Application of LLDT-8 in preparation of medicine for treating polymyositis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of LLDT-8 in preparation of a medicine for treating polymyositis, LLDT-8 reduces expression levels of MHC-I and NLRC5 in muscular tissues and inhibits activation of CD8 + T cells through targeted regulation of an NLRC5 / MHC-I pathway, so that infiltration of the CD8 + T cells in the muscular tissues is reduced, muscular inflammation is remarkably inhibited, and phenotypes of polymyositis diseases are improved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the use of LLDT-8 in the preparation of a medicament for treating polymyositis. Background Art

[0002] Polymyositis is an autoimmune inflammatory myopathy that affects striated muscle due to immune system disorders. The exact etiology of polymyositis is still unclear. Its onset is insidious and it is extremely easy to be misdiagnosed and delay treatment. In addition, the disease progresses rapidly and even affects multiple organs, seriously affecting the physical and mental health of patients. In polymyositis patients, the major histocompatibility complex MHC-I molecules are abnormally expressed in muscle fibers, accompanied by a large number of inflammatory cell infiltrations in muscle tissues, mainly CD8 + T cells, and CD4 + T cells, dendritic cells and macrophages can also be seen. The MHC-I / CD8 complex is considered to be a characteristic pathological marker of polymyositis. Studies have found that MHC-I molecules are highly expressed in muscle fibers under inflammatory injury or certain pathological conditions. In polymyositis patients, abnormal up-regulation of MHC-I antigen already exists in muscle fibers even at the early stage of the disease or far from the lesion, accompanied by inflammatory cell infiltration mainly composed of CD8 + T cells. When MHC-I in skeletal muscle cells is overexpressed, transgenic mice show muscle inflammatory responses, muscle atrophy and significant decline in muscle strength. Further mechanism studies have found that CD8 + T cells invade myotubes with up-regulated MHC-I, directly bind to MHC-I molecules on the surface of muscle fibers through receptors on the surface of T cells, and form CD8 + T / CD8 complex, resulting in the activation of CD8 + T cells; the latter secrete perforin, granzyme, etc. to cause cell lysis and directly kill muscle cells. Activated T cells also release various cytokines such as interferon-γ, interleukin-1 and tumor necrosis factor, promoting the up-regulation of MHC-I molecules in muscle fibers and enhancing the killing effect of CD8 + T cells, resulting in difficult-to-heal muscle damage. At present, there is still a lack of effective therapeutic drugs for polymyositis.

[0003] NLR family CARD domain-containing protein 5 (NLRC5 for short) is a key regulator of MHC-I-dependent immune responses. Abnormal elevation of NLRC5 leads to inflammation or autoimmune responses in the body. As a cytoplasmic protein, NLRC5 translocates into the nucleus to form a complex with the SXY structure on the MHC-I promoter, regulating MHC-I transcriptional expression. At the same time, NLRC5 regulates the expression of other genes retaining the SXY module. Both MHC-I and its light chain β2M, antigen peptide transporter TAP1 / 2, and antigen-processing immunoproteasome Psmb8 / 9 retain the SXY module to varying degrees and bind to NLRC5 through this module to regulate the antigen presentation process.

[0004] (5R)-5-Hydroxytriptolide (LLDT-8 for short) is a structural derivative of triptolide and is used to treat inflammatory autoimmune diseases due to its unique anti-inflammatory and immunosuppressive effects. Studies have shown that it has therapeutic effects on various animal models of autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, immune liver injury, and pulmonary fibrosis. LLDT-8 also has an anti-transplant rejection effect. In the mouse allogeneic heterotopic heart transplant rejection reaction, LLDT-8 significantly prolongs the survival of mice in the acute graft-versus-host disease (aGVHD) model by promoting immune reconstruction. However, it has not been found that LLDT-8 inhibits the activation of CD8 + T cells and reduces the infiltration of CD8 + T cells in the muscle tissue of polymyositis mice and improves the disease phenotype of polymyositis. Summary of the Invention

[0005] The object of the present invention is to provide an application of LLDT-8 in the preparation of a drug for treating polymyositis in view of the above problems existing in the prior art.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] Application of LLDT-8 in the preparation of a drug for treating polymyositis.

[0008] The drug for treating polymyositis is a preparation that down-regulates the expression levels of MHC-I and NLRC5 in muscle tissue and inhibits the activation of CD8 + T cells.

