Use of a recombinant protein for the preparation of a medicament for inhibiting muscle atrophy

Abstract

The application relates to application of a recombinant protein to preparation of a medicine for inhibiting muscle atrophy, and belongs to the field of biological medicines. The application discloses application of a recombinant protein AGGF1 to preparation of a medicine for inhibiting muscle atrophy. The recombinant AGGF1 protein can inhibit mouse muscle atrophy (peripheral nerve injury muscle atrophy and cachexia muscle atrophy) and loss of skeletal muscle cell protein content. Therefore, the recombinant AGGF1 protein can be used for preparation of an anti-muscle atrophy medicine and treatment of muscle atrophy caused by lumbar intervertebral disc herniation and cachexia.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, relates to the application of a recombinant protein in the preparation of drugs that inhibit muscle atrophy. Background Technology

[0002] Skeletal muscular atrophy (SMA) is defined as a loss of skeletal muscle mass. Its main characteristics include a reduction in the cross-sectional area of ​​skeletal muscle fibers and protein content, as well as a loss of muscle strength. SMA is a common and debilitating condition associated with many chronic diseases, including neuromuscular disorders, cancer, cachexia, diabetes, chronic obstructive pulmonary disease (COPD), heart failure, and kidney failure. SMA is associated with increased morbidity and mortality, prolonged hospital stays, and poor quality of life. Peripheral nerve injury (PNI) is a leading cause of SMA and is commonly associated with puncture wounds, direct trauma, crush injuries, falls, war, fractures, gunshot wounds, obstetric trauma, tears, and dislocations. However, treatment options for SMA are limited, and there are no effective drug therapies available. Summary of the Invention

[0003] In view of the above-mentioned defects or improvement needs of the prior art, the present invention provides a recombinant protein and its application, the purpose of which is to prepare an anti-muscle atrophy drug through the AGGF1 recombinant protein, effectively control the aggravation of muscle atrophy and clinical intervention.

[0004] According to the present invention, an application of a recombinant protein is provided for the preparation of a drug to inhibit muscle atrophy; the amino acid sequence of the recombinant protein is as follows:

[0005] (1) As shown in the amino acid sequence of SEQ ID NO: 1; or

[0006] (2) An amino acid sequence that shares 80% to 100% homology with the amino acid sequence defined by SEQ ID NO: 1 and encodes a protein with the same function; or

[0007] (3) The amino acid sequence defined by SEQ ID NO: 1 is modified by adding, deleting or replacing one or more amino acids to have an amino acid sequence with the same activity as the protein shown in the sequence SEQ ID NO: 1.

[0008] Preferably, the muscle atrophy is skeletal muscle atrophy.

[0009] Preferably, the skeletal muscle is the gastrocnemius and / or soleus muscle.

[0010] Preferably, the muscle atrophy is peripheral nerve injury-related muscle atrophy or cachectic muscle atrophy.

[0011] Preferably, the cachectic muscular atrophy is muscular atrophy secondary to wasting diseases;

[0012] Preferably, the wasting disease is a malignant tumor.

[0013] Preferably, the muscle atrophy is muscle atrophy caused by denervation.

[0014] Preferably, the muscle atrophy is caused by lumbar disc herniation.

[0015] Preferably, the muscle atrophy is leg muscle atrophy caused by lumbar disc herniation.

[0016] Preferably, the recombinant protein inhibits muscle atrophy by slowing down the ubiquitination and degradation of muscle cells.

[0017] Preferably, the recombinant protein inhibits muscle atrophy by suppressing the loss of protein content in skeletal muscle cells.

[0018] In summary, compared with the prior art, the technical solutions conceived by this invention have the following main advantages: the recombinant AGGF1 protein provided by this invention can protect muscle cells by slowing down the ubiquitination degradation pathway in skeletal muscle cells, increasing the cross-sectional area of ​​muscle bundles and related proteins, and inhibiting muscle atrophy. Therefore, the His-tagged recombinant AGGF1 protein of this invention can be used to prepare anti-muscle atrophy drugs for the treatment of muscle atrophy diseases. Attached Figure Description

[0019] Figure 1 yes Aggf1 Haploid deficiency (heterozygote) Aggf1 + / - This increases muscle atrophy in male mice 7 days after denervation. (8-week-old control and...) Aggf1 + / - Mice were used for surgery. Seven days after denervation, samples were collected from the control group and... Aggf1 + / - Tendon-to-tendon dissection of hind limb skeletal muscles (including gastrocnemius and soleus) in mice was used for weight measurement. Further dissection of the gastrocnemius and soleus muscles was performed for histological staining and Western blot analysis. (a, b) Representative images of hind limb skeletal muscles (a: gastrocnemius and b: soleus) (n = 6 / group) a: scale bar = 5 mm. (c) Analysis of dystrophin in gastrocnemius muscle sections (left, scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (middle, scale bar = 50 μm), and Sirius red staining analysis (right, scale bar = 50 μm) in immunofluorescence control group and... Aggf1 + / -The mean muscle fiber count, F4 / 80 signal, and fibrotic area of ​​denervated and quantified muscle fibers in mice after 7 days are shown below the images (n = 6 / group). (d) Immunofluorescence analysis of dystrophin in soleus muscle sections (left, scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (middle, scale bar = 50 μm), and Sirius red staining analysis (right, scale bar = 50 μm) in the control group and in Aggf1+ / - mice after 7 days of denervation and quantified mean muscle fiber count (CSA), F4 / 80 signal, and fibrotic area are shown below the images (n = 6 / group). (e) WT and Aggf1 + / - Representative Western blot analysis of MyHC, α-actin, p-p65, MuRF1, and MAFbx in mouse gastrocnemius muscle, and quantification of Western blot images (n = 6 / group). (f) WT and Aggf1+ / - Representative Western blot analysis and quantification of MyHC, α-actin, p-p65, MuRF1, and MAFbx in mouse soleus muscle (n = 6 / group). All error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significance.

[0020] Figure 2 yes Aggf1 Haploid deficiency (heterozygote) Aggf1 + / - (a) Gastrocnemius muscle atrophy was exacerbated 7 days after denervation in mice. (a) WT and glutathione levels 7 days after denervation Aggf1 + / - H&E analysis of mouse gastrocnemius muscle sections and quantification of right-sided mean fibrous CSA. Scale bar = 20 μm. (b) WT and Aggf1 + / - Western blot analysis and quantification of right-side blot images of collagen I, collagen III, p-JNK, LC-3B, cleaved Caspase 3, and cleaved PARP 1 in mouse gastrocnemius muscle (n = 6 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01. ns indicates no significance.

[0021] Figure 3 yes Aggf1 Haploid deficiency (heterozygote) Aggf1 + / - (a) Seven days after denervation in mice, soleus muscle atrophy was exacerbated. (a) WT and Aggf1 + / -H&E analysis of mouse soleus muscle sections and quantification of right-sided mean muscle fiber CSA. Scale bar = 20 μm. (b) WT and Aggf1 + / - Western blot analysis and quantification of right-side blot images of collagen I, collagen III, p-JNK, LC-3B, cleaved Caspase 3, and cleaved PARP 1 in mouse soleus muscle (n = 6 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01. ns indicates no significance.

