Use of an ER beta agonist in the preparation of a medicament for treating Duchenne muscular dystrophy

Abstract

The present invention provides an application of an ER beta agonist in the preparation of a drug for treating Duchenne muscular dystrophy, belonging to the field of pharmaceutical technology. Experiments of the present invention show that after treatment with the ER beta agonist diarylpropionitrile (DPN), the damaged area of muscle decreases and the inflammatory infiltration reduces; meanwhile, DPN can enhance the skeletal muscle strength function of B10-DMD-KO mice, alleviate the muscle tissue damage caused by the self-gene defect of B10-DMD-KO mice, promote the formation of new muscle fibers and accelerate their maturation, ultimately promote the regeneration of skeletal muscle fibers and the recovery of skeletal muscle strength, and can be used to improve the muscle regeneration disorder caused by Duchenne muscular dystrophy, providing a theoretical basis for the development of drugs for treating Duchenne muscular dystrophy. In addition, compared with the existing technical means, DPN has the advantages of lower treatment cost and less side effects, and thus has good application prospects in the preparation of drugs for treating Duchenne muscular dystrophy.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to the application of ER beta agonists in the preparation of drugs for treating Duchenne muscular dystrophy, specifically to the application of diarylpropionitrile in the preparation of drugs for treating muscle regeneration defects and / or muscle weakness caused by Duchenne muscular dystrophy. Background Art

[0002] Duchenne muscular dystrophy (DMD) is a genetic disease caused by the deletion of the gene encoding dystrophin. Dystrophin connects the cytoskeleton and the extracellular matrix to maintain the stability of the muscle cell membrane. Currently, the main treatment strategies for DMD patients are divided into etiological treatment and symptomatic treatment: Etiological treatment mainly includes AAV-mediated microgene replacement, AAV-mediated follistatin expression, and exon skipping at the RNA level. However, so far, gene therapy still faces problems such as a narrow scope of drug application, being only effective for patients with specific mutation types, high cost, and difficult R & D. For example, in 2016, the US Food and Drug Administration (FDA) approved the injection of Exondys 51 (eteplirsen) for the treatment of patients with Duchenne muscular dystrophy (DMD). Eteplirsen can only be used to treat patients diagnosed with exon 51 mutations in the dystrophin gene, and this group accounts for only about 13% of the total number of DMD patients. Even the latest gene therapy-based drug, Elevidys, approved by the FDA on June 23, 2023, still has problems such as a narrow treatment window (only for DMD patients aged 4 - 5 years), high cost ($3.2 million per injection), and significant safety risks (the immunogenicity of AAV itself); Symptomatic treatment mainly focuses on glucocorticoid treatment to improve muscle function and control inflammation, but its effect is difficult to last for a long time, cannot significantly extend the survival period of patients, and there are also relatively serious side effects, resulting in poor patient compliance.

[0003] Estrogen is deeply involved in the regulation of motor organs (bones and skeletal muscles). Its deficiency can lead to loss of muscle mass. Supplementary therapy with estrogen has been proven by research to promote the regeneration of skeletal muscles after injury. The knockout of estrogen receptor β rather than α can lead to impaired proliferation ability of skeletal muscle satellite cells, and the up-regulation of ER beta has been confirmed in various skeletal muscle injury models. It has been found in research that in acute muscle injury induced by cardiotoxin (CTX), the expression level of ER beta in the tibialis anterior (TA) is significantly up-regulated; in the study of treating mdx mice with tamoxifen, a significant up-regulation of ER beta in the tamoxifen treatment group was found. However, the reason for the up-regulation of ER beta in the acute muscle injury model and mdx mice lacks further research, and the role played by ER beta in myoblast differentiation and the formation of new muscle fibers remains unclear. Diarylpropionitrile (DPN) is a selective ER beta agonist. It has been reported that it can inhibit the secretion and expression of macrophage RANTES after LPS activation by inhibiting the activation of nuclear factor κB. DPN has also been reported as an ER beta agonist, which has the effect of protecting hippocampal neurons from glutamate-induced cell death and does not activate the adverse effects of estrogen in reproductive organs. Therefore, the small molecule DPN that specifically activates ER beta has certain pharmacological effects and drug-forming value. At the present stage, it is of great significance to find small molecule compounds that can effectively treat DMD patients with less side effects. Summary of the Invention

[0004] The object of the present invention is to provide a new use of an ER beta agonist in the preparation of a drug for treating Duchenne muscular dystrophy. In the present invention, DPN promotes muscle regeneration to increase the grasping force of skeletal muscles by specifically activating ER beta1 in ER beta, and can also reduce skeletal muscle inflammation, ultimately improving muscle weakness and / or muscle atrophy caused by Duchenne muscular dystrophy.

[0005] The object of the present invention is achieved by the following technical solutions:

[0006] The application of an ER beta agonist in the preparation of a drug for treating Duchenne muscular dystrophy.