[0009] The drug for treating polymyositis is a preparation that down-regulates the expression levels of antigen presentation factors β2M, Psmb8, Tap1, and Tap2 in muscle tissue.

[0010] The polymyositis is induced by intraperitoneal injection of Bordetella pertussis while immunizing with an emulsion of myosin and Freund's adjuvant.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] The present invention explores the application of LLDT-8 in the preparation of drugs for treating polymyositis. Research has confirmed that LLDT-8 targets and regulates the NLRC5 / MHC-I pathway, down-regulates the expression levels of MHC-I and NLRC5 in muscle tissues, and inhibits the activation of CD8 + T cells, thereby reducing the infiltration of CD8 + T cells in muscle tissues, and further significantly inhibiting muscle inflammation and improving the disease phenotype of polymyositis. Description of the Drawings

[0013] Figure 1 Shows the general characterization test results of mice in each group on the 18th day.

[0014] Figure 2 Shows the body weight test results of mice in each group at different times.

[0015] Figure 3 Shows the Lennon A score results of mice in each group at different times.

[0016] Figure 4 Shows the average muscle endurance test results of mice in each group at different times.

[0017] Figure 5 Shows the muscle endurance change rate test results of mice in each group at different times.

[0018] Figure 6 Shows the average muscle grip force test results of mice in each group at different times.

[0019] Figure 7 Shows the muscle pain threshold test results of mice in each group at different times.

[0020] Figure 8 Shows the HE staining results of muscle tissues of mice in each group.

[0021] Figure 9 Shows the pathological score results of muscle tissues of mice in each group.

[0022] Figure 10 Shows the positive expression of pathogenic CD8 + T cells in muscle tissues of mice in each group.

[0023] Figure 11 Shows the MHC-I mRNA expression levels in muscle tissues of mice in each group.

[0024] Figure 12 Shows the positive expression of MHC-I in muscle tissues of mice in each group.

[0025] Figure 13 Quantitative analysis of the protein level of MHC-I in the muscle tissues of each group of mice.

[0026] Figure 14 The expression level of NLRC5 mRNA in the muscle tissues of each group of mice.

[0027] Figure 15 The expression levels of antigen presentation-related factors β2M, Psmb8, Psmb9, Tap1, and Tap2 mRNA in the muscle tissues of each group of mice.

[0028] Figure 16 For the protein levels of MHC-I and NLRC5 in IFN-γ-induced C2C12 cells detected by Western blot.

[0029] In the above figure, # P < 0.05, ## P < 0.01, ### P < 0.001; * P < 0.05, ** P < 0.01, *** P < 0.001. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] Next, a polymyositis mouse model was constructed by immunizing mice with an antigen to explore the effect of LLDT-8 on inhibiting the activation of CD8 + T cells by targeting the regulation of the NLRC5 / MHC-I pathway, thereby reducing the infiltration of CD8 + T cells in the muscle tissues of polymyositis mice and improving the disease phenotype of polymyositis.

[0032] 1 Materials and instruments

[0033] 1.1 Animals

[0034] Healthy female BALB / c mice, 6 - 8 weeks old, weighing 18 - 22 g, SPF grade, were intensively housed in the SPF barrier environment of the Phase I Animal House of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Laboratory Animal License: SCXK(Shanghai)2018 - 0009; Feeding environment: temperature 20 - 25 °C, humidity 50 - 70%, 12 h light / dark cycle. The feed was given after disinfection, and the mice had free access to food and clean water; After one week of adaptive feeding, when the actual body weight reached 18 g, animal modeling could be carried out;

[0035] Female guinea pigs (400 - 480 g), 10 weeks old, clean grade, were housed in the Phase I Clean Animal House of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The mice had free access to food and clean water. Laboratory Animal License: SCXK(Beijing)2016 - 0011, used for the preparation of skeletal muscle myosin;

[0036] All operations on all experimental animals during the experiment were approved by the International Animal Welfare and the Experimental Animal Management Committee of the Institute of Materia Medica (Experiment No.: 2019 - 12 - ZJP - 113).

[0037] 1.2 Drugs and Reagents

[0038] LLDT - 8: Provided by Shanghai Pharmaceutical Group Co., Ltd., with a purity greater than 98.5%; Molecular formula is C 20 H 24 O7, molecular weight 376.39, in the form of white powder, and the chemical structural formula is shown as follows:

[0039]

[0040] Methylprednisolone (abbreviation: MP): CAS: 83 - 43 - 2, molecular weight 374.47.