[0022] Figure 4 yes Aggf1 Haploid deficiency (heterozygote) Aggf1 + / - Two days after denervation in male mice, some signals for muscle atrophy were activated. Eight-week-old control mice and... Aggf1 + / - Mice were used for surgery. Two days after denervation, samples were collected from the control group and... Aggf1 + / - Tendon-to-tendon dissection of hind limb skeletal muscles (including gastrocnemius and soleus) in mice was used for body weight measurement. Further dissection of the gastrocnemius and soleus muscles was performed for histological staining and Western blot analysis. (a, b) Representative photographs and quality of hind limb skeletal muscles (a: gastrocnemius and b: soleus) (n = 6 / group) a: scale bar = 5 mm. (c) Immunofluorescence analysis of dystrophin (left, scale bar = 20 μm) Immunohistochemical analysis of F4 / 80 (middle, scale bar = 50 μm) and Sirius red staining analysis of gastrocnemius muscle sections (right, scale bar = 50 μm) Control group and Aggf1 + / - Two days after denervation, the mean muscle fibers, F4 / 80 signal, and fibrotic areas of the mice are shown below the images (n = 6 / group). (d) Immunofluorescence analysis of dystrophin in soleus muscle sections (left, scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (middle, scale bar = 50 μm), and Sirius red staining analysis (right, scale bar = 50 μm) are shown below the images (n = 6 / group), compared with the control group and... Aggf1 + / - Mice were analyzed 2 days after denervation. (e) WT and Aggf1 + / - Representative proteomic blot analysis of MyHC, α-actin, p-p65, MuRF1, and MAFbx in mouse gastrocnemius muscle, and quantification of proteomic blot images around the panel (n = 6 / group). (f) WT and Aggf1 + / -Representative Western blot analysis of MyHC, α-actin, p-p65, MuRF1, and MAFbx in mouse soleus muscle, and quantification of peripanel blot images (n = 6 / group). All error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significance.

[0023] Figure 5 yes Aggf1 Haploid deficiency (heterozygote) Aggf1 + / - Two days after denervation in mice, fibrosis was induced and autophagy in the gastrocnemius muscle was inhibited. (a) Effects on WT and gluteal muscle two days after denervation. Aggf1 + / - H&E analysis was performed on gastrocnemius muscle sections from mice, and the mean fibrous subtraction angiography (CSA) of the right side was quantified. Scale bar = 20 μm. (b) WT and Aggf1 + / - Western blot analysis of collagen I, collagen III, p-JNK, and LC-3B in mouse gastrocnemius muscle and quantification of right-side blot images (n = 6 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (ns, not significant).

[0024] Figure 6 yes Aggf1 Haploid deficiency (heterozygote) Aggf1 + / - Two days after denervation in mice, fibrosis was induced and autophagy in soleus muscle was inhibited. (a) Effects on WT and [other muscle groups] two days after denervation. Aggf1 + / - H&E analysis was performed on soleus muscle sections from mice, and the mean fibrous subtraction angiography (CSA) of the right side was quantified. Scale bar = 20 μm. (b) WT and Aggf1 + / - Western blot analysis and quantification of right-side blot images of collagen I, collagen III, p-JNK, and LC-3B in mouse soleus muscle (n = 6 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (ns, not significant).

[0025] Figure 7 yes Aggf1 Haploid deficiency (heterozygote) Aggf1 + / - This increased muscle atrophy in male mice injected with mouse lung cancer cells (cachexia model). The results showed that the 8-week-old control and... Aggf1 + / - Mice were used to develop a cachexia model. Twenty-one days after injection of lung cancer cells into mice, the results were observed in control and... Aggf1 + / - The hindlimb skeletal muscles (including the gastrocnemius and soleus) of mice were separated from tendon to tendon for body weight measurement. The gastrocnemius and soleus muscles were further separated for histological staining and Western blot analysis. (a, b) Body weight changes and hindlimb skeletal muscle mass (n = 6 / group) a: Scale bar = 5 mm. (c) Analysis of dystrophin in gastrocnemius muscle sections (left, scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (middle, scale bar = 50 μm), and Sirius red staining analysis (right, scale bar = 50 μm) on immunofluorescence of mouse lung cancer cells injected 21 days after the control group and... Aggf1 + / - Comparison of mice, and quantification of mean muscle fibers, F4 / 80 signal, and fibrotic areas are shown below the images (n = 6 / group). (d) Analysis of dystrophin in soleus muscle sections (left, scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (middle, scale bar = 50 μm), and Sirius red staining analysis (right, scale bar = 50 μm) in immunofluorescence of mouse lung cancer cells injected 21 days after the control group and... Aggf1 + / - Comparisons between mice, and quantified mean muscle fibers, F4 / 80 signal, and fibrotic areas are shown below the images (n = 6 / group). (e) WT and Aggf1 + / - Representative proteomic blot analysis of MyHC, α-actin, p-p65, MuRF1, and MAFbx in mouse gastrocnemius muscle, and quantification of surrounding proteomic blot images (n = 6 / group). (f) WT and Aggf1 + / - Representative Western blot analysis of MyHC, α-actin, p-p65, MuRF1, and MAFbx in mouse soleus muscle, and quantification of surrounding Western blot images (n = 6 / group). All error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significance.

[0026] Figure 8 yes Aggf1 Haploid deficiency (heterozygote) Aggf1 + / - (a) Exacerbated gastrocnemius muscle atrophy in cachectic mice. (a) 21 days after LLC injection, WT and Aggf1 + / -H&E analysis of mouse gastrocnemius muscle sections and quantification of right-sided mean fibrous subcutaneous absorptiometry (CSA). Scale bar = 20 μm. (b) WT and Aggf1 + / - Western blot analysis and quantification of right-side blot images of collagen I, collagen III, p-JNK, LC-3B, cleaved Caspase 3, and cleaved PARP 1 in mouse gastrocnemius muscle (n = 6 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. (ns, not significant).

[0027] Figure 9 yes Aggf1 Haploid deficiency (heterozygote) Aggf1 + / - (a) Exacerbated soleus muscle atrophy in cachectic mice. (a) 21 days after LLC injection, the effects on WT and Aggf1 + / - H&E analysis of mouse soleus muscle sections and quantification of right-sided mean fibrous subcutaneous absorptiometry (CSA). Scale bar = 20 μm. (b) WT and Aggf1 + / - Western blot analysis and quantification of right-side blot images of collagen I, collagen III, p-JNK, LC-3B, cleaved Caspase 3, and cleaved PARP 1 in mouse soleus muscle (n = 6 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

[0028] Figure 10AGGF1 protein treatment can prevent the inflammatory response, fibrosis, and muscle atrophy caused by denervation. Twenty-four hours after the last injection (7 days after denervation), hind limb skeletal muscle of mice was isolated, weighed, and quantified. The gastrocnemius and soleus muscles were further isolated for paraffin sectioning. (a, b) Representative photographs of hind limb skeletal muscle and its mass are shown on the right (n = 5 / group). All error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. **P < 0.01. a: Scale bar = 5 mm. (c) Immunofluorescence analysis of dystrophin (scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (scale bar = 50 μm), and Sirius red staining analysis of gastrocnemius muscle sections (scale bar = 50 μm). Quantification of mean myofibril CSA, F4 / 80 signal, and fibrotic regions is shown below the image (n = 5 / group). Error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P<0.05, **P<0.01. (d) Immunofluorescence analysis of dystrophin (scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (scale bar = 50 μm), and Sirius red staining analysis of soleus muscle sections (scale bar = 50 μm). Mean myofiber CSA, F4 / 80 signal, and fibrotic region were quantified below the images (n = 5 / group). Error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P<0.05, **P<0.01. (e) Representative Western blot analysis and quantification of Western blot images and surrounding Western blot images of MyHC, α-actin, p-p65, MuRF1, and MAFbx in gastrocnemius muscle (n = 5 / group). (f) Western blot analysis of MyHC, α-actin, p-p65, MuRF1, and MAFbx in soleus muscle and quantification of surrounding Western blot images (n = 5 / group). All error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01.