[0007] As a preferred solution, the ER beta agonist is the ER beta small molecule agonist diarylpropionitrile, and its structural formula is as follows:

[0008]

[0009] As a preferred embodiment, the dosage of the diarylpropionitrile for mice is 0.25 - 1 mg / kg / d, and the dosage for adults is 1.21 mg / d - 4.88 mg / d (calculated based on 60 kg).

[0010] As a preferred embodiment, it includes the application of diarylpropionitrile in the preparation of a drug for treating muscle regeneration defects and / or muscle weakness caused by Duchenne muscular dystrophy.

[0011] As a preferred embodiment, it includes the application of diarylpropionitrile in the preparation of a drug for treating muscle regeneration defects and / or muscle weakness caused by Duchenne muscular dystrophy caused by the absence of dystrophin.

[0012] In the present invention, the therapeutic effect of the ER beta agonist DPN was investigated in acute skeletal muscle injury and B10 - DMD - KO mice. The results showed that after treatment with the ER beta agonist DPN, the damaged area of the muscle decreased and the inflammatory infiltration reduced; at the same time, DPN could enhance the skeletal muscle strength function of B10 - DMD - KO mice, reduce the muscle tissue damage caused by the self - gene defect of B10 - DMD - KO mice, and promote the formation of new muscle fibers and accelerate their maturation. Ultimately, it could promote skeletal muscle fiber regeneration and skeletal muscle strength recovery, and could be used to improve the muscle regeneration disorder caused by Duchenne muscular dystrophy, providing a theoretical basis for the development of drugs for treating Duchenne muscular dystrophy. In addition, compared with the existing technical means, DPN has the advantages of lower treatment cost and fewer side effects. 2 Description of the Drawings Description of the Drawings

[0013] Figure 1 It is the graph of the body weight and TA muscle weight coefficient of each group of mice with acute muscle injury induced by BaCl2 in the examples of the present invention;

[0014] Figure 2 It is the HE staining result graph of the TA cross - section of each group of mice with acute muscle injury induced by BaCl2 in the examples of the present invention;

[0015] Figure 3 It is the detection graph of the inflammatory factor level of TA of each group of mice with acute muscle injury induced by BaCl2 in the examples of the present invention;

[0016] Figure 4 It is the result graph of the macrophage level of TA of mice with acute muscle injury induced by BaCl2 in the examples of the present invention (200×);

[0017] Figure 5a It is the weight gain of B10 - DMD - KO mice in the examples of the present invention;

[0018] Figure 5bIt is the graph of the weight coefficients of GAS, TA, and testis of B10-DMD-KO mice in the embodiments of the present invention;

[0019] Figure 6 It is the graph of the muscle grip strength level of B10-DMD-KO mice in the embodiments of the present invention;

[0020] Figure 7 It is the graph of the determination of the hanging rod time of B10-DMD-KO mice in the embodiments of the present invention;

[0021] Figure 8a It is the graph of the detection result of the CK content of B10-DMD-KO mice in the embodiments of the present invention;

[0022] Figure 8b It is the graph of the detection result of the LDH content of B10-DMD-KO mice in the embodiments of the present invention;

[0023] Figure 9 It is the graph of the H&E staining result of the cross-section of GAS of B10-DMD-KO mice in the embodiments of the present invention;

[0024] Figure 10 It is the staining graph (200×) of macrophages (using CD68 as a marker) in GAS of B10-DMD-KO mice in the embodiments of the present invention;

[0025] Figure 11 It is the staining graph (200×) of MHC of mature muscle fibers in GAS of B10-DMD-KO mice in the embodiments of the present invention;

[0026] Figure 12a It is the staining graph (200×) of neonatal muscle fibers in GAS of B10-DMD-KO mice in the embodiments of the present invention;

[0027] Figure 12b It is the staining graph (200×) of neonatal muscle fibers in DIA of B10-DMD-KO mice in the embodiments of the present invention;

[0028] Figure 12c It is the statistical graph of the proportion of the positive area of the stained neonatal muscle fibers in GAS and DIA of B10-DMD-KO mice in the embodiments of the present invention;

[0029] Figure 13a It is the protein levels of PAX7, MyoD, and ER beta1 in GAS of B10-DMD-KO mice in the embodiments of the present invention;

[0030] Figure 13b It is the statistical graph of the protein levels of PAX7, MyoD, and ER beta1 in GAS of B10-DMD-KO mice in the embodiments of the present invention;

[0031] Figure 14a WB bands showing the effect of DPN on the expression levels of ER beta1 and MyoD in C2C12 cells in the examples of the present invention;

[0032] Figure 14b Statistical chart showing the effect of DPN on the expression levels of ER beta1 and MyoD in C2C12 cells in the examples of the present invention;

[0033] Figure 15 Statistical chart showing the effect of si ER beta1 on the expression levels of ER beta1 and MyoD in C2C12 cells promoted by DPN in the examples of the present invention;

[0034] Figure 16 Fluorescence images showing the changes in the expression level of MYH in C2C12 cells after si ER beta1 in the examples of the present invention;

[0035] Figure 17 WB bands and statistical chart showing the changes in the expression level of MYH in C2C12 cells after si ER beta1 in the examples of the present invention. Detailed implementation manners

[0036] For better understanding of the content of the present invention by those skilled in the relevant technical fields, the following detailed description is given of the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are provided. However, the content of the present invention is not limited to the following examples. The following explains the role of the ER beta agonist in the drug for Duchenne muscular dystrophy in the present invention through experiments.