[0041] 1.3 Instruments

[0042] Mouse grip strength wire mesh (32.5 cm × 32.5 cm wire mesh);

[0043] Blade homogenizer: Model T18DS25, IKA, T18 digital ULTRA TuRRAX, rpm; X1000, Germany;

[0044] Digital push - pull force gauge: SF - 500, Shunkeda;

[0045] pH meter: Model PB - 10, Sartorius Scientific Instruments (Beijing) Co., Ltd.;

[0046] Centrifuge: Model Eppendorf 58102;

[0047] CO2 Incubator: Model HERAcell vios 160i, Thermo Company, Germany;

[0048] Autoclave: Model MLS-3781L-PC, Panasonic, Japan;

[0049] Electric Thermostatic Drying Oven: Model CS101-2EB, Hengda Instruments, Chongqing Sida Experimental Instrument Co., Ltd.;

[0050] Digital Control Ultrasonic Cleaner: Model KQ5200DE, Kunshan Ultrasonic Instrument Co., Ltd.;

[0051] Inverted Microscope: Model CKX41SF, OLMPUS Corporation, Tokyo, Japan;

[0052] Biological Safety Cabinet: Model Hfsafe-1500LC, Shanghai Lishen Scientific Instrument Co., Ltd.;

[0053] Vortex Shaker: Model QT-1, Shanghai Qite Analytical Instrument Co., Ltd.;

[0054] Magnetic Stirrer: Model MS-H-S, Scilogex;

[0055] Electronic Analytical Balance: Model ME104, METTLER TOLEDO Company;

[0056] Beta Counter: MicroBeta Trilux, PerkinElmer;

[0057] Automatic Biochemical Analyzer: Toshiba Series 40FR;

[0058] HITACHI Automatic Biochemical Analyzer: HITACHI Company;

[0059] Experimental Animal Asphyxiator: Model SMQ-1, Shanghai Tianhuan Technology Development Co., Ltd.;

[0060] 2 Methods

[0061] 2.1 Animal Modeling, Grouping and Drug Administration

[0062] (1) Extraction of Guinea Pig Skeletal Muscle Myosin

[0063] First, guinea pigs weighing approximately 450 g were euthanized, and the skin of their limbs was disinfected with alcohol. The guinea pigs were fixed on a low-temperature operating table, and skeletal muscles of the limbs were excised using scissors and forceps, and then quickly placed in pre-cooled physiological saline to prevent myosin degradation. On the low-temperature operating table, the fascia, blood vessels, and nerve tissues around the muscles were removed, blotted dry with absorbent paper, weighed, placed in a 50 mL centrifuge tube, and 3 mL of homogenization buffer (0.3 M KCl, 0.15 M sodium phosphate buffer) was added per gram of muscle. Using a blade homogenizer at a speed of 10,000 rpm per second, the whole process was carried out intermittently to avoid protein degradation caused by excessive temperature. After the muscle was fully homogenized, the muscle tissue homogenate was transferred to a 1.5 mL EP tube, centrifuged at 12,000 rpm at 4 °C for 30 - 45 min, and the supernatant was collected. After filtration, 3 mL was taken into a 15 mL centrifuge tube, diluted with pre-cooled ddH2O at a ratio of 1:4, the above 15 mL centrifuge tube was mixed well and centrifuged at 3,900 rpm at 4 °C for 30 min, and the supernatant was discarded to obtain the precipitate. The precipitate was weighed and dissolved in an appropriate amount of 0.5 mol / L KCl solution and mixed well. Protein quantitative detection was carried out by the BCA method, the protein concentration was calculated, and the extracted myosin was aliquoted and stored for later use according to the usage amount, and it should be used up within 1 month to prevent protein degradation.

[0064] (2) Modeling method

[0065] 100 μL of purified guinea pig skeletal muscle myosin containing 1.0 mg was fully emulsified with an equal volume of complete Freund's adjuvant to prepare the immunizing antigen. Each BALB / c mouse was inoculated with 200 μL of the emulsifier each time, immunized 2 times with a 1-week interval; the first time was intramuscular injection in the left hind limb, and the second time was subcutaneous injection at the base of the tail. Each time during immunization, pertussis toxin (500 ng / 200 μL physiological saline) was injected intraperitoneally to obtain a polymyositis mouse model.