[0029] Figure 11 AGGF1 protein treatment reduced gastrocnemius muscle atrophy in mice 7 days after denervation. (a) Hematologic and epithelial analysis (H&E) of gastrocnemius muscle sections and quantification of mean CSA of right-sided muscle fibers (n = 5 / group). (b) Western blot analysis of cleaved Caspase3, cleaved PARP1, p-JNK, LC-3B, and AGGF1 in gastrocnemius muscle and quantification of right-sided Western blot images (n = 5 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

[0030] Figure 12 AGGF1 protein treatment reduced soleus muscle atrophy in mice 7 days after denervation. (a) Hematologic and epithelial analysis (H&E) of soleus muscle sections and quantification of mean CSA of right-sided muscle fibers (n = 5 / group). (b) Western blot analysis of cleaved Caspase3, cleaved PARP1, p-JNK, LC-3B, and AGGF1 in soleus muscle and quantification of right-sided Western blot images (n = 5 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

[0031] Figure 13AGGF1 protein treatment inhibited the inflammatory response and fibrosis induced by denervation. Twelve hours after the last injection (2 days after denervation), hind limb skeletal muscle of mice was isolated, weighed, and quantified. The gastrocnemius and soleus muscles were further isolated for paraffin sections. (a, b) Representative photographs of hind limb skeletal muscle and its mass are shown on the right (n = 5 / group). Error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. ns indicates no significance. a: Scale bar = 5 mm. (c) Immunofluorescence analysis of dystrophin (scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (scale bar = 50 μm), and Sirius red staining analysis of gastrocnemius muscle sections (scale bar = 50 μm). c: Dystrophin, F4 / 80, Sirius red, scale bar = 100 μm. Quantification of mean myofibril CSA, F4 / 80 signal, and fibrotic region below the image (n = 5 / group). Error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. (d) Immunofluorescence analysis of dystrophin (scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (scale bar = 50 μm), and Sirius red staining analysis of soleus muscle sections (scale bar = 50 μm). Mean myofiber CSA, F4 / 80 signal, and fibrotic region were quantified below the images (n = 5 / group). Error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. (e) Representative Western blot analysis and quantification of MyHC, α-actin, p-p65, MuRF1, and MAFbx in the gastrocnemius muscle, and quantification of surrounding Western blot images (n = 5 / group). (f) Western blot analysis and quantification of MyHC, α-actin, p-p65, MuRF1, and MAFbx in the soleus muscle (n = 5 / group). All error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01.

[0032] Figure 14Autophagy in mouse gastrocnemius muscle was induced two days after denervation by AGGF1 protein treatment. (a) Hematologic and epithelial analysis (H&E) of gastrocnemius muscle sections and quantification of mean CSA of right-sided muscle fibers (n = 5 / group). (b) Western blot analysis of p-JNK and LC-3B in gastrocnemius muscle and quantification of right-sided Western blot images (n = 5 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01. ns, not significant.

[0033] Figure 15 Autophagy in mouse soleus muscle was induced two days after denervation by AGGF1 protein treatment. (a) Hematologic and epithelial analysis (H&E) of soleus muscle sections and quantification of mean CSA of right myofibrils (n = 5 / group). (b) Western blot analysis of p-JNK and LC-3B in soleus muscle and quantification of right-side Western blot images (n = 5 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01. ns, not significant.

[0034] Figure 16AGGF1 protein treatment prevented inflammatory responses, fibrosis, and muscle atrophy induced by injection of lung cancer cells in mice. Twenty-four hours after the last injection (21 days after lung cancer cell injection), hind limb skeletal muscle was isolated, weighed, and quantified. The gastrocnemius and soleus muscles were further isolated for paraffin sectioning. (a, b) Representative photographs of hind limb skeletal muscle and its mass are shown on the right (n = 5 / group). All error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. **P < 0.01. a: Scale bar = 5 mm. (c) Immunofluorescence analysis of dystrophin (scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (scale bar = 50 μm), and Sirius red staining analysis of gastrocnemius muscle sections (scale bar = 50 μm). Quantification of mean myofibril CSA, F4 / 80 signal, and fibrotic regions below the panel (n = 5 / group). Error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P<0.05, **P<0.01. (d) Immunofluorescence analysis of dystrophin (scale bar = 20 μm), immunohistochemical analysis of F4 / 80 (scale bar = 50 μm), and Sirius red staining analysis of soleus muscle sections (scale bar = 50 μm). Mean myofiber CSA, F4 / 80 signal, and fibrotic region were quantified below the panel (n = 5 / group). Error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P<0.05, **P<0.01. (e) Representative Western blot analysis and quantification of Western blot images of MyHC, α-actin, p-p65, MuRF1, and MAFbx in gastrocnemius muscle, and quantification of Western blot images around the panel (n = 5 / group). (f) Western blot analysis of MyHC, α-actin, p-p65, MuRF1, and MAFbx in soleus muscle and quantification of peripanel blot images (n = 5 / group). All error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01.

[0035] Figure 17After 21 days of cachexia, AGGF1 protein treatment reduced gastrocnemius muscle atrophy in mice. (a) Hematologic and epithelial analysis (H&E) of gastrocnemius muscle sections and quantification of mean CSA of right-sided muscle fibers (n = 6 / group). (b) Western blot analysis of cleaved Caspase3, cleaved PARP1, p-JNK, LC-3B, and AGGF1 in gastrocnemius muscle and quantification of right-sided Western blot images (n = 5 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

[0036] Figure 18 After 21 days of cachexia, AGGF1 protein treatment reduced soleus muscle atrophy in mice. (a) Hematologic and epithelial analysis (H&E) of soleus muscle sections and quantification of mean right-sided myofiber CSA (n = 6 / group). (b) Western blot analysis of cleaved Caspase3, cleaved PARP1, p-JNK, LC-3B, and AGGF1 in soleus muscle and quantification of right-sided Western blot images (n = 5 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

[0037] Figure 19 AGGF1 inhibits the TWEAK-Fn14 signaling pathway by interacting with TWEAK in vitro and in vivo. (a, c) Representative Co-IP analysis of AGGF1 and TWEAK. (b, d) Co-IP shows that AGGF1 knockdown enhances the interaction between TWEAK and Fn14 (n = 3 or 6 / group). Western blot analysis shows that AGGF1 inhibits the interaction between TWEAK and Fn14 in a dose-dependent manner (n = 3 or 6 / group). (e, f) Representative Western blot analysis of p-p65 in the C2C12 cell line and quantitative data shown on the right (n = 3 / group). All error bars represent mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01, ns indicates no significance.

[0038] Figure 20 shows a significant increase in AGGF1 expression in the gastrocnemius muscle after denervation. (a) Immunohistochemical analysis of AGGF1 in the gastrocnemius muscle 7 days after denervation. a: Scale bar = 100 μm. AGGF1 protein expression in A is quantified and plotted on the right. (n = 6 / group). (b) Western blot analysis of AGGF1 from the gastrocnemius muscle of mice with and without denervation, with quantified data shown on the right (n = 6 / group). (ce) Relative mRNA levels of Aggf1, Tnfrsf12 (encoding TWEAK), and Tnfrsf12a (encoding Fn14) in the denervated gastrocnemius muscle and sham-operated group 7 days after surgery in 8-week-old C57BL / 6 mice (n = 6 / group). (f) Relative protein expression of TWEAK and Fn14 in denervated gastrocnemius muscle of 8-week-old C57BL / 6 mice 7 days after surgery, in the sham-operated + EB group and the denervated + EB group (n = 6 / group). All error bars represent mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student's t-test. **P < 0.01, ns indicates no significance.

