structural proteins, including Dsg1, Dsg3, and PG, in cells.

[0014] The present invention is further configured such that the drug inhibits the secretion of inflammatory factors TNF-α, IFN-γ, IL-6 and IL-8.

[0015] The present invention is further configured such that: the roflumilast is the only active ingredient of the drug.

[0016] The present invention is further configured such that the drug is used alone or in combination with an oral hormone, specifically prednisone.

[0017] The present invention is further configured such that the drug is administered by topical application.

[0018] The present invention is further configured such that the drug is a topical dosage form.

[0019] The present invention is further configured such that the external dosage form is a semi-solid dosage form.

[0020] The present invention is further configured such that the semi-solid dosage form is an ointment.

[0021] In summary, the present invention has the following beneficial effects:

[0022] This invention breaks through the limitations of the single hypothesis of "steric hindrance" that has long been relied upon in traditional pemphigus (PV) pathogenesis research. It moves beyond the traditional framework that explains the pathogenesis solely from the perspective of the blockage of binding between autoantibodies and epidermal cell adhesion molecules. For the first time, it establishes a multidimensional, interconnected pathological mechanism analysis perspective involving "endoplasmic reticulum stress-apoptosis-inflammatory cascade activation," deepening the scientific understanding of the occurrence, development, and persistent nature of PV lesions from the deeper dimension of intracellular homeostasis imbalance. This mechanism clarifies the decisive role of inhibiting apoptosis and endoplasmic reticulum stress in the PV lesion repair process, filling the cognitive gap at the intracellular regulatory pathway level in existing PV pathogenesis research, and providing new theoretical support for the screening of innovative therapeutic targets for PV.

[0023] This invention, for the first time, systematically validated the efficacy of roflumilast in treating pemphigus (PV) using animal and human keratinocyte in vitro models. Multiple parallel controlled experiments confirmed that topical application of specific doses of roflumilast can downregulate endoplasmic reticulum stress and inhibit keratinocyte apoptosis, thereby blocking the cascade release of downstream inflammatory factors and ultimately promoting PV lesion healing. This achievement provides a novel topical targeted therapy option for PV clinical treatment, particularly suitable for PV patients who are intolerant to traditional hormone therapy, have contraindications to hormone use, or require long-term maintenance of topical therapy. It significantly expands the range of available clinical medications for PV and possesses extremely high clinical translational value and application prospects.

[0024] The topical roflumilast treatment for PV established in this invention can achieve precise intervention of local lesions without relying on high-dose systemic hormones or immunosuppressants. While ensuring the therapeutic effect, it significantly reduces the risk of infection, metabolic disorders and other systemic side effects caused by systemic medication. It provides a safer new path for the long-term standardized treatment of PV and also provides a new research paradigm that can be referenced for the development of local targeted drugs for other autoimmune bullous skin diseases. Attached Figure Description

[0025] Figure 1 This is a technical roadmap of the in vitro cell experiment in Embodiment 1 of the present invention;

[0026] Figure 2 This is a technical roadmap of the animal experiments in Embodiment 2 of the present invention;

[0027] Figure 3This invention relates to the effects of FK506, Roflumilast, and Pdn on HaCaT cell proliferation in Example 1. Cells were treated for 24 hours with different concentrations of FK506 (0, 5 nM, 10 nM, 50 nM, 100 nM, 300 nM, 500 nM, 1 μM, 5 μM), Roflumilast (0, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 5 μM, 50 μM, 100 μM, 200 μM), and Prednisone (0, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM), and cell proliferation activity was measured using the MTT assay. Data are expressed as mean ± SE of four independent experiments. Compared with the normal culture group (con), P>0.05, **p<0.01, ***p<0.001, and there was no statistically significant difference in ns.

[0028] Figure 4 In Example 1 of this invention, PV-IgG inhibited the expression of HaCaT desmosome structural proteins to establish an in vitro cell model. After the cells reached 30% confluence, PV-IgG was added for further treatment for 48 hours. The normal culture group (Control) served as a parallel control. The expression of Dsg3, Dsg1, PG, DP, and PKP3 was detected by Western blotting, with GAPDH used as an internal control. Compared with the Control group, *P<0.05, **p<0.01, ***P<0.001, and there was no statistically significant difference in ns. The expression of desmosome structural proteins decreased with increasing PV-IgG concentration.

[0029] Figure 5 This invention relates to the effects of PV-IgG and drug treatment on the fluorescence expression of Dsg3 and PG in HaCaT cells, as described in Example 1. HaCaT cells were treated with PV-IgG (1 mg / mL) and the corresponding drug groups for 48 h, followed by immunofluorescence staining with anti-Dsg3 and anti-PG antibodies, respectively. Cell nuclei were stained with DAPI (blue). In the control group, linear Dsg3 fluorescence was observed distributed along the cell membrane. However, after PV-IgG treatment, Dsg3 and PG became granular and fragmented. Drug treatment restored fluorescence expression (scale bar: 20 µm), and the fluorescence intensity of both Dsg3 and PG was significantly higher than that in the PV group (*P<0.05, **p<0.01).

[0030] Figure 6This invention relates to the effects of PV-IgG and drug treatment on the expression of Dsg3 and PG in HaCaT cells in Example 1. After HaCaT cells were treated with PV-IgG (1 mg / mL) and the corresponding drug group for 48 h, proteins were extracted for Western blot experiments. After PV-IgG treatment, the expression of connexins in HaCaT cells decreased. Rof, FK506 and Pdn could significantly increase and restore the expression levels of desmosome structural proteins such as Dsg1, Dsg3 and PG in PV model cells (*P<0.05, no statistically significant difference in ns).

[0031] Figure 7 This invention relates to the effects of PV-IgG and drug treatment on HaCaT cell apoptosis in Example 1. HaCaT cells were treated with PV-IgG (1 mg / mL) and the corresponding drug for 48 hours, followed by cell digestion and flow cytometry analysis. PV-IgG treatment significantly increased the early apoptosis rate of HaCaT cells, while the drug treatment group showed a significant decrease in the early apoptosis rate. Data are expressed as the mean ± SE of three independent experiments. Compared with the normal culture group (con), *p<0.05, **p<0.01, and there was no statistically significant difference in ns.

[0032] Figure 8 This invention relates to the effects of PV-IgG and drug treatment on the expression of apoptotic proteins in HaCaT cells in Example 1. HaCaT cells were treated with PV-IgG (1 mg / mL) and the corresponding drug group for 48 h, and then proteins were extracted for Western blot analysis. After PV-IgG treatment, the expression of apoptotic proteins in HaCaT cells increased, while the expression of apoptotic proteins in the drug treatment group decreased (*p<0.05, **p<0.01, no statistically significant difference in ns).

[0033] Figure 9 This invention relates to the effects of PV-IgG and drug treatment on the expression of inflammatory factors in HaCaT cells in Example 1. HaCaT cells were treated with PV-IgG (1 mg / mL) and the corresponding drug group for 48 h, and the cell supernatant was collected for ELISA experiments. After PV-IgG treatment, the expression of inflammatory factors TNF-α, IFN-γ, IL-6, and IL-8 in HaCaT cells increased, while the expression of inflammatory factors decreased in the drug treatment group (***p<0.001).

[0034] Figure 10 This relates to the establishment of a passive metastasis model of pemphigus in mice in Example 2 of this invention. Recombinant Dsg3 protein, 20 μg / subcutaneous injection / mouse, was subcutaneously injected into the back of newborn mice to establish a passive metastasis model of pemphigus. Compared with the healthy control group, typical erythema, vesicles and extensive erosions were observed on the back skin and mucous membrane areas of the pemphigus disease group.

[0035] Figure 11In Example 2 of this invention, Rof improved the clinical symptoms of pemphigus mice and reduced antibody titer levels. Note: After 5 consecutive days of treatment in both the single-drug group and the combination drug group, the skin lesions on the back of the mice in the treatment group were significantly improved, and the wound inflammation appearance score and antibody titer were significantly reduced, which were lower than those in the disease group (***p<0.001, no statistically significant difference in ns).

[0036] Figure 12 In Example 2 of this invention, Rof reduced the expression of inflammatory factors in the serum of pemphigus mice. After 5 consecutive days of treatment in both the single-drug group and the combination-drug group, mouse serum was collected for ELISA analysis. The expression of inflammatory factors TNF-α, IFN-γ, IL-6, and IL-8 in the serum of pemphigus mice was significantly increased (***p<0.001), while the expression level of inflammatory factors in the treatment group was significantly decreased (***p<0.001, no statistically significant difference in ns).

[0037] Figure 13 In Example 2 of this invention, Rof improves the pathological tissue of pemphigus mouse skin lesions; HE staining showed that the skin tissue of the pemphigus disease group mice had obvious intraepidermal vesicle formation, acantholysis and a large number of inflammatory cell infiltrations; while the epidermal structure of the roflumilast treatment group was relatively intact, the degree of vesicles and acantholysis was significantly reduced, and the inflammatory infiltration was significantly reduced.

[0038] Figure 14 This relates to the effect of Rof in inhibiting apoptosis of pemphigus mouse skin lesions in Example 2 of this invention; immunohistochemistry and TUNEL staining showed that the expression of apoptotic proteins surged in the skin lesions of pemphigus mice, and the number of TUNEL-positive cells increased significantly; Rofurazone treatment significantly reduced the occurrence of apoptosis (***p<0.001, no statistically significant difference in ns).

[0039] Figure 15 In Example 2 of this invention, endoplasmic reticulum stress may be involved in the development and progression of pemphigus. Transcriptome sequencing (RNA-Seq) analysis was performed on mice from each group, revealing significant differential gene expression between pemphigus lesions and the control group. Pathway enrichment analysis indicated significant activation of endoplasmic reticulum stress and unfolded protein response-related signaling pathways. Volcano plot analysis showed upregulation of endoplasmic reticulum stress-related genes (Hspa8, etc.) in the PV group. GSEA indicated upregulation of the endoplasmic reticulum stress (ERS) pathway in the PV group, with NES values ​​greater than 1.

[0040] Figure 16In Example 3 of this invention, endoplasmic reticulum stress proteins were highly expressed in the pemphigus skin lesion area. The endoplasmic reticulum stress proteins (CHOP, ATF4, IRE1) were detected by IHC method. The results showed that normal control skin epithelial cells hardly expressed them or only showed scattered weak expression in the basal layer, while the expression of endoplasmic reticulum stress marker proteins was significantly increased in PV skin lesion tissue (***p<0.001).

[0041] Figure 17 In Example 3 of this invention, the expression of apoptotic proteins in the pemphigus skin lesion area was increased. IHC detection of apoptotic proteins (Bax, Caspase 3) showed that normal control skin epithelial cells showed almost no expression or scattered weak expression, while the expression of apoptotic proteins was significantly increased in PV skin lesion tissue (***p<0.001). Detailed Implementation

[0042] The following is in conjunction with the appendix Figure 1-17 The present invention will be described in further detail below.

[0043] The full names of the abbreviations used in this invention are shown in Table 1:

[0044] Table 1: List of Abbreviations

[0045]

[0046] Example 1: Investigating the mechanism of action of roflumilast in inhibiting keratinocyte apoptosis, remodeling the desmosome cytoskeleton, and improving pemphigus through in vitro cell experiments.

[0047] 1. Collection of patient serum and skin specimens

[0048] The serum and clinical skin samples used in the experiment were reviewed and approved by the Ethics Committee of Guangzhou Skin Disease Hospital (NO.201802). Blood and skin samples were collected by Guangzhou Skin Disease Hospital from October 2022 to October 2025 from outpatients of patients diagnosed with pemphigus vulgaris.

[0049] The inclusion and exclusion criteria are as follows:

[0050] Inclusion criteria: ① Age ≥ 18 years. ② Clinically positive Nikolsky sign, pathological intraepidermal bullae / acantholysis, and serum Dsg1 / Dsg3 ratio for pemphigus. ③ No systemic drug treatment within the past month. ④ No serious organ or neuropsychiatric disorders. ⑤ Female not pregnant or lactating.

[0051] Exclusion criteria: ① Pregnant women; ② Those whose pemphigus cannot be ruled out due to drug-induced pemphigus; ③ Those with malignant tumors or major organ dysfunction.

[0052] Serum samples were collected from 10 patients clinically and pathologically diagnosed with pemphigus vulgaris. Patient information is shown in Table 1. 5 mL of whole blood was collected, allowed to stand at room temperature for 1 hour, and then centrifuged at 3000 rpm for 5 minutes. The supernatant serum was collected and placed in a new EP tube, stored at -80°C. The Dsg1 / 3 antibody titers of all collected patient serum samples were determined by ELISA and recorded. Referring to previous literature, to reduce individual variability, the serum was mixed and IgG was purified before use. PV-IgG was quantified using a BCA kit, and gradient concentrations were set for subsequent experiments.

[0053] The pemphigus lesion tissue used in this experiment was collected from the outpatient clinic of Guangzhou Skin Disease Hospital from October 2022 to October 2025. A total of 10 pemphigus vulgaris lesions were collected, and the normal control group was obtained from normal tissue next to the lesions.

[0054] Table 2: Description of patients with pemphigus vulgaris

[0055]

[0056] Note: PDAI: Pemphigoid disease area index; Dsg1 reference range: <14 negative, 14-20 suspicious, 20 positive; Dsg3 reference range: <7 negative, 7-20 suspicious, ≥20 positive.

[0057] Cell experiment grouping and processing:

[0058] Normal control group (Control): 10% FBS DMEM medium;

[0059] Healthy serum group (Healthy-IgG): Purified serum IgG from healthy individuals was added to DMEM medium;

[0060] PV-serum group (PV-IgG): Purified pemphigus patient serum IgG was added to DMEM medium;

[0061] Drug treatment: Under the above conditions, different concentrations of Roflumilast, Prednisone, and Tacrolimus (FK506) were added.

[0062] 2. Cell resuscitation and culture

[0063] 2.1 Cell resuscitation

[0064] (1) Before using the biosafety cabinet, turn on the UV lamp for 20 min to disinfect, thaw the complete culture medium (90% DMEM, 1% antibiotics, 10% FBS), and preheat it to 37℃ for later use.

[0065] (2) Take out the cryopreservation tube and place it in a 37°C constant temperature water bath for 5 minutes to thaw the cells. Be careful to seal the tube opening to prevent contamination.

[0066] (3) Add 5 mL of culture medium to a 15 mL centrifuge tube, aspirate the frozen solution, transfer the solution to the centrifuge tube with a pipette and mix well. Centrifuge at 1000 rpm for 3 min and discard the supernatant.

[0067] (4) Add 5 mL of complete culture medium, resuspend by pipetting, transfer the cell suspension to a 10 cm cell culture dish containing 5 mL of complete culture medium, mix by cross-shaking, and incubate in a 5% CO2, 37℃ incubator.

[0068] (5) After culturing for 24 hours, observe the cell adhesion under a microscope and select an appropriate time for passage culture based on the cell growth status.

[0069] 2.2 Cell passage and cryopreservation

[0070] (1) Observe the growth of HaCaT cells daily. Once the cells have fused to 80%-90%, they can be passaged.

[0071] (2) When passaged, aspirate the culture medium, add 1×PBS along the wall of the dish and shake to wash (do not directly rinse the cells), discard the PBS, and repeat 2-3 times.

[0072] (3) Add an appropriate amount of 0.25% EDTA-trypsin to a 10cm culture dish, adding it to the wall. Incubate for 6 minutes. When the sludge-like substance is visible to the naked eye or the intercellular spaces become larger and the cells become rounder under a microscope, the digestion is complete. Add twice the volume of complete culture medium to stop the digestion. Transfer the suspension to a new 15mL centrifuge tube, balance it, and centrifuge at 1000rpm for 3 minutes.

[0073] (4) Discard the supernatant, add 2ml of complete culture medium and gently pipette to mix, resuspend the cells, transfer them to a new culture dish at a ratio of 1:2 to 1:4, add 10mL of complete culture medium to a 10cm culture dish, observe the cell density, place in a 37℃, 5% CO2 incubator, observe daily, and change the medium every other day.

[0074] (5) Cell cryopreservation: Add an appropriate amount of cryopreservation solution to the cell pellet, pipette and resuspend the cells, transfer them to cryopreservation tubes, store at -80℃, and transfer to liquid nitrogen for storage after 24-48 hours.

[0075] 3. MTT assay for cell proliferation activity

[0076] (1) Seed 100 μL of cell suspension (approximately 6000 cells / well) into each well of a 96-well plate and incubate overnight in an incubator. Supplement the surrounding and blank wells with 1×PBS.

[0077] (2) After observing cell adhesion the next day, discard the original culture medium and add different concentrations of complete culture medium containing Roflumilast (0, 5nM, 10nM, 50nM, 100nM, 500nM, 5μM, 50μM, 100μM, 200μM), FK506 (0, 5nM, 10nM, 50nM, 100nM, 300nM, 500nM, 1μM, 5μM), and Prednisone (0, 0.1nM, 1nM, 10nM, 100nM, 1μM, 10μM, 100μM, 1mM) to the wells and continue incubation for 24 hours.

[0078] (3) Add 20 μL of 5 mg / mL MTT solution to each well of the 96-well plate, shake gently, and incubate in an incubator for 4 hours.

[0079] (4) Terminate the culture, discard the MTT solution, add 150 μl of dimethyl sulfoxide to each well, and place on a shaker to shake at low speed for 5-10 min to fully dissolve the crystals.

[0080] (5) Use an ELISA reader to measure absorbance at OD490nm.

[0081] Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%

[0082] Absorbance As of experimental wells (absorbance of wells containing cells, drugs, and MTT)

[0083] Absorbance of blank wells (Absorbance of wells containing culture medium and MTT)

[0084] Absorbance Ac of control wells (absorbance of wells containing cells and MTT)

[0085] The results show that ( Figure 3 Within the established effective concentration range, none of the above drugs exhibited significant toxic effects on HaCaT cell proliferation (P>0.05). Based on the results of this preliminary experiment and in conjunction with relevant literature, to avoid drug toxicity interference and ensure efficacy, the drug intervention concentrations uniformly determined in subsequent experiments were: PV-IgG (1 mg / mL), Rof (50 nM), tacrolimus (100 nM), and prednisone (30 μM).

[0086] 4. Purification of patient serum proteins and establishment of an in vitro pemphigus cell model

[0087] (1) Buffer preparation: Equilibration / washing buffer: 0.15M NaCl, 20mM Na2HPO4, pH7.0;

[0088] Elution Buffer: 0.1M glycine, pH 3.0; Neutralization Solution: 1M Tris-HCl, pH 8.5; Filter with a 0.22µm or 0.45µm filter membrane before use.

[0089] (2) Serum IgG purification: Take an appropriate amount of rProtein A / G Add the beads to a 50ml centrifuge tube, centrifuge at 800rpm for 5min, and discard the supernatant. Add 5mL of equilibration buffer to resuspend the packing material, ensuring the packing material and target protein are in the same buffer system. Centrifuge at 800rpm for 5min, discard the supernatant, and repeat twice. Add the mixed patient serum to the equilibrated packing material, mix well, and resuspend the packing material. Mix at room temperature, centrifuge at 800rpm for 5min after about 30min, and collect the supernatant for later testing. Add 5mL of washing buffer to the packing material to suspend it and wash it to remove non-specifically adsorbed proteins. Centrifuge at 800rpm for 5min, aspirate and discard the supernatant, and repeat twice. Add 5 column volumes of elution buffer to the centrifuge tube, mix well, and then place it in a rotary mixer at room temperature. Centrifuge at 800rpm for 5min after 10min, aspirate the supernatant, and collect the elution fraction, which is the target antigen. Collect the supernatant into a new centrifuge tube and immediately add one-tenth of a volume of neutralization solution to adjust the pH of the elution fraction to 7.0-8.0 for subsequent experiments.

[0090] (3) Establishment of the pemphigus cell model: 2 ml of cell suspension (approximately 250,000 cells / well) was seeded into each well of a 6-well plate and cultured overnight in an incubator. The next day, after observing cell adhesion, culture medium containing gradient concentrations of PV-IgG (0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL) was added and incubated for 48 h. After washing 2-3 times with PBS, the cells were digested and collected. The cells were centrifuged at 1000 rpm for 3 min and the supernatant was discarded. The remaining cell pellet was used for subsequent experiments.

[0091] 5. Western blotting to determine the expression of different proteins

[0092] (1) Cell collection: Discard the cell culture medium, wash 3 times with 1xPBS, digest with trypsin for 6 min, add complete culture medium to stop digestion, centrifuge at 1000 rpm for 5 min, discard the supernatant and keep the precipitate.

[0093] (2) Lysis of proteins: Cell collection and lysis were performed entirely on ice. An appropriate amount of RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors was added to each tube according to the number of cells. After thorough mixing, the tubes were incubated on ice for 30 minutes. Every 10 minutes, the tubes were vortexed for 10 seconds. After balancing, the tubes were centrifuged at 13,000 rpm for 15 minutes.

[0094] (3) Protein denaturation: The supernatant after centrifugation is the protein extract. Take 20 μL of the protein extract to determine the protein concentration. Add 1 / 4 volume of 5× Loading Buffer to the remaining protein extract in the centrifuge tube, heat in a 95℃ metal bath for 10 min to denature, and after the liquid has completely cooled, divide it into portions and store at -20℃.

[0095] (4) Protein concentration determination (BCA method)

[0096] ① Preparation of BCA working solution

[0097] Based on the number of samples, add 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1) to prepare an appropriate amount of BCA working solution, and mix thoroughly.

[0098] ② Determination of standard products

[0099] Dilute 10 μL of protein standard (5 mg / mL BSA) to 50 μL, resulting in a final concentration of 1 mg / mL. Store the diluted protein standard at -20°C for extended periods. Deionized water or 1*PBS can be used as the diluent for this standard solution. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to each well of a 96-well plate, and then add diluent to bring the total volume to 20 μL. Add an appropriate volume of sample to each well of the 96-well plate; if the sample volume is less than 20 μL, add diluent to bring the total volume to 20 μL. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min. Measure the absorbance at 562 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0100] (5) Gel electrophoresis

[0101] ① Rubber Mixing

[0102] Prepare the gel, install it horizontally in the vertical electrophoresis tank, fill the tank and the space between the glass plates with electrophoresis buffer, gently pull out the comb vertically upwards, and rinse the sample wells with a syringe.

[0103] ② Sample loading and electrophoresis

[0104] Arrange the sample wells according to different experimental groups. Slowly add 5 to 20 μL of protein and pre-stained marker to each well. Set the electrophoresis tank voltage to 80 to 90 V. When the protein sample is observed to form a line in the stacking gel, the electrophoresis tank voltage can be increased to 120 V. Electrophoresis can be stopped when the bromophenol blue reaches the bottom of the separating gel.

[0105] ③ Transfer membrane

[0106] Arrange the necessary components in sequence according to the power electrode direction (positive electrode, absorbent cotton, filter paper, activated PVDF membrane, gel, filter paper, absorbent cotton, negative electrode), avoiding air bubbles when placing the gel layer and PVDF. Install it horizontally in the transfer tank, fill the tank with pre-cooled transfer buffer, and add ice cubes and an ice box to maintain a low temperature. Set the transfer tank voltage to 300mA and set the timer for 1-1.5 hours.

[0107] ④ Closed

[0108] Remove the PVDF membrane from the "sandwich" structure and place it in an antibody incubator containing 5% BSA for 2 hours at room temperature.

[0109] ⑤ Incubate primary antibody

[0110] The primary antibody was diluted with the primary antibody diluent at an appropriate ratio; after blocking, the diluted primary antibody working solution was poured into the incubation tank and incubated overnight at 4°C.

[0111] ⑥ Washing film

[0112] After the primary antibody incubation is complete, wash three times with 1×TBST Buffer, 15 min each time.

[0113] ⑦ Incubate secondary antibodies

[0114] Dilute the secondary antibody according to an appropriate ratio. After washing, add the diluted secondary antibody working solution to the incubation tank and incubate at room temperature for 1 hour.

[0115] ⑧ Washing film

[0116] After the secondary antibody incubation is complete, wash three times with 1×TBST Buffer, 15 min each time.

[0117] 9. Development

[0118] Equal volumes of luminescent liquid A and liquid B were mixed thoroughly. Then, the luminescent liquid was dropped onto a PVDF film, and the film was placed in a developing instrument for exposure. The results were analyzed for grayscale using ImageJ.

[0119] Western blot results showed that, compared with the normally cultured control group, the expression levels of desmosome structural proteins such as Dsg1, Dsg3, DP, PG, and PKP3 in cells treated with PV-IgG were significantly reduced, and the reduction was clearly concentration-dependent. Figure 4 This confirms the successful establishment of an in vitro cell model of pemphigus.

[0120] 6. Immunofluorescence staining

[0121] (1) Place the fluorescent crawling plate (Nest) into the 24-well plate, passage and seed the cells. After the cells have reached 60-70% confluence, treat them according to different groups.

[0122] (2) After adhesion, the culture medium is aspirated and the cells are washed 2-3 times with 1×PBS.

[0123] (3) Fixation: Add 4% paraformaldehyde and fix at room temperature for 10 min, then wash with 1×PBS 3 times, 5 min each time.

[0124] (4) Punching: Remove PBS at room temperature, treat with 0.1% Triton X-100 in PBS punching agent for 10 min, then wash with 1×PBS 3 times, 5 min each time.

[0125] (5) Blocking: Block with 5% BSA (in PBS) at room temperature for 1 hour, depending on the antibody species.

[0126] (6) Incubate primary antibody: Take the blocking solution, add the primary antibody diluted with antibody diluent, 200 μL / well, and use the negative control as the blocking solution. Incubate overnight at 4°C.

[0127] (7) Incubation of secondary antibody: Wash 3 times with 1×PBST (0.1% Tween20 in PBS) for 5 min each time. Select a suitable secondary antibody according to the species of the primary antibody. Dilute the secondary antibody at 1:500-1:800 and incubate at room temperature in the dark for 1 h.

[0128] (8) Mounting: Wash with PBST 3 times, 5 min each time. Place the glass slide with the cell culture side down on a glass slide with anti-quenching mounting medium (containing DAPI) to avoid the formation of air bubbles.

[0129] (9) Taking pictures: Use a confocal microscope to take pictures, and adjust the taking pictures parameters and conditions according to the fluorescence intensity.

[0130] Immunofluorescence assay ( Figure 5 The results showed that, compared with the control group, Dsg3 and PG on the cell membrane of the PV-IgG (1 mg / mL) treatment group became granular and fragmented, with significantly reduced fluorescence intensity (P<0.01), and significantly weakened intercellular connections. After intervention with drugs such as Rof, the fluorescence intensity of Dsg3 and PG was significantly increased compared with the PV group (P<0.05), Dsg3 recovered to linear distribution, and the morphology of intercellular connections was close to that of the normal control. Western blot quantitative results ( Figure 6 Further confirmation showed that Rof, FK506 and Pdn could significantly enhance and restore the expression levels of desmosome structural proteins such as Dsg1, Dsg3 and PG in PV model cells (P<0.05).

[0131] 7. Apoptosis detection by flow cytometry

[0132] (1) Preparation of cell samples:

[0133] Digest cells with trypsin and collect the cell culture medium into a centrifuge tube. Centrifuge at approximately 1000×g for 3-5 minutes to pellet the cells. Carefully aspirate the supernatant, leaving approximately 30 μl of culture medium to avoid removing the cells. Add approximately 1 ml of pre-chilled PBS (4°C), resuspend the cells, centrifuge again to pellet the cells, and carefully aspirate the supernatant. Add another 1 ml of pre-chilled PBS (4°C), resuspend the cells, and centrifuge again to pellet the cells.

[0134] (2) Dilute Binding Buffer (4mL Binding Buffer + 12mL deionized water) with deionized water at a ratio of 1:3.

[0135] (3) Resuspend the cells with 250 μl Binding Buffer and adjust their concentration to 1 × 10⁶ / mL.

[0136] (4) Take 100 μl of cell suspension into a 5 mL flow cytometer and add 5 μl of Annexin V-PE and 10 μl of 7-AAD solution.

[0137] (5) After mixing, incubate at room temperature in the dark for 15 minutes.

[0138] (6) Add 400 μl PBS to the reaction tube and analyze by flow cytometry (FACS).

[0139] Flow cytometry detection ( Figure 7 The results showed that, compared with the control group, PV-IgG (1 mg / mL) stimulation significantly increased the early apoptosis rate of HaCaT cells (P<0.01). After intervention with Rof, FK506, and Pdn, the early apoptosis rate was significantly lower than that in the PV group (P<0.05). Western blot analysis of key proteins in the apoptosis pathway (…) Figure 8 The results showed that PV-IgG treatment significantly upregulated the protein expression of Caspase-3, Cleaved-Caspase-3, Caspase-7, Bax, and Fas, and downregulated the expression of Bcl-2. All drug intervention groups effectively inhibited the activation of Caspase-3 and Caspase-7, significantly reduced the Bax / Bcl-2 ratio, and inhibited Fas expression (P<0.05), confirming the anti-apoptotic mechanism of the drugs at the molecular level.

[0140] 2.8 ELISA kit for detecting TNF-α, IFN-γ, IL-6, and IL-8 expression

[0141] (1) Collect cell supernatant: After collecting the liquid, centrifuge at 1000×g for 20 minutes at 2-8℃ to remove impurities and cell debris. Take the supernatant for testing.

[0142] (2) Set up standard wells, blank wells, and sample wells separately. Add 100 μL of serially diluted standard to the standard wells.

[0143] Add 100 μL of standard and sample diluent to the blank wells and 100 μL of the sample to be tested to the remaining wells. Cover the microplate with a membrane and incubate at 37°C for 90 minutes.

[0144] (3) Discard the liquid in the wells, add 100 μL of biotinylated antibody working solution to each well, cover the plate with a membrane, and incubate at 37°C for 1 hour.

[0145] (4) Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350 μL of washing buffer to each well, soak for 1 minute, aspirate or shake off the liquid in the microplate, and pat dry. Repeat this washing step 3 times.

[0146] (5) Add 100 μL of HRP enzyme conjugate working solution to each well, cover the microplate with a membrane, and incubate at 37°C for 30 minutes.

[0147] (6) Shake off all the liquid in the hole and wash the plate 5 times.

[0148] (7) Add 90 μL of substrate solution (TMB) to each well, cover the microplate with a membrane, and incubate at 37°C in the dark for about 15 minutes. Stop the incubation when a clear gradient appears in the standard wells (a clear blue gradient appears in the first 4 wells).

[0149] (8) Add 50 μL of stop solution to each well to terminate the reaction.

[0150] (9) Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader and perform calculations.

[0151] Supernatants from each group of cells were collected after 48 hours of culture for ELISA detection. Figure 9 The results showed that, compared with the control group, the concentrations of inflammatory factors TNF-α, IFN-γ, IL-6, and IL-8 secreted by cells in the PV-IgG treatment group were significantly increased (P<0.001). After intervention with Rof, FK506, and Pdn, the expression levels of the above four pro-inflammatory factors were significantly reduced compared with the PV group (P<0.001), confirming that these drugs have a clear anti-inflammatory effect in the in vitro pemphigus model.

[0152] Example 2: Study on the therapeutic effect and mechanism of action of roflumilast in a pemphigus animal model through animal experiments.

[0153] 1. Establishment of a passive transfer model of pemphigus in mice

[0154] Newborn BALB / c mice were selected, ensuring clear provenance and good health. All animal experiments were conducted according to protocols approved by the institution's animal ethics committee and in accordance with animal welfare principles. Recombinant mouse Dsg3 protein was subcutaneously injected into newborn mice to induce the production of anti-Dsg3 antibodies.

[0155] Specific method: Purified recombinant murine Dsg3 protein was used as the antigen. Newborn mice (usually 1-3 days after birth) were subcutaneously injected with Dsg3 protein on their backs at a dose of 20 μg / day per mouse; q2w, immunized 3 times.

[0156] After the final immunization, mouse serum was collected, and the titer of anti-Dsg3 antibody was detected by ELISA to confirm the successful establishment of the passive metastasis model of pemphigus. Simultaneously, the skin manifestations of the mice were observed, such as the presence of pemphigus-like lesions like blisters and erosions.

[0157] Compared with healthy controls, newborn pemphigus mice in the pemphigus disease group, after receiving subcutaneous injection of recombinant mouse Dsg3 protein (20 μg / mouse), exhibited typical erythema, vesicles, and extensive erosions on their dorsal skin and mucous membranes. This phenotype successfully replicated the core clinical features of human pemphigus vulgaris, confirming the successful establishment of the passive transfer animal model. Figure 10 ).

[0158] 2. Experimental grouping and drug administration

[0159] Mice that successfully developed the model were randomly divided into the following groups:

[0160] (1) Healthy control group: injected with an equal volume of physiological saline or PBS.

[0161] (2) Pemphigus disease group (PV): No other intervention was performed, and the natural development process of the disease was observed.

[0162] (3) Pemphigus + Placebo group: The same dose of petroleum jelly as the treatment group was given.

[0163] (4) Pemphigus + oral hormone group (PV+poPdn): oral hormones were administered. According to the pemphigus treatment guidelines, the oral hormone dose for humans is 1 mg / kg / d. Based on body surface area, the oral dose for mice is 12.33 μg / g / d.

[0164] (5) Pemphigus + topical roflumilast group (PV+ext.Rof): Topical roflumilast ointment only.

[0165] (6) Pemphigus + oral hormones + topical roflumilast group (PV+poPdn+ext.Rof): On the basis of oral hormone therapy, topical roflumilast ointment was applied.

[0166] (7) Pemphigus + oral hormones + topical hormones group (PV+poPdn+ext.PdnPV+poPdn+ext.Pdn): In addition to oral hormone therapy, topical hydrocortisone cream was applied.

[0167] 3. Observation indicators and detection methods

[0168] During the experiment, the following indicators were observed and recorded regularly: Body weight: Mouse body weight was measured regularly to observe changes in body weight; Physical signs: Mouse mental state, activity level, and feeding status were observed; Appearance: Detailed records of mouse skin lesions were kept, including the number, size, and distribution of blisters, and the degree of erosion; Wound inflammation appearance score was used to score mouse skin lesions and assess the severity of the disease; Blood routine, liver and kidney function, and other toxicological and biochemical indicators; Serum samples were collected, and the expression levels of inflammatory factors IL-6, IL-8, TNF-α, and IFN-γ were detected using ELISA to assess the degree of inflammatory response in each group; Histopathological examination: HE staining: Mouse skin tissue was taken and HE staining was performed to observe morphological changes, including acantholysis and inflammatory cell infiltration; Immunofluorescence staining: Immunofluorescence staining was performed to detect the expression and distribution of specific proteins in skin tissue and analyze the disease mechanism; TUNEL apoptosis detection: TUNEL assay was used to detect cell apoptosis in skin tissue and assess the effect of drugs on cell apoptosis.

[0169] Appearance recordings and quantitative scores after 5 days of continuous treatment showed that, compared with the pemphigus disease group and the placebo group, the Rof treatment group mice had a significantly reduced number of new skin lesions, faster skin lesion healing, and a significantly lower wound inflammation appearance score (P<0.001). Furthermore, ELISA results indicated that Rof intervention significantly reversed the abnormally elevated anti-Dsg3 antibody titer in the serum of pemphigus model mice. Figure 11 ).

[0170] ELISA results showed that the expression levels of inflammatory factors such as TNF-α, IFN-γ, IL-6, and IL-8 in the serum of pemphigus disease mice were significantly higher than those in the healthy control group, suggesting that a strong inflammatory response was deeply involved in the disease process. After RoF intervention, the levels of the above-mentioned serum inflammatory factors were significantly lower than those in the pemphigus disease group and the placebo group (P<0.001), confirming that the drug has a potent anti-inflammatory effect in vivo. Figure 12 ).

[0171] 4. Hematoxylin-eosin staining (H&E)

[0172] (1) After the sample is fixed for 12 hours, rinse it with running water for 1 hour and place it in 75% ethanol for long-term preservation.

[0173] (2) Ethanol dehydration: The tissue was dehydrated stepwise with ethanol solutions of different concentrations: 70%, 80%, 90%, 95%, 100%, 100%, each ethanol solution for 40 min.

[0174] (3) Transparency: The tissue was soaked in three xylene solutions in sequence for 1 hour each.

[0175] (4) Wax immersion: The tissue was immersed in three paraffin baths in sequence for 1 hour each.

[0176] (5) Embedding: Pour liquid paraffin into the mold box, then place the tissue block soaked in paraffin flat at the bottom, making sure the cut surface is facing down. After the paraffin solidifies, remove the embedding frame. After it has completely cooled and hardened, trim the paraffin block. Leave an appropriate amount of paraffin around the tissue for sectioning.

[0177] (6) Sectioning: Fix the pre-cooled wax block on the paraffin microtome, making the cut surface of the wax block parallel to the blade. The blade tilt angle is usually 15°. Rotate the rotary feeder to adjust the section thickness to 4μm, cutting into sections of uniform thickness. Hold a brush in your left hand and rotate the microtome handle with your right hand. After the section is brought out, gently lift it with the brush and then gently tweeze it with tweezers, placing it face up in the slide box with water at about 45°C. After it is flattened, retrieve the section. Attach the section: Hold one end of the glass slide in your left hand and vertically immerse it in water to attach the section. Use tweezers in your right hand to assist in pushing it, attaching it to two-thirds of the way up the glass slide.

[0178] (7) Baking: After the slices are attached, let them air dry slightly, bake them on a 65℃ baking machine for 1 hour, and then bake them in an oven for 2 hours.

[0179] (8) Dewaxing: Paraffin sections were sequentially placed in xylene I for 10 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and 50% ethanol for 5 min for gradient dewaxing.

[0180] (9) HE staining: stain with hematoxylin for 1 min, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, then blue with 1% ammonia solution for 1 min, rinse with running water for a few seconds, stain with eosin solution for a few seconds, rinse with running water. After drying, take pictures under a microscope.

[0181] HE staining histopathological examination showed ( Figure 13In the pemphigus disease group, the skin tissue of mice showed obvious intraepidermal vesicle formation, acantholysis, and a large number of inflammatory cell infiltrations; while the epidermal structure of the roflumilast treatment group was relatively intact, the degree of vesicle and acantholysis was significantly reduced, and the inflammatory infiltration was significantly reduced.

[0182] 5. Immunohistochemistry and tissue immunofluorescence

[0183] Dehydration: The fixed tissue was removed and dehydrated by sequentially applying a series of ethanol solutions: 75% ethanol for 1 h → 85% ethanol for 1 h → 90% ethanol for 1 h → 95% ethanol for 1 h → anhydrous ethanol for 1 h → anhydrous ethanol for 1 h → xylene I for 30 min → xylene II for 30 min → paraffin I for 1 h → paraffin II for 1 h → paraffin III for 1 h.

[0184] (2) Embedding: Place the molten paraffin into the embedding frame. Before the paraffin solidifies, remove the tissue from the dehydration box, place it into the embedding frame according to the embedding requirements, and affix the corresponding label. Place it on a -20℃ freezing table to cool. After the paraffin block solidifies, remove it from the embedding frame and trim it. Place the embedded paraffin block in a 4℃ refrigerator for setting, and determine the setting time based on subsequent experiments.

[0185] (3) Sectioning: After 10 hours of curing, remove the sections from the 4℃ refrigerator and fix them on the microtome. First, coarsely cut a 1mm thick paraffin section to determine the tissue section surface, and then finely cut it into 5μm paraffin sections. Use small tweezers to place the cut sections into 45℃ constant temperature water to spread them out. After the sections are spread out flat, use a glass slide to take the sections out of the water and let them air dry naturally before baking them in a 37℃ oven.

[0186] (4) Dewaxing and hydration of sections: Before dewaxing, the sections were baked in an oven at 60°C for 20 minutes; the sections were immersed in fresh xylene for 5 minutes, repeated 3 times; the sections were immersed in anhydrous ethanol for 5 minutes, repeated 2 times; the sections were immersed in 95% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes in sequence, and rinsed with PBS 3 times, 5 minutes each time.

[0187] (5) Antigen retrieval: After the 1×EDTA antigen retrieval solution is heated to boiling in the microwave, the slide is immersed in the solution and the temperature is set to low for 15 minutes. After the slide is removed and cooled naturally, it is washed three times with PBS for 5 minutes each time, and the slide is dried with absorbent paper.

[0188] (6) Blocking endogenous peroxidase: Place the slices in 3% hydrogen peroxide solution and incubate at room temperature for 10 min to block endogenous peroxidase; then wash with PBS 3 times for 5 min each time and wipe dry.

[0189] (7) Antibody hybridization: 5% BSA was added to the tissue of each slide and the slide was blocked at room temperature for 20 min. The BSA solution was removed and 50 μl of diluted antibody (dilution ratio: 1:100) was added to each slide and the slide was incubated overnight at 4°C. After the slide was removed from 4°C, it was placed at room temperature for 45 min to warm up. The slide was washed 3 times with PBS for 5 min each time. The PBS was removed and 50-100 μl of diluted secondary antibody was added to each slide and the slide was incubated at room temperature for 30 min. The slide was washed 3 times with PBS for 5 min each time. The PBS was removed and 50-100 μl of freshly prepared DAB solution was added to each slide and the color development was observed under a microscope. After the color development was complete, the slide was rinsed with distilled water to stop the color development.

[0190] (8) Hematoxylin counterstaining: Place the sections in hematoxylin for counterstaining, observe under a microscope and control the staining time; place the stained sections in 1% hydrochloric acid alcohol for differentiation (1s), and rinse with tap water.

[0191] (9) Dehydration and clearing: Immerse the sections in 75% ethanol for 10 min → 85% ethanol for 10 min → 95% ethanol for 10 min → anhydrous ethanol for 10 min in sequence to dehydrate; take out the sections and put them in xylene for 5 min, repeat 3 times.

[0192] (10) Mounting and photographing: Use neutral resin to mount the slides, and be careful not to generate air bubbles; observe and photograph under an optical microscope.

[0193] (11) Immunofluorescence: Cover the tissue with 5% BSA and block at room temperature for 20 min. Remove the blocking solution, add 1 ml of primary antibody (diluted according to the instructions) and incubate overnight at 4°C; recover the primary antibody and wash 3 times with TBST, 5 min each time. Add 1 ml of secondary antibody (diluted according to the instructions) and incubate on a shaker (50 rpm) at room temperature in the dark for 1 hour; discard the secondary antibody and wash 3 times with TBST, 5 min each time. After staining with DAPI for 5 minutes, remove the DAPI and wash 3 times with TBST, 5 min each time; fix the slide with Fluoroshield®™ containing 4′,6-diamino-2-phenylindole (DAPI) and photograph under a fluorescence microscope.

[0194] (12) Use ImageJ software to analyze fluorescence intensity.

[0195] 6. TUNEL staining for apoptosis detection

[0196] After washing each group of treated tissues once with PBS, fix them with 4% paraformaldehyde for 30 minutes. After fixation, wash with PBS and incubate with 0.3% Triton X-100 at room temperature for 5 minutes.

[0197] Table 3: Preparation of TUNEL test solution

[0198]

[0199] Wash twice with PBS, add 100 μl of TUNEL detection solution to the sample, and incubate at 37ºC in the dark for 60 minutes. Wash three times with PBS, observe and photograph under a fluorescence microscope.

[0200] Immunohistochemistry and TUNEL staining further indicated that ( Figure 14 In the pemphigus group of mice, the expression of apoptotic proteins surged at the skin lesions, and the number of TUNEL-positive cells increased significantly; roflumilast treatment significantly reduced the occurrence of apoptosis, confirming that it plays a key protective role in vivo by inhibiting keratinocyte apoptosis.

[0201] 7. RNA-seq suggests that endoplasmic reticulum stress may be involved in the development and progression of pemphigus.

[0202] RNA-seq sequencing was performed on skin lesions from a pemphigus model and healthy skin controls. The results showed a large number of differentially expressed genes between the two groups. Further pathway enrichment analysis revealed that signaling pathways related to endoplasmic reticulum stress (ERS) and unfolded protein responses were significantly activated in the pemphigus group. Volcano plot analysis showed that ERS-related genes (such as Hspa8) were upregulated in the PV group. GSEA results confirmed the upregulation of the ERS pathway in the PV group, with NES values ​​greater than 1. These genomic results suggest that the ERS pathway is deeply involved in the occurrence and development of pemphigus. Figure 15 ).

[0203] Example 3: Clinical Validation

[0204] 1. Abnormally elevated levels of intracellular plasma reticulum stress marker proteins in skin lesions of pemphigus patients.

[0205] Immunohistochemistry (IHC) was used to detect the expression of endoplasmic reticulum stress core marker proteins (CHOP, ATF4, and IRE1) in skin lesions and surrounding normal skin tissue of clinical PV patients. The results showed that normal control skin epithelial cells showed almost no expression or only scattered weak expression in the basal layer, while the expression of these endoplasmic reticulum stress marker proteins was significantly increased in the epithelial cells of PV lesion tissue. Figure 16 ).

[0206] 2. Increased expression of apoptosis proteins in the skin lesions of clinical patients with pemphigus.

[0207] Immunohistochemistry (IHC) was used to detect the expression of apoptosis marker proteins (Bax, Caspase 3) in skin lesions of clinical PV patients and normal skin tissue. The results showed that normal control skin epithelial cells showed almost no expression or only scattered weak expression, while the expression of apoptosis proteins was significantly increased in the epithelial cells of PV lesion tissue. Figure 17 ).

[0208] All experimental data were statistically analyzed using SPSS software, and the gray values ​​of Western blot bands were measured using ImageJ software. Quantitative data were expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA or t-tests were used for comparisons between groups. If the quantitative data conformed to a normal distribution, a chi-square test was used. A p-value < 0.05 was considered statistically significant.

[0209] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. The application of roflumilast in the preparation of drugs for the treatment of pemphigus, characterized by: The drug is used to downregulate endoplasmic reticulum stress and inhibit keratinocyte apoptosis.

2. The use of roflumilast according to claim 1 in the preparation of a pemphigus treatment drug, characterized in that: The drug significantly reduces the apoptosis rate of pemphigus-induced keratinocytes by downregulating the expression of endoplasmic reticulum stress marker proteins and inhibiting the Caspase family and Fas-mediated pro-apoptotic cascade.

3. The use of roflumilast according to claim 2 in the preparation of a pemphigus treatment drug, characterized in that: The endoplasmic reticulum stress marker proteins are CHOP, ATF4, and IRE1.

4. The use of roflumilast according to claim 2 in the preparation of a pemphigus treatment drug, characterized in that: The Caspase family includes Caspase-3, Cleaved-Caspase-3, and Caspase-7.

5. The use of roflumilast according to claim 4 in the preparation of a pemphigus treatment drug, characterized in that: The drug significantly upregulated the protein expression of Caspase-3, Cleaved-Caspase-3, Caspase-7, Bax, and Fas, and downregulated the expression of Bcl-2.

6. The use of roflumilast according to claim 1 in the preparation of a pemphigus treatment drug, characterized in that: The drug can significantly enhance and restore the expression levels of desmosome structural proteins, including Dsg1, Dsg3, and PG, in cells.

7. The use of roflumilast according to claim 6 in the preparation of a pemphigus treatment drug, characterized in that: The drug inhibits the secretion of inflammatory factors TNF-α, IFN-γ, IL-6, and IL-8.

8. The use of roflumilast according to claim 7 in the preparation of a pemphigus treatment drug, characterized in that: Roflumilast is the only active ingredient in the drug.

9. The use of roflumilast according to claim 8 in the preparation of a pemphigus treatment drug, characterized in that: The drug may be used alone or in combination with an oral hormone, specifically prednisone.

10. The use of roflumilast according to claim 9 in the preparation of a pemphigus treatment drug, characterized in that: The drug is administered via topical application.

11. The use of roflumilast according to claim 9 in the preparation of a pemphigus treatment drug, characterized in that: The drug is a topical dosage form.

12. The use of roflumilast according to claim 9 in the preparation of a pemphigus treatment drug, characterized in that: The topical dosage form is a semi-solid dosage form.

13. The use of roflumilast according to claim 9 in the preparation of a pemphigus treatment drug, characterized in that: The semi-solid dosage form is a cream.

Images