[0066] (3) Grouping and administration

[0067] All mice were divided into the following 5 groups according to their basal body weight, limb muscle endurance, limb muscle grip strength, etc.:

[0068] Normal group (Normal, n = 8): Not modeled, normal diet;

[0069] Model group (Vehicle, n = 8): The modeling method is not described here again. During modeling, 0.2% HPMC (hydroxypropyl methylcellulose) solvent (abbreviated as HPMC) was given by gavage for treatment, and the administration dose was 0.2 mL / d;

[0070] Positive drug group (MP, n = 8): The modeling method is not described here again. During modeling, 10 mg / kg MP was given by gavage for treatment;

[0071] LLDT-8 low-dose group (LLDT-8-L, n = 8): The modeling method will not be elaborated here. During modeling, 0.0625 mg / kg LLDT-8 was given by gavage for treatment, and the solvent was 0.2% HPMC;

[0072] LLDT-8 high-dose group (LLDT-8-H, n = 8): The modeling method will not be elaborated here. During modeling, 0.125 mg / kg LLDT-8 was given by gavage for treatment, and the solvent was 0.2% HPMC;

[0073] Since the model has an acute onset, obvious pathological manifestations will occur during the first immunization and modeling, and the modeling time is only 18 days. Therefore, drug intervention was given during modeling, and relevant detections were carried out on the 0th day, 7th day, 14th day, and 18th day respectively.

[0074] 2.2 Mouse cell culture and differentiation

[0075] C2C12 mouse myoblasts were provided by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences; C2C12 cell culture: Cultured in DMEM high-glucose medium with 10% FBS overnight in an incubator at 37°C and 5% CO2, and the medium was changed every two days. Cell passage culture: Add trypsin for digestion and termination treatment, collect the cells and centrifuge, discard the supernatant, add fresh complete medium, and continue to culture according to the aforementioned cell culture steps.

[0076] 3 Index detection

[0077] 3.1 Observation of mouse clinical manifestations

[0078] The body weights of the mice were recorded on the 0th day, 7th day, 14th day, and 18th day respectively. The general manifestations of the mice were observed daily, including hair, coat color, mental state, resting posture, activity, etc., and recorded according to the Lennon A scoring standard.

[0079] Table 1 Lennon A scoring standard

[0080] Score Symptom manifestation 0 points No obvious myasthenia manifestation 1 point Unable to bite or cry out 2 points Humped back position at rest, head drooping, forelimbs flexed, tremors while walking 3 points Severe myasthenia, not crying out, weight loss, even muscle atrophy, difficulty breathing, on the verge of death

[0081] 3.2 Collection and evaluation of limb muscle strength

[0082] Limb muscle endurance detection: The inverted screen experiment was used to detect the muscle endurance level of the mice on the 0th day, 7th day, 14th day, and 18th day respectively; Inverted screen experiment method: Place the mouse in the center of the barrier. After 30 - 60 s of adaptation, immediately invert the mouse, confirm that the mouse grasps the wire mesh with its four limbs, and record the duration before the mouse falls at the same time. Repeat the detection 3 times and take the average value as the muscle endurance level of the mouse;

[0083] Detection of limb muscle grasping force: A digital push-pull force gauge was connected to a wire mesh as a grasping force instrument for the experiment. The grasping force levels of mice were detected on the 0th day, 7th day, 14th day, and 18th day respectively. Experimental method of the grasping force instrument: Confirm that the mouse grasps the wire mesh with its limbs. The detector holds the mouse's tail and pulls it backward, and the grasping force of the mouse's limbs is detected by the grasping force instrument. The detection is repeated 3 times and the average value is taken as the grasping force level of this mouse's muscle.

[0084] 3.3 Detection of muscle pain threshold

[0085] Mouse hot plate method: First, preheat the hot plate instrument to the set temperature of 55 °C. After the temperature is constant, place the mouse in the hot plate instrument and cover it. Start timing until the mouse shows the behavior of licking its paw for the first time. The time interval from when the mouse is placed on the hot plate to when it shows the licking paw action for the first time is used as the pain threshold of the mouse. The detection is repeated 3 times and the average value is taken as the limb muscle pain threshold of this mouse.