[0039] Figure 21AGGF1 protein therapy can reverse the downregulation of myoglobin in the injured leg muscles of patients with lumbar disc herniation. (a) Representative H&E staining images of leg muscle fibers from patients with lumbar disc herniation (n = 10 / group, injury group: injured leg muscles from patients with lumbar disc herniation; control group: normal leg muscles from patients with lumbar disc herniation). a: scale bar = 100 μm. (b) Western blot analysis of AGGF1 in leg muscles from patients with lumbar disc herniation and quantification of the right-side Western blot image (n = 10 / group, C: control group; I: injury group). Error bars represent mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student's t-test. **P < 0.01. (c) Representative immunohistochemical images of AGGF1 in leg muscles from patients with lumbar disc herniation and quantification of AGGF1 positive signal in sections and data on the right side (n = 10 / group). (d) Western blot analysis of Fn14 and TWEAK in leg muscles of patients with lumbar disc herniation and quantification of right-side blot images (n = 4 / group). Error bars represent mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student's t-test. **P < 0.01. Error bars represent mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student's t-test. **P < 0.01. (e) Western blot analysis of MyHC and α-actin, MuRF1 and MAFbx in leg muscle tissue isolated and cultured after AGGF1 treatment in patients with lumbar disc herniation and quantification of right-side blot images (n = 4 / group, EB: elution buffer). Error bars represent mean ± SD. Statistical significance was assessed using a one-way ANOVA test. *P < 0.05, **P < 0.01.

[0040] Figure 22 AGGF1 protein treatment increased the cross-sectional area of ​​myotubes in leg muscle damage in patients with lumbar disc herniation. (a) H&E staining and Ki67 immunostaining of leg muscle sections from patients with lumbar disc herniation treated with AGGF1 (50 μl x 50 ng / μl) or EB (50 μl) for 3 days. (b) H&E staining and Ki67 immunostaining of leg muscle sections from patients with lumbar disc herniation treated with AGGF1 (50 μl x 50 ng / μl) or EB (50 μl) for 10 days.

[0041] Figure 23AGGF1 protein treatment partially reversed the downregulation of myoprotein in the injured leg muscles of patients with lumbar disc herniation. (a) Western blot analysis of MyHC and α-actin in myotubes of leg muscles isolated and cultured for 10 days after AGGF1 treatment (50 μl x 50 ng / μl) or EB (50 μl), and quantitative Western blot images at the bottom (n = 10 / group). Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA. *P < 0.05, **P < 0.01.

[0042] Figure 24 This describes the interaction of the truncated protein AGGF1 with TWEAK in the C2C12 cell line. (a) Structure of the AGGF1 protein and the four deleted domains of the AGGF1 plasmid. (b) Immunoprecipitation Co-IP analysis of AGGF1 and TWEAK (removal of the Coiled-coil domain, OCRE domain, FHA domain, and G-patch domain).

[0043] Figure 25 This is a diagram illustrating the mechanism by which AGGF1 protein inhibits muscle atrophy. In a mouse model of muscle atrophy, Fn14 levels are upregulated. Sustained overactivation of TWEAK-Fn14 signaling leads to activation of NF-κB (p65 phosphorylation) and Caspase-3. NF-κB activation increases MuRF1 levels. MuRF1 mediates muscle protein degradation, ultimately leading to muscle atrophy. Caspase-3 mediates apoptosis, also contributing to muscle protein loss. Treatment with AGGF1 protein blocks the interaction between TWEAK and Fn14, inhibits overactivation of TWEAK / Fn14 signaling, suppresses Caspase-3 activation, reduces NF-κB (p65) activation, and downregulates MuRF1 expression. All of these factors result in inhibition of the ubiquitin-proteasome system (UPS) and caspase signaling, thereby preventing muscle protein degradation and loss. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other. Without departing from the above-described technical concept and spirit of this invention, various equivalents, substitutions, modifications, or alterations made based on ordinary technical knowledge and common practices in the art all fall within the scope of protection of this invention.

[0045] To effectively control the occurrence and clinical intervention of muscle atrophy, this invention aims to elucidate the pathogenesis of muscle atrophy and find effective intervention methods. This invention provides an in vitro purified recombinant protein, namely AGGF1 protein, for the preparation of an anti-muscle atrophy drug with therapeutic effects on muscle atrophy. The recombinant protein provided by this invention has the following nucleic acid sequence encoding it:

[0046] (1) A protein encoded by the nucleic acid sequence shown in SEQ ID No. 2; or

[0047] (2) A nucleic acid sequence that encodes a protein with 80% to 100% homology to the nucleic acid sequence defined by SEQ ID No. 2; or

[0048] (3) A sequence derived from (1) with equivalent activity by adding, deleting or replacing one or more amino acid codons of the nucleic acid sequence shown in SEQ ID No.2.

[0049] The recombinant protein, named AGGF1, is a novel gene encoded by Klippel-Trenaunay syndrome (KTS), discovered and cloned through gene linkage localization analysis. AGGF1 protein exhibits extremely high expression levels in endothelial cells, smooth muscle cells, and osteoblasts. Furthermore, high expression levels of AGGF1 can be detected in blood vessels of various tissues. Chicken embryo villus sampling experiments revealed that the recombinant AGGF1 protein, like VEGF, possesses a strong pro-angiogenic capacity. Moreover, injection of the AGGF1 naked plasmid significantly improved blood perfusion in the ischemic hindlimbs of mice and inhibited tissue necrosis in the ischemic hindlimbs.

[0050] The cloning method for the recombinant protein gene that inhibits muscle atrophy in this invention uses the forward primer shown in SEQ ID NO: 3 and the reverse primer shown in SEQ ID NO: 4 to clone the AGGF1 gene from human cDNA using PCR.

[0051] In this invention, a recombinant plasmid expressing a recombinant protein gene that inhibits muscle atrophy is provided. The AGGF1 gene is linked to a His tag sequence and inserted into the multiple cloning restriction site of the expression vector pet28a to obtain the pet28a-AGGF1 plasmid, which is the recombinant plasmid.

[0052] This invention discloses a recombinant protein that inhibits muscle atrophy, the amino acid sequence of which is as follows:

[0053] (1) As shown in the amino acid sequence of SEQ ID NO: 1; or

[0054] (2) An amino acid sequence that is 80% to 100% homologous to the amino acid sequence defined by SEQ ID NO: 1 and encodes a protein with the same function; or

[0055] (3) The amino acid sequence defined by SEQ ID NO: 1 is modified by adding, deleting or replacing one or more amino acids, and the expression product has an amino acid sequence with the same activity as the protein expressed by sequence SEQ ID NO: 1.

[0056] The preparation method of the recombinant protein that inhibits muscle atrophy in this invention includes the following steps:

[0057] (1) After linking the human AGGF1 gene with the His tag sequence, insert it into the multiple cloning restriction site of the prokaryotic expression vector pet28a to construct the recombinant plasmid pet28a-AGGF1 containing the AGGF1 gene.

[0058] (2) Transfect the recombinant plasmid described in step (1) into Escherichia coli BL21(DE3) and obtain positive cell clones with antibiotic resistance by screening for kanamycin resistance;

[0059] (3) After the positive cell clones obtained in step (2) are expanded and cultured, the recombinant protein that inhibits muscle atrophy is obtained after separation and purification.

