Application of O-GlcNAc glycosylation in resistance of myoblasts to disuse atrophy

Abstract

The invention relates to application of O-GlcNAc glycosylation in disuse atrophy resistance of myoblasts, and further relates to a method for improving disuse atrophy resistance of myoblasts, and the method comprises the step of improving the level of O-GlcNAc glycosylation of myoblasts. In the myoblasts with waste atrophy, the O-GlcNAc glycosylation level is obviously reduced, the atrophy of the myoblasts caused by waste conditions can be inhibited by up-regulating the O-GlcNAc glycosylation, and the atrophy of the myoblasts caused by the waste conditions can be intensified by down-regulating the O-GlcNAc glycosylation, so that the myoblasts with waste atrophy can be inhibited. And a new path is provided for detection and treatment of disuse atrophy of myoblasts and muscular tissues.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of O-GlcNAc glycosylation in the resistance of myoblasts to disuse atrophy. Background Art

[0002] Disuse muscle atrophy is a condition in which muscles lose size and strength due to prolonged inactivity or reduced activity. This condition can affect multiple parts of the body, including the thighs, calves, arms, and face. The most direct manifestations of muscle atrophy are a decrease in muscle mass, size, cross-sectional area, and changes in muscle fiber type. Under disuse, changes in muscle fiber type are primarily manifested by a decrease in the number of type I slow-twitch fibers and an increase in the number of type II fast-twitch fibers and mixed fibers.

[0003] The mechanism of disuse muscular atrophy is complex, involving imbalance between muscle protein synthesis and degradation, mitochondrial changes, enhanced apoptosis, and increased oxidative stress levels. The maintenance of muscle mass depends on the balance between protein synthesis and degradation rates. The essence of disuse muscular atrophy is weakened protein synthesis and increased protein degradation. In addition, under disuse, significant changes occur in mitochondria in atrophic muscle fibers, such as increased mitochondrial DNA mutations.

[0004] O-GlcNAc glycosylation is an important protein post-translational modification (PTM) that involves the attachment of a single N-acetylglucosamine (GlcNAc) to a serine or threonine residue of a protein via a β-glycosidic bond. This modification is widely present on thousands of proteins in the nucleus, cytoplasm, cell membrane, and mitochondria, including protein kinases, transcription factors, and coactivators. O-GlcNAc glycosylation is dynamic and reversible, and it regulates a variety of key cellular processes such as cell cycle and apoptosis by affecting protein stability, subcellular localization, and activity.

[0005] Currently, there is no research on the association between disuse muscular atrophy and O-GlcNAc glycosylation. Summary of the invention

[0006] In the study, our team found that the level of O-GlcNAc glycosylation was significantly decreased in disuse-induced atrophy myoblasts, and upregulation of O-GlcNAc glycosylation could inhibit the atrophy of myoblasts caused by disuse conditions, while downregulation of O-GlcNAc glycosylation could aggravate the atrophy of myoblasts caused by disuse conditions.

[0007] Based on the above findings, the present invention provides the application of O-GlcNAc glycosylation level in detecting disuse atrophy of myoblasts.

[0008] The present invention also provides the use of an O-GlcNAc glycosylation level enhancing agent in enhancing the resistance of myoblasts to disuse atrophy.

[0009] In a specific embodiment, the O-GlcNAc glycosylation level increasing agent is an O-GlcNAc glycosylase inhibitor.

[0010] The present invention also provides a method for improving the resistance of myoblasts to disuse atrophy, comprising the step of improving the O-GlcNAc glycosylation level of the myoblasts.

[0011] In a specific embodiment, the O-GlcNAc glycosylation level of the myoblasts is increased by adding an O-GlcNAc glycosylation level-enhancing agent to the culture environment of the myoblasts.

[0012] In a specific embodiment, the O-GlcNAc glycosylation level increasing agent is an O-GlcNAc glycosylase inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Effects of Thiamcet-G and OSMI-1 on the O-GlcNAc glycosylation level of myoblasts (mean±SD, n=8). A: Western blot results; B: Changes in O-GlcNAc glycosylation level; NC: negative control. **P<0.01.

[0014] Figure 2 The effects of simulated disuse conditions on the O-GlcNAc glycosylation level and the expression of O-GlcNAc glycosyltransferase and hydrolase proteins in normal myoblasts and O-GlcNAc up-regulated / down-regulated myoblasts (mean±SD, n=8). A: Western blot results; B: Changes in O-GlcNAc glycosylation level and the expression of O-GlcNAc glycosyltransferase and hydrolase proteins. NC: negative control; OGA: O-GlcNAc hydrolase; OGT: O-GlcNAc transferase; RPM: random position meter simulated disuse conditions. **P<0.01.