[0037] Intramuscular injection of barium chloride (BaCl 2 ) injection solution was used to induce acute skeletal muscle injury: An acute skeletal muscle injury model mouse induced by BaCl 2 was used in the present invention. The skeletal muscle injury induced by BaCl 2 was caused by inhibiting the efflux of potassium ions, resulting in long-term depolarization of muscle fibers, and then activating the L-type voltage-gated calcium channels in the muscle membrane. The release from the sarcoplasmic reticulum (the specialized endoplasmic reticulum of muscle fibers) and the influx of extracellular fluid led to an increase in the concentration of Ca 2+ , triggering proteolysis, and ultimately leading to the degradation of contractile proteins and cell membranes. The experimental mice used in the present invention were male C57BL / 6J mice, purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Experimental data were analyzed using GraphPad Prism 9 statistical software and expressed as Mean±SEM. For comparison between multiple experimental groups, one-way analysis of variance (One Way ANOVA) was used to analyze the significant differences between groups. * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and ns represents no statistically significant difference.

[0038] Example 1: Specific experimental protocol for treating acute muscle injury in C57BL / 6J mice with DPN

[0039] 1. Experimental grouping and drug administration monitoring

[0040] In this example, a total of 64 C57BL / 6J mice with a body weight of 19 - 21 g were used. The C57BL / 6J mice were randomly divided into 8 groups according to body weight stratification: wild group (n = 8) and wild drug administration group (n = 8), 2 - day model group (n = 8) and 2 - day drug administration group (n = 8), 5 - day model group (n = 8) and 5 - day drug administration group (n = 8), 7 - day model group (n = 8) and 7 - day drug administration group (n = 8). The model groups were injected with 50 μl of 1.2% (w / v) BaCl 2 into both TA of the mice for modeling; the drug administration groups were all given DPN at 1 mg / kg / d.

[0041] The specific experimental protocol is shown in Table 1:

[0042] Table 1 Specific grouping and drug administration scheme of experimental animals

[0043]

[0044] During the experiment, the body weight of the mice was recorded every day, and the state of the mice was observed;

[0045] One day before dissection, the mice were fasted overnight for 12 h. After anesthesia with isoflurane, the eyeballs of each group of mice were taken for blood sampling, and the tibialis anterior (TA) was taken after decapitation. One side of the TA was frozen - embedded, and the other side was stored at - 80 °C to detect the mRNA levels of inflammatory factors IL - 1β, IL - 10, TNF - α and macrophage marker CD68. The embedded muscle was used for frozen sectioning, and the thickness of the section was 10 μm. The sections of each group were used for histopathological observation (HE staining).

[0046] The body weight and TA muscle weight coefficient of each group of mice are as Figure 1 shown, as Figure 1 shown, the body weight results of each group of mice suggest that there is no significant difference between the model group and the drug administration group. The TA muscle weight coefficient of the mice in the 2 - day DPN drug administration group was significantly decreased compared with the 2 - day model group, indicating that DPN may reduce the inflammatory response after injury and thus relieve the swelling and weight increase caused by muscle damage.

[0047] 2. Histopathological observation

[0048] In this example, histopathological observation was performed using hematoxylin-eosin (HE) staining. After 7 days of modeling, the mice were sacrificed, and the fresh TA was quickly embedded in OCT embedding medium and then placed in a cryostat for sectioning. The section thickness was 10 μm for HE staining, and after staining, the sections were sealed with neutral balsam. Finally, the sections were observed and images were collected under a BX53 (Olympus, Japan) microscope. Figure 2 These are the results of histopathological observation.

[0049] As Figure 2 shown, in the 2-day model group, a large number of muscle fibers were edematous and necrotic, and there was a large amount of inflammatory cell infiltration. In the DPN-administered 2-day group, the inflammatory infiltration decreased and the necrotic area of muscle fibers decreased. At 5 days of acute injury, some muscle fibers with nuclei in the center had formed, and the inflammatory infiltration was still severe. After administration of DPN, a large number of new muscle fibers appeared and the inflammatory infiltration was alleviated.