[0060] Example 1

[0061] Preparation of recombinant AGGF1 protein: The AGGF1 protein expression plasmid pet28-AGGF1 was expressed in *E. coli* BL21(DE3). The expression strain was cultured overnight in 20 ml of kanamycin-resistant LB medium. The next day, this 20 ml medium was added to 1 L of kanamycin-resistant LB medium and cultured at 37°C with shaking for 2.5 hours. Then, 1 mM IPTG was added to induce protein expression. IPTG is a highly stable lactose analog that can inhibit lac repressor protein and induce the synthesis of β-galactosidase. This enzyme promotes lactose utilization. IPTG can be used to induce the expression of target genes regulated by the lac operon. After IPTG induction, *E. coli* continued to grow in the medium for 5.5 hours. The medium was then centrifuged at 4000 rpm, 4°C, for 10 min. If the protein was not immediately isolated, the *E. coli* pellet was stored at -20°C. The recombinant protein was purified using the QIAexpressionist high-level expression and Qiagen hexahistidine-labeled protein purification manual. Prepare 6 ml of buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM Mildazole, 0.05% Tween-20, pH 8.0), and add a protease inhibitor mixture (6 µg / ml chymostatin, 1 µg / ml E64, 2 µg / ml aprotinin, 0.5 µg / ml phosphoramidon, 1 µg / ml pepstatin A, 5 µg / ml leupeptin, 5 µg / ml antipain, 0.1 mM benzamidine (Sigma)). Add the buffer to the E. coli pellet after centrifugation in 1 L of culture medium, resuspend the cells, add 1 mg / ml lysozyme (Invitrogen) to the suspension, and incubate on ice for 30 minutes. Disrupt the suspension using the ultrasonic probe of an ultrasonic disruptor. Use 300 W power, 10 s for each sonication session, 10 s interval, for 6 cycles. The fragmented product was transferred to an EP tube and centrifuged at 12,000 rpm, 4°C, for 50 min. The supernatant was collected and stored on ice. Ni-NTA agarose beads were used to purify his-tag-tagged proteins from the supernatant. Ni-NTA metal chelate affinity chromatography has a very high affinity for biomolecules tagged with hexahistine. Ni-NTA beads were washed twice with 5 ml of EB solution, and the supernatant was added to the beads (1 ml of supernatant per bead). The mixture was incubated overnight at 4°C on a shaker. The next day, the supernatant was discarded by centrifugation. The beads were washed three times with buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 0.05% Tween-20, pH 8.0) to reduce non-specific binding proteins.The 6X-his tag-labeled protein was eluted with 1 ml of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM Mimidazole, 0.05% Tween-20, pH 8.0). The eluted protein was dialyzed twice, and then dialyzed in 1X EB at 4°C using Spectra / Por dialysis tubing (Spectrum Laboratories, Inc., USA). After dialysis, the purified protein was aliquoted and stored at -80°C, which is the recombinant protein provided by this invention.

[0062] Example 2

[0063] Preparation of AGGF1 homologous recombinant proteins: Proteins containing 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, and 80% homology in the amino acid sequence of AGGF1 recombinant proteins (homological proteins obtained through AGGF1 protein sequence truncation or mutation) were prepared. Using eukaryotically expressed pcDNA3.1-AGGF1 as a template, the full-length AGGF1 gene and various truncated fragments were amplified by PCR. The obtained truncated fragments were cloned into the pet28 protein expression plasmid after double enzyme digestion. The constructed full-length AGGF1 and each truncated plasmid were transformed into Ecoli BL21(DE3). IPTG was used to induce the expression of the truncated AGGF1 gene fusion protein, and Western blotting was used to identify the expression of the truncated AGGF1 gene fusion protein. The preparation method is the same as in Example 1.

[0064] Example 3

[0065] Application of the recombinant protein synthesized in Example 1 in the preparation of an anti-muscle atrophy drug.

[0066] Wild-type (WT) mice and Aggf1 + / - ( Aggf1 Haploid deficiency mice were divided into 4 groups: sham-operated wild-type group (Sham + WT), sham-operated... Aggf1 + / - Group (Sham+) Aggf1 + / - ), Denervation wild-type group (Denervation +WT), Denervation Aggf1 + / - Group (Denervation+) Aggf1 + / -Six mice were used in each group. Seven days after the denervation surgery, the muscle function of the mice was evaluated using pathological sections.

[0067] like Figure 1 As shown: Compared to WT mice with muscle atrophy, Aggf1 + / - Mice exhibited more severe muscle atrophy phenotype and related protein degradation, from Figure 1 It can be seen from this that Aggf1 + / - The muscle mass of muscular atrophy mice is much smaller than that of WT muscular atrophy mice.

[0068] Furthermore, in this embodiment of the invention, the sham-operated wild-type group (Sham + WT) underwent sham surgery. Aggf1 + / - Group (Sham+) Aggf1 + / - ), Denervation wild-type group (Denervation +WT), Denervation Aggf1 + / - Group (Denervation+) Aggf1 + / - Compared to WT muscular atrophy mice, Aggf1 + / - Muscle mass was significantly reduced in muscular atrophy mice. Immunofluorescence and H&E staining of dystrophin showed... Aggf1 + / - Muscular atrophy mice significantly reduced CSA in muscle fibers ( Figure 1 , Figure 2 , Figure 3 Immunohistochemical analysis of gastrocnemius and soleus muscle sections showed that... Aggf1 + / - Muscular atrophy mice showed significantly increased inflammation, as evidenced by increased F4 / 80 staining signal in these mice. Figure 1 Sirius red staining indicates Aggf1 + / - Muscular atrophy mice showed a significant increase in collagen accumulation in the denervated gastrocnemius and soleus muscles. Figure 2 and Figure 3 Furthermore, Western blot analysis indicates that... Aggf1 + / - Muscular atrophy mice exhibited exacerbated muscle protein loss in the gastrocnemius and soleus muscles induced by denervation. Furthermore, we found that 7 days after denervation, Aggf1 + / - Significant differences in MyHC and α-actin protein levels were observed in the gastrocnemius and soleus muscle tissues of muscular atrophy mice. Similar analyses indicated that... Aggf1 + / -In muscular atrophy mice, denervation for 7 days exacerbated the increase in phosphorylated p65, cleaved caspase-3, and cleaved PARP levels in the gastrocnemius muscle. Figure 1 , Figure 2 , Figure 3 Compared to the WT group 7 days after denervation, Aggf1 + / - Muscular atrophy mice showed a significant reduction in autophagy-related proteins LC-3B and phosphorylated JNK in hind limb muscle tissue. Figure 2 and Figure 3 Compared to the WT muscular atrophy mouse group 7 days after denervation, Aggf1 + / - Muscular atrophy mice showed significantly increased protein expression of the ubiquitination-associated ligase MuRF1 in hind limb muscles (gastrocnemius and soleus). Seven days after denervation, compared to the WT muscular atrophy mouse group, Aggf1 + / - There were no significant differences in the levels of another ubiquitination-associated enzyme, MAFbx, in the hind limb muscles (gastrocnemius and soleus) of muscular atrophy mice. These data suggest that... Aggf1 + / - Muscular atrophy mice exacerbate muscle atrophy in denervated mouse models by increasing inflammation, apoptosis, and fibrosis.

[0069] Example 4

[0070] Application of the recombinant protein synthesized in Example 1 in the preparation of an anti-muscle atrophy drug.

[0071] Wild-type (WT) mice and Aggf1 + / - ( Aggf1 Haploid deficiency mice were divided into 4 groups: sham-operated wild-type group (Sham + WT), sham-operated... Aggf1 + / - Group (Sham+) Aggf1 + / - ), Denervation wild-type group (Denervation +WT), Denervation Aggf1 + / - Group (Denervation+) Aggf1 + / - Six mice were used in each group. Two days after the denervation surgery, the muscle function of the mice was evaluated using pathological sections.