[0015] Figure 3 To simulate the effect of disuse conditions on the O-GlcNAc glycosylation distribution of normal myoblasts and O-GlcNAc up-regulated / down-regulated myoblasts. NC: negative control; RPM: random position meter simulated disuse conditions.

[0016] Figure 4The effect of simulated disuse conditions on the mRNA expression of O-GlcNAc glycosyltransferase and hydrolase in normal myoblasts and O-GlcNAc up-regulated / down-regulated myoblasts (mean±SD, n=8). NC: negative control; OGA: O-GlcNAc hydrolase; OGT: O-GlcNAc transferase; RPM: random position meter simulated disuse conditions. **P<0.01.

[0017] Figure 5 To simulate the effects of disuse conditions on the O-GlcNAc glycosylation levels of myosin heavy chain, Akt and AMPK in normal myoblasts and O-GlcNAc up-regulated / down-regulated myoblasts (Western blot results). NC: negative control; MHC: myosin heavy chain; RPM: random position meter to simulate disuse conditions.

[0018] Figure 6 The effects of simulated disuse conditions on myotube diameter and myosin heavy chain in normal myoblasts and O-GlcNAc up-regulated / down-regulated myoblasts (mean±SD, n=8). A: Changes in myotube diameter; B: Changes in myosin heavy chain. NC: negative control; MHC: myosin heavy chain; OGA: O-GlcNAc hydrolase; OGT: O-GlcNAc transferase; RPM: random position meter simulated disuse conditions. **P<0.01; *P<0.05.

[0019] Figure 7 The effect of simulated disuse conditions on the OGlcNAc glycosylation levels of Akt and AMPK in normal myoblasts and O-GlcNAc up-regulated / down-regulated myoblasts (mean±SD, n=8). NC: negative control; RPM: random position meter simulated disuse conditions. *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0020] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0021] 1. Establishment of a disuse muscular atrophy model using rat myoblasts

[0022] The rat myoblasts were cultured using a random rotation machine (RPM) to establish a disuse muscular atrophy model.

[0023] O-GlcNAc down-regulated myoblasts were obtained by adding OGT inhibitor (OSMI-1) to the culture medium, and O-GlcNAc up-regulated myoblasts were obtained by adding OGA inhibitor (Thiamet-G) to the culture medium. DMSO was added to the control group. Western blot was used to detect the O-GlcNAc glycosylation level of each group. The results showed that the O-GlcNAc glycosylation level in the O-GlcNAc up-regulated group was significantly increased, and the O-GlcNAc glycosylation level in the O-GlcNAc down-regulated group was significantly decreased ( Figure 1 ), indicating that O-GlcNAc up-regulated / down-regulated myoblasts were successfully constructed.

[0024] 2. Effects of simulated disuse conditions on the O-GlcNAc glycosylation level and distribution in normal myoblasts and O-GlcNAc upregulated / downregulated myoblasts. Western blot was used to detect the O-GlcNAc glycosylation level.

[0025] The results are as follows Figure 1 As shown in the figure, after simulated disuse, the O-GlcNAc glycosylation levels of normal myoblasts and O-GlcNAc up-regulated / down-regulated myoblasts decreased significantly, but the O-GlcNAc glycosylation level of O-GlcNAc up-regulated myoblasts was significantly higher than that of normal myoblasts, and the O-GlcNAc glycosylation level of O-GlcNAc down-regulated myoblasts was significantly lower than that of normal myoblasts ( Figure 2 ).

[0026] Lectin fluorescence histochemical staining was used to detect the distribution of O-GlcNAc glycosylation.

[0027] The results are as follows Figure 2 As shown, O-GlcNAc glycosylation is distributed throughout the myotubes; after simulated disuse, the fluorescence intensity of lectin staining of normal myoblasts and O-GlcNAc upregulated / downregulated myoblasts decreased, but the fluorescence intensity of O-GlcNAc glycosylation upregulated myoblasts was significantly higher than that of normal myoblasts, and the fluorescence intensity of O-GlcNAc glycosylation downregulated myoblasts was significantly lower than that of normal myoblasts.

[0028] The above results indicate that the level of OGlcNAc glycosylation significantly decreased in the disuse atrophy of myoblasts caused by random positioning apparatus simulating disuse conditions, suggesting that O-GlcNAc glycosylation may play an important role in disuse-induced muscle atrophy of myoblasts.

[0029] 3. Effects of simulated disuse conditions on O-GlcNAc glycosyltransferase (OGT) and hydrolase (OGA) in normal myoblasts and O-GlcNAc up-regulated / down-regulated myoblasts

[0030] Real-time PCR and Western blot were used to detect the changes in mRNA and protein expression of O-GlcNAc glycosyltransferase and hydrolase.

[0031] The results showed that compared with normal myoblasts, the mRNA and protein expressions of O-GlcNAc transferase and hydrolase in O-GlcNAc up-regulated / down-regulated myoblasts did not change significantly; after simulated disuse, the mRNA and protein expressions of O-GlcNAc transferase in normal myoblasts and O-GlcNAc up-regulated / down-regulated myoblasts decreased significantly, while the mRNA and protein expressions of O-GlcNAc hydrolase did not change significantly ( Figure 2 , Figure 4 ).

[0032] This indicates that in the myoblast disuse model, changes in the level of myoblast O-GlcNAc glycosylation are related to the expression of O-GlcNAc glycosyltransferase.

[0033] 4. Effects of simulated disuse conditions on the morphology and myosin heavy chain expression of normal myoblasts and O-GlcNAc up-regulated / down-regulated myoblasts

[0034] Detect changes in the transverse diameter of myotubes. Figure 6 As shown, compared with normal myoblasts, the transverse diameter of myotubes in O-GlcNAc upregulated / downregulated myoblasts did not change significantly; after simulated disuse, the transverse diameter of myotubes in O-GlcNAc upregulated / downregulated myoblasts was significantly reduced, but the transverse diameter of myotubes in O-GlcNAc glycosylation upregulated myoblasts was significantly larger than that in normal myoblasts, and the diameter of myotubes in O-GlcNAc glycosylation downregulated myoblasts was significantly smaller than that in normal myoblasts.

[0035] Real-time PCR and Western blot were used to detect the changes in myosin heavy chain mRNA and protein expression. Figure 5 As shown, compared with normal myoblasts, there was no significant change in the globin heavy chain of O-GlcNAc up-regulated / down-regulated myoblasts; after simulated disuse, the globin heavy chain of O-GlcNAc up-regulated / down-regulated myoblasts was significantly reduced, but the globin heavy chain of O-GlcNAc glycosylation up-regulated myoblasts was significantly larger than that of normal myoblasts, and the globin heavy chain of O-GlcNAc glycosylation down-regulated myoblasts was significantly smaller than that of normal myoblasts.

[0036] The above results indicate that upregulating O-GlcNAc glycosylation can inhibit the atrophy of myoblasts induced by disuse, while downregulating O-GlcNAc glycosylation can aggravate the atrophy of myoblasts induced by disuse, which further clarifies that OGlcNAc glycosylation plays an important role in the disuse atrophy of myoblasts.

[0037] 5. Effects of simulated disuse conditions on the O-GlcNAcylation levels of Akt and AMPK in normal myoblasts and O-GlcNAc upregulated / downregulated myoblasts

[0038] O-GlcNAc-glycosylated proteins were extracted by lectin, and the O-GlcNAc-glycosylation levels of Akt and AMPK were detected by Western blot. The results showed that compared with normal myoblasts, the O-GlcNAc-glycosylation level of AMPK in O-GlcNAc-upregulated myoblasts was significantly increased, and the O-GlcNAc-glycosylation level of AMPK in O-GlcNAc-downregulated myoblasts was significantly decreased; after simulated disuse, the O-GlcNAc-glycosylation level of AMPK in normal myoblasts and O-GlcNAc-upregulated / downregulated myoblasts was significantly decreased, but the O-GlcNAc-glycosylation level of AMPK in O-GlcNAc-glycosylated upregulated myoblasts was significantly higher than that in normal myoblasts, and the O-GlcNAc-glycosylation level of AMPK in O-GlcNAc-downregulated myoblasts was significantly lower than that in normal myoblasts ( Figure 5 , Figure 7 ).

[0039] These results suggest that O-GlcNAcylation may be involved in disuse atrophy of myoblasts by regulating Akt and AMPK.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of O-GlcNAc glycosylation level in detecting disuse atrophy of myoblasts.

2. Application of O-GlcNAc glycosylation level enhancing reagents in improving the resistance of myoblasts to disuse atrophy.

3. The use according to claim 2, characterized in that: The O-GlcNAc glycosylation level enhancing agent is an O-GlcNAc glycosyl hydrolase inhibitor.

4. A method for improving the resistance of myoblasts to disuse atrophy, characterized in that: The method comprises the step of increasing the O-GlcNAc glycosylation level of the myoblasts.

5. The method according to claim 4, characterized in that The O-GlcNAc glycosylation level of the myoblasts is increased by adding an O-GlcNAc glycosylation level increasing reagent to the culture environment of the myoblasts.

6. The method according to claim 5, characterized in that The O-GlcNAc glycosylation level enhancing agent is an O-GlcNAc glycosyl hydrolase inhibitor.

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