[0050] 3. Detection of mRNA levels of inflammatory factors such as IL-1β and TNF-α in TA tissue

[0051] RT-qPCR was used to detect the mRNA levels of inflammatory factors (IL-1β, IL-10, TNF-α) and macrophage marker (CD68) in TA tissue. After the animals were sacrificed, the fresh TA was quickly placed on dry ice and then stored in a -80°C refrigerator. Approximately 20 mg of TA from each group was used for RNA extraction, reverse transcribed into cDNA for later measurement. The specific results are as Figure 3 shown.

[0052] The PCR results showed that after 2 days of barium chloride injection injury in the model group, the levels of inflammatory factors increased significantly, while in the DPN treatment group on the corresponding days, the mRNA expression levels of inflammatory factors could be significantly reduced.

[0053] 4. Detection of immunofluorescence results of CD68

[0054] CD68 is a surface marker of macrophages. Immunofluorescence experiments were performed to stain CD68 in each group to investigate the inflammatory infiltration of skeletal muscle in B10-DMD-KO mice after administration of DPN. The fresh TA was embedded in OCT for freezing and then sectioned using a cryostat with a section thickness of 10 μm. The sections of each group were fixed with paraformaldehyde for 15 minutes, washed three times with PBS (5 minutes each time), blocked in 5% goat serum and 3% BSA solution for 1 hour. After blocking, the sections were stained with CD68 and Laminin antibodies. Laminin was used to label the cell outline, and the sections were placed in a 4°C refrigerator overnight. After rewarming for 1.5 h the next day, the sections were incubated in a 37°C oven for 0.5 h, washed three times with PBS (5 minutes each time), stained with the corresponding fluorescent secondary antibody for 2 h, and finally sealed with an anti-fluorescence quencher (containing DAPI) and observed and images were collected under an FV3000; asFigure 4 It is the image result.

[0055] Such as Figure 4 The immunofluorescence results of CD68 showed that in the model group on the 5th day, there was still severe inflammatory infiltration five days after injury, and it was in a clustered form. In the group administered DPN for 5 days, the inflammatory infiltration was reduced and showed scattered dots.

[0056] Example 2: Pharmacodynamic evaluation experimental protocol for treating 5-week-old B10-DMD-KO mice with DPN

[0057] In this example, male model mice C57BL / 10ScSnJGpt-DMDem3Cd4 / GPT (hereinafter referred to as B10-DMD-KO mice) representing DMD disease were used. There was a deletion mutation in exon 4 of the DMD gene in B10-DMD-KO mice, resulting in their inability to produce functional dystrophin. Dystrophin is crucial for the connection between the cytoskeleton and the extracellular matrix. Eventually, B10-DMD-KO mice showed symptoms similar to those of DMD patients. Compared with normal wild-type mice C57BL / 10ScSnJ, B10-DMD-KO mice showed enlarged and disordered muscle fiber structures, weakened skeletal muscle function, mainly weakened skeletal muscle tone; B10-DMD-KO mice also showed continuous inflammatory cell infiltration in muscle tissues, accompanied by severe fibrosis and muscle fiber necrosis; in this example, due to the loss of Dystrophin function in B10-DMD-KO mice, the sarcolemma was damaged, and creatine kinase was released from skeletal muscle tissue, further manifested as an increase in serum creatine kinase levels. In this example, the male model mice C57BL / 10ScSnJGpt-DMDem3Cd4 / GPT were from Jicui Yakang.

[0058] Pharmacodynamic evaluation of DPN in treating 5-week-old B10-DMD-KO mice. The specific experimental protocol is as follows: In this example, B10-DMD-KO mice were intraperitoneally administered DPN starting from the fifth week of age, with the dosage set at 0.25 mg / kg / d and 1.00 mg / kg / d for 21 days. On the 14th day of administration, the skeletal muscle function of the mice was measured using the hanging rod test and muscle grip strength test respectively. After the last administration on the 21st day of administration, the mice were fasted for 12 h. On the 22nd day, blood was collected by enucleating the eyeballs after anesthesia with isoflurane, and the mice were sacrificed by cervical dislocation. The gastrocnemius muscle (GAS), tibialis anterior muscle (TA), soleus muscle (SOL), and diaphragm (DIA) were taken. One side was used for cryoembedding, and the other side was stored frozen at -80 °C. The cryoembedded tissue was used for frozen sectioning, with the section thickness of 10 μm. The sections of each group were used for histopathological observation (HE staining), investigation of the positive area of neonatal myofibers eMyHC (immunofluorescence staining of eMyHC), and investigation of the positive area of mature myofibers (immunofluorescence staining of MHC); the other side was used for WB to measure ER beta1, PAX7, and myogenic regulatory factor (MyoD).