[0072] like Figure 4 As shown: Compared to WT mice with muscle atrophy, Aggf1 + / - Mice did not exhibit more severe muscle atrophy phenotypes and related protein degradation, from Figure 4 It can be seen from this that Aggf1+ / - The muscle weight of muscular atrophy mice was not significantly different from that of WT muscular atrophy mice, but Aggf1 + / - Mice with muscular atrophy exhibited more severe early signs of muscular atrophy, such as inflammation, apoptosis, and fibrosis in the muscle tissue.

[0073] Furthermore, in this embodiment of the invention, the sham-operated wild-type group (Sham + WT) underwent sham surgery. Aggf1 + / - Group (Sham+) Aggf1 + / - ), Denervation wild-type group (Denervation +WT), Denervation Aggf1 + / - Group (Denervation+) Aggf1 + / - Compared to WT muscular atrophy mice, Aggf1 + / - There was no significant difference in muscle mass between the muscular atrophy mice. Immunofluorescence and H&E staining of dystrophin showed... Aggf1 + / - Muscular atrophy mice did not show significant changes in myofibril CSA (Cellular Surface Antibody). Figure 4 , Figure 5 , Figure 6 Immunohistochemical analysis of gastrocnemius and soleus muscle sections showed that... Aggf1 + / - Muscular atrophy mice showed significantly increased inflammation, as evidenced by increased F4 / 80 staining signal in these mice. Figure 4 Sirius red staining indicates Aggf1 + / - Muscular atrophy mice showed a significant increase in collagen accumulation in the denervated gastrocnemius and soleus muscles. Furthermore, Western blot analysis indicated that… Aggf1 + / - Loss of muscle protein in the gastrocnemius and soleus muscles of muscular atrophy mice induced by denervation. Furthermore, we found that 2 days after denervation... Aggf1 + / - There were no significant differences in the levels of MyHC and α-actin proteins in the gastrocnemius and soleus muscles of muscular atrophy mice. Similar analyses suggest that... Aggf1 + / - Two days after denervation, muscular atrophy mice showed increased levels of phosphorylated p65, cleaved caspase-3, and cleaved PARP in the gastrocnemius and soleus muscles. Figure 4 , Figure 5 , Figure 6 Compared to the WT group 2 days after denervation, Aggf1 + / -Muscular atrophy mice showed a significant reduction in autophagy-related proteins LC-3B and phosphorylated JNK in hind limb muscle tissue. Figure 5 and Figure 6 Compared with the WT muscular atrophy mouse group 2 days after denervation, Aggf1 + / - Muscular atrophy mice showed significantly increased protein expression of the ubiquitination-associated ligase MuRF1 in hind limb muscles (gastrocnemius and soleus). Two days after denervation, compared to the WT muscular atrophy mouse group, Aggf1 + / - There were no significant differences in the levels of another ubiquitination-associated enzyme, MAFbx, in the hind limb muscles (gastrocnemius and soleus) of muscular atrophy mice. These data suggest that... Aggf1 + / - Muscular atrophy mice exacerbate muscle atrophy in denervated mouse models by increasing inflammation, apoptosis, and fibrosis.

[0074] Example 5

[0075] Application of the recombinant protein synthesized in Example 1 in the preparation of an anti-muscle atrophy drug.

[0076] Wild-type (WT) mice and Aggf1 + / - ( Aggf1 Haploid deficiency mice were divided into 4 groups: control wild-type group (PBS + WT), control group, and control group. Aggf1 + / - Group (PBS+) Aggf1 + / - ), cachexia wild-type group (LLC + WT), cachexia Aggf1 + / - Group (LLC+) Aggf1 + / - Six mice were used in each group. Twenty-one days after the cachexia model was established, muscle function in the mice was assessed using pathological sections.

[0077] like Figure 7 As shown: Comparison of cachectic muscular atrophy WT mice, Aggf1 + / - Mice exhibited more severe muscle atrophy phenotype and related protein degradation, from Figure 7 It can be seen from this that Aggf1 + / - The muscle mass of muscular atrophy mice is much smaller than that of WT muscular atrophy mice.

[0078] Furthermore, in this embodiment of the invention, the control wild-type group (PBS + WT) and the control group... Aggf1 + / - Group (PBS+) Aggf1 + / -), cachexia wild-type group (LLC + WT), cachexia Aggf1 + / - Group (LLC+) Aggf1 + / - Compared to WT muscular atrophy mice, Aggf1 + / - Muscle mass was significantly reduced in muscular atrophy mice. Immunofluorescence and H&E staining of dystrophin showed... Aggf1 + / - Muscular atrophy mice showed a significant reduction in CSA in muscle fibers. Immunohistochemical analysis of gastrocnemius and soleus muscle sections indicated that... Aggf1 + / - Muscular atrophy mice showed significantly increased inflammation, as evidenced by increased F4 / 80 staining signal in these mice. Figure 7 , Figure 8 , Figure 9 Sirius red staining indicates Aggf1 + / - In muscular atrophy mice, collagen accumulation in the gastrocnemius and soleus muscles induced by cachexia was significantly increased. Furthermore, Western blot analysis showed that... Aggf1 + / - In muscular atrophy mice, the loss of muscle protein in the gastrocnemius and soleus muscles was exacerbated. Furthermore, we found... Aggf1 + / - Significant differences were found in the levels of MyHC and α-actin proteins in the gastrocnemius and soleus muscle tissues of muscular atrophy mice. Similar analyses indicate that... Aggf1 + / - In a cachexia model, muscular atrophy mice exhibited increased levels of phosphorylated p65, cleaved caspase-3, and cleaved PARP in the gastrocnemius muscle. Figure 7 , Figure 8 , Figure 9 Compared with the cachectic WT mouse group, Aggf1 + / - Muscular atrophy mice showed a significant reduction in autophagy-related proteins LC-3B and phosphorylated JNK in hind limb muscle tissue. Figure 8 and Figure 9 Compared with the WT muscular atrophy mouse group, Aggf1 + / - Muscular atrophy mice showed significantly increased protein expression of the ubiquitination-associated ligase MuRF1 in hind limb muscles (gastrocnemius and soleus). Compared with the WT muscular atrophy mouse group, Aggf1 + / - There were no significant differences in the levels of another ubiquitination-associated enzyme, MAFbx, in the hind limb muscles (gastrocnemius and soleus) of muscular atrophy mice. These data suggest that... Aggf1 + / -Muscular atrophy mice exacerbate muscle atrophy in denervated mouse models by increasing inflammation, apoptosis, and fibrosis.

[0079] Example 6

[0080] Application of the recombinant protein synthesized in Example 1 in the preparation of an anti-muscle atrophy drug.

[0081] C57BL / 6 mice were randomly divided into three groups: sham surgery with EB (Sham + EB), denervation with EB (Denervation + EB), and denervation with recombinant AGGF1 protein (Denervation + AGGF1), with six mice in each group. EB was used as the protein eluent. Seven days after denervation, muscle function was assessed using pathological sections. Twelve hours after surgery, mice were injected with recombinant AGGF1 protein or the same dose of EB as controls via intramuscular injection once daily for one week. The protein dosage was 0.25 mg / kg.

[0082] like Figure 10 As shown: Comparing mice with muscle atrophy treated with recombinant AGGF1 protein and mice with muscle atrophy treated with control EB, AGGF1 protein significantly inhibited muscle atrophy and related protein degradation. Figure 10 As can be seen, the muscle mass of muscular atrophy mice treated with recombinant AGGF1 protein was much greater than that of muscular atrophy mice treated with control EB.