[0086] 3.4 HE staining and immunohistochemical analysis of muscle tissue

[0087] Terminal treatment of mice: Fix the mouse, separate and cut off the proximal muscles of the four limbs. Fix the upper end of the quadriceps femoris of the right hind limb of the mouse in 10% formaldehyde, which will be used for pathological detection later, and the rest are quickly placed in a -80 °C ultra-low temperature refrigerator for storage.

[0088] HE staining: Cross-sectional sections are made for each muscle sample, with a section thickness of 5 - 8 μm. First, perform conventional dewaxing, that is, treat with xylene I and xylene II in sequence. Then perform gradient rehydration with alcohol, that is, treat with absolute ethanol I, absolute ethanol II, 95% ethanol, 85% ethanol, and 70% ethanol in sequence. Then rinse with tap water and double-distilled water 3 times respectively. Stain with hematoxylin, and then wash with tap water and double-distilled water 3 times again. Subsequently, perform hydrochloric acid-alcohol differentiation treatment, and then repeatedly wash with tap water and double-distilled water until the cell nucleus turns blue under the microscope. Dehydrate with gradient alcohol, stain with eosin; dehydrate and mount the slides, and mount the slides with neutral gum.

[0089] Immunohistochemical analysis of muscle tissue: The experimental method refers to the literature "The Role of NLRP3 Inflammasome and Its Downstream Factors in the Pathogenesis of Idiopathic Inflammatory Myopathy" by author Yin Xi. The immunohistochemical score is graded according to the number and area of muscle fibers involved in positive expression: Grade 0 has no lesions; Grade 1 involves 1 - 5 fibers, and the positive expression area is 0 - 25%; Grade 2 involves 6 - 30 muscle fibers, and the positive expression area is 26 - 50%; Grade 3 involves the entire muscle fiber bundle, and the positive expression area is 51 - 75%; Grade 4 involves more than 1 muscle fiber bundle or the entire muscle tissue, and the positive expression area is 76 - 100%.

[0090] 3.5 Real-time fluorescence quantitative reverse transcription polymerase chain reaction qRT-PCR

[0091] (1) Extraction of total RNA from tissues and cells

[0092] Total RNA extraction from tissues: Take 50 mg of mouse muscle tissue, add 1 mL of Trizon lysis buffer, homogenize using a homogenizer at 70 HZ for 30 s, repeating 3 times; operate according to the instructions of the RNA extraction kit. Centrifugation conditions: 4 °C, 12,000 rpm, pre-cooled in advance, centrifuge for 5 min and take the supernatant. Add 200 μL of chloroform to the supernatant and transfer to a new RNase-free centrifuge tube, shake vigorously for 15 s, and let stand for 3 min; centrifuge again for 10 min, take the upper colorless aqueous phase, with a volume of approximately 500 μL; transfer the aqueous phase to adsorption column CR3, slowly add 250 μL of absolute ethanol and mix well, centrifuge for 30 s, and discard the waste liquid; add protein removal solution RD, centrifuge for 30 s, and discard the waste liquid; add 500 μL of washing solution RW again, let stand for 2 min and then centrifuge for 30 s, and discard the waste liquid; place the adsorption column in a 2 mL collection tube, centrifuge for 2 min to remove the residual liquid; air-dry the adsorption column in the laminar flow hood, place the adsorption column CR3 in a new 1.5 mL centrifuge tube, add 30 - 50 μL of RNase-Free ddH2O to extract RNA, let stand for 2 min, centrifuge for 2 min, and store the centrifuge tube containing RNA at -80 °C for future measurement.

[0093] Total RNA extraction from cells: After the cell culture in the 6-well plate is completed, wash the cells with sterile PBS; add 1 mL of lysis buffer RZ to each well, for adherent cells, a cell scraper can be used to assist, aspirate the mixed liquid into a 1.5 mL EP tube, let stand for 5 min, centrifuge at 12,000 rpm, 4 °C for 10 min, take the supernatant and divide it for storage at -80 °C in the refrigerator for future measurement.

[0094] (2) RNA concentration measurement

[0095] Dilute the extracted RNA sample 100-fold, that is, 1 μL of RNA stock solution + 99 μL of ddH2O, mix well, and use a spectrophotometer to detect the RNA concentration and the absorbance at wavelengths of 260 / 280 nm and 230 / 280 nm; store the remaining samples at 4 °C temporarily for reverse transcription.