[0059] There were a total of 18 B10-DMD-KO mice, 4 - 5 weeks old, male, and 6 male wild-type mice C57BL / 10ScSnJ of the same age. The B10-DMD-KO mice were divided into three groups according to body weight: model group (n = 6), low-dose DPN administration group (n = 6), and high-dose DPN administration group (n = 6). The low dose of the DPN group was 0.25 mg / kg / d, and the high dose of the DPN group was 1.00 mg / kg / d. The specific experimental protocol is shown in Table 2:

[0060] Table 2 Specific grouping and dosing regimens of experimental animals

[0061]

[0062] During the experiment, the mice were administered at a fixed time point every day, and the morphology of the mice was observed and the body weight was recorded. The body weight results are as Figure 5a shown, and the muscle weight coefficient results are as Figure 5b shown. The body weight of each group of mice increased steadily without significant differences. Compared with the wild-type mice, the muscles of the mice in the model group were swollen and had a higher muscle weight coefficient. DPN could significantly reduce the muscle weight coefficient of the TA of the mice at both the dosages of 0.25 mg / kg and 1.00 mg / kg. When the DPN dosage was 0.25 mg / kg, it could also significantly reduce the muscle weight coefficient of the GAS of the mice, thus improving the phenomenon of muscle pseudohypertrophy in the model mice.

[0063] 1. Determination of muscle grip strength and limb grip strength

[0064] On the 14th day after administration, the muscle grip strength of the forelimbs of mice was measured using a muscle grip strength meter (YLS-13A, Jinan Yiyan Technology Development Co., Ltd.) to measure the maximum muscle grip strength of the forelimbs of each group of mice. Each mouse was measured 3 times and the average value was taken. The results are shown in Table 3 and Figure 6 as follows:

[0065] Table 3 Statistical table of muscle grip strength measurement results of B10-DMD-KO mice

[0066] Grouping con DMD-KO 0.25 mg / kg / d 1.00 mg / kg / d Mean±SEM. 12.63±0.5548 <![CDATA[5.633±0.2939 **** > <![CDATA[9.945±0.2173 #### > <![CDATA[11.45±0.5236 #### >

[0067] Compared with the con group, **** P<0.0001; compared with the DMD-KO group, #### P<0.0001.

[0068] The results of muscle grip strength measurement showed that the DPN administration group could enhance the muscle grip strength of B10-DMD-KO mice, indicating that after administration of DPN, the skeletal muscle function of B10-DMD-KO mice could be improved.

[0069] Measurement of hanging rod time: On the 14th day after administration, the limb grip strength of mice was measured. A metal ring made of a 2-mm-diameter iron wire and a 37-cm-high bracket were used, and a foam board was placed under the bracket (to prevent the mice from being injured when they fell). After the mice grasped the iron ring with their two forelimbs, the timing started. If the mice could hang for 600 s, they were carefully removed from the iron ring and put back into the mouse cage; if the mice fell before this time, they were given up to two chances to hang again, and the final hanging time of each mouse was recorded. The hanging rod index of each mouse = mouse body weight (g) * hanging duration (s). The results are shown in Table 4 and Figure 7 as follows:

[0070] Table 4 Statistical table of hanging rod time measurement results of B10-DMD-KO mice (mean ± SEM)

[0071] Grouping con DMD-KO 0.25 mg / kg / d 1.00 mg / kg / d Mean±SEM. 14300±325.6 <![CDATA[1773±247.1 **** > <![CDATA[3147±127.7 ## > <![CDATA[3628±241.2 ### >

[0072] Compared with the con group, **** P<0.0001; compared with the DMD-KO group, ## P<0.01, ### P<0.001.

[0073] The results of the hanging rod index showed that the hanging index of the mice in the DPN administration group increased significantly, indicating that DPN at a dose of 0.25 - 1.00 mg / kg could significantly enhance the limb strength of B10-DMD-KO mice and improve the skeletal muscle strength function of B10-DMD-KO mice.

[0074] 2. Measurement of blood biochemical indexes creatine kinase CK and lactate dehydrogenase LDH

[0075] Twenty-one days after administration, the mice were sacrificed on the 22nd day (fasted for 12 h before sacrifice), and anatomical samples were taken. After anesthesia with isoflurane, blood was collected by eye enucleation. The serum was allowed to stand for two hours and then centrifuged at 4 °C at a speed of 4000 rpm. The upper layer of serum was taken to measure the contents of CK and LDH; fresh GAS, TA, and testis were weighed, and the body weight of the mice and the muscle weight coefficients of each tissue were as Figure 3 shown. The contents of serum CK and LDH are shown in Table 5 and Figure 8a and Figure 8b shown as follows:

[0076] Table 5 Serum CK and LDH levels in B10-DMD-KO mice (mean ± SEM)

[0077] Grouping con DMD-KO 0.25 mg / kg / d 1.00 mg / kg / d CK 1665±483.8 <![CDATA[9135±380.8 **** > <![CDATA[6945±791.6 p＝0.09 > <![CDATA[5481±751.7 ## > LDH 1194±117.3 <![CDATA[11515±1724 **** > <![CDATA[6991±865.4 p＝0.06 > <![CDATA[5069±1390 ## >

[0078] Compared with the con group, **** P < 0.0001; compared with the DMD-KO group, ## P < 0.01.

[0079] The results showed that compared with the wild type, the CK and LDH in the B10-DMD-KO mouse model group were significantly up-regulated (P < 0.001). After administration of DPN, the CK and LDH in the serum could be reduced. The dose of 1.0 mg / kg / d could significantly reduce the values of CK and LDH (P < 0.01), indicating that DPN has a significant therapeutic effect on the excessive CK and LDH caused by muscle fiber necrosis in B10-DMD-KO mice.

[0080] 3. Histopathological observation

[0081] For histopathological observation, HE staining was used. The mice were sacrificed on the 21st day, and fresh GAS was quickly frozen and embedded in OCT, and then placed in a cryostat for sectioning. The section thickness was 10 μm. The sections of each group were used for HE staining, sealed with neutral gum, and then observed and photographed under a BX53 microscope. The results were as Figure 9 shown.

[0082] The HE results showed that there were a large number of inflammatory cell infiltrations in the GAS of the B10-DMD-KO model group mice. The muscle fibers were round and swollen in shape, severely necrotic, and of different sizes. The muscle fibers showed nuclear centralization and obvious loss, while in the GAS of each DPN-administered group, a significant reduction in inflammatory infiltration, recovery of the morphology of skeletal muscle fibers, and orderly and closer arrangement of skeletal muscle fibers could be observed.

[0083] 4. Detection of CD68 immunofluorescence results

[0084] The inflammatory infiltration in the skeletal muscle of B10-DMD-KO mice was mainly manifested as inflammatory infiltration mainly composed of macrophages. In the immunofluorescence experiment, the macrophage surface marker CD68 was selected for staining to investigate the effect of DPN administration on the inflammatory cell infiltration in the skeletal muscle of B10-DMD-KO mice. Fresh TA was cryo-embedded in OCT and sectioned, and the section thickness was 10 μm. The sections of each group were fixed with paraformaldehyde for 15 minutes, washed three times with PBS (5 minutes each time), blocked in 5% goat serum and 3% BSA solution for 1 hour. After blocking, they were stained with CD68 and Laminin antibodies, placed in a refrigerator at 4 °C overnight. After rewarming for 1.5 h the next day, they were incubated in an oven at 37 °C for 0.5 h, washed three times with PBS (5 minutes each time), stained with the corresponding fluorescent secondary antibody for 2 h, and finally sealed with an anti-fluorescence quencher (containing DAPI), and placed under the FV3000 for observation and image acquisition. The results are as Figure 10 shown.

[0085] The immunofluorescence results of CD68 showed that compared with the wild type, the infiltration of macrophages in the TA of the model group mice was more severe, and the expression of CD68 decreased after DPN administration.

[0086] 5. Detection of immunofluorescence results of MHC (Myosin heavy chain, sarcomere)

[0087] In the immunofluorescence experiment, MHC was selected for staining of mature muscle fibers to investigate the effect of DPN administration on the morphology and quantity of mature muscle fibers in the skeletal muscle of B10-DMD-KO mice. Fresh GAS was cryo-embedded in OCT and sectioned, and the section thickness was 10 μm. The sections of each group were fixed with paraformaldehyde for 15 minutes, washed three times with PBS (5 minutes each time), blocked in 5% goat serum and 3% BSA solution for 1 hour. After blocking, they were stained with MHC and Laminin antibodies, placed in a refrigerator at 4 °C overnight. After rewarming for 1.5 h the next day, they were incubated in an oven at 37 °C for 0.5 h, washed three times with PBS (5 minutes each time), stained with the corresponding fluorescent secondary antibody for 2 h, and finally sealed with an anti-fluorescence quencher (containing DAPI), and placed under the FV3000 for observation and image acquisition. The results are as Figure 11 shown.

[0088] The MHC results showed that compared with the wild type group, the mature muscle fibers of GAS in the model group mice were severely necrotic and there was a phenomenon of loss of mature muscle fibers. The muscle fibers were round. After DPN administration, the morphology of the mature muscle fibers was complete and the arrangement was relatively neat.

[0089] 6. Immunofluorescence staining of eMyHC (Myosin heavy chain, embryonic / myosin-3)

[0090] For the immunofluorescence experiment, neonatal myofibers eMyHC and Laminin were selected for staining to investigate the effects of DPN administration on the neonatal myofibers and fiber morphology of skeletal muscles in B10-DMD-KO mice. Fresh GAS and DIA were quickly frozen and embedded in OCT and then sectioned, with the section thickness being 10 μm. The sections of each group were fixed with paraformaldehyde for 15 minutes, washed three times with PBS (5 minutes each time), blocked in 5% goat serum and 3% BSA solution for 1 hour. After blocking, they were stained with eMyHC and Laminin antibodies respectively, placed in a refrigerator at 4°C overnight, rewarmed for 1.5 h the next day and then incubated in an oven at 37°C for 0.5 h, washed three times with PBS (5 minutes each time), stained with the corresponding fluorescent secondary antibody for 2 h, and finally sealed with an anti-fluorescence quencher (containing DAPI), placed under the FV3000 for observation and image acquisition. Image J was used to statistically analyze the positive area, and the results are as Figure 12a 、 Figure 12b and Figure 12c shown.

[0091] The results of eMyHC showed that there were relatively severe differentiation disorders in B10-DMD-KO mice themselves (rare neonatal myofibers were seen in GAS and DIA), while the expression of eMyHC (positive area) in each DPN-administered group of mice increased significantly, indicating that DPN could relieve the phenomenon of skeletal muscle regeneration disorder in B10-DMD-KO mice, significantly promote the formation of neonatal myofibers, provide sufficient neonatal myofibers for the subsequent maturation of myofibers, and ultimately restore skeletal muscle strength.

[0092] 7. Detection of protein levels of myogenic regulatory factors

[0093] Frozen GAS of each group of mice was selected for protein extraction. RIPA strong lysis buffer added with protease inhibitor and phosphatase inhibitor was used for tissue lysis. After the muscle was homogenized in a grinder, it was placed on ice for lysis for 30 minutes, and then centrifuged at 12,000×g at 4°C for 15 minutes in a centrifuge. The supernatant was aspirated, the protein concentration was measured and quantified using a BCA kit, and then the protein mother liquor was diluted with normal saline to 5 μg / μL. 5×loading buffer was added, vortexed and mixed evenly, and then heated in a metal bath at 95°C for 15 minutes. After returning to room temperature, it was stored at -80°C. The samples were used for Western blot experiments to detect the protein levels of PAX7, myogenic regulatory factor (MyoD), and ER beta1. Among them, according to the literature report, MyoD, as a myogenic regulatory factor that dominates muscle regeneration, plays a promoting role in the subsequent myosin development, specifically manifested in that the proximal promoter of the myosin gene contains an E-box that responds to MyoD, and the results are as Figure 13a and Figure 13b shown.

[0094] The WB results showed that DPN administration could significantly promote the expression levels of PAX7 and myogenic regulatory factor (MyoD), and also increased the expression level of ER beta1. Combining the results of Examples 8 and 9, it was shown that the ER beta-specific agonist DPN could improve the myogenic differentiation disorder in B10-DMD-KO mice, promote the increase in the level of MyoD, and further promote the formation of neonatal myofibers, improving the phenomenon of loss of mature myofibers in the model group mice.

[0095] 8. Effect of DPN on the expression level of MyoD in myoblasts

[0096] Mouse C2C12 myoblasts were selected as the research object to investigate whether DPN could promote the expression level of MyoD in C2C12 cells in the proliferation phase. When the C2C12 cells grew to about 90% of the surface area of the large dish, the cells were digested, the cell precipitate was centrifuged and resuspended, counted, and a certain volume of cell suspension was pipetted into a new proliferation medium. After mixing, 2 ml of the cell-containing medium was added to each well of a 6-well plate. When the cells grew to 30 - 40%, different doses of DPN were given, and after continued culture for 36 - 48 hours, the cells were lysed with SDS containing protease inhibitors and phosphatase inhibitors, and the corresponding volume of 5×loading was added. After mixing, it was heated in a 95°C metal bath for 15 minutes. After the protein lysate returned to room temperature, it was stored in an -80°C refrigerator, and Western blot experiments were performed. The results were as Figure 14a and Figure 14b .

[0097] The WB results showed that 50 nM DPN could significantly promote the expression level of MyoD, and at the same time, the protein level of ER beta1 also increased synchronously.

[0098] 9. Effect of Si ER beta1 on the effect of DPN in promoting MyoD expression

[0099] Mouse C2C12 myoblasts were selected as the research object to investigate whether the decrease in the protein level of ER beta1 would cause a decrease in the expression level of MyoD, and whether DPN promoted the expression level of MyoD through ER beta1. When the C2C12 cells grew to about 90% of the surface area of the large dish, the cells were digested, the cell pellet was resuspended by centrifugation, counted, and a certain volume of cell suspension was pipetted into a new proliferation medium. After mixing, 2 ml of the cell-containing medium was added to each well of a 6-well plate. After the cells adhered, within 24 hours of spotting the plate, when the cell density was 30-40%, small interfering experiments were performed on C2C12 cells using si ERbeta 1. After 8 hours, the original medium was discarded, and the proliferation medium was added and the cells were cultured for another 48 hours. The cells were lysed with SDS containing protease inhibitors and phosphatase inhibitors, and the corresponding volume of 5×loading was added. After mixing, the mixture was heated in a metal bath at 95°C for 15 minutes. After the protein lysate returned to room temperature, it was stored in a -80°C refrigerator. Western blot experiments were performed, and the results were as Figure 15 .

[0100] The WB results showed that the administration of 50 nM DPN could significantly promote the expression level of MyoD, while after si ER beta1, the promoting effect of DPN on the expression level of MyoD was inhibited. This indicates that DPN promotes the expression of MyoD through ER beta1. Moreover, the decrease in the protein level of ER beta1 would cause the inhibition of MyoD expression.

[0101] 10. Effect of Si ER beta1 on the expression level of MYH (Muscle embryonic myosin antibody) during the differentiation period of myoblasts

[0102] Mouse C2C12 myoblasts were selected as the research object to investigate whether DPN could promote muscle differentiation, i.e., the expression level of MYH, through ER beta1. When the C2C12 cells grew to about 90% of the surface area of the large dish, the cells were digested, the cell pellet was resuspended by centrifugation, counted, and a certain volume of cell suspension was aspirated into a new proliferation medium. A certain volume of cell resuspension was aspirated into a 12-well plate containing cell slides. When the growth area of mouse myoblasts on the slide surface exceeded about 90%, they were induced to differentiate into myotubes. The complete medium in the plate was discarded, and the cells were washed 1-2 times with PBS buffer. An appropriate amount of differentiation medium was added to the culture plate to induce differentiation, and the differentiation state of myoblasts was observed at any time. The myoblasts were changed the medium once every 24 h, and after 96 h, they were fixed in paraformaldehyde for 15 minutes. Washed three times with PBS, 5 minutes each time. The cells were permeabilized with a permeabilizing solution of 0.5% Triton X-100 for 15 minutes. Washed three times with PBS, 5 minutes each time, blocked in 5% goat serum and 3% BSA solution for 1 hour. After blocking, stained with MYH and ERβ antibodies, placed in a 4°C refrigerator overnight, rewarmed for 2 h the next day, then washed three times in PBS (5 minutes each time), stained with the corresponding fluorescent secondary antibody for 2 h, and finally sealed with an anti-fluorescence quencher (containing DAPI). Observed and imaged with FV3000, and the results were as Figure 16 shown.

[0103] When the C2C12 cells grew to about 90% of the surface area of the large dish, the cells were digested, the cell pellet was resuspended by centrifugation, counted, and a certain volume of cell suspension was aspirated into a new proliferation medium. After mixing, 2 ml of cell-containing medium was added to each well of a 6-well plate. When the cells grew to 60-80%, small interfering experiments were performed on C2C12 cells using si ERbeta 1. After 8 h, the original medium was discarded, and the differentiation medium was added to continue differentiation for 72 h. Then, the cells were lysed with SDS containing protease inhibitors and phosphatase inhibitors, the corresponding volume of 5×loading was added, mixed well, and heated in a 95°C metal bath for 15 minutes. After the protein lysate returned to room temperature, it was stored in an -80°C refrigerator for Western blot experiments, and the results were as Figure 17 shown.

[0104] The results of immunofluorescence and WB showed that administration of 50 nM DPN could significantly promote the expression level of MYH, while after si ERbeta1, the promoting effect of DPN on the expression level of MYH was inhibited. This indicates that DPN exerts its promoting effect on MYH expression through ER beta1. Moreover, a decrease in the protein level of ER beta1 will lead to hindered formation of MYH. In summary, in the present invention, DPN promotes the expression level of MyoD by specifically activating ER beta1 to promote the formation of neonatal muscle fibers, thereby increasing mature muscle fibers and ultimately improving the grasping force of skeletal muscle. In addition, it can also reduce skeletal muscle inflammation. Therefore, the ER beta specific agonist DPN in the present invention has the pharmacological effect of improving muscle weakness and / or muscle atrophy caused by Duchenne muscular dystrophy.

[0105] The above are only the preferred embodiments of the present invention, and are not limited to the above embodiments. Any equivalent modifications, equivalent substitutions, and improvements made by those of ordinary skill in the art according to the disclosure of the present invention shall be included in the protection scope recorded in the claims.

Claims

1. Use of an ER beta agonist in the preparation of a medicament for treating Duchenne muscular dystrophy, characterized in that, the ER beta agonist is the ER beta small molecule agonist diarylpropionitrile, and its structural formula is as follows:

2. The use according to claim 1, characterized in that, the adult administration dose of the diarylpropionitrile is 1.21 - 4.88 mg / d.

3. The use according to claim 1, characterized in that, the medicament is for the muscle regeneration defect and / or muscle weakness symptoms of Duchenne muscular dystrophy.

4. The use according to claim 3, characterized in that, the Duchenne muscular dystrophy is caused by the absence of dystrophin.

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