[0083] Furthermore, in this embodiment of the invention, sham surgery was performed on the EB group (Sham+EB), denervation surgery was performed on the EB group (Denervation+EB), and denervation surgery was performed on the recombinant AGGF1 protein group (Denervation+AGGF1). Compared with the control EB treatment group, the muscle mass of the muscular atrophy mice treated with recombinant AGGF1 protein was significantly improved. Immunofluorescence and H&E staining of dystrophin showed that AGGF1 protein treatment significantly delayed the reduction of myofibril CSA (muscular dystrophy). Figure 10 , Figure 11 , Figure 12 Immunohistochemical analysis of gastrocnemius and soleus muscle sections showed that AGGF1 protein treatment significantly reduced inflammation, as indicated by reduced F4 / 80 staining signal in skeletal muscle atrophy mice. Figure 10Sirius red staining showed that AGGF1 protein treatment significantly reduced collagen accumulation in the gastrocnemius and soleus muscles induced by denervation. Furthermore, Western blot analysis indicated that AGGF1 protein treatment reversed the loss of muscle protein in the gastrocnemius and soleus muscles induced by denervation at 7 days. Additionally, we found significant differences in MyHC and α-actin protein levels in the gastrocnemius and soleus muscles between the intramuscularly injected AGGF1 protein group and the EB group at 7 days post-denervation. Similar analyses showed that AGGF1 protein treatment reversed the increase in phosphorylated p65, cleaved caspase-3, and cleaved PARP levels in the gastrocnemius and soleus muscles 7 days after denervation. Figure 10 , Figure 11 , Figure 12 Compared with the EB group 7 days after denervation, AGGF1 protein treatment significantly increased the levels of autophagy-related protein LC-3B and phosphorylated JNK in hindlimb muscle tissue. Figure 11 and Figure 12 Compared with the control EB treatment 7 days after denervation, AGGF1 protein treatment in mice significantly reduced the protein expression of the ubiquitination-associated ligase MuRF1 in the hind limb muscles (gastrocnemius and soleus). Seven days after denervation, there was no significant difference in the expression of another ubiquitination-associated enzyme, MAFbx, in the hind limb muscles (gastrocnemius and soleus) of AGGF1-treated mice compared with the control EB group. These data indicate that AGGF1 protein therapy successfully alleviated muscle atrophy in a denervated mouse model by inhibiting inflammation, apoptosis, and fibrosis.

[0084] Example 7

[0085] Application of the recombinant protein synthesized in Example 1 in the preparation of an anti-muscle atrophy drug.

[0086] C57BL / 6 mice were randomly divided into three groups: sham surgery with EB (Sham + EB), denervation with EB (Denervation + EB), and denervation with recombinant AGGF1 protein (Denervation + AGGF1), with six mice in each group. EB was used as the protein eluent. Two days after denervation, muscle function was assessed using pathological sections. Twelve hours after surgery, mice were injected with recombinant AGGF1 protein or the same dose of EB as controls via intramuscular injection once daily for one week. The protein dosage was 0.25 mg / kg.

[0087] like Figure 13As shown: Comparing muscular atrophy mice treated with recombinant AGGF1 protein with those treated with control EB protein, AGGF1 protein significantly inhibited early symptoms of muscular atrophy, such as inflammation, apoptosis, and fibrosis. Furthermore, in this embodiment, sham surgery was performed on the EB group (Sham+EB), denervation surgery on the EB group (Denervation+EB), and denervation surgery on the recombinant AGGF1 protein group (Denervation+AGGF1). Compared to the control EB treatment group, there was no significant difference in muscle mass in muscular atrophy mice treated with recombinant AGGF1 protein. Immunofluorescence and H&E staining of dystrophin showed no significant change in CSA (muscular dystrophy) of myofibrils after AGGF1 protein treatment. Figure 13 , Figure 14 , Figure 15 Immunohistochemical analysis of gastrocnemius and soleus muscle sections showed that AGGF1 protein treatment significantly reduced inflammation, as indicated by reduced F4 / 80 staining signal in skeletal muscle atrophy mice. Figure 13 Sirius red staining showed that AGGF1 protein treatment significantly reduced collagen accumulation in the gastrocnemius and soleus muscles induced by denervation. Furthermore, Western blot analysis indicated no significant difference in MyHC and α-actin protein levels in the gastrocnemius and soleus muscles between the intramuscularly injected AGGF1 protein group and the EB group. Similar analyses showed that AGGF1 protein treatment reversed the increase in phosphorylated p65, cleaved caspase-3, and cleaved PARP levels in the gastrocnemius and soleus muscles 2 days after denervation. Figure 13 , Figure 14 , Figure 15 Compared with the EB group 2 days after denervation, AGGF1 protein treatment significantly increased the levels of autophagy-related protein LC-3B and phosphorylated JNK in hindlimb muscle tissue. Figure 14 and Figure 15 Compared with the control EB treatment 2 days after denervation, AGGF1 protein treatment in mice significantly reduced the protein expression of the ubiquitination-associated ligase MuRF1 in the hind limb muscles (gastrocnemius and soleus). Two days after denervation, there was no significant difference in the level of another ubiquitination-associated enzyme, MAFbx, in the hind limb muscles (gastrocnemius and soleus) of AGGF1-treated mice compared with the control EB group. These data indicate that AGGF1 protein therapy successfully alleviated muscle atrophy in a denervated mouse model by inhibiting inflammation, apoptosis, and fibrosis.

[0088] Example 8

[0089] Application of the recombinant protein synthesized in Example 1 in the preparation of anti-muscle atrophy drugs

[0090] C57BL / 6 mice were randomly divided into three groups: a control group given EB (EB+EB), a cachexia group given EB (LLC+EB), and a cachexia group given recombinant AGGF1 protein (LLC+AGGF1), with six mice in each group. LLC represents mouse lung cancer cells. Twenty-one days after LLC injection, muscle function was assessed using pathological sections. Twelve hours after surgery, mice were intraperitoneally injected with recombinant AGGF1 protein or the same dose of EB as a control, starting from day seven of LLC injection, once daily for two weeks. The protein dosage was 0.25 mg / kg.

[0091] like Figure 16 As shown: Comparing mice with muscle atrophy treated with recombinant AGGF1 protein and mice with muscle atrophy treated with control EB, AGGF1 protein significantly inhibited muscle atrophy and related protein degradation. Figure 16 As can be seen, the muscle mass of muscular atrophy mice treated with recombinant AGGF1 protein was much greater than that of muscular atrophy mice treated with control EB.