[0096] (3) Reverse transcription of RNA to synthesize cDNA

[0097] Premixing: Add the reaction solution in the appropriate proportion according to Table 2, adjust the actual concentration of the RNA sample to the same final concentration, and incubate at 42 °C for 2 min to remove the residual DNA in the sample;

[0098] Table 2 Proportion of reverse transcription reaction solution addition

[0099] Reaction solution Addition amount 5×gDNA digester Buffer 2 μL gDNA digester 1 μL <![CDATA[RNase-free ddH2O]]> To a final volume of 10 μL in the system RNA sample 1 ng - 5 μg

[0100] Reverse transcription reaction: Add an equal volume of 2× of the reaction solution with the final volume ΠSuperMix plus, pipette and mix well; incubate under the following conditions: 25°C, 5 min; 42°C, 30 min; 85°C, 5 min;

[0101] (4) Real-time quantitative PCR

[0102] Perform real-time quantitative PCR reaction according to the PCR system shown in Table 3 and the PCR program shown in Table 4:

[0103] Table 3 Proportion of each component added in the PCR system

[0104]

[0105] Table 4 PCR program

[0106]

[0107] 3.6 Western blot detection

[0108] (1) Preparation of protein samples: Aspirate and discard the cell supernatant, wash with PBS, add 120 μL / well of SDS lysis buffer containing protease inhibitor, transfer the lysed protein to a 1.5 mL EP tube, centrifuge at 4°C, 12,000 rpm for 10 min, and take the supernatant; perform protein quantification using a BCA kit, adjust the protein concentration to be consistent, aliquot and store at -80°C for later use.

[0109] (2) Detection of protein expression by protein immunoblotting: Place two clean glass slides opposite each other in the slot, add the required reagents in sequence according to the ratio to prepare 10% separating gel and 5% stacking gel, insert the comb, and let it stand for about 30 min until the gel solidifies. Place the prepared gel slot into the electrophoresis tank according to the instructions, slowly add electrophoresis buffer to be much higher than the slot; load samples according to the groups, after loading, cover the lid; connect the Bio-RAD electrophoresis instrument, set the parameters to 80 V for 30 min; switch to 120 V and 60 min of current for sample separation; transfer the gel with separated protein to the middle NC membrane between three layers of wet filter paper on the upper and lower covers, pay attention to discharging air bubbles, fix the clips, insert the transfer membrane tank, put ice bags into the transfer membrane tank, pour transfer membrane buffer into the tank, the liquid level is slightly lower than the upper horizontal plane of the plate, cover the lid, set the parameters to 100 V and electrophorese for 90 min; place the transferred NC membrane in a tray, add Ponceau S staining for 1 - 2 min, obtain the target band according to the need, wash the membrane twice with ddH2O, shake on a shaker until the Ponceau S is washed off; add blocking solution, incubate at room temperature on a shaker for 60 min; add primary antibody according to the dilution factor respectively, incubate overnight on a shaker in a 4°C cold room; wash with TBST washing solution 3 - 5 times, 10 min each time; add secondary antibody (1:20000), incubate on a shaker at room temperature for 1 h; wash with TBST washing solution 5 times, 5 - 10 min each time; add luminescent solution, expose and take pictures with the instrument, and analyze the bands.

[0110] 4 Result Analysis

[0111] 4.1 LLDT-8 Improves the Clinical Manifestations of Mice with Polymyositis Model

[0112] The general manifestations, body weights at different times, and Lennon A scores at different times of mice in each group are shown as follows Figures 1 to 3 It can be seen that at the end of the experiment, the mice in the normal group had good mental states, normal activities, and smooth and shiny hair; the hair of the model group was erected; compared with the model group, the mental states of the mice in the positive drug group and the high-dose and low-dose LLDT-8 groups improved, and the hair luster was somewhat restored. During the whole experiment, the body weights of the mice in the normal group did not change significantly; the body weights of the mice in the model group decreased significantly from the start of modeling to after the second immunization, and showed an upward trend to the end point as the disease model stabilized, and the Lennon A score was significantly higher than that of the normal group. After the first immunization with antigen, the change in body weight of the mice in the positive drug group was not significantly different from that of the model group. After the second immunization, the body weight was significantly lower than that of the model group. Although it also showed an upward trend at the end of the experiment, the body weight was still lower than that of the model group, and the Lennon A score was not significantly different from that of the model group. Compared with the model group, there was no significant difference in body weight in the high-dose and low-dose LLDT-8 groups. After the second immunization, the Lennon A scores of the high-dose and low-dose LLDT-8 groups were significantly lower than those of the model group, indicating that the pathological features showed a gradually alleviating and recovering state.

[0113] 4.2 LLDT-8 Dose-dependently Improves the Limb Muscle Endurance and Muscle Tension Levels of Mice with Polymyositis Model

[0114] The average muscle endurance and average muscle grip strength of mice in each group at different times are shown as follows Figure 4 、 Figure 6 respectively, and the change rate of muscle endurance of mice is calculated as shown in Figure 5 Combined with Figures 4 to 5It can be seen that the limb muscle endurance of the mice in the model group decreased significantly after the initial immunization. By the end of the experiment, the limb muscle endurance and limb muscle tension were still significantly lower than those of the normal group. Compared with the model group, the limb muscle endurance of the mice in the positive drug group was significantly improved on the 4th day after the initial immunization, the change rate of muscle endurance was significantly shortened, and the muscle tension was also alleviated compared with the model group. On the 4th, 11th, and 18th days of modeling, the limb muscle endurance, the change rate of muscle endurance, and the muscle grasping force value of the mice in the positive drug group were significantly higher than those of the model mice. From the 4th day of drug administration to the end of the second immunization, the muscle endurance of the mice in the low-dose LLDT-8 group showed an upward trend, and the change rate of muscle endurance was significantly better than that of the model group on the 4th, 11th, and 18th days of modeling; the four muscle tensions of the mice in the low-dose LLDT-8 group were also significantly higher than those of the model group. The muscle endurance of the mice in the high-dose LLDT-8 group was significantly higher than that of the model group from the 4th day of modeling, and it was still significantly increased compared with the model group on the 11th and 18th days; the change rate of muscle endurance in this group also showed the same trend, and the change rate of muscle endurance was significantly lower than that of the model group. The limb muscle tension gradually recovered and was better than that of the model group on the 4th, 11th, and 18th days of modeling. From the above experimental results, it can be seen that LLDT-8 can dose-dependently improve the limb muscle endurance and muscle tension levels of mice.

[0115] 4.3 LLDT-8 prolongs the first foot licking time of mice with polymyositis model

[0116] The detection results of the myalgia threshold of mice in each group at different times are as follows Figure 7 shown. The first foot licking time of the mice in the model group from the 4th day of modeling to the second immunization and from the second immunization to the end of the experiment was significantly lower than that of the normal group, indicating that the model mice themselves had myalgia symptoms and were more sensitive to pain under hot plate stimulation, significantly shortening the foot licking time. There was no obvious alleviation in the positive drug group compared with the model group. However, in the low-dose and high-dose LLDT-8 groups, starting from the 4th day of modeling, there was a trend of gradually prolonging the first foot licking time. Especially after the second immunization, the first foot licking time could be significantly prolonged, indicating an obvious analgesic effect.

[0117] 4.4 LLDT-8 improves the pathological damage of muscle tissue in mice with polymyositis model

[0118] The HE staining results of the muscle tissue and the pathological scoring results of the muscle tissue of mice in each group are as follows Figure 8 and Figure 9 shown. The muscle fibers and muscle bundles of the right hind limb quadriceps femoris of the mice in the normal group were intact. Compared with the normal group, the muscle fibers of the mice in the model group were atrophied and necrotic, accompanied by a large number of inflammatory cell infiltrations. The positive drug group could significantly reduce the infiltration of inflammatory cells and improve the muscle damage degree of mice with polymyositis. Both the low-dose and high-dose LLDT-8 groups could significantly reduce the infiltration of inflammatory cells in the muscle tissue and improve the muscle damage degree of mice with polymyositis.

[0119] 4.5 LLDT-8 Reduces the Infiltration of Pathogenic T Cells in the Muscle Tissue of Mice with Polymyositis

[0120] The quadriceps femoris of the right hindlimb of mice in each group was taken, and the positive expression of pathogenic CD8 + T cells in the muscle tissue was analyzed by immunohistochemistry, and the results were as Figure 10 shown. In the muscle tissue of normal group mice, the infiltration of CD8 + T cells was less, and the muscle fibers were polygonal or oval. In the muscle tissue of model group mice, the infiltration area of CD8 + T cells in the muscle tissue increased, and the positive expression sites of CD8 + T cells increased significantly, accompanied by severe damage to muscle fibers. In the muscle tissue of positive drug group mice, the positive expression sites of CD8 + T cells decreased significantly. Both the low-dose and high-dose groups of LLDT-8 significantly reduced the positive expression sites of CD8 + T cells, and the improvement effect was more significant in the high-dose group of LLDT-8.

[0121] 4.6 LLDT-8 Downregulates the Expression of MHC-I in the Muscle Tissue of Mice with Polymyositis

[0122] The expression levels of MHC-I mRNA in the muscle tissue of mice in each group were obtained as Figure 11 shown. Since the regulatory mechanism of LLDT-8 was explored, the data of the positive drug group were not included. It can be Figure 11 seen that after the administration of LLDT-8, the expression of MHC-I mRNA in the muscle tissue can be downregulated. To further verify this result, the positive expression of MHC-I in the muscle tissue of mice in each group was analyzed by immunohistochemistry, and the protein level of MHC-I in the muscle tissue was quantitatively analyzed. The results were as Figure 12 and Figure 13 shown respectively, and it was found that the expression of MHC-I in the muscle tissue of model group mice was abnormally upregulated, while there was a tendency to reduce its expression after the administration of LLDT-8.

[0123] 4.7 LLDT-8 Downregulates the Expression of NLRC5 in the Muscle Tissue of Mice with Polymyositis

[0124] The expression levels of NLRC5 mRNA in the muscle tissue of mice in each group were obtained as Figure 14 shown, and it can be seen that after the administration of LLDT-8, the expression of NLRC5 in the muscle tissue can be significantly downregulated.

[0125] 4.8 LLDT-8 Downregulates the Expression of Antigen Presentation-related Factors in the Muscle Tissue of Mice with Polymyositis

[0126] Since NLRC5 can not only affect the expression of MHC-I, but also regulate the expression of factors related to the MHC-I pathway of antigen presentation, the mRNA expression levels of antigen presentation-related factors β2M, Psmb8, Psmb9, Tap1, and Tap2 in the muscle tissues of mice in each group were detected, and the results are as Figure 15 shown. It can be seen that the mRNA expression levels of β2M, Tap1, Tap2, Psmb8, and Psmb9 in the muscle tissues of the model group mice were significantly higher than those of the normal group; after administration of LLDT-8, the expression levels of antigen presentation factors β2M, Psmb8, Tap1, and Tap2 mRNA were significantly decreased, and the expression level of Psmb9 mRNA was inhibited to a certain extent. The above results indicate that LLDT-8 can inhibit the activation of the NLRC5 / MHC-I downstream pathway.

[0127] 4.9 LLDT-8 inhibits the activation of the NLRC5 / MHC-I pathway induced by IFN-γ in C2C12 cells

[0128] Since normal muscle fibers do not express MHC-I, MHC-I expression can be induced by IFN-γ. Using undifferentiated C2C12 mouse myoblasts as the research object in vitro, an in vitro cell system of IFN-γ acting on C2C12 cells was constructed. After LLDT-8 was intervened for 15 min and 30 min respectively in this in vitro cell system, the cells were collected to extract proteins, and the protein levels of MHC-I and NLRC5 in the cells were detected by Western blot to investigate the regulatory effect of LLDT-8 on the NLRC5 / MHC-I pathway. The detection results are as Figure 16 shown, and GAPDH in the figure is the internal reference. From Figure 16 it can be seen that LLDT-8 (200 nM) can significantly down-regulate the protein levels of MHC-I and NLRC5, indicating that LLDT-8 can inhibit the activation of the NLRC5 / MHC-I pathway induced by IFN-γ in C2C12 cells.

Claims

1. Application of LLDT-8 in the preparation of drugs for the treatment of polymyositis.

2. The use according to claim 1, characterized in that: The drug for treating polymyositis is to downregulate the expression levels of MHC-I and NLRC5 in muscle tissue, inhibit CD8 + Preparations for T cell activation.

3. The use according to claim 2, characterized in that: The drug for treating polymyositis is used to down-regulate the expression levels of antigen presenting factors β2M, Psmb8, Tap1 and Tap2 in muscle tissue.

4. The use according to claim 1, characterized in that: The polymyositis refers to polymyositis induced by immunization with an emulsion of myosin and Freund's adjuvant and intraperitoneal injection of Bordetella pertussis.

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