Use of inhibitors of lactic acid modification of lysine 249 of hsp60 protein

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of an inhibitor for inhibiting lactic acid modification of lysine at the 249th position of HSP60 protein. The application provides application of the inhibitor for inhibiting lactic acid modification of lysine at the 249th position of HSP60 protein in preparation of a medicine for preventing and / or treating psoriasis, and an amino acid sequence of the HSP60 protein (heat shock protein 60) is shown as SEQ ID NO. 1. The application provides a brand-new, mechanism-driven psoriasis treatment target, lactic acid modification of lysine at the 249th position of HSP60 protein. By inhibiting the specific modification, the purpose of precise intervention and treatment from an upstream of the disease is achieved by stabilizing mitochondrial function and blocking intrinsic immune activation triggered by metabolic stress.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of an inhibitor that inhibits the lactation modification of lysine at position 249 of the HSP60 protein. Background Technology

[0002] Psoriasis is a skin disease associated with immune activation. Its pathological mechanism involves complex interactions between the innate and adaptive immune systems. Histologically, it is characterized by excessive proliferation and abnormal differentiation of keratinocytes (KCs), dermal inflammatory infiltration, and angiogenesis. Psoriasis has a high incidence rate, with a global prevalence of 2%–3%, showing an increasing trend annually. Its recurrence rate can be as high as 98.4%, and it is often accompanied by chronic diseases, disfigurement, disability, and related complications, severely impacting and burdening the physical and mental health of patients. Currently, in recent years, biologics, represented by TNF-α and IL-17 antibodies, have been increasingly used in the clinical treatment of moderate to severe psoriasis, achieving good results. However, these biologics still have limitations in therapeutic efficacy and a high recurrence rate. Summary of the Invention

[0003] To address the limitations of existing biological agents used in the clinical treatment of psoriasis, which suffer from limited efficacy and high relapse rates, this invention aims to provide a novel strategy for the prevention or treatment of psoriasis. Its core lies in the first-time proposal and validation of "inhibiting the lactation modification of lysine at position 249 of heat shock protein 60 (HSP60)" as an effective means of treating psoriasis, and provides the application of an inhibitor that inhibits the lactation modification of lysine at position 249 of the HSP60 protein. To achieve the above objective, this invention adopts the following technical solution.

[0004] The first objective of this invention is to provide an inhibitor that inhibits the lactation modification of lysine at position 249 of the HSP60 protein in the preparation of a medicament for the prevention and / or treatment of psoriasis, wherein the amino acid sequence of the HSP60 protein (heat shock protein 60) is shown in SEQ ID NO.1 (searched in the Uniprot database by ID number P10809).

[0005] The inhibitor is a targeted inhibitor that acts directly on the K249 site of the HSP60 protein.

[0006] This invention reveals a complete "metabolic-mitochondrial-immune" signaling pathway that plays a crucial role in the pathogenesis of psoriasis: In psoriatic lesions, high expression of lactate dehydrogenase A (LDHA) leads to intracellular lactate accumulation, which in turn triggers lactation modification of the mitochondrial protein HSP60 at lysine position 249. This modification directly weakens the binding of HSP60 to the mitochondrial membrane pore regulatory protein CypD, resulting in abnormal opening of the mitochondrial membrane permeability transition pore, releasing mitochondrial DNA into the cytoplasm, subsequently activating the cGAS-STING innate immune pathway, and ultimately driving a persistent inflammatory response. Any method or substance capable of inhibiting lactation modification at position K249 of the HSP60 protein can be used to prepare drugs for the prevention or treatment of psoriasis.

[0007] This invention targets and inhibits the lactation modification of lysine at position 249 of the HSP60 protein, thereby intervening in the core pathological mechanism of psoriasis at the level of metabolic and immune interaction, thus solving the problem of easy relapse with existing biological agents. While existing biological agents such as TNF-α, IL-17, and IL-23 inhibitors can effectively block inflammatory signaling pathways, they mainly act on the regulation of immune cell activity and are difficult to eliminate skin-resident memory T cells (TRM) and keratinocytes (EpSCs) with inflammatory memory. Furthermore, they cannot correct the abnormally enhanced glycolysis in the lesion site leading to lactic acid accumulation and its mediated lactation and other epigenetic modifications. Therefore, the median relapse time after drug withdrawal is only a few months. In contrast, HSP60 is pathologically highly expressed in psoriatic lesions and promotes abnormal proliferation of keratinocytes. The lactation modification of its lysine 249 position is a key functional regulatory site involved in maintaining the inflammatory microenvironment and metabolic reprogramming. This modification promotes the opening of the mitochondrial membrane permeability transition pore by weakening the binding of HSP60 to mitochondrial membrane porin CypD, leading to the release of mitochondrial DNA and activation of the cGAS-STING innate immune pathway, thereby driving a persistent inflammatory response. This invention intervenes at a key upstream node of metabolic stress and immune activation by specifically inhibiting lactation at this site. This can stabilize mitochondrial function and reduce mtDNA leakage, thereby blocking the vicious cycle of "metabolic abnormality-mitochondrial damage-immune activation". This strategy directly targets the early stage of psoriasis pathogenesis, rather than just inhibiting downstream inflammatory signals. Therefore, it has the potential to overcome the clinical challenges of limited treatment efficacy and easy relapse after drug withdrawal of existing therapies.

[0008] Furthermore, the inhibitor is selected from at least one of the following: Direct inhibitors: Substances used to directly block the transfer of lactate groups to the K249 site of the HSP60 protein or to competitively occupy this modification site.

[0009] Indirect inhibitors: substances used to reduce lactation modification at the K249 site of the HSP60 protein by lowering intracellular lactate concentration.

[0010] Functional substitutes: substances used to mimic the non-lactated state or enhance the binding stability of HSP60 to cyclophilic protein D to counteract the harmful effects of lactation.

[0011] Furthermore, the direct inhibitor is selected from small molecule compounds, targeting peptides, antibodies or their antigen-binding fragments, or nucleic acid aptamers.

[0012] Furthermore, the indirect inhibitor is a lactate dehydrogenase A (LDHA) inhibitor, used to inhibit upstream lactate production.

[0013] Furthermore, the functional substitute is selected from: The HSP60 K249R mutant mimics the non-lactated state.

[0014] A molecule that enhances the stability of HSP60 binding to cyclic protein D (CypD).

[0015] Furthermore, the inhibitors include lactate dehydrogenase A (LDHA) inhibitors.

[0016] Furthermore, the lactate dehydrogenase A (LDHA) inhibitor indirectly inhibits lactation modification at the K249 site of the HSP60 protein by reducing lactate levels.

[0017] Furthermore, the lactate dehydrogenase A (LDHA) inhibitor is selected from any one or more of GSK2837808A, AZ-33, sodium oxamate, FX-11, LDHA-IN-3 (compound 2), (R)-GNE-140, and Galloflavin.

[0018] Furthermore, the drug is a pharmaceutical composition and also includes a DHA inhibitor.

[0019] Furthermore, the DHA inhibitor is a topical administration formulation or a keratinocyte-specific delivery formulation.

[0020] Furthermore, the DHA inhibitor is a small molecule compound, siRNA, shRNA, or a CRISPR gene editing system.

[0021] Furthermore, the nucleotide sequence of the siRNA is shown in SEQ ID NO.2.

[0022] Furthermore, the nucleotide sequence of the shRNA is shown in SEQ ID NO.3.

[0023] Furthermore, the drug is a pharmaceutical composition that also includes a cGAS-STING pathway inhibitor.

[0024] A second objective of this invention is to provide a medicament for the prevention and / or treatment of psoriasis, wherein the active ingredient of the medicament is a lactate dehydrogenase A inhibitor, as well as a DHA inhibitor and / or a cGAS-STING pathway inhibitor.

[0025] Furthermore, the drug also includes vitamin D analogs or glucocorticoids for topical treatment of psoriasis.

[0026] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides the application of an inhibitor that inhibits the lactation modification of lysine at position 249 of the HSP60 protein in the preparation of drugs for the prevention and / or treatment of psoriasis. The target provided by this invention has high specificity and a well-defined mechanism, allowing intervention at the upstream stages of psoriasis pathogenesis, thereby achieving a more fundamental therapeutic effect and reducing the risk of recurrence. By targeting and inhibiting the lactation modification of lysine at position 249 of the HSP60 protein, this invention can intervene in the core pathological mechanism of psoriasis at the metabolic and immune interaction level, thus solving the problem of easy recurrence with existing biologics. While existing biologics such as TNF-α, IL-17, and IL-23 inhibitors can effectively block inflammatory signaling pathways, they primarily regulate the activity of immune cells and are unable to eliminate skin-resident memory T cells (TRMs) and ephemeral keratinocytes (EpSCs) with inflammatory memory. Furthermore, they cannot correct the abnormally enhanced glycolysis at the lesion site leading to lactic acid accumulation and its mediated lactation and other epigenetic modifications. Therefore, the median relapse time after drug withdrawal is only a few months. This study demonstrates that HSP60 expression is significantly increased in psoriatic lesions, and the lactation modification of lysine at position 249 is a key molecular event regulating keratinocyte proliferation, mitochondrial function, and the maintenance of the inflammatory microenvironment. This modification promotes the opening of the mitochondrial membrane permeability transition pore by weakening the binding of HSP60 to mitochondrial membrane porin CypD, leading to the release of mitochondrial DNA and activation of the cGAS-STING innate immune pathway, thereby driving a persistent inflammatory response. This invention intervenes at a key upstream node of metabolic stress and immune activation by specifically inhibiting HSP60 K249 lactation. This stabilizes mitochondrial function and reduces mtDNA leakage, thereby breaking the vicious cycle of "metabolic abnormality-mitochondrial damage-immune activation." This strategy directly targets the early stages of psoriasis pathogenesis, rather than merely inhibiting downstream inflammatory signals, and therefore has the potential to overcome the clinical challenges of limited efficacy and high relapse rates after discontinuation of existing therapies.

[0028] 2. Compared with existing psoriasis treatment technologies, this invention has the following significant advantages and beneficial effects: (1) Source intervention with better therapeutic potential: Existing therapies mostly target downstream inflammatory factors, while this invention targets the "molecular switch" that connects metabolic abnormalities and immune activation upstream, which is expected to intervene at an earlier stage of disease occurrence, achieve more fundamental treatment, and reduce the recurrence rate.

[0029] (2) Precise target and fewer potential side effects: This invention targets a specific post-translational modification site that occurs under pathological conditions, rather than the widely expressed protein itself. This precision helps to design highly selective drugs, avoids widespread interference with normal physiological functions, and thus improves treatment safety.

[0030] (3) Clear mechanism to guide drug development: We have elucidated the complete molecular pathway from target to therapeutic effect (HSP60 K249la→mPTP→mtDNA→cGAS-STING), which provides clear biomarkers and validation systems for drug screening (such as establishing screening models based on HSP60-CypD binding) and efficacy evaluation (such as detecting cytoplasmic mtDNA).

[0031] (4) Providing novel treatment strategies and combination therapy possibilities: This target opens up a new direction for the development of psoriasis drugs. At the same time, based on the upstream and downstream relationships of this pathway, it can be used in combination with LDHA inhibitors or cGAS-STING inhibitors to form a synergistic treatment plan.

[0032] 3. The technical solution of this invention is: any substance or method capable of specifically inhibiting lactation modification at the K249 site of the HSP60 protein can be used to prepare a drug for the prevention or treatment of psoriasis. The "inhibition" includes, but is not limited to:

[0033] (1) Direct inhibition: Directly block the transfer of lactate groups to the K249 site of HSP60 protein through small molecule compounds, targeting peptides, antibodies or their fragments, nucleic acid aptamers, etc., or competitively occupy the modification site.

[0034] (2) Indirect inhibition: By inhibiting the activity or expression of LDHA, a key enzyme in upstream lactate production, the intracellular lactate concentration is reduced, thereby reducing the occurrence of this lactation modification from the root.

[0035] (3) Functional substitution: The harmful effects of lactation can be counteracted by introducing HSP60 mutants that simulate the non-lactated state (such as K249R) or molecules that enhance the binding stability of HSP60 and CypD.

[0036] 4. The purpose of this invention is to provide a novel, mechanism-driven lactation modification of lysine 249 of the HSP60 protein, a therapeutic target for psoriasis. By inhibiting this specific modification, the aim is to stabilize mitochondrial function and block innate immune activation triggered by metabolic stress, thereby achieving precise intervention and treatment from the upstream of the disease. Attached Figure Description

[0037] Figure 1 In this invention, lactic acid promotes mPTP opening and mtDNA release; wherein: a) promotes mPTP opening with added lactic acid; b represents a relative quantitative analysis of a; c indicates that the mPTP opening level is higher in HEKa cells overexpressing LDHA than in NC cells; d represents a relative quantitative analysis of c; e represents the kit detection of mPTP opening level. The results show that increasing lactate (LDHA overexpression or exogenous LA) can increase mPTP opening. f is a schematic diagram showing that mPTP opening leads to increased mtDNA release from mitochondria and a decrease in mitochondrial membrane potential. g represents the mitochondrial membrane potential of the JC-1 control group (NC), psoriasis cell model group (M5), LDHA overexpressing cells, LDHA knockdown cells, and cells with added LA intervention; membrane potential changes are shown in monomeric and aggregate forms, scale bar = 200µm. h represents a quantitative analysis of the membrane potential changes in g, where increases in M5 and lactate both lead to a decrease in membrane potential. i represents the detection of mtDNA release in LDHA-knocked cells and under the addition of LA; j represents the detection of mtDNA release in LDHA-overexpressing cells; K~m represent the cGAS-STING-NF-κB signal transduction status under different treatments (LDHA knockdown / overexpression, addition of M5 or lactate) detected by Western blot.

[0038] Figure 2 This is the identification process of the key target HSP60 K249 lactation in this invention; wherein: a is the experimental flowchart, which includes protein extraction, enzymatic digestion, labeling and enrichment of LDHA-overexpressing cells and control HEKa cells, and lactation mass spectrometry detection and analysis. b is a volcano plot showing protein changes in LDHA-overexpressing cells and control HEKa cells as detected by lactation mass spectrometry. c represents the Western blot analysis of changes in pan-lactic acidification (L-lactylation) in LDHA-overexpressing cells and control cells; d represents the GO function analysis of differentially expressed proteins identified in LDHA-overexpressing cells and control HEKa cells; e represents the subcellular localization of differentially expressed proteins identified in LDHA-overexpressing cells and control HEKa cells, including mitochondrial localization, nuclear localization, and membrane localization; f shows the localization of mitochondria (TOMM20 label, green) and pan-lactylation (L-lactylation, red) detected by immunofluorescence staining, with a scale bar of 50µm; g is a volcano plot focusing on mitochondrial histones among the differentially expressed proteins identified in (e); h represents a list and scores of lactotropic proteins that show significant changes in mitochondria in LDHA-overexpressing cells and control HEKa cells. i represents GO analysis, which is the GO analysis of proteins that are significantly upregulated in the mitochondria of LDHA-overexpressing cells compared to control cells.

[0039] Figure 3 This invention aims to identify all lactation sites of HSP60; wherein: a is an identification map of lactation at each lysine site, including K72, K125, K133, K156, K202, K249 / 250, and K523; b represents the co-ip results, which showed a significant increase in HSP60 lactation in LDHA-overexpressing cells. Data are presented as mean ± standard error (n = 3 independent biological samples), * p <0.05,** p <0.01, *** p <0.001, no statistically significant difference in ns, two-tailed t-test / two-way ANOVA combined with Bonferroni correction;

[0040] c shows the immunofluorescence staining of skin tissue sections from healthy controls (Normal) and psoriasis patients (Psoriasis), namely DAPI (nuclei, blue), HSP60 (red), pan-lactic acidification (L-lactylation, yellow), and fused Merge images, with a scale bar of 100µm.

[0041] Figure 4 In this invention, the HSP60 K249 mutation delactation can prevent the weakening of HSP60 binding to CypD; wherein: a is a schematic diagram of the components of mPTP, including ATP synthase, CypD, Ant and Pic, among which HSP60 can reduce the opening of mPTP by antagonizing CypD; b shows that in LDHA-overexpressing HEKa cells, the binding degree of HSP60 to CypD is reduced; c represents the GSE151177 single-cell dataset (data from the GEO database) used in quasi-time series analysis. Ldha Quantify changes in HSP60 (HSPD1) and CypD (PPIF) in single-cell data of dorsal skin tissue from epidermal-specific knockout mice. d is a schematic diagram of the docking between HSP60 and CypD; e is a schematic diagram of constructing a stable HEKa cell line overexpressing HSP60 at different sites; f shows the HSP60 co-IP results of stable cells with mutations at 8 different sites; g is used to detect mtDNA release in HEKa cells with LDHA knockdown or overexpression and cells treated with M5 by immunofluorescence staining. dsDNA is marked in green and TOMM20 is marked in red (mitochondria). The scale bar is 50 μm. h represents the detection of four HSP60 mutant cell lines using Duolink@PL neighbor-to-neighbor connectivity technology. K133R HSP60 K156R HSP60 K249 HSP60 K523 ) and control HSP60 WT After treating cell lines with M5 and LA lactate, respectively, the interaction between HSP60 and CypD was examined. I~k represent the quantitative analysis of the immunofluorescence results in h, respectively; Data are presented as mean ± standard error (n = 3 independent biological samples), * p <0.05,** p <0.01, *** p <0.001, no statistically significant difference in ns, two-tailed t-test / two-way ANOVA combined with Bonferroni correction.

[0042] Figure 5 This invention provides in vivo efficacy validation of targeting HSP60 K249; wherein: a is the experimental flowchart: WT wild-type mice were selected and injected with Hspd1 (HSP60) WT type and Mut type (i.e. K249R mutant) AAV virus, respectively. After 2 weeks, IMQ-induced psoriasis model was used for verification and detection. b is AAV-Hspd1 fused with GFP (green fluorescent protein). Fluorescence microscopy of skin tissue sections showed green fluorescence, proving that it was successfully expressed. c shows representative photos of the skin on the backs of mice in each group from day 1 to day 5; d represents the PASI assessment of the skin lesion status on the back of mice in each group; e represents the change in body weight of mice in each group; f represents Hspd1 after IMQ stimulation. WT and Hspd1 Mut Comparative photographs of spleen size in mice; multiple samples in each row are parallel samples; g represents the spleen weight and weight ratio relative to body weight of mice in each group; h represents the H&E staining of the skin on the back of mice in each group. h represents the Hspd1 levels detected by flow cytometry after IMQ stimulation. WT and Hspd1 Mut Changes in the number of Treg cells in mouse spleen; i represents the Hspd1 levels after IMQ stimulation, measured by flow cytometry. WT and Hspd1 Mut Changes in the number of Th17 cells in mouse spleen. Data are presented as mean ± standard error (n = 6 independent biological samples), * p <0.05,** p <0.01, *** p <0.001, no statistically significant difference in ns, two-tailed t-test / two-way ANOVA combined with Bonferroni correction.

[0043] Figure 6 This is a schematic diagram of an alternative embodiment of the present invention; wherein: a is a small molecule inhibitor (such as a specific compound) that binds near the K249 site of the HSP60 protein to prevent lactation; b is a monoclonal antibody that specifically binds to lactated HSP60 to neutralize its function or label it for clearance. c is siRNA or shRNA downregulating LDHA mRNA, reducing lactate production; d is a stable peptide that enhances the interaction between HSP60 and CypD, resisting dissociation caused by lactation. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0045] Example 1 1. Construction of a cell model of psoriasis HEKa cells were stimulated for 24 h with M5 (IL-1α, IL-17A, IL-22, Oncostain M, and TNF-α) to simulate the inflammatory environment of psoriasis. The working concentration of M5 was 10 ng / mL. HEKa cells were seeded in 6-well plates, and after simultaneous adhesion, complete culture medium containing M5 was added for 24 h. The viability of HEKa cells was observed, and proteins and RNA were extracted from the cells for subsequent Western blotting and qRT-PCR experiments.

[0046] M5 is sourced from PeproTech.

[0047] HEKa cells are derived from the healthy human epidermal keratinocyte cell line HEKa (ATCC).

[0048] 2. mPTP Openness Detection The degree of mPTP opening was assessed using a mitochondrial permeability transition pore assay kit and by observing the degree of mitochondrial swelling. The kit method was performed according to the instructions.

[0049] Adding the fluorescent probe Calcein AM, a nonpolar dye, to HEKa cells allows for fluorescent staining of live cells. Calcein AM enters the cell via passive transport and accumulates in cytoplasmic components, including mitochondria. Intracellularly, Calcein AM is hydrolyzed by esterases to remove methyl acetyl, generating the non-membrane-permeable polar fluorescent dye Calcein (or Fluorexon). This causes Calcein to remain within the cell and mitochondria, resulting in strong green fluorescence in the cytoplasm, including mitochondria. CoCl2 is then added to quench the green fluorescence of Calcein in the cytoplasm. Under normal circumstances, the mitochondrial mPTP is closed, preventing CoCl2 from entering the mitochondria. Therefore, in normal cells, only Calcein in the cytoplasm is quenched. However, in pathological conditions, the mPTP is partially open, allowing CoCl2 to enter the mitochondria and quench Calcein thereto as well. The green fluorescence signal of Calcein is detected using fluorescence microscopy, and the intensity of the fluorescence signal is used to determine the degree of mPTP opening (e.g., ...). Figure 1 (e in the text).

[0050] The degree of mitochondrial swelling was determined at 25°C under de-energized conditions, and was assessed by monitoring the decrease in light scattering caused by mitochondrial swelling (recording the change in absorbance at 520 nm). In the experimental system, mitochondria were incubated for 2 minutes in a buffer (pH 7.2) at a concentration of 0.2 mg protein / ml. The buffer composition was as follows (in mmol / L): 150 KSCN, 20 Mops, 10 Tris, and 2-azotriacetic acid, supplemented with 2 µmol / L A23187, 0.5 µmol / L rotenone, and 0.5 µmol / L antimycin A. Mitochondrial swelling was initiated by adding 0.91 mmol / L CaCl2 to achieve a free [Ca²⁺] concentration of 80 µmol / L in the buffer system. The results are as follows. Figure 1 a~ Figure 1 As shown in d.

[0051] 3. Extraction and Western blot detection of total cellular protein (1) Extraction of total cellular protein a) Protein extraction was performed on HEKa cells (LDHA overexpression, knockdown, and control) after they reached 80% confluence. RIPA lysis buffer and PMSF (phenylmethylsulfonyl fluoride) were mixed thoroughly in advance at a volume ratio of 100:1 and placed on ice.

[0052] b) Add 0.5 mL of lysis buffer to each 10 cm dish and lyse on ice for 10 min.

[0053] c) Centrifuge at 4℃ and 12000 rpm for 15 min, carefully aspirate the supernatant, and transfer it to another EP tube to obtain the total cell protein solution.

[0054] d) BCA method for protein concentration determination: Mix reagent A and reagent B at a volume ratio of 50:1. Add 98 μL to each well of a 96-well plate, followed by 2 μL of the extracted protein and standard protein (0 μg / μL, 0.25 μg / μL, 0.5 μg / μL, 1 μg / μL, and 2 μg / μL). Mix thoroughly by shaking on a shaker, then incubate at 37°C for 30 min. Finally, measure the OD using a spectrophotometer. 562nm The protein concentration was calculated based on the standard curve.

[0055] e) Add 1 / 4 volume of 5× loading buffer to the protein, boil at 95℃ for 10 min, and then store at -20℃ for later use.

[0056] (2) SDS-PAGE electrophoresis a) Prepare the lower gel for polyacrylamide gel electrophoresis. After the gel solidifies, pour in the upper gel, insert a clean and dry comb to ensure there are no air bubbles, and let it stand for 30 minutes to allow the stacking gel to polymerize.

[0057] b) Fix the gel in the protein electrophoresis tank, adjust the loading amount according to the total cell protein concentration to ensure that each well contains the same amount of protein (20 μg), and add 5 µL of pre-stained protein molecular weight marker to determine the molecular weight of the cell protein to be tested.

[0058] c) Electrophoresis: After adding the sample, electrophoresis at 15mA for 30 min, then at 25mA for about 60 min, until the bromophenol blue reaches the bottom of the separating gel and then electrophoresis is stopped.

[0059] d) Remove the gel and place it in the transfer buffer.

[0060] (3) Transfer membrane a) Prepare two sheets of filter paper and one PVDF membrane, with a size similar to that of the SDS-PAGE gel.

[0061] b) The PVDF membrane needs to be soaked in methanol for 1 minute first, and then soaked in transfer buffer for later use.

[0062] c) Place filter paper-gel-PVDF membrane-filter paper in sequence from cathode to anode on the transfer device, ensuring there are no air bubbles between each layer.

[0063] d) Constant current 230mA for 90min for membrane transfer.

[0064] (4) Closed The PVDF membrane was sealed at room temperature for 1 hour with a sealing solution of 5% (m / v) skim milk powder prepared with 1×TBST.

[0065] (5) Antibody incubation a) Cut the PVDF membrane into strips of appropriate width according to different molecular weights.

[0066] b) After rinsing the PVDF membrane with 1×TBST, add the primary antibody prepared to the required concentration and incubate overnight at 4°C. "Overnight" refers to a time ≥12 hours.

[0067] c) Recover the primary antibody and rinse the PVDF membrane three times with 1×TBST for 10 min each time.

[0068] d) Add the secondary antibody diluted 1:3000 in 1×TBST and incubate at room temperature for 1 h.

[0069] e) Rinse the membrane three times with 1×TBST for 10 minutes each time, then drain the liquid.

[0070] (6) Development (operated in a darkroom) a) Take equal volumes of chemiluminescent reagent A and B, mix them thoroughly before use to obtain the luminescent solution.

[0071] b) Spread the prepared luminescent solution evenly on the PVDF membrane. After reacting for 1 minute, blot the luminescent solution with filter paper, place the film, and adjust the exposure time from 2 seconds to 1 minute according to the exposure effect of different protein bands.

[0072] c) Remove the film, immerse it in the developing solution, rinse with water, fix it, and then scan and analyze it after drying.

[0073] (7) Membrane regeneration strip method a) The PVDF film after luminescence is rinsed with 1×TBST for 5 min.

[0074] b) Add 2 mL of membrane regeneration solution and incubate at room temperature for 30 min.

[0075] c) Rinse with 1×TBST for 5 minutes for 3 times.

[0076] d) Re-block, incubate with antibody, and develop, as in steps (4), (5) and (6).

[0077] This section detected LDHA, cGAS, STING, p-NF-κB / NF-κB, L-lactylation, CypD, HIS-tagged proteins, and the nuclear internal control Histone H3, as well as the total internal controls Actin, α-TUBULIN, or GAPDH.

[0078] 4. Extraction of total RNA from cells, reverse transcription into cDNA, and qRT-PCR. (1) Extraction of total RNA from cells a) HEKa cells (LDHA overexpression, knockdown, and control) were cultured in six-well plates to 80% confluence. Pipe tips, pipette boxes, EP tubes, etc., used in the experiment were soaked in DEPC overnight to remove RNase.

[0079] b) Discard the culture medium in the six-well plate and wash twice with PBS. The PBS was purchased from Gibco, with a pH of 7.4 and a concentration of 1×.

[0080] c) Add 1 mL of Trizol to a six-well plate and let stand for 2 min. Use a pipette to blow the mixture a few times, then transfer it to a 1.5 mL EP tube. Gently invert the tube 10 times and place it on ice for 5 min.

[0081] d) Add 200 μL of chloroform to a 1.5 mL EP tube, invert 10 times to mix thoroughly until it becomes milky white, place on ice for 5 min, and then centrifuge at 12000 rpm for 15 min at 4 °C.

[0082] e) Carefully transfer the colorless and transparent supernatant (about 300 μL) to a new 1.5 mL EP tube, add an equal volume of isopropanol, gently invert 10 times, and then place on ice for 20 min.

[0083] f) Centrifuge at 4℃ and 12000rpm for 15min. A white precipitate will be visible at the bottom of the tube. Carefully discard the supernatant.

[0084] g) Add 1 mL of pre-cooled 75% (v / v) ethanol to a 1.5 mL EP tube to wash the precipitate, and then centrifuge at 7500 rpm for 5 min at 4 °C.

[0085] h) Discard the supernatant, dry at room temperature for 10 min, and the white precipitate at the bottom will become transparent. Add 50 μL of DEPC water, mix well, and place at 55℃ for 10 min to allow the RNA to dissolve completely.

[0086] i) Determination of RNA purity and concentration: Add 1 μL of extracted RNA to 99 μL of DEPC water (diluted 100 times), and measure the OD on a spectrophotometer. 260nm and OD 280nm Value, OD2 60nm / OD 280nm A value between 1.9 and 2.0 indicates high RNA purity. Total RNA concentration (µg / mL) = A 260nm The RNA product was diluted by 40 times to prepare for subsequent reverse transcription into cDNA experiments. The remaining RNA product was stored at -80°C for later use.

[0087] (2) Reverse transcription reaction a) Add the reagents to an RNase-free PCR tube on ice according to the reverse transcription system, mix well, and then centrifuge briefly.

[0088] b) Place in a PCR instrument and reverse transcription conditions: 25℃, 10 min; 42℃, 30 min; 85℃, 5 min.

[0089] c) Store the reverse transcription product at -20℃ for later use.

[0090] (3) Real-time quantitative PCR (qRT-PCR) a) Add 80 μL of DEPC water (i.e., dilute to 5 times) to 20 μL of reverse-transcribed cDNA.

[0091] b) On ice, prepare a 10 μL reaction mixture in an RNase-free 0.1 mL PCR 8-tube strip as follows: 1 μL cDNA, 5 μL SYBR Green Master Mix, 0.5 μL each of forward and reverse primers, and 3 μL DEPC water.

[0092] 5. Construction of LDHA knockdown and overexpression cell lines Cell knockdown models were established using both siRNA and shRNA. The siRNA was obtained from Shanghai Jima Biotechnology Co., Ltd., and the shRNA from Sigma-Aldrich. The pLKO.1-LDHA-shRNA recombinant plasmid was constructed, transformed, screened, and identified. Stable transfected strains were obtained through lentiviral packaging and screening. Specific procedures are as follows:

[0093] The pLKO.1-LDHA-shRNA recombinant plasmid (approximately 500 μg / mL) was dissolved in 500 μL of Opti-MEM medium, with the volume ratio of LDHA recombinant plasmid, lentiviral membrane plasmid, and packaging plasmid being 2 μL:2 μL:2 μL, respectively. Then, 6 μL of X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland) was added to the mixture, and the mixture was incubated at room temperature for 15 minutes. The mixture was then added to 293FT cells, and the supernatant was collected for further infection of HEKa cells for 5 days. Stable transfected cells were selected using puromycin. Finally, the protein and mRNA levels of LDHA were detected by Western blot and qRT-PCR. Specific results are shown in [link to results]. Figure 1 k and Figure 1 m. siRNA was transfected when the cell density reached 40%, using Lipofectamine® RNAiMAX (Invitrogen) as the transfection reagent.

[0094] The pLKO.1-LDHA-shRNA recombinant plasmid was purchased from Miaoling Biotechnology Co., Ltd.

[0095] Opti-MEM medium was purchased from Gibco.

[0096] The lentiviral membrane plasmid refers to pMD2.G, which originates from Addgene.

[0097] The packaging plasmid refers to psPAX2, which is derived from Addgene.

[0098] 293FT cells were purchased from the American Type Culture Collection (ATCC).

[0099] The sequence of the siRNA is shown in SEQ ID NO.2: 5′-CUCUAAAGGAUCAGCUGAUTT-3′.

[0100] The sequence of the shRNA is shown in SEQ ID NO.3: 5′-GGCAAAGACTATAATGTAT-3′.

[0101] The LDHA recombinant plasmid, specifically the LDHA overexpression plasmid, was purchased from Miaoling Biotechnology Co., Ltd. Viral coating and HEKa cell infection were performed using the same method as for the pLKO.1-LDHA-shRNA recombinant plasmid, with a volume ratio of 2 μL:2 μL:2 μL for the LDHA overexpression plasmid, lentiviral membrane plasmid, and packaging plasmid, respectively. Then, 6 μL of X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland) was added to the mixture, and the mixture was incubated at room temperature for 15 minutes. The mixture was then added to 293FT cells, and the supernatant was collected for further HEKa cell infection for 5 days. Stable transfected cells were selected using puromycin. Finally, the protein and mRNA levels of LDHA were detected by Western blot and qRT-PCR. Specific results are shown in [link to results]. Figure 1 k~ Figure 1 m in the text.

[0102] Example 2 1. Mass spectrometry experimental method for lactation modification HEKa cells were collected and lysed with 500 μL of Lysis buffer (containing 1% (m / v) protease inhibitor). Lysis was accelerated by sonication on ice, and after standing for 30 min, the cells were centrifuged at 20000g, 4°C for 10 min. The supernatant was collected for BCA quantification and leveled. 30 μg of the supernatant was then subjected to SDS-PAGE for quality control. 1.8 mg of protein was taken from each HEKa cell sample (10 cm culture dish), and DTT was added to reduce disulfide bonds. The reaction was carried out at 30°C for 60 min, followed by the addition of IAM to block free thiol groups. The reaction was carried out at room temperature in the dark for 45 min, after which the protein was precipitated with acetone overnight and washed. TEAB was added, and the protein precipitate was dissolved by sonication on ice. Trypsin was added, and the reaction was carried out at 37°C overnight. The peptides were desalted using a C18 SPE column, and the eluted peptides were dried using a vacuum concentrator. Each HEKa cell protein sample was labeled according to the TMT reagent instructions. The peptides were reconstituted and dried, and lactated peptides were enriched from the total peptides using lactating resin (Anti-L-lactyllysine antibody conjugated agarose beads, Lot# WM102, Micron biolab). The bound lactated peptides were eluted and dried in a vacuum concentrator, reconstituted and dried again, desalted with C18 ZipTip, and dried again to obtain the enriched lactated peptides.

[0103] Subsequently, the enriched peptides were analyzed using liquid chromatography-mass spectrometry (LC-MS). Each pre-separated component was dissolved in LC solution A, centrifuged at 20,000g for 2 min, and the supernatant was transferred to a sample vial. The enriched peptides were then analyzed using LC-MS for 1 h. The parameters of the nanoliter ultra-high performance liquid chromatography system were set as follows:

[0104] A / B solutions: 100% (v / v) H2O, 0.1% (v / v) FA / 80% (v / v) ACN, 0.1% (v / v) FA; loading solution: 0.1% (v / v) TF; column oven temperature: 40℃, flow rate: 250µL / min, gradient program: 0~6min, 2%~10% (v / v) B; 6min~51min, 10%~20% (v / v) B; 51min~58min, 20%~80% (v / v) B; 58min~62min, 80% (v / v) B; 62min~63min, 80%~2% (v / v) B; 63min~70min, 2% (v / v) B.

[0105] The mass spectrometer used was a Thermo Scientific Q Exactive HF, with a spray voltage of 2.0 kV, capillary temperature of 250 °C, MS1 scan range of 350 m / z to 1800 m / z, MS1 resolution of 60,000, MS1 AGC 1E6, MS1 maxIT 50 ms, MS2 scan range from 110 m / z, MS2 resolution of 30,000, MS2 AGC 1E5, MS2 maxIT 200 ms, Top 15 precursor ion selection, precursor ion selection threshold of 1E5, separation window ±0.7 m / z, fragmentation energy HCD, 26% NCE, and dynamic exclusion on, 6 s.

[0106] The obtained mass spectrometry Raw format files were directly searched using Maxquant (v.1.5.2.8). The search parameters were set as follows: the database was the protein database for this species; the restriction enzyme digestion method was set to Trypsin / P; 10-plex TMT quantification was used, with a maximum allowed number of missed cleavage sites of 2, an FDR of less than or equal to 0.01, and a primary precursor ion mass error tolerance of 10 ppm; the secondary fragment ion mass error tolerance was set to 0.02 Da; the fixed modification was set to cysteine ​​alkylation; the variable modifications were set to methionine oxidation, daamidation (NQ), N-terminal acetylation of the protein, and lysine lactation (Kla). The overall experimental procedure is as follows: Figure 2 As shown in a, the enrichment analysis results include the overall protein volcano map ( Figure 2 b) GO analysis ( Figure 2 d) Sublocation analysis ( Figure 2(e) Mitochondrial protein volcano diagram ( Figure 2 g) and GO analysis ( Figure 2 (i in the text).

[0107] 2. Extraction and Western blot detection of total cellular protein The methods and procedures in this section are the same as described above, and LDHA, L-lactylation, HSP60, and the nuclear internal reference Histone H3, as well as the total internal references Actin and α-TUBULIN, were detected respectively. Figure 2 c in Figure 3 (b) The results showed that LDHA overexpression significantly enhanced the overall lactation and HSP60 lactation levels.

[0108] 3. Immunofluorescence staining of cells and tissues HEKa cells were seeded on sterile coverslips (24-well plates) and cultured until 80% confluence.

[0109] (1) Fixation: Remove the culture medium and wash once quickly with PBS. Add 4% (w / v) PFA and fix at room temperature for 15 min. Wash 3 times with PBS, 5 min each time.

[0110] (2) Permeation: Add 0.1% (v / v) Triton X-100 (prepared with PBS) and incubate at room temperature for 15 min. Wash with PBS 3 times, 5 min each time.

[0111] (3) Blocking: Add blocking solution (5% (w / v) BSA + 0.03% (v / v) / PBS) and block at room temperature for 1 h.

[0112] (4) Primary antibody incubation: Dilute the primary antibody with blocking buffer, Rabbit anti-L-lactylation: 1:200 Mouse anti-Tomm 20: 1:200 (v / v), and incubate overnight (12h) at 4°C in the dark.

[0113] (5) Recover the primary antibody and wash with PBS 3 times, 10 min each time.

[0114] (6) Secondary antibody incubation: Dilute the secondary antibody with blocking buffer (prepare in the dark): Alexa Fluor 594 anti-rabbit (red): 1:300, Alexa Fluor 488 anti-mouse (green): 1:300. Incubate at room temperature in the dark for 1 hour, then wash 3 times with PBS for 10 minutes each time (in the dark).

[0115] (7) DAPI nuclear staining: Add DAPI working solution (1 μg / mL in PBS), incubate at room temperature in the dark for 10 min, and wash twice with PBS for 5 min each time.

[0116] (8) Mounting: Place 2 μL of anti-quenching mounting medium on a glass slide. Carefully remove the coverslip with tweezers, place it cell-side down on the mounting medium, avoiding air bubbles, and seal the edges with clear nail polish. Incubate in the dark for 20 minutes to allow the mounting medium to harden, then it can be examined under a microscope or stored for a short period at 4°C in the dark. Results in this section are shown below. Figure 2 Immunofluorescence staining showed that after LDHA overexpression (OE), mitochondria and lactation colocalized to some extent compared with the NC control.

[0117] Paraffin-embedded tissues need to be dewaxed before antigen retrieval and re-staining. The specific procedures are as follows:

[0118] (1) Dewaxing: xylene 2×10min, 100% (v / v) ethanol: 2×5min, 95% (v / v) ethanol: 5min, 90% (v / v) ethanol: 5min, 80% (v / v) ethanol: 5min, 70% (v / v) ethanol: 5min, PBS: 2×5min.

[0119] (2) Antigen retrieval The retrieval solution was 0.01M citrate buffer (pH 6.0). Sections were placed in the retrieval solution, heated to boiling in a water bath, maintained for 10 min, and then allowed to cool naturally to room temperature for 60 min. The sections were washed with PBS 2×5 min each time. Afterward, permeabilization-blocking-incubation with primary and secondary antibodies-DAPI staining-mounting were performed as described above for cell culture. Results in this section are shown below. Figure 3 The results showed that the skin lesions of psoriasis patients had significantly upregulated lactation levels compared with normal controls, and co-localization showed lactation of HSP60.

[0120] Example 3 1. Endogenous co-IP detection (1) Cell processing and lysis: When the HEKa NC control group and LDHA overexpression group cells reached 90% confluence, they were washed twice with PBS. 500 μL of pre-chilled lysis buffer was added to each 10 cm dish, and the cells were lysed on ice for 30 min with occasional shaking. The cells were scraped off and transferred to EP tubes, and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected, with 100 μL reserved for input and the remainder for IP.

[0121] (2) Pre-cleaning: Take Protein A / G beads and wash them 3 times with lysis buffer. Add 30 μL of beads to each sample tube and incubate at 4 °C for 60 min by rotation. Centrifuge, collect the supernatant (discard the beads), and transfer it to a new tube.

[0122] (3) Antibody binding (IP): IgG control group: 2 μg of Normal Mouse IgG added; IP experimental group: 2 μg of anti-HSP60 added. Both groups were incubated at 4℃ for 12 h.

[0123] (4) Binding beads: Add 40 μL of washed Protein A / G beads to each tube. Incubate by rotation at 25°C for 1 h.

[0124] (5) Washing: Wash twice with pre-cooled lysis buffer and once with PBS.

[0125] (6) Elution and sample preparation: Aspirate the last wash buffer and add 100 μL of 1× SDS loading buffer (containing β-ME). Boil at 95℃ for 10 min. Centrifuge briefly, take the supernatant, load it onto a gel for subsequent Western blot experiments. Figure 4 Figure a shows a schematic diagram of HSP60 and the mitochondrial membrane structure mPTP (mitochondrial membrane permeability transition pore) containing CypD. According to the IP results, the interaction between HSP60 and CypD was significantly reduced after LDHA overexpression. Figure 4 (b) in the middle.

[0126] 2. Bioinformatics Analysis (1) Autodock molecular docking: CypD and HSP60 protein Protein-protein docking of CypD and HSP60 was performed using AutoDock Vina. First, the 3D structures of CypD and HSP60 were preprocessed using AutoDock Tools (ADT) to remove water molecules and non-protein ligands, add polar hydrogen atoms, and calculate Gasteiger charges. Docking simulations were performed using default parameters, retaining the top 10 conformations with the lowest binding free energy (ΔG, kcal / mol) for subsequent analysis. The binding modes, including hydrogen bonds, hydrophobic interactions, and key residues at the binding interface, were visualized and analyzed using PyMOL and Discovery Studio. The eight lactation sites of HSP60 were also displayed in the docking structure, as shown below. Figure 4 As shown in d.

[0127] (2) Single-cell data analysis: HSP60 and CypD Normalized expression analysis of genes during pseudotime of keratinocyte differentiation The single-cell transcriptome data were processed and analyzed using the Seurat (v4) workflow in R. After quality control, standardization, and normalization, principal component analysis (PCA) and graph clustering were used to identify keratinocyte subpopulations. Cell clusters were annotated as basal keratinocytes and spinous keratinocytes based on classic marker genes (basal cells: KRT5 / KRT14; spinous cells: KRT1 / KRT10). Monocle 3 was used for pseudo-time trajectory analysis to reconstruct the differentiation trajectory from basal cells to spinous cells. The normalized and normalized expression levels of HSPD1 (encoding HSP60) and PPIF (encoding CypD) were extracted (Seurat “data” slots or log-normalized counts), and the dynamic changes in gene expression along the pseudo-time axis were visualized using line graphs. Figure 4 As shown in c in the figure.

[0128] 3. Construction of Quick change point mutation plasmid Based on the HSP60 sequence, primers were designed to mutate lysine (K) at positions 72, 125, 133, 156, 202, 249, 250, and 523 to arginine (R), simulating delactation. A mutant recombinant plasmid was constructed using PCR, and the product expressed by the recombinant vector was HSP60Mut. The PCR reaction system consisted of: 200 ng template, 0.5 μL each of upper and lower primers, 10 μL 5× reaction buffer, 4 μL dNTPs, 1 μL Q5 DNA high-fidelity enzyme, and ddH2O to a final volume of 50 μL. PCR reaction conditions were set according to the Q5 DNA high-fidelity enzyme protocol. After the PCR reaction, [the following was added]... Dpn I. Restriction endonucleases (the plasmid to be mutated will be methylated, and can be used) Dpn I. Digest the template plasmid (retaining the mutant plasmid) and the corresponding buffer solution, and incubate at 37°C for 4 hours. Afterwards, use a DNA recovery kit for gel extraction. Transform 100 ng of the sample into plasmids according to the desired concentration, incubate for ≥18 hours, and then pick single clones for assaying. Expand the culture of samples with normal sequencing results, extract plasmids, and transform them into HEKa cells for subsequent experiments. The construction results are shown below. Figure 4 e~ Figure 4 As shown in f in the figure.

[0129] The sequence of HSP60 is shown in SEQ ID NO.1: .

[0130] Mutant recombinant plasmids are constructed by cloning mutated HSP60 cDNA into the pcDNA3.1 vector. They originated from Sangon Biotech Co., Ltd. Results in this section are shown below. Figure 4 e and Figure 4 f in the text.

[0131] 4. Cell immunofluorescence staining Consistent with the methods described above, the results in this section show that mitochondrial DNA leakage exists in LDHA-overexpressing (OE) cells and M5-treated cells. Figure 4 g in (the middle part).

[0132] 5. PLA Proximity Marker (Duolink@ Kit) (1) Preparation and fixation of HEKa-HSP60 mutant and control cells: Cells were crawled onto slides until 70% confluence. The culture medium was aspirated and the cells were washed once quickly with PBS. The cells were fixed with 4% (w / v) PFA at room temperature for 15 min. The cells were washed three times with PBS for 5 min each time.

[0133] (2) Permeability: 0.1% (v / v) Triton X-100 (PBS) at room temperature for 10 min. Wash twice with PBS, 5 min each time.

[0134] (3) Sealing (PLA-specific sealing): Add PLA Blocking Solution and seal in a humidified chamber at 37°C for 60 minutes.

[0135] (4) Primary antibody incubation (rabbit anti + mouse anti): The primary antibody was diluted with Antibody Diluent (PLA-compatible): Rabbit anti-CypD, 1:200, Mouse anti-HSP60, 1:200. After mixing, it was dropped onto the slide and incubated overnight (16 h) in a humidified chamber at 4°C in the dark. Wash with Wash Buffer A for 3 × 5 min.

[0136] (5) PLA probe incubation (PLUS+MINUS): Dilute the PLA probe with Antibody Diluent, and mix PLUS (anti-rabbit) and MINUS (anti-mouse) at a ratio of 1:5 according to the instructions. Add the mixture dropwise and incubate in a humidified chamber at 37°C for 60 min (protected from light). Wash twice with Wash Buffer A, 5 min each time.

[0137] (6) Ligation reaction: Prepare Ligation working solution: Ligation Solution + Ligase (50:1), add dropwise, and humidify at 37°C for 30 min. Wash twice with Wash Buffer A, 2 min each time.

[0138] (7) Rolling circle amplification: Prepare the amplification working solution, add amplification solution + polymerase (25:1) dropwise, and humidify at 37°C for 100 min in the dark. Wash once with Wash Buffer B for 10 min each time, and then wash once quickly with PBS.

[0139] (8) DAPI nuclear staining and mounting: Add anti-quenching mounting medium containing DAPI (1 μg / mL) and mount at room temperature in the dark for 1 min. Seal the edges with nail polish and cure in the dark for 10 min before imaging.

[0140] PLA labeling experiments were conducted to determine the effects of different lactation sites on HSP60 in mutant cell lines and under different treatment conditions (M5, lactate LA). K249R It can significantly increase the binding of HSP60 and CypD ( Figure 4 h~ Figure 4 k in the middle.

[0141] Example 4 1. Establishment of an IMQ-induced mouse model of psoriasis C57BL / 6J mice were randomly divided into 3 groups of 6 mice each. The specific grouping and treatment methods are as follows: (1) AAV- Hspd1 WT Virus control group: AAV- was injected subcutaneously at 2-5 points. Hspd1 WT Virus, injection titer ≥5E+12GC / mL. Hair removal begins on day 13 after injection, and no further treatment is required thereafter.

[0142] (2) AAV- Hspd1 WT Model group (IMQ group): AAV- was injected subcutaneously at 2-5 points. Hspd1 WT Virus, injection titer ≥5E+12GC / mL. Hair removal begins on day 13 after injection, followed by daily application of 5% IMQ cream (Sichuan Mingxinlidi) to the back for 5 consecutive days.

[0143] (3) AAV- Hspd1 Mut Virus control group: AAV- was injected subcutaneously at 2-5 points. Hspd1 Mut Virus, injection titer ≥5E+12GC / mL. Hair removal begins on day 13 after injection, and no further treatment is required thereafter.

[0144] (4) AAV- Hspd1 Mut Model group (IMQ group): AAV- was injected subcutaneously at 2-5 points. Hspd1 Mut Virus, injection titer ≥5E+12GC / mL. Hair removal begins on day 13 after injection, followed by daily application of 5% IMQ cream (Sichuan Mingxinlidi) to the back for 5 consecutive days.

[0145] The C57BL / 6J mice were purchased from Cyagen Biosciences.

[0146] AAV- Hspd1 WT The virus originated from Qingke Biotechnology Company.

[0147] AAV- Hspd1 Mut The virus originated from Qingke Biotechnology Company.

[0148] flow chart( Figure 5 a) shows the experimental procedure for establishing an IMQ-induced psoriasis model and AAV virus intervention. This overexpressed virus contains fused GFP and is capable of autofluorescence, such as... Figure 5 As shown in b. The backs of mice in each group were dehaired using electric hair clippers and depilatory cream, with a dehairing area of ​​2cm × 2cm. A mouse model of psoriasis was established after hair removal. The back skin of mice in each group was photographed after hair removal and before and after treatment. After treatment, mice in each group were sacrificed by cervical dislocation, and the back skin was harvested. The skin tissue was washed with cold PBS and fixed in 4% (v / v) paraformaldehyde. The spleen was harvested from the abdominal cavity, washed with cold PBS, dried, weighed, and photographed. Representative photographs are shown in Figure 1. Figure 5 c and Figure 5 As shown in f, the spleen weight and relative weight are as follows: Figure 5 As shown in g in the figure.

[0149] The PBS was purchased from Gibco, with a pH of 7.4 and a concentration of 1×.

[0150] Psoriasis Skin Lesion Area and Severity Index (PASI Score) in mice: The severity of each lesion was assessed using three indicators: erythema, infiltration, and epidermal desquamation / scaling. Each indicator was scored from 0 to 4, where 0 = none, 1 = mild, 2 = moderate, 3 = severe, and 4 = extremely severe. The total score was the PASI score. Photos were taken and the PASI scores for each group of mice were calculated. The results are as follows: Figure 5 As shown in d in the figure. Mouse weight was recorded daily, and the results are presented in [the figure]. Figure 5 In the middle of e.

[0151] 2. HE staining of mouse tissue (1) Processing of clinical mouse skin specimens The specimens obtained during the IMQ modeling experiment (mouse skin tissue, liver tissue, and kidney tissue from each group) were thoroughly rinsed with physiological saline to remove blood and contaminants. The tissue blocks were then placed in 4% (v / v) paraformaldehyde and fixed for 24 hours.

[0152] (2) Preparation of glass slides a) After ultrasonically cleaning the glass slide with detergent, immerse it in a potassium dichromate-concentrated sulfuric acid mixture for 24 hours.

[0153] b) After removing the potassium dichromate-concentrated sulfuric acid cleaning solution (chromic acid cleaning solution) from the acid tank, rinse it with distilled water until completely clean, and then place it in an oven at 60°C to dry overnight. "Overnight" refers to a time ≥12 hours.

[0154] c) To prevent tissue from falling off, the slides need to be treated with 3-aminopropyl-3-ethoxysilane (APES). Dilute the APES stock solution with acetone at a volume ratio of 1:50 to prepare a working solution. Place the cleaned slides into the newly prepared APES working solution and leave them for 30 seconds before removing them.

[0155] d) Remove the slide, pause for 10 seconds, then place it in acetone solution for 30 seconds to rinse away any unbound APES working solution.

[0156] e) Place the slides in the oven to dry for another 2 hours, then pack them into boxes for later use.

[0157] (3) Paraffin embedding and tissue sectioning a) Embedding tissue: First, add liquid paraffin to a small pathological embedding mold (2cm×2cm×0.5cm). After cooling slightly, place the skin tissue to be embedded in the paraffin and arrange it neatly. Then, cover the plastic mold box and add 1mL of liquid paraffin. Cool it to make it solid.

[0158] b) Sectioning: Remove the embedded tissue from the small pathological embedding mold, place it on a paraffin microtome, adjust the section thickness to 4μm, and section continuously.

[0159] c) Place the sections in a 60℃ oven and bake for 90 minutes, then transfer them to a 37℃ oven and incubate overnight to obtain skin tissue sections. "Overnight" refers to an incubation time of ≥12 hours.

[0160] (4) HE staining steps a) Stain skin tissue sections in hematoxylin solution for several minutes, then separate the colors in acid and ammonia solutions for 10 seconds each.

[0161] b) Rinse with running water for 1 hour, then immerse in distilled water for a short time.

[0162] c) Dehydrate in 70% (v / v) and 90% (v / v) alcohol for 10 minutes each.

[0163] d) Stain with alcohol-eosin staining solution for 3 minutes.

[0164] e) Dehydrate the sections with ethanol, clear them with xylene, and mount them with neutral resin.

[0165] (5) Determination of HE staining results, microscopic examination, and image acquisition and analysis. The HE staining results of mouse skin tissue in this section are as follows: Figure 5 As shown in h.

[0166] 3. Flow cytometry detection of Th17 and Treg cell counts in mouse spleen tissue (1) Th17 cell detection procedure (IL-17A PE / CD4 FITC) a) In vitro stimulation and protein transport blockade: IMQ group, AAV- Hspd1 WT and AAV- Hspd1 Mut Mouse spleen single-cell suspension, cell concentration adjusted to 1×10 6Cells / mL. Add cell stimulation mixture: PMA 50ng / mL + Ionomycin 1μg / mL + Brefeldin A 10μg / mL, and incubate at 37℃, 5% CO2 for 4h to induce cytokine synthesis and block its secretion.

[0167] b) Surface staining: Collect cells and wash once with PBS containing 2% (v / v) FBS. Add CD4 FITC fluorescent antibody (1:200) and incubate at 4°C in the dark for 30 min. After staining, wash twice with PBS to remove unbound antibodies.

[0168] c) Fixation and permeabilization: Add the Fixation / Permeabilization working solution from the transcription factor permeabilization kit (eBioscience) and incubate at 4°C in the dark for 60 min. Then wash twice with Permeabilization Buffer to complete the cell membrane permeabilization process.

[0169] d) Intracellular cytokine staining: Add IL-17A PE fluorescent antibody (1:200) to the permeabilization buffer and incubate at 4°C in the dark for 30 min. Wash twice with permeabilization buffer, and finally resuspend the cells in 2 mL PBS for instrumental analysis.

[0170] e) Flow cytometry analysis: Using CD4⁺T lymphocytes as the gate, analyze the proportion of IL-17A⁺ cells, which is the percentage of Th17 cells among CD4⁺T cells.

[0171] (2) Treg cell detection procedure (FOXP3 PE / CD25 APC) a) Surface staining: Take IMQ group, AAV- Hspd1 WT and AAV- Hspd1 Mut Mouse spleen single-cell suspension was washed once with PBS containing 2% (v / v) FBS. CD25 APC fluorescent antibody (1:200) was added and incubated at 4°C in the dark for 30 min. The cells were then washed twice with PBS to remove unbound antibody.

[0172] b) Fixation and permeabilization: Add the Fixation / Permeabilization working solution from the Foxp3 permeabilization kit and incubate at 4°C in the dark for 60 min. Wash twice with Permeabilization Buffer to permeate the cell membrane.

[0173] c) Intracellular transcription factor staining: Add FOXP3 PE fluorescent antibody (1:200) to the permeabilization buffer and incubate at 4°C in the dark for 60 min. Wash twice with permeabilization buffer, resuspend in PBS, and then perform detection.

[0174] d) Flow cytometry analysis: Using CD4⁺ T lymphocytes as the gating gate, the proportion of CD25⁺ and FOXP3⁺ double-positive cells was analyzed, representing the percentage of Treg cells among CD4⁺ T cells. The results in this section confirm that HSP60K249R can effectively reduce inflammation in IMQ mice, manifested as a decrease in Th17 cells and an increase in Treg cells. Figure 5 (i in the text).

[0175] The experimental results above show that lactate treatment or LDHA overexpression can significantly promote mPTP opening, while inhibiting LDHA or HSP60 K249 lactation can effectively inhibit this process.

[0176] Example 5 The invention also includes alternative embodiments, including small molecule inhibitors (such as a specific compound) binding near the K249 site of the HSP60 protein to prevent lactation. Figure 6 (a) A monoclonal antibody that specifically binds to lactated HSP60, neutralizing its function or labeling it for clearance ( Figure 6 (b) siRNA or shRNA downregulates LDHA mRNA, reducing lactate production ( Figure 6 (c) A stabilizing peptide enhances the interaction between HSP60 and CypD, resisting dissociation caused by lactation. Figure 6 (d in the text)

[0177] In summary, the technical solution of this invention encompasses a multi-level therapeutic strategy ranging from upstream metabolic intervention to downstream protein function regulation. The aforementioned alternative implementation schemes can be implemented independently or combined according to the disease type and patient condition. Those skilled in the art should understand that conventional modifications, equivalent substitutions, or optimizations made to the specific embodiments, without departing from the core inventive point of this invention—namely, treating related diseases by regulating the lactation level of lysine at position 249 of the HSP60 protein or its interaction—should fall within the scope of protection of this invention. Furthermore, the intervention methods described in this invention are not limited to the molecular types listed in the examples; any bioactive molecule or therapeutic method capable of achieving the above-mentioned technical effects is within the scope of protection of this invention.

[0178] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0179] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. The use of an inhibitor of lactation modification of lysine at position 249 of the HSP60 protein in the preparation of drugs for the prevention and / or treatment of psoriasis, characterized in that, The amino acid sequence of the HSP60 protein is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The inhibitors include lactate dehydrogenase A inhibitors.

3. The application according to claim 2, characterized in that, The lactate dehydrogenase A inhibitor is selected from any one or more of GSK2837808A, AZ-33, sodium oxalate, FX-11, LDHA-IN-3, (R)-GNE-140, and Galloflavin.

4. The application according to claim 1 or 2, characterized in that, The drug is a pharmaceutical composition, which also includes a DHA inhibitor.

5. The application according to claim 4, characterized in that, The DHA inhibitor is a small molecule compound, siRNA, shRNA, or CRISPR gene editing system.

6. The application according to claim 5, characterized in that, The nucleotide sequence of the siRNA is shown in SEQ ID NO.

2.

7. The application according to claim 5, characterized in that, The nucleotide sequence of the shRNA is shown in SEQ ID NO.

3.

8. The application according to claim 1 or 2, characterized in that, The drug is a pharmaceutical composition, which also includes cGAS-STING pathway inhibitors.

9. A medicine for the prevention and / or treatment of psoriasis, characterized in that, The active ingredient of the drug is a lactate dehydrogenase A inhibitor, as well as a DHA inhibitor and / or a cGAS-STING pathway inhibitor.

10. The medicament according to claim 9, characterized in that, The medication also includes vitamin D analogs or glucocorticoids used for topical treatment of psoriasis.

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