[0092] Furthermore, in this embodiment of the invention, the control group was given EB (EB+EB), the cachexia group was given EB (LLC+EB), and the cachexia group was given recombinant AGGF1 protein (LLC+AGGF1). Compared with the control group treated with EB, the muscle mass of the muscular atrophy mice treated with recombinant AGGF1 protein was significantly improved. Immunofluorescence and H&E staining of dystrophin showed that AGGF1 protein treatment significantly delayed the reduction of myofibrils CSA (cell subarachnoid fibrosis). Figure 16 , Figure 17 , Figure 18 Immunohistochemical analysis of gastrocnemius and soleus muscle sections showed that AGGF1 protein treatment significantly reduced inflammation, as indicated by reduced F4 / 80 staining signal in muscular atrophy mice. Figure 16 Sirius red staining showed that AGGF1 protein treatment significantly reduced collagen accumulation in the gastrocnemius and soleus muscles induced by cachexia. Furthermore, Western blot analysis indicated that AGGF1 protein treatment reversed the loss of muscle protein in the gastrocnemius and soleus muscles caused by denervation at 7 days. Additionally, we found significant differences in MyHC and α-actin protein levels in the gastrocnemius and soleus muscles between the intramuscularly injected AGGF1 protein group and the EB group at day 21 of cachexia. Similar analyses showed that AGGF1 protein treatment reversed the increase in phosphorylated p65, Cleaved Caspase-3, and Cleaved PARP levels in the gastrocnemius muscle during cachexia. Figure 16 , Figure 17 , Figure 18Compared with the cachectic EB group, AGGF1 protein treatment significantly increased the levels of autophagy-related proteins LC-3B and phosphorylated JNK in hind limb muscle tissue. Figure 17 and Figure 18 Compared with the control EB treatment, AGGF1 protein treatment in mice significantly reduced the protein expression of the ubiquitination-associated ligase MuRF1 in the hind limb muscles (gastrocnemius and soleus). At 21 days of cachexia modeling, there was no significant difference in the levels of another ubiquitination-associated enzyme, MAFbx, in the hind limb muscles (gastrocnemius and soleus) of AGGF1-treated mice compared with the control EB group. These data indicate that AGGF1 protein therapy successfully alleviated skeletal muscle atrophy in a cachexia mouse model by inhibiting inflammation, apoptosis, and fibrosis.

[0093] Example 9

[0094] Application of the recombinant protein synthesized in Example 1 in the preparation of an anti-muscle atrophy drug.

[0095] First, immunoprecipitation analysis revealed the TWEAK interaction of AGGF1 in C2C12 cells ( Figure 19 Similarly, in denervated muscle, endogenous AGGF1 interacts with endogenous TWEAK. Furthermore, Co-IP analysis showed that knockdown of Aggf1 expression enhanced the interaction between TWEAK and Fn14 in C2C12 cells. On the other hand, AGGF1 protein treatment in C2C12 cells significantly reduced the interaction between TWEAK and Fn14 in a dose-dependent manner. Similarly, in vivo, the interaction between TWEAK and Fn14 in denervated muscle was enhanced in Aggf1+ / - mice, and this effect was reversed by AGGF1 protein treatment.

[0096] Phosphorylated p65 is a major downstream signaling molecule in TWEAK-mediated skeletal muscle mass loss. Therefore, we determined the role of AGGF1 in NF-κB activation in C2C12 cells. C2C12 cells were treated with AGGF1 protein or EB for 24 hours under normal or starvation (serum-free) conditions. Similar to AGGF1 treatment in atrophic mice, AGGF1 treatment significantly reduced p-p65 levels in starved C2C12 cells (mimicking muscle atrophy), but not under normal conditions. Figure 19Overexpression of TWEAK in C2C12 cells eliminated the inhibitory effect of AGGF1 on p-p65 under starvation. Similar results were obtained in C2C12 cells treated with TNF-α or IFN-γ (mimicking muscle atrophy).

[0097] In summary, these data support the view that AGGF1 inhibits TWEAK-Fn14 signaling and NF-κB p-p65 activation and MuRF1 by competitively binding to TWEAK.

[0098] Example 10

[0099] Application of the recombinant protein synthesized in Example 1 in the preparation of an anti-muscle atrophy drug.

[0100] AGGF1 expression remodeling in denervated mouse muscles and leg muscles of patients with lumbar disc herniation ( Figure 20 and Figure 21 Lumbar disc herniation often leads to sciatic nerve injury and unilateral upper leg skeletal muscle atrophy. Western blot and immunostaining analyses showed significantly increased AGGF1 levels in leg muscles of patients with lumbar disc herniation and in denervated muscles of mice. Fn14, but not TWEAK, was significantly upregulated in atrophied muscles from mice and patients with muscle atrophy. AGGF1 protein treatment had no significant effect on Fn14 and TWEAK levels in denervated mouse muscles. Figure 20 and Figure 21 )

[0101] AGGF1 protein therapy can reverse the loss of myoprotein in the leg muscles of patients with lumbar disc herniation.

[0102] Compared to the uninjured side, the CSA muscle fibers in the injured leg muscle portion of the patient were significantly reduced ( Figure 21 and Figure 22 We isolated and cultured thigh muscles from injured and uninjured control legs from patients and treated the samples with AGGF1 protein or EB for 3 or 10 days. H&E staining showed that muscle fibers remained viable when the samples were treated with AGGF1 for 3 days, and AGGF1 treatment significantly improved muscle fiber viability by increasing CSA (carotene-associated fatty acid). Figure 22 Ki67 immunostaining did not detect a positive Ki67 signal in myotubules of muscle treated with AGGF1 or EB, indicating that AGGF1 does not induce muscle regeneration in atrophied muscle. Figure 22 Compared to the normal counterpart, MuRF1 and MAFbx levels were significantly upregulated on the injured side, but no difference was found in TWEAK. Figure 21AGGF1 protein treatment significantly reduced MuRF1 levels in the muscles of injured patients, but did not reduce MAFbx levels. Compared to the uninjured side, the levels of myoprotein MyHC and α-actin were significantly reduced on the injured side. Figure 21 Treatment with AGGF1 protein partially reversed the downregulation of these skeletal muscle proteins. Similar results were obtained in cultured muscle treated with AGGF1 for 10 days. Figure 23 Furthermore, H&E staining revealed severe damage to muscle fibers cultured for 10 days, while AGGF1 treatment significantly improved muscle fiber function by increasing CSA. These data suggest that AGGF1 protein treatment can alleviate muscle protein loss in human patients with skeletal muscle atrophy.

[0103] Example 11

[0104] The potential application of the truncated recombinant protein synthesized in Example 2 in the preparation of anti-muscle atrophy drugs.

[0105] Immunoprecipitation analysis showed that the truncated AGGF1 protein, lacking each of the four domains, still interacted with TWEAK in C2C12 cells. Figure 24 These data suggest that the truncated AGGF1 protein may still have an inhibitory effect on muscle atrophy.

[0106] In summary, the His-tagged recombinant AGGF1 protein of this invention can inhibit muscle atrophy in mice by antagonizing muscle protein atrophy, improving muscle function, inhibiting ubiquitination and degradation of muscle cells, and ultimately inhibiting muscle atrophy. Figure 25 Therefore, the recombinant AGGF1 protein containing the His tag of the present invention can be used to prepare drugs against muscle atrophy for the treatment of muscle atrophy-related diseases.

[0107] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of a recombinant protein, characterized in that, The recombinant protein is used for preparing a medicine for inhibiting muscle atrophy; the recombinant protein inhibits muscle atrophy by slowing down ubiquitination and degradation of muscle cells, thereby inhibiting loss of protein content in skeletal muscle cells; and the amino acid sequence of the recombinant protein is shown as SEQ ID NO:

1.

2. Use according to claim 1, wherein The muscle atrophy is skeletal muscle atrophy.

3. Use according to claim 2, wherein the compound is ###0002### The skeletal muscle is gastrocnemius and / or soleus.

4. The use according to claim 1, wherein The muscle atrophy is peripheral nerve injury muscle atrophy or cachexia muscle atrophy.

5. The use according to claim 4, wherein the compound is ###0002### The cachexia muscle atrophy is muscle atrophy secondary to a wasting disease.

6. Use according to claim 5, wherein The wasting disease is a malignant tumor.

7. The use according to claim 1, wherein The muscle atrophy is denervation-induced muscle atrophy.

8. The use according to claim 1, wherein The muscle atrophy is muscle atrophy caused by lumbar intervertebral disc herniation.

9. Use according to claim 8, wherein the compound is ###0002### The muscle atrophy is leg muscle atrophy caused by lumbar intervertebral disc herniation